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WO2001068141A2 - Dispersions de conjugues polypeptidiques - Google Patents

Dispersions de conjugues polypeptidiques Download PDF

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Publication number
WO2001068141A2
WO2001068141A2 PCT/DK2001/000182 DK0100182W WO0168141A2 WO 2001068141 A2 WO2001068141 A2 WO 2001068141A2 DK 0100182 W DK0100182 W DK 0100182W WO 0168141 A2 WO0168141 A2 WO 0168141A2
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WO
WIPO (PCT)
Prior art keywords
polypeptide
amino acid
conjugate
group
residue
Prior art date
Application number
PCT/DK2001/000182
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English (en)
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WO2001068141A8 (fr
Inventor
Christian Karsten Hansen
Original Assignee
Maxygen Aps
Maxygen Holdings Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/DK2000/000471 external-priority patent/WO2001015736A2/fr
Priority claimed from PCT/DK2000/000631 external-priority patent/WO2001036001A2/fr
Application filed by Maxygen Aps, Maxygen Holdings Ltd. filed Critical Maxygen Aps
Priority to AU42310/01A priority Critical patent/AU4231001A/en
Publication of WO2001068141A2 publication Critical patent/WO2001068141A2/fr
Publication of WO2001068141A8 publication Critical patent/WO2001068141A8/fr

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    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion

Definitions

  • the present invention relates to novel dispersions of a conjugate comprising a polypeptide variant and a non-polypeptide moiety, and pulmonary delivery of therapeutically effective amounts of said dispersion for the treatment of diseases.
  • Polypeptides including proteins, are used for a wide range of applications, includ- ing industrial uses and therapy.
  • Drawbacks commonly associated with the use of polypeptides for applications involving contact with humans or animals is that the polypeptides may give rise to an immune response, the polypeptides need to be administered frequently due to low half- lives and/or the discomfort and difficulty experienced in connection with injection of polypep- tide-based pharmaceuticals.
  • Other low molecular weight, non-proteinaceous compounds, including corticosteroids and cromolyn sodium have been administered systemically via pulmonary absorption.
  • the more distal levels of branching form the transitional and respiratory zones, comprised of respiratory bronchioles, alveolar ducts, and alveoli, where gas exchange and pulmonary absorption occur.
  • the respiratory zone in contrast to the conducting zone, is non-ciliated and comprised of a single cell layer.
  • each alveolus is in intimate association with numerous blood-bearing capillaries bringing oxygen-depleted blood from distal body tissues.
  • This air-blood barrier is comprised of the alveolar epithelium, the capillary endo- thelium, and the lymph-filled interstitial space separating these two cell layers.
  • the mean thick- ness of the air-blood barrier in humans is 2.2 micrometers [Gehr et al. (1978), Resp. Physiol., vol. 32, pp. 121-140], while the alveolar epithelium itself can be as thin as 0.05 micrometers.
  • adjacent cells overlap and are bound by non-leaky tight junctions, which, in conjunction with the non-leaky single cell layer comprising the capillary endothelium, limits the movement of fluids, cells, salts, proteins, and numerous other macromolecules from the blood and intercellular spaces into the lumen of the alveoli. Most molecules, including proteins and polypeptides, must be actively or passively transported across this barrier in the absence of lung injury.
  • alveolar macrophages migrate from the blood across the air-blood barrier. These macrophages can phagocytose inhaled particles that reach the alveoli. These phagocytes may then migrate back into the lymphatic channels or to the base of the bronchial tree to be swept out of the lung by the ciliary rejection current. Additionally, other cell types, such as neutro- phils and lymphocytes, can move into the alveoli from the blood to combat infection.
  • Pulmonary delivery of pharmaceutical compositions intended for systemic administration to patients requires the deposition of a therapeutically active substance from a res- ervoir containing that active ingredient to an area of the patient's lungs capable transferring that substance, either actively or passively, to the patient's blood.
  • the deposition is best accomplished by propelling a preparation comprised of an aqueous aerosol or solid particles containing the active ingredient into the lungs of the patient.
  • Particle size is an important consideration in achieving particle deposition in the distal lung regions. Porush et al., reported that to reach the alveoli, small particles should be 0.5 micrometers to 7 micrometers in diameter [(I960) Amer. Pharm. Assoc. Sci. Ed., vol. 49, p. 70]. Later, the preferred particle size for such deposition was reported to be less than 5 micrometers in diameter [Newman et al., (1983) Thorax, vol. 38, p. 881].
  • Utsumi et al. PCT Patent Application No. WO 91/16038) disclosed the preparation of an aero- sol composition comprised of solid, micronized human interferon or interleukin for pulmonary administration. In their preparation, the particles ranged from 0.5 micrometers to 10 micrometers in median diameter.
  • Devices capable of depositing aerosolized formulations in the alveoli of a patient include nebulizers, metered dose inhalers, and powder inhalers. Other devices suitable for di- recting the pulmonary administration of dispersions are also known in the art. All such devices require the use of formulations suitable for the dispensing of an aerosol.
  • aerosols can be comprised of either solutions (both aqueous and non-aqueous) or solid particles.
  • Nebulizers are useful in producing aerosols from solutions, while metered dose inhalers, dry powder inhalers, etc. are effective in generating small particle aerosols.
  • each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in protein-based drug therapy.
  • Attempts have been made to reduce the immunogenicity of polypeptides and/or increase the half-life thereof.
  • One of the most widespread strategies has been to shield epitopes of the polypeptide (which give rise to the undesired immune response) with polymer molecules, such as polyethylene glycol (PEG), conjugated to the polypeptide.
  • PEG polyethylene glycol
  • modified polypeptide with reduced allergenicity comprise a parent polypeptide with a molecular weight in the range of 10-100 KDa conjugated to a polymer with a molecular weight in the range of 1-60 KDa.
  • polypeptide to be modified may be a variant of a parent enzyme that has additional attachment groups, such as amino groups, in comparison to the parent enzyme.
  • WO 98/35026 discloses polypeptide-polymer conjugates having added and/or removed one or more selected attachment groups for coupling polymer molecules on the surface of the three dimensional structure of the polypeptide.
  • site-directed mutagenesis it is suggested to add attachment groups for the polymer molecules at predetermined locations of the polypeptide surface in an attempt to increase the number of polymer molecules, which may be attached in comparison to the corresponding parent polypeptide.
  • site-directed mutagenesis it is suggested to remove attachment groups at or close to the active site of the polypeptide allegedly to avoid too extensive PEGylation near the active site, which may lead to decreased activity of the polypeptide.
  • WO 98/48837 relates to single-chain antigen-binding polypeptide-polyalkylene oxide conjugates with a reduced antigenicity and an increased half-life in the blood stream.
  • the single chain antigen-binding polypeptide to be modified may include one or more inserted Cys or Lys capable of polyalkylene oxide conjugation at certain predetermined sites. Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems, 9(3,4): 249-304 (1992) is a review article disclosing the state of the art in relation to the uses and properties of PEG-linked polypeptides.
  • WO 96/12505 discloses conjugates of a polypeptide with a low molecular weight lipophilic compound, which are reported to have improved pharmacological properties.
  • WO 94/13322 discloses a process for the preparation of a conjugate between a polymer and a first substance having a biological activity mediated by a domain thereof, wherein, during conjugation, the domain of the first substance is protected by a second substance which is removed after conjugation has taken place. It is stated that by using the method the biological activity of the first substance is fully preserved in contrast to the conventional conjugation processes, which normally lead to polymer conjugates with reduced biological activity.
  • WO 93/1 189 relates to a method of preparing proteolytic enzyme-PEG adducts in which the proteolytic enzyme is linked to a macromolecularized inhibitor when reacted with PEG so as to block the active site of the enzyme and thereby preventing that PEG is bound at or near the active site.
  • WO 97/11957 discloses a process for improving the in vivo function of a polypep- tide, in particular factor NIH, by shielding exposed targets of said polypeptide, in which method the polypeptide is immobilized by interaction with a group-specific adsorbent carrying ligands manufactured by organic-chemical synthesis, a biocompatible polymer is activated and conjugated to the immobilized polypeptide and the conjugate is eluted from the adsorbent.
  • WO 97/47751 discloses various forms for modification of a D ⁇ Ase, e.g. by con- jugation to a polymer, a sugar moiety or an organic derivatizing agent.
  • WO 9420069 discloses dispersions of particles consisting of a conjugate of human Met-G-CSF and polyethylene glycol. It is speculated that other dispersions comprising PEGylated versions of SCF, EPO, GM-CSF, CSF-1, IL-1, IL-2 and IL-6 may be prepared. However, no variants of these proteins with added or removed attachment sites for polyethylene glycol are disclosed.
  • US 5354934 discloses pulmonary delivery of erythropoietin, but does not disclose dispersions comprising conjugates comprising variants having added or removed attachment sites and polyethylene glycol. There is, however, still a need for needle-free methods for administering therapeutically effective amounts of proteins to mammals, including human beings.
  • the conjugates described herein form dispersions without undergoing aggregation (or at least they have a reduced tendency to form aggregates compared to the wild-type protein).
  • An additional advantage is that such conjugates have a higher bioavailability deriving from their lower molecular weight/size in contrast to the aggregation products that result from formulation of their non-conjugated counterparts.
  • the conjugates have reduced immunogenecity due to lower or no formation of aggregations products.
  • the conjugates disclosed herein may have extended half -lives.
  • pulmonary administration can be accomplished by directing a stream of a therapeutically effective amount of the dispersion into the respiratory tract of the inhaling mammal.
  • substantial amounts of the conjugate of the dispersion are thereby deposited in the lung and absorbed from the lung into the bloodstream, resulting in prolonged periods of elevated levels of the active conjugate in the lungs and or the circulatory system, and/or in therapy with a conjugate with lower immunogenicity than the corresponding non-conjugated polypeptide.
  • Pulmonary administration of the dispersion thus provides an effective non-invasive alternative to the systemic and local pulmonary delivery of long half -life and/or reduced immunogenicity polypeptide-comprising drugs.
  • the present invention relates to a dispersion of particles containing a conjugate comprising at least one non-polypeptide moiety covalently attached to a polypeptide variant, wherein the amino acid sequence of said polypeptide variant differs from that of the corresponding wild-type human polypeptide in that at least one amino acid residue comprising an attachment group for said non-polypeptide moiety has been introduced and/or removed.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the dispersion of the invention and a pharmaceutically acceptable carrier.
  • the present invention also relates to methods for pulmonary administration of such conjugates and dispersions as well as to such dispersion for use as a medicament.
  • the present invention relates to a method for administering a therapeutically effective amount of the conjugate to the lung and/or the circulatory system of a mammal comprising administering to the lung(s) a dispersion of particles of the invention, optionally in a pharmaceutically acceptable carrier.
  • the present invention relates to a method for pulmonary administration of the conjugate, the method comprising depositing an effective amount of said conjugate, optionally in a pharmaceutically acceptable carrier, in the lungs of a mammal in need thereof by providing means for inhaling the dispersion of the invention.
  • the present invention relates to a method for delivering the conjugate into blood circulation of a patient, said method comprising providing from 1 to 10 individual dosages of the dispersion of the invention in a gas stream to be inhaled into the alveolar region of the patient's lungs, wherein the particles of the dispersion dissolve in the fluid lining of the alveolar regions of the lungs and the conjugate is systemically absorbed into patient blood circulation.
  • a total of 0.1 to 25 mg of the conjugate is delivered in total in this manner, more preferably, 0.5 to 10 mg.
  • Such delivery may be accomplished through the use of a mechanical device.
  • Examples of mechanical devices useful in accordance with the methods of the invention include metered dose inhalers, powder inhalers, and nebulizers, par- ticularly jet nebulizers and ultrasonic nebulizers to which devices this invention is also directed.
  • dispersion and "a dispersion of particles” shall have the meaning commonly accepted in the art.
  • conjugate is intended to indicate a heterogeneous molecule formed by the covalent attachment of one or more polypeptides to one or more non-polypeptide moieties, such as polymer molecules, lipophilic compounds, sugar moieties or organic derivatizing agents (as defined below).
  • the conjugate is soluble at relevant concentrations and conditions, i.e. soluble in physiological fluids such as blood.
  • conjugated polypeptide conjugates invention include glycosylated and/or PEGylated polypeptides.
  • covalent attachment means that the polypeptide and the non- polypeptide moiety are either directly covalently joined to one another, or else are indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties.
  • non-conjugated polypeptide may be used about the polypeptide part of the conjugate.
  • non-polypeptide moiety means a molecule that is capable of conjugating to an attachment group of the polypeptide.
  • Preferred examples of such molecules include polymer molecules, sugar moieties, lipophilic compounds, or organic deri- vatizing agents.
  • the non-polypeptide moiety is linked to the polypeptide part of the conjugate through an attachment group of the polypeptide.
  • the non-polypeptide moiety may be directly co- valently joined to the attachment group or it may be indirectly covalently joined to the attachment group through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties.
  • polymer molecule is defined as a molecule formed by covalent linkage of two or more monomers, wherein none of the monomers is an amino acid residue, except where the polymer is human albumin or another abundant plasma protein.
  • polymer may be used interchangeably with the term “polymer molecule”. Except where the number of polymer molecule(s) in the conjugate is expressly indicated every reference to "a polymer”, “a polymer molecule”, “the polymer” or “the polymer molecule” contained in a conjugate or otherwise used in the present invention shall be a reference to one or more polymer molecule(s) in the conjugate.
  • Carbohydrate molecules attached by in vivo glycosylation are referred to herein as a "sugar moiety".
  • sugar moieties such as polymer molecule(s) or sugar moieties in the conjugate
  • every reference to "a non-polypeptide moiety" contained in a conjugate or otherwise used in the present invention shall be a reference to one or more non-polypeptide moieties, such as polymer molecule(s) or sugar moieties.
  • attachment group is intended to indicate an amino acid residue group capable of coupling to a non-polypeptide moiety such as a polymer molecule or a sugar moiety suitable for use in the construction of the conjugate.
  • a non-polypeptide moiety such as a polymer molecule or a sugar moiety suitable for use in the construction of the conjugate.
  • attachment group is used in an unconventional way to indicate one, two or all three of the amino acid residues constituting an N- glycosylation site (with the sequence N-X-S/T, wherein X is any amino acid residue except proline, N is asparagine and S/T is either serine or threonine).
  • N-X-S/T wherein X is any amino acid residue except proline
  • N is asparagine
  • S/T is either serine or threonine
  • amino acid residue com- prising an attachment group for the non-polypeptide moiety as used in connection with alterations of the amino acid sequence of the parent polypeptide is to be understood as one, two or all of the amino acid residues constituting an N-glycosylation site is/are to be altered in such a manner that either a functional N-glycosylation site is introduced into the amino acid sequence or removed from said sequence.
  • amino acid names and atom names e.g. CA, CB, NZ
  • N, O, C, etc are used as defined by the Protein DataBank (PDB) which are based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residue names, atom names etc.), Eur. J. Biochem., 138, 9-37 (1984) together with their correc- tions in Eur. J. Biochem., 152, 1 (1985).
  • PDB Protein DataBank
  • amino acid residue is intended to indicate an amino acid residue contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (lie or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Tip or W), and tyrosine (Tyr or Y) residues.
  • C17 indicates that position 17 in a given amino acid sequence is occupied by a cysteine residue.
  • C17S indicates that a cysteine residue in position 17 has been replaced with a serine residue.
  • Alternative substitutions are indicated with a "/”, e.g. K32D E means that a lysine in position 32 is substituted with either aspartic acid or glutamic acid.
  • Multiple substitutions are indicated with a "+”, e.g.
  • K143N+N145S/T means that a lysine residue in position 143 is substituted with an asparagine residue and that an asparagine residue in position 145 is sub- stituted with either a serine or a threonine residue.
  • the insertion of an additional amino acid residue, such as insertion of an alanine residue after G124 is indicated by G124GA.
  • a deletion of an amino acid residue is indicated by an asterix. For example, deletion of a glycine in position 124 is indicated by G124*.
  • the term "differs from” as used in connection with specific mutations is intended to allow for additional differences being present apart from the specified amino acid difference.
  • the wild-type human polypeptide may comprise other substitutions that are not related to introduction and/or removal of such amino acid residues.
  • the amino acid alterations disclosed herein aimed at remov- ing and/or introducing attachment sites for the non-polypeptide moiety it will be understood that the amino acid sequence of the polypeptide may, if desired, contain other alterations that need not be related to introduction or removal of attachment sites, i.e. other substitutions, insertions or deletions.
  • substitutions may, for example, include truncation of the N- and/or C-terminus by one or more amino acid residues, or addition of one or more extra residues at the N- and/or C- terminus, e.g. addition of a methionine residue at the N-terminus as well as "conservative amino acid substitutions", i.e. substitutions performed within groups of amino acids with similar characteristics, e.g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids. Preferred substitutions in the present invention may in particular be selected from the conservative substitution groups listed in the table below.
  • nucleotide sequence is intended to indicate a consecutive stretch of two or more nucleotide molecules.
  • the nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
  • an IFNG variant shall mean a polypeptide with an amino acid sequence which differs from that of human BFNG (SEQ ID NO:l), but which retains some or all of the therapeutically relevant characteristics of human IFNG.
  • protein sequence family is used in its conventional meaning, i.e. to indicate a group of polypeptides, which are related to each other by having an amino acid se- quence, which exhibits a sufficient degree of identity to a given amino acid sequence to allow alignment of the sequences, e.g. using the CLUSTALW program.
  • the degree of identity should preferably, be at least 75%, e.g. at least 80%, such as 85%, more preferably at least 90%, e.g. at least 95%, such as at least 97%.
  • the protein sequence family may also be referred to herein as the "polypeptide sequence family”. Sequence families are available, e.g. from the PFAM fami- lies, version 4.0, or may be prepared by use of a suitable computer program such as
  • PCR polymerase chain reaction
  • PCR generally refers to a method for amplification of a desired nucleotide sequence in vitro, as described, for example, in US 4,683,195.
  • the PCR method involves repeated cycles of primer extension synthesis, using oligonucleotide primers capable of hybridising preferentially to a template nucleic acid.
  • Cell Cell
  • host cell cell
  • cell line cell culture
  • Transformation and “transfection” are used interchangeably to refer to the process of introducing DNA into a cell.
  • operably linked refers to the covalent joining of two or more nucleotide sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences can be performed.
  • the nucleotide sequence encoding a presequence or secretory leader is operably linked to a nucleotide sequence for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide: a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • operably linked means that the nu- cleotide sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used, in conjunction with standard recombinant DNA methods.
  • introduce is primarily intended to mean substitution of an existing amino acid residue, but may also mean insertion of an additional amino acid residue.
  • the term "remove” is primarily intended to mean substitution of the amino acid residue to be removed for another amino acid residue, but may also mean deletion (without substitution) of the amino acid residue to be removed.
  • immunogenicity as used in connection with a given substance is in- tended to indicate the ability of the substance to induce a response from the human immune system.
  • the immune response may be a cell or antibody mediated response (see, e.g., Roitt: Essential Immunology (8 th Edition, Blackwell) for further definition of immunogenicity).
  • reduced immunogenicity is intended to indicate that the conjugate of the dispersion of the present invention gives rise to a measurably lower immune response than a reference molecule.
  • the term "functional in vivo half -life” is the time in which 50% of a biological function of interest of the drug is retained, when tested in vivo, e.g. in the cause of a treatment of a mammal with the complex.
  • the term "serum half -life” is used in its normal meaning, i.e. the time in which half of the complex circulate in the plasma or bloodstream prior to being cleared.
  • Alternatively used terms include "plasma half-life", “circulating half -life”, “serum clearance”, “plasma clearance” and "clearance half -life”.
  • the serum half- life may be used as an indication of the improved property of the complex of the invention.
  • parent polypeptide or "parent protein” is intended to indicate the molecule to be modified in accordance with the present invention.
  • the parent polypeptide or parent protein is normally the wild-type human polypeptide or protein.
  • the conjugate contained in the dispersion of the invention may have reduced immunogenicity, and/or enhanced in vivo, and/or serum half -life and/or a reduced tendency to from aggregates as compared to the corresponding parent or variant polypeptide. Furthermore, the conjugate may have at least part of the therapeutically relevant properties and or activities, qualitatively and/or quantitatively, of the corresponding parent wild-type human polypeptide.
  • the conjugate has improved properties as compared to a conjugate comprising the corresponding polypeptide, or variant thereof, not hav- ing introduced or removed attachment groups for non-polypeptide moieties, in terms of receptor-binding/signalling, therapeutic efficacy, in vivo and/or serum half-life, and/or immunogenicity, when the conjugates compared comprise similar non-polypeptide moieties linked to the polypeptide moieties of the conjugates by similar chemical linkages.
  • the particles of said dispersion have aerodynamic properties in the lung corresponding to particles having a mass median diameter of less than 10 micrometers, preferably of between 0.5 and 5 micrometers, most preferably of between 1.5 and 3.5 micrometers. Preferably, the particles have a density of about 1 g/ cm 2 and the above-mentioned mass median di- ameters.
  • the particles of the dispersion may be in aqueous or non-aqueous form, e.g. in the form of a powder, optionally in a pharmaceutically acceptable carrier.
  • the conjugate of the dispersion of the invention exhibiting part or all of the activity of the parent polypeptide is administered at a dose approximately paralleling that employed in therapy with known commercial preparations of the parent polypeptide or more to compen- sate for only partial up-take from the lungs to the circulatory system.
  • the exact dose to be administered depends on the circumstances. Normally, the dose should be capable of preventing or lessening the severity or spread of the condition or indication being treated.
  • an effective amount of a dispersion of the invention depends, inter alia, upon the disease, the dose, the administration schedule, whether the dispersion com- prising the conjugate is administered alone or in conjunction with other therapeutic agents, the serum half -life of the compositions, and the general health of the patient.
  • the dispersion of the invention is preferably administered in a composition in- eluding a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient means a carrier or excipient that does not cause any untoward effects in patients to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art.
  • the conjugate of the dispersion of the invention can be formulated into pharmaceutical compositions by well-known methods. Suitable formulations are described by Remington's Pharmaceutical Sciences by E.W.Martin.
  • composition of the dispersion containing the conjugate may be formulated in a variety of forms, including liquid, lyophilized, powder or any other suitable form.
  • the preferred form will depend upon the particular indication being treated and will be apparent to one skilled in the art.
  • each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in conjugate therapy.
  • Each formulation is also typically specific to the conjugate to be delivered as a dispersion. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
  • Conjugate formulations suitable for use with a nebulizer will typically comprise conjugate dissolved in water at a concentration of, e.g., about 0.01 to 25 mg of conjugate per ml of solution, preferably about 0.1 to 10 mg/ml.
  • the formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure), and/or human serum albumin ranging in concentration from 0.1 to 10 mg ml.
  • buffers which may be used are sodium acetate, citrate and glycine.
  • the buffer will have a composition and molarity suitable to adjust the solution to a pH in the range of 3 to 9.
  • buffer molarities of from 1 mM to 50 mM are suitable for this purpose.
  • sugars which can be utilized are lactose, maltose, mannitol, sorbitol, trehalose, and xylose, usually in amounts ranging from 1% to 10% by weight of the formulation.
  • the nebulizer formulation may also contain a surfactant to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.
  • a surfactant to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.
  • Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty acid esters. Amounts will generally range between 0.001% and 4% by weight of the formulation.
  • An especially preferred surfactant for purposes of this invention is polyoxyethylene sorbitan monooleate.
  • nebulizers suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo., the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado, and the AERx pulmonary drug delivery system manufactured by Aradigm Corporation, Hayward, California. Dry Formulation For Metered Dose Inhalers and Powder Inhalers
  • Conjugate formulations for use with a metered dose inhaler device will generally comprise a finely divided powder.
  • This powder may be produced by lyophilizing and then milling a liquid conjugate formulation and may also contain a stabilizer such as human serum al- bumin (HSA). Typically, more than 0.5% (w/w) HSA is added.
  • HSA human serum al- bumin
  • one or more sugars or sugar alcohols may be added to the preparation if necessary. Examples include lactose maltose, mannitol, sorbitol, sorbitose, trehalose, xylitol, and xylose.
  • the amount added to the formulation can range from about 0.01 to 200% (w/w), preferably from approximately 1 to 50%, of the conjugate present. Such formulations are then lyophilized and milled to the desired particle size.
  • the properly sized particles are then suspended in a propellant with the aid of a surfactant.
  • the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroefhanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof.
  • Suitable surfactants include sorbitan tri- oleate and soya lecithin. Oleic acid may also be useful as a surfactant. This mixture is then loaded into the delivery device.
  • Such conjugate formulations for powder inhalers will comprise a finely divided dry powder containing conjugate and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50% to 90% by weight of the formulation.
  • the particles of the powder shall have aerodynamic properties in the lung. This typically corresponds to particles with a density of about 1 g/cm having a median diameter less than 10 micrometers, preferably between 0.5 and 5 micrometers, most preferably of between 1.5 and 3.5 micrometers.
  • the powders for these devices may be generated and/or delivered by methods dis- closed in US 5997848, US 5993783, US 5985248, US 5976574, US 5922354, US 5785049 and US 55654007 which are hereby incorporated by reference.
  • Mechanical devices for the administration of the dispersion of the invention may be generated and/or delivered by methods dis- closed in US 5997848, US 5993783, US 5985248, US 5976574, US 5922354, US 5785049 and US 55654007 which are hereby incorporated by reference.
  • the pharmaceutical composition containing the conjugate may be administered by a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those of skill in the art.
  • Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn JJ nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Nentolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts; the "standing cloud” device of Inhale Therapeutic Systems, Inc., San Carlos, California; the AIR inhaler manufactured by Alkermes, Cambridge, Massachusetts; and the AERx pulmonary drug delivery system manufactured by Aradigm Corporation, Hayward, California.
  • the conjugate contained in the dispersion of the invention is the result of a generally new strategy for developing improved proteins. More specifically, by removing or introducing an amino acid residue comprising an attachment group for the non-polypeptide moiety it is possible to specifically adapt the polypeptide so as to make the molecule more susceptible to conjugation to the non-polypeptide moiety of choice, to optimize the conjugation pattern (e.g. to ensure an optimal distribution of non-polypeptide moieties on the surface of the variant molecule) and thereby obtain a new conjugate molecule, which has the activity, or a similar activity, of the parent polypeptide and in addition one or more improved properties as compared to the parent polypeptide.
  • the conjugates disclosed herein have a higher specific activity as compared to the corresponding conjugates comprising at least one non-polypeptide moiety, such as at least one PEG group, covalently attached to a wild-type human polypeptide.
  • the specific activity of the conjugates disclosed herein is at least 10% higher than the specific activity of the corresponding conjugate compris- ing at least one non-polypeptide moiety covalently attached to a wild-type human polypeptide, such as at least 20%, e.g. at least 30%, more preferably at least 40%, such as at least 50%, e.g. at least 60%, most preferably at least 70%, such as at least 80%, e.g. at least 90%.
  • more than one amino acid residue of the polypeptide is altered, e.g. the alteration embraces removal as well as introduction of amino acid residues comprising attachment sites for the non-polypeptide moiety of choice.
  • the polypeptide variant may comprise other substitutions which are not related to introduction and/or removal of amino acid residues comprising an attachment group for the non-polypeptide moiety.
  • the variant of the may differ from the corresponding wild-type human polypeptide by 1-15 amino acid residues which may comprise, and/or be comprised in, attachment groups for the non-polypeptide moiety, be it by way of introduction or removal of attachment groups, optionally both by removal and introduction of attachment groups.
  • attachment groups for linking to non-polypeptide polymers such as lysine and cysteine residues, may be removed or introduced.
  • attachment groups for non-polypeptide moieties such as sugar residues having the Asn-glycosylation consensus attachment site of Asn-X-Ser/Thr-Y, where X and Y are any amino acid residue except Pro, may be introduced or removed.
  • the sequence of the variant may differ from the corresponding polypeptide by way of a sequence difference in an epitope.
  • the position of the polypeptide to be modified is preferably located at the surface of the polypeptide, and more preferably occupied by an amino acid residue which has more than 25% of its side chain exposed to the solvent, preferably more than 50% of its side chain exposed to the solvent. Such positions may be identified on the basis of an analysis of a 3D structure of the human wild-type polypeptide as described in the Examples herein.
  • amino acid residue comprising an attachment group for a non-polypeptide moiety is selected on the basis of the nature of the non-polypeptide moiety part of choice and, in most instances, on the basis of the method in which conjugation between the polypeptide and the non-polypeptide moiety is to be achieved.
  • the non-polypeptide moiety is a polymer molecule such as a polyethylene glycol or polyalkylene oxide derived molecule
  • amino acid residues capable of functioning as an attachment group may be selected from the group consisting of lysine, cysteine, aspartic acid, glutamic acid and arginine.
  • a suitable activated molecule is, e.g., mPEG-SPA from Shearwaters Polymers or oxycarbonyl-oxy-N-dicarboxyimide-PEG (US 5,122,614).
  • the amino acid residue comprising an attachment group for a non-polypeptide moiety is preferably selected so that the modification in question does not significantly alter or impair the function of the molecule (e.g. the catalytic activity, the ability to bind to receptors, etc.).
  • the conjugate may have a molecular weight above 60 kD, more preferably above 67 kD or above 70kDa, up to about 200 kD, more preferably up to about 100 kD, e.g., as measured by SDS-PAGE according to Laemmli, U.K. , Nature Vol 227 (1970), p680-85.
  • polypeptide part of the conjugate will typically have an amino acid sequence having more than 90% identity with the amino acid sequence of the parent wild-type human polypeptide, preferably more than 95%, such as more than 96%.
  • polypeptide part of the conjugate will typically have an amino acid sequence having more than 97% identity with the amino acid sequence of the parent wild-type human polypeptide, such as more than 98%, e.g. more than 99% identity.
  • Amino acid sequence homology/identity is conveniently determined from aligned sequences, using e.g. the ClustalW program, version 1.8, June 1999, using default parameters (Thompson et al., 1994, ClustalW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research, 22: 4673-4680) or from the PFAM families database version 4.0 (http://pfam.wustl.edu/) (Nucleic Acids Res. 1999 Jan 1; 27(l):260-2) by use of GENEDOC version 2.5 (Nicholas, K.B., Nicholas H.B. Jr., and Deerfield, D.W. ⁇ . 1997 GeneDoc: Analysis and Visualization of Genetic Variation, EMBNEW.NEWS 4: 14; Nicholas, K.B. and Nicholas H.B. Jr. 1997 GeneDoc: Analysis and Visualization of Genetic Variation).
  • the total number of amino acid residues to be altered will typically not exceed 15.
  • the polypeptide part of the conjugate differs in 1-15 amino acid residues from the parent polypeptide sequence such as in 1-8 or 2-8 amino acid residues, e.g. in 1-5 or 2-5 amino acid residue from the parent polypeptide sequence.
  • the polypeptide variant comprises an amino acid sequence which differs from the parent polypeptide sequence in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.
  • the polypeptide part of the conjugate exhibiting parent polypeptide activity may comprise an amino acid sequence, which is a hybrid molecule between the parent polypeptide and another homologous polypeptide optionally containing one or more additional substitutions introduced into the hybrid molecule.
  • Such a hybrid molecule may contain an amino acid sequence, which differs in more than 15 amino acid residues from the amino acid sequence of the parent polypeptide.
  • one difference between the amino acid sequence of the polypeptide variant and the parent polypeptide is that at least one and preferably more, e.g. 1- 15, amino acid residues comprising an attachment group for the non-polypeptide moiety has been introduced, preferably by substitution, into the amino acid sequence.
  • the polypeptide part is boosted or otherwise altered in the content of the specific amino acid residues to which the non-polypeptide moiety of choice binds whereby a more efficient, specific and/or extensive conjugation is achieved.
  • the total number of amino acid residues comprising an attachment group for the non-polypeptide of choice is increased a greater propor- tion of the polypeptide molecule is shielded and thus a lower immune response will result.
  • a higher functional in vivo half-life is normally obtained. In most cases the introduction of an amino acid residue will be by way of substitution of an amino acid residue.
  • one difference between the amino acid sequence of the variant and the parent polypeptide amino acid sequence is that at least one and preferably more, e.g. 1-15, amino acid residues comprising an attachment group for the non- polypeptide moiety has/have been removed, preferably by substitution, from the amino acid sequence.
  • the term "functional site” is intended to indicate one or more amino acid residues which is/are essential for or otherwise involved in the function or performance of the polypeptide. Such amino acid residues are "located at" the functional site.
  • the functional site may be determined by methods known in the art and is preferably identified by analysis of a structure of the polypeptide complexed to a relevant receptor.
  • the amino acid sequence of the variant differs from the amino acid sequence of the parent wild-type human polypeptide in that a) at least one amino acid residue comprising an attachment group for the non-polypeptide moiety and present in the parent polypeptide amino acid sequence has been removed, preferably by substitution, and b) at least one amino acid residue comprising an attachment group for the non-polypeptide moiety has been introduced into the amino acid sequence.
  • This embodiment is considered of particular interest in that it is possible to specifically design the polypeptide variant so as to obtain an optimal conjugation to the non-polypeptide moiety of choice.
  • the exact number of amino acid residues and the type of amino acid residues to be re- moved depend, i.e., on the desired nature and degree of conjugation (e.g. the identity of the non-polypeptide moiety, whether and where in the polypeptide conjugation should be avoided, etc.).
  • the removal, preferably by replacement, of amino acid residues comprising attachment sites for the non-polypeptide of choice may be illustrated as follows:
  • the non- polypeptide moiety is one which binds to a lysine residue, e.g. the ⁇ -amino group thereof, such as a polymer molecule, e.g. based on polyethylene glycol, a lipophilic group or a sugar moiety
  • the polypeptide part may comprise an amino acid sequence, which differs from the parent polypeptide sequence shown in the removal of at least one lysine residue, whereby conjugation of these residues can be avoided.
  • the lysine residue(s) may be replaced with any other amino acid residue, but is preferably replaced by an arginine or a glutamine.
  • the non-polypeptide moiety of the conjugate is a molecule which has lysine as an attachment group, i.e. the amino acid sequence of the polypeptide vari- ant, which forms part of the conjugate, differs from the amino acid sequence of the parent wild- type human polypeptide in that at least one lysine residue, such as 1-15 lysine residues, in particular 1-10, 1-6 or 2-4 lysine residues, has been introduced and/or removed.
  • amino acid sequence of the variant polypeptide part of the conjugate differs from the amino acid sequence of the parent wild-type human polypeptide in that at least one lysine residue, such as 1-15 lysine residues, in particular 1-10, 1-6 or 2-4 lysine residues, has been introduced, preferably by substitution.
  • at least one lysine residue such as 1-15 lysine residues, in particular 1-10, 1-6 or 2-4 lysine residues, has been introduced, preferably by substitution.
  • the amino acid sequence of the variant polypeptide part of the conjugate differs from the amino acid sequence of the parent wild-type human polypeptide in that at least one lysine residue, such as 1-15 lysine residues, in particular 1-10, 1-6 or 2-4 lysine residues, has been removed, preferably by substitution.
  • the lysine residue may be substituted with any other amino acid residue, but is preferably substituted with R, Q, N or H, more preferably R
  • amino acid sequence of the variant polypeptide part of the conjugate differs from the amino acid sequence of the parent wild-type human polypeptide in that at least one lysine residue has been introduced, preferably by substitution, and that at least one lysine residue has been removed, preferably by substitution.
  • non-polypeptide moiety of the conjugate may be any molecule which, when using the given conjugation method has lysine as an attachment group (such as a sugar moiety, a lipophilic group or an organic de- rivatizing agent), it is preferred that the non-polypeptide moiety is a polymer molecule.
  • the polymer molecule may be any of the molecules mentioned in the section entitled "Conjugation to a polymer molecule", but is preferably selected from the group consisting of linear or branched polyethylene glycol or polyalkylene oxide. Most preferably, the polymer molecule is mPEG-SPA or mPEG-SC described in WO 90/13540. Conjugate where the non-polypeptide moiety is a molecule which has an acid group as an attachment group
  • the non-polypeptide moiety of the conjugate is a molecule which has an acid group as an attachment group, i.e. the amino acid sequence of the polypep- tide variant, which forms part of the conjugate, differs from the amino acid sequence of the parent wild-type human polypeptide in that at least one aspartic acid residue or at least one glutamic acid residue, such as 1-15 aspartic acid residues or glutamic acid residues, in particular 1- 10, 1-6 or 2-4 aspartic acid residues or glutamic acid residues, has been introduced and/or removed.
  • the amino acid sequence of the variant polypeptide part of the conjugate differs from the amino acid sequence of the parent wild-type human polypeptide in that at least one aspartic acid residue or glutamic acid residue, such as 1-15 aspartic acid residues or glutamic acid residues, in particular 1-10, 1-6 or 2-4 aspartic acid residues or glutamic acid residues, has been introduced, preferably by substitution.
  • at least one aspartic acid residue or glutamic acid residue such as 1-15 aspartic acid residues or glutamic acid residues, in particular 1-10, 1-6 or 2-4 aspartic acid residues or glutamic acid residues, has been introduced, preferably by substitution.
  • those positions occupied by K, R, Q or N in the parent polypeptide has been substituted for an aspartic acid residue or a glutamic acid residue.
  • the amino acid sequence of the variant polypeptide part of the conjugate differs from the amino acid sequence of the parent wild-type human polypeptide in that at least one aspartic acid residue or glutamic acid residue, such as 1-15 aspartic acid residues or glutamic acid residues, in particular 1-10, 1-6 or 2-4 aspartic acid residues or glutamic acid residues, has been removed, preferably by substitution.
  • the lysine residue may be substituted with any other amino acid residue, but is preferably substituted with R, Q, N or H, more preferably R
  • amino acid sequence of the variant polypeptide part of the conjugate differs from the amino acid sequence of the parent wild-type human polypeptide in that at least one aspartic acid residue or glutamic acid residue has been introduced, preferably by substitution, and that at least one aspartic acid residue or glutamic acid residue has been removed, preferably by substitution.
  • non-polypeptide moiety of the conjugate according to this aspect of the invention can be any non-polypeptide moiety with such property, it is presently preferred that the non-polypeptide moiety is a polymer molecule or an organic derivatizing agent, in particular a polymer molecule, and the conjugate is prepared, e.g., as described by Sakane and Pardridge, Pharmaceutical Research, Vol. 14, No. 8, 1997, pp 1085-1091.
  • the non-polypeptide moiety of the conjugate is a molecule which has cysteine group as an attachment group, i.e. the amino acid sequence of the polypeptide variant, which forms part of the conjugate, differs from the amino acid sequence of the parent wild-type human polypeptide in that at least one cysteine residue, such as 1-15 cys- teine residue, in particular 1-10, 1-6 or 2-4 cysteine residue, has been introduced.
  • cysteine residue is introduced into at least one position that is occupied by a T or a S residue.
  • non-polypeptide moiety of the conjugate according to this aspect of the invention may be any molecule which, when using the given conjugation method has cysteine as an attachment group it is preferred that the non-polypeptide moiety is a polymer molecule.
  • the polymer molecule may be any of the molecules mentioned in the section entitled "Conjugation to a polymer molecule", but is preferably selected from the group consisting of linear or branched polyethylene glycol or another polyalkylene oxide.
  • the polymer molecule is PEG, such as VS-PEG.
  • the conjugation between the poly- peptide and the polymer may be achieved in any suitable manner, e.g. as described in the section entitled "Conjugation to a polymer molecule", e.g. in using a one step method or in the stepwise manner referred to in said section.
  • the non-polypeptide moiety of the conjugate is a sugar moiety, i.e. the amino acid sequence of the polypeptide variant, which forms part of the conjugate, differs from the amino acid sequence of the parent wild-type human polypeptide in that at least one glycosylation site, such as 1-15 glycosylation sites, in particular 1-10, 1-6, or 2-4 glycosylation sites, have been introduced or removed by way of introduction or removal of amino acid residue(s) constituting a part of a glycosylation site.
  • at least one glycosylation site such as 1-15 glycosylation sites, in particular 1-10, 1-6, or 2-4 glycosylation sites, have been introduced or removed by way of introduction or removal of amino acid residue(s) constituting a part of a glycosylation site.
  • a non-naturally occurring glycosylation site has been introduced, or a non-naturally occurring glycosylation site has been introduced in combination with the removal of a natural occurring glycosylation site.
  • the introduced glycosylation site may be an O- glycosylation site or an N-glycosylation site.
  • the glycosylation site is an in vivo O- glycosylation site or an in vivo N-glycosylation site, in particular an in vivo N-glycosylation site.
  • the in vivo glycosylation site is preferably intro- pokerd into a position wherein only one mutation is required to create the site (i.e. where any other amino acid residues required for creating a functional glycosylation site is already present in the molecule).
  • the amino acid sequence of the polypeptide variant, which forms part of the conjugate differs from the amino acid sequence of the parent wild- type human polypeptide in that at least one glycosylation site, such as 1-15 glycosylation sites, in particular 1-10, 1-6, or 2-4 glycosylation sites, have been introduced by way of introduction amino acid residue(s) constituting a part of a glycosylation site, i.e. a glycosylation site having the sequence N-S-S/T has been introduced into the amino acid sequence by substitution of the relevant amino acid residues, X being any amino acid residue except P.
  • at least one glycosylation site such as 1-15 glycosylation sites, in particular 1-10, 1-6, or 2-4 glycosylation sites, have been introduced by way of introduction amino acid residue(s) constituting a part of a glycosylation site, i.e. a glycosylation site having the sequence N-S-S/T has been introduced into the amino acid sequence by substitution of the relevant amino acid residues,
  • the polypeptide variant in order to prepare a conjugate according to this embodiment the polypeptide variant must be expressed in a glycosylating host cell capable of attaching sugar moieties at the glycosylation site(s) or alternatively subjected to in vitro glycosylation.
  • glycosylating host cells are given in the section further below entitled "Coupling to a sugar moiety”.
  • conjugates which are particular suited for forming dispersions according to the invention, and therefore particularly suitable for being administered via the pulmonary route are conjugates, which have a low tendency to form aggregates.
  • the present inventors have developed a preliminary test, which may be used for the initial assessment of which conjugates fulfil these requirements. This test is described in the experimental part and is entitled the "Aggregation Test”.
  • the dispersion of particles contains a conjugate comprising at least one non-polypeptide moiety covalently at- tached to a polypeptide variant, wherein the amino acid sequence of said polypeptide variant differs from that of the corresponding wild-type human polypeptide in that at least one amino acid residue comprising an attachment group for said non-polypeptide moiety has been introduced and/or removed and wherein said conjugate, when tested in the "Aggregation Test", de- fined herein, has a tendency to form aggregates which has been reduced by at least 10% as compared to corresponding wild-type human polypeptide or compared to the corresponding polypeptide variant.
  • the tendency of the conju- gate to form aggregates has been reduced by at least 20%, e.g. by at least 30%, such as by at least 40%, more preferably by at least 50%, e.g. by at least 60%, such as by at least 70%, most preferably by at least 80, e.g. by at least 90%, such as by at least at least 95%.
  • the amino acid sequence of the variant may be mutated or further mutated so as to destroy or otherwise inactivate an epitope present in the parent polypeptide.
  • Epitopes of the parent polypeptide may be identified by use of methods known in the art, also known as epitope mapping, see, e.g.
  • IgGl antibodies from specific antisera towards the parent polypeptide are purified by immuno- precipitation and the reactive phages are identified by immunoblotting.
  • the sequence of the oligopeptide can be determined followed by localization of the sequence on the linear and/or 3D-structure of the parent polypeptide. The thereby identified region on the structure constitutes an epitope that then can be selected as a target region for introduction of an attachment group for the non-polypeptide moiety.
  • the variant of the conjugate may contain further substitutions.
  • the conjugate may contain further sugar moieties as a con- sequence of the variant thereof being expressed in a glycosylating host cell and glycosylation achieved at the wild-type glycosylation sites.
  • Non-polypeptide moiety of the conjugate of the dispersion of the invention may contain further sugar moieties as a con- sequence of the variant thereof being expressed in a glycosylating host cell and glycosylation achieved at the wild-type glycosylation sites.
  • non-polypeptide moiety of the conjugate is preferably selected from the group consisting of a polymer molecule, a lipophilic compound, a sugar moiety (e.g. by way of in vivo glycosylation) and an organic derivatizing agent. All of these agents may confer desirable properties to the polypeptide variant part of the conjugate, in particular a reduced immunogenicity and/or and increased functional in vivo half -life.
  • the variant part of the conjugate is normally conjugated to only one type of non-polypeptide moiety, but may also be conjugated to two or more different types of non-polypeptide moieties, e.g.
  • conjugation to two or more different non-polypeptide moieties may be done simultaneous or sequentially.
  • the variant and the lipophilic compound may be conjugated to each other, either directly or by use of a linker.
  • the lipophilic compound may be a natural compound such as a saturated or unsaturated fatty acid, a fatty acid diketone, a terpene, a prostaglandin, a vitamine, a carotenoide or steroide, or a synthetic compound such as a carbon acid, an alcohol, an amine and sulphonic acid with one or more alkyl-, aryl-, alkenyl- or other multiple unsaturated com- pounds.
  • the conjugation between the variant and the lipophilic compound, optionally through a linker may be done according to methods known in the art, e.g. as described by Bodanszky in Peptide Synthesis, John Wiley, New York, 1976 and in WO 96/12505.
  • the polymer molecule to be coupled to the variant may be any suitable polymer molecule, such as a natural or synthetic homo-polymer or heteropolymer, typically with a molecular weight in the range of 300-20,000 Da, more preferably in the range of 500-10,000 Da, even more preferably in the range of 500-5000 Da.
  • suitable polymer molecule such as a natural or synthetic homo-polymer or heteropolymer, typically with a molecular weight in the range of 300-20,000 Da, more preferably in the range of 500-10,000 Da, even more preferably in the range of 500-5000 Da.
  • homo-polymers include a polyol (i.e. poly-OH), a polyamine (i.e. poly-NH 2 ) and a polycarboxylic acid (i.e. poly-COOH).
  • a hetero-po- lymer is a polymer, which comprises one or more different coupling groups, such as, e.g., a hy- droxyl group and an
  • suitable polymer molecules include polymer molecules selected from the group consisting of polyalkylene oxide (PAO), including polyalkylene glycol (PAG), such as polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG) and polypropylene glycol (PPG), PEG-glycidyl ether (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), brancedPEGs, poly-vinyl alcohol (PVA), poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextran including carboxymethyl-dextran, or any other biopolymer suitable for reducing immunogenicity and/or increase functional in vivo half-life.
  • PAO polyalkylene oxide
  • PAG polyalkylene glycol
  • PEG polyethylene glycol
  • mPEG methoxypolyethylene glycol
  • PPG poly
  • polyalkylene glycol-derived polymers of value for modification of polypeptides/variants for therapeutic applications is general biocompatibility.
  • the polymers have various water solubility properties and are non-toxic. They are non-antigenic and non-immunogenicity. They have long circulation half-life in the blood and are easily excreted from living organisms.
  • PEG and especially mPEG is used since such polymer molecules have only few reactive groups capable of cross-linking compared, e.g., to polysaccharides such as dex- tran, and the like.
  • a non-toxic polymer molecule such as (m)polyethylene glycol ((m)PEG) is of interest since the chemistry for its covalent coupling to attachment group(s) of the variant is relatively simple because it has only one reactive end capable of conjugating with the variant. Consequently, the risk of cross-linking is eliminated and the resulting conjugates are more homogeneous and the reaction of the polymer molecules with the variant is easier to control.
  • Acti- vated mPEG with a functional group reactive towards amines on a protein molecule, is used in most cases.
  • the hy- droxyl end groups of the polymer molecule must be provided in activated form, i.e. with reactive functional groups.
  • the polymer molecules are purchased in activated form, e.g. from Shearwater Polymers, Huntsville, Alabama, USA.
  • the polymer molecules can be activated by conventional methods known in the art, e.g. as disclosed in WO 90/13540.
  • the conjugation of the variant and the activated polymer molecules is conducted by use of any conventional method, e.g. as described in the following references (which also describe suitable methods for activation of polymer molecules): R.F. Taylor, (1991), "Protein immobilisation.
  • the PEGylation may be designed so as to introduce an optimal num- ber of polymer molecules. For instance, if a substantial increase of the functional in vivo half- life or a substantial decrease of immunogenicity is desirable it may be desirable to conjugate more and larger polymer molecules to the variant since in general, the more polymer molecules attached per variant molecule the greater the extension of the half-life and the greater reduction of immunogenicity.
  • extensive PEGylation may be employed when it is not criti- cal to maintain a close to intact function of the conjugate, since a normally observed drawback of too extensive PEGylation is that the function of the conjugate is reduced.
  • the extensive PEGylation is conveniently performed according to the embodiment of the invention wherein a functional site of the variant is blocked during PEGylation. If, on the other hand, it is critical to maintain a high function of the conjugate and less critical to obtain a substantially increased functional in vivo half-life and/or reduced immunogenicity, the PEGylation should be designed so as to allow for a less extensive PEGylation.
  • the polymer conjugation is performed under conditions aiming at reacting all available polymer attachment groups with polymer molecules.
  • the molar ratio of activated polymer molecules to variant is 200-1, preferably 100-1, such as 10-1 or 5-1 in order to obtain optimal reaction.
  • linker it is also contemplated according to the invention to couple the polymer molecules to the variant through a linker.
  • Suitable linkers are well known to the skilled person.
  • a preferred example is cyanuric chloride (Abuchowski et al, (1977), J. Biol. Chem., 252, 3578-3581; US 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.
  • the coupling of a sugar moiety may take place in vivo or in vitro.
  • the nucleotide sequence encoding the variant part of the conjugate must be inserted in a glycosylating, eukaryotic expression host.
  • the expression host cell may be selected from fungal (filamentous fungal or yeast), insect or animal cells or from transgenic plant cells.
  • the host cell is a mammalian cell, such as a COS-1 cell, CHO cell, BHK or HEK cell, e.g. HEK293, or an insect cell, such as an SF9 cell, or a yeast cell, e.g. Saccharomyces cerevisiae, Pichia pastoris or any other suitable glycosylating host, e.g. as described further below.
  • a mammalian cell such as a COS-1 cell, CHO cell, BHK or HEK cell, e.g. HEK293, or an insect cell, such as an SF9 cell, or a yeast cell, e.g. Saccharomyces cerevisiae, Pichia pastoris or any other suitable glycosylating host, e.g. as described further below.
  • Covalent in vitro coupling of glycosides to amino acid residues of the variant may be used to modify or increase the number or profile of carbohydrate substituents.
  • the sugar(s) may be attached to a) arginine and histidine, b) free car- boxyl groups, c) free sulfhydryl groups such as those of cysteine, d) free hydroxyl groups such as those of serine, threonine, tyrosine or hydroxyproline, e) aromatic residues such as those of phenylalanine or tryptophan or f) the amide group of glutamine.
  • amino acid residues constitute examples of attachment groups for a sugar moiety, which may be introduced and/or removed in the variant of the conjugate.
  • Suitable methods of in vitro coupling are described, for example in WO 87/05330 and in Aplin et al., CRC Grit Rev. Biochem., pp. 259-306, 1981.
  • Covalent modification of the variant exhibiting part or all of the activity of the parent polypeptide may be performed by reacting (an) attachment group(s) of the variant with an organic derivatizing agent.
  • organic derivatizing agent Suitable derivatizing agents and methods are well known in the art. For example, cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or car- boxyamidomethyl derivatives.
  • Cysteinyl residues also are derivatized by reaction with bromo- trifluoroacetone, oc-bromo- ⁇ -(4-imidozoyl)propionic acid, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3- diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Suitable reagents for derivatizing -amino-containing residues include imi- doesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and nin- hydrin.
  • Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine guanidino group.
  • the helper molecule is one, which specifically recognizes a functional site of the variant, such as a receptor.
  • the helper molecule may be an antibody, in particular a monoclonal antibody recognizing the variant exhibiting part or all of the activity of the parent polypeptide.
  • the helper molecule may be a neutralizing monoclonal antibody.
  • the variant is allowed to interact with the helper molecule before effecting conjugation. This ensures that the functional site of the variant is shielded or protected and conse- quently unavailable for derivatization by the non-polypeptide moiety such, as a polymer. Following its elution from the helper molecule, the conjugate between the non-polypeptide moiety and the variant can be recovered with at least a partially preserved functional site.
  • the helper molecule is first covalently linked to a solid phase such as column packing materials, for instance Sephadex or agarose beads, or a surface, e.g. reaction vessel. Subsequently, the variant is loaded onto the column material carrying the helper molecule and conjugation carried out according to methods known in the art, e.g. as described in the sections above entitled "Conjugation to .".
  • This procedure allows the conjugate to be separated from the helper molecule by elution.
  • the conjugate is eluted by conventional techniques under physico-chemical conditions that do not lead to a substantive degradation of the conjugate.
  • the fluid phase containing the conjugate is separated from the solid phase to which the helper molecule remains covalently linked.
  • the helper molecule may be derivatized with a second molecule (e.g. biotin) that can be recognized by a specific binder (e.g. streptavidin).
  • the specific binder may be linked to a solid phase thereby allowing the separation of the conjugate from the helper mole- cule-second molecule complex through passage over a second helper-solid phase column which will retain, upon subsequent elution, the helper molecule-second molecule complex, but not the conjugate.
  • the conjugate may be released from the helper molecule in any appropriate fashion.
  • De-protection may be achieved by providing conditions in which the helper molecule dissociates from the functional site of the variant to which it is bound.
  • a complex be- tween an antibody to which a polymer is conjugated and an anti-idiotypic antibody can be dissociated by adjusting the pH to an acid or alkaline pH.
  • the variant is expressed, as a fusion protein, with a tag, i.e. an amino acid sequence or peptide stretch made up of typically 1-30, such as 1-20 amino acid residues.
  • a tag i.e. an amino acid sequence or peptide stretch made up of typically 1-30, such as 1-20 amino acid residues.
  • the tag is a convenient tool for achieving conjugation between the tagged variant and the non-polypeptide moiety.
  • the tag may be used for achieving conjugation in microtiter plates or other carriers, such as paramagnetic beads, to which the tagged variant can be immobilised via the tag.
  • the conjugation to the tagged variant in, e.g., microtiter plates has the advantage that the tagged variant can be immobilised in the microtiter plates directly from the culture broth (in principle without any purification) and subjected to conjugation. Thereby, the total number of process steps (from expression to conjugation) can be reduced.
  • the tag may function as a spacer molecule ensuring an improved accessibility to the immobilised variant to be conjugated.
  • the conjugation using a tagged variant may be to any of the non-polypeptide moieties disclosed herein, e.g. to a polymer molecule such as PEG.
  • the identity of the specific tag to be used is not critical as long as the tag is capable of being expressed with the variant and is capable of being immobilised on a suitable surface or carrier material.
  • suitable tags are commercially available, e.g. from Unizyme Laboratories, Denmark.
  • the tag may any of the following sequences: His-His-His-His-His-His-His Met-Lys-His-His-His-His-His-His-His-His-His-His-His-His-His-His
  • Antibodies against the above tags are commercially available, e.g. from ADI, Aves Lab and Research Diagnostics.
  • the subsequent cleavage of the tag from the variant may be achieved by use of commercially available enzymes.
  • variant part of a conjugate may be produced by any suitable method known in the art. Such methods include constructing a nucleotide sequence encoding the variant and expressing the sequence in a suitable transformed or transfected host.
  • variants of the conjugate of the dispersion of the invention may be pro- prised, albeit less efficiently, by chemical synthesis or a combination of chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.
  • the nucleotide sequence a variant may be constructed by isolating or synthesizing a nucleotide sequence encoding the parent polypeptide and then changing the nucleotide se- quence so as to effect introduction (i.e. insertion or substitution) or deletion (i.e. removal or substitution) of the relevant amino acid residue(s).
  • nucleotide sequence is conveniently modified by site-directed mutagenesis in accordance with well-known methods.
  • the nucleotide sequence is prepared by chemical synthesis, e.g. by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired variant, and preferably selecting those codons that are favored in the host cell in which the recombinant variant will be produced.
  • oligonucleotides are designed based on the amino acid sequence of the desired variant, and preferably selecting those codons that are favored in the host cell in which the recombinant variant will be produced.
  • several small oligonucleotides coding for portions of the desired variant may be synthesized and assembled by PCR, ligation or ligation chain reaction (LCR).
  • LCR ligation or ligation chain reaction
  • the individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • the nucleotide sequence encoding the variant is inserted into a recombinant vector and operably linked to control sequences necessary for expression of the variant in the desired transformed host cell.
  • control sequences necessary for expression of the variant in the desired transformed host cell It should of course be understood that not all vectors and expression control sequences function equally well to express the nucleotide sequence encoding a variant described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it or be able to integrate into the chromosome.
  • the vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
  • an expression control sequence a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compati- bility with the nucleotide sequence encoding the variant, particularly as regards potential secondary structures.
  • Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the nucleotide sequence, their secretion characteristics, their ability to fold the variant correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the nucleotide sequence.
  • the recombinant vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromo- somal replication, e.g. a plasmid.
  • the vector is one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector, in which the nucleotide sequence encoding the variant is operably linked to additional segments required for transcription of the nucleotide sequence.
  • the vector is typically derived from plasmid or viral DNA.
  • suitable expression vectors for expression in the host cells mentioned herein are commercially available or described in the literature.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus.
  • Specific vectors are, e.g., pCDNA3.1(+) ⁇ Hyg (Invitro- gen, Carlsbad, CA, USA) and pCI-neo (Stratagene, La Jola, CA, USA).
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pBR322, pET3a and pET12a (both from Novagen Inc., Wl, USA), wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g.
  • Useful ex- pression vectors for yeast cells include the 2 ⁇ plasmid and derivatives thereof, the POTl vector (US 4,931,373), the ⁇ JSO37 vector described in (Okkels, Ann. New York Acad. Sci. 782, 202- 207, 1996) and pPICZ A, B or C (Invitrogen).
  • Useful vectors for insect cells include pVL941, pBG311 (Gate et al., "Isolation of the Bovine and Human Genes for Mullerian Inhibiting Substance And Expression of the Human Gene In Animal Cells", Cell, 45, pp. 685-98 (1986), pBluebac 4.5 and pMelbac (both available from Invitrogen).
  • vectors for use in this invention include those that allow the nucleotide sequence encoding the variant to be amplified in copy number.
  • amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, "Construction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient Expression", Mol. Cell. Biol, 2, pp. 1304-19 (1982)) and glutamine synthetase ("GS”) amplification (see, e.g., US 5,122,464 and EP 338,841).
  • the recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question.
  • An example of such a sequence is the SV40 origin of replication.
  • suitable sequences enabling the vector to replicate are the yeast plasmid 2 ⁇ replication genes REP 1-3 and origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reduc- tase (DHFR) or the Schizosaccharomyces pombe TPI gene (described by P.R. Russell, Gene 40, 1985, pp. 125-130), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • selectable markers include amdS, pyrG, arcB, niaD, sC.
  • control sequences is defined herein to include all components, which are necessary or advantageous for the expression of the variant.
  • Each control sequence may be native or foreign to the nucleic acid sequence encoding the variant.
  • Such control sequences in- elude, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activating sequence, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter.
  • expression control sequences may be used in the present invention.
  • useful expression control sequences include the expression control sequences associ- ated with structural genes of the foregoing expression vectors as well as any sequence known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • control sequences for directing transcription in mammalian cells include the early and late promoters of SV40 and adenovirus, e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovirus immediate-early gene promoter (CMV), the human elongation factor l (EF-l ⁇ ) promoter, the Droso- phila minimal heat shock protein 70 promoter, the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC) promoter, the human growth hormone terminator, S V40 or adenovirus Elb region polyadenylation signals and the Kozak consensus sequence (Kozak, M. JMol Biol 1987 Aug 20;196(4):947-50).
  • adenovirus 2 major late promoter e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalo
  • a synthetic intron may be inserted in the 5' untranslated region of the nucleotide sequence encoding the variant of interest.
  • An example of a synthetic intron is the synthetic intron from the plasmid pCI-Neo (available from Promega Corporation, Wl, USA).
  • control sequences for directing transcription in insect cells include the polyhedrin promoter, the P10 promoter, the Auto grapha californica polyhedrosis virus basic protein promoter, the baculovirus immediate early gene 1 promoter and the baculovirus 39K delayed-early gene promoter, and the S V40 polyadenylation sequence.
  • control sequences for use in yeast host cells include the promoters of the yeast ⁇ -mating system, the yeast triose phosphate isomerase (TPI) promoter, promoters from yeast glycolytic genes or alcohol dehydogenase genes, the ADH2-4c promoter and the inducible GAL promoter.
  • TPI yeast triose phosphate isomerase
  • control sequences for use in filamentous fungal host cells include the ADH3 promoter and terminator, a promoter derived from the genes encoding As- pergillus oryzae TAKA amylase triose phosphate isomerase or alkaline protease, an A. niger a- amylase, A. niger or A. nidulans glucoamylase, A. nidulans acetamidase, Rhizomucor miehei aspartic proteinase or lipase, the TPI1 terminator and the ADH3 terminator.
  • control sequences for use in bacterial host cells include promoters of the lac system, the trp system, the TAC or TRC system and the major promoter regions of phage lambda.
  • the nucleotide sequence encoding a variant exhibiting part or all of the parent polypeptide activity may or may not also include a nucleotide sequence that encode a signal peptide.
  • the signal peptide is present when the variant is to be secreted from the cells in which it is expressed. Such signal peptide, if present, should be one recognized by the cell chosen for expression of the variant.
  • the signal peptide may be homologous (e.g. be that normally associated with the parent polypeptide) or heterologous (i.e. originating from another source) to the variant or may be homologous or heterologous to the host cell, i.e.
  • the signal peptide may be prokaryotic, e.g. derived from a bacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian, or insect or yeast cell.
  • the presence or absence of a signal peptide will, e.g., depend on the expression host cell used for the production of the variant, the variant to be expressed (whether it is an intracellular or intracellular variant) and whether it is desirable to obtain secretion.
  • the signal peptide may conveniently be derived from a gene encoding an Aspergillus sp.
  • amylase or glucoamylase a gene encoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosa lipase.
  • the signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral -amylase, A. niger acid-stable amylase, or A. niger glucoamylase.
  • the signal peptide may conveniently be de- rived from an insect gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipoki- netic hormone precursor, (cf.
  • a preferred signal peptide for use in mammalian cells is that of the parent human polypeptide apparent from the examples hereinafter or the murine Ig kappa light chain signal peptide (Coloma, M (1992) J. Imm. Methods 152:89-104).
  • suitable signal peptides have been found to be the ⁇ -factor signal peptide from S. cereviciae. (cf . US 4,870,008), the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase signal peptide (cf. L.A.
  • Any suitable host may be used to produce the variant part of the conjugate, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. Examples of bacterial host cells in- elude grampositive bacteria such as strains of Bacillus, e.g. B. brevis orB.
  • a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thome, 1987, Journal of Bacteriology 169: 5771-5278).
  • filamentous fungal host cells examples include strains of Aspergillus, e.g. A. oryzae, A. niger, or A. nidulans, Fusarium or Trichoderma.
  • Fungal cells may be trans- formed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se.
  • Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and US 5,679,543.
  • Suitable methods for transforming Fusarium species are described by Malardier et al, 1989, Gene 78: 147-156 and WO 96/00787.
  • Yeast may be transformed using the procedures described by Becker and Guar- ente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al, 1983, Journal of Bacteriology 153: 163; and Hinnen et al, 1978, Proceedings of the Na- tional Academy of Sciences USA 75: 1920.
  • yeast host cells examples include strains of Saccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Kluyveromyces, Pichia, such as P. pastoris or P. methano- lica, Hansenula, such as H. Polymorpha or Yarrowia.
  • suitable insect host cells include a Lepidoptora cell line, such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (High Five) (US 5,077,214). Transformation of insect cells and production of heterologous polypeptides/variants therein may be performed as described by Invitrogen.
  • Lepidoptora cell line such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (High Five) (US 5,077,214). Transformation of insect cells and production of heterologous polypeptides/variants therein may be performed as described by Invitrogen.
  • Suitable mammalian host cells include Chinese hamster ovary (CHO) cell lines, (e.g. CHO-Kl; ATCC CCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL- 1573)), as well as plant cells in tissue culture.
  • COS Green Monkey cell lines
  • BHK Baby Hamster Kidney
  • HEK 293 ATCC CRL- 1573
  • Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, electroporation, DEAE-dextran mediated transfection, liposome-mediated transfection, viral vectors and the transfection method described by Life Technologies Ltd, Paisley, UK using Lipofectamin 2000. These methods are well known in the art and e.g. described by Ausbel et al. (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA. The cultivation of mammalian cells are conducted according to established methods, e.g. as disclosed in (Animal Cell Biotechnology, Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc, Totowa, New Jersey, USA and Harrison MA and Rae IF, General Techniques of Cell Culture, Cambridge University Press 1997).
  • the cells are cultivated in a nutrient medium suitable for production of the variant using methods known in the art.
  • the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the variant to be expressed and or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art.
  • Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g. , in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.
  • the resulting variant may be recovered by methods known in the art.
  • the variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.
  • the variants may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofo- cusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • Methods for purifying variants exhibiting part or all of the activity of a parent polypeptide may be found in literature concerning purification of the parent polypeptide.
  • the biological activity of the conjugates and/or variants exhibiting part or all of the activity of the parent polypeptide can be assayed by any suitable method known in the art applicable to the corresponding parent polypeptide.
  • composition of the invention may be administered in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the dispersion with the conju- gate, either concurrently or in accordance with any other acceptable treatment schedule. In addition, the dispersion of the invention may be used as an adjunct to other therapies.
  • the present invention provides compositions and methods for treating a mammal in need thereof with the dispersion of the invention.
  • the invention in a further aspect relates to a method of treating a mammal having circulating antibodies against the parent polypeptide, which method comprises administering a dispersion comprising a conjugate which has the bioactivity of the parent polypeptide and which does not react with said antibodies.
  • the dispersion is preferably a dispersion as described herein and the mammal is preferably a human being.
  • the mammals to be treated may suffer from any of the diseases commonly treated with the parent polypeptide or conjugates comprising the parent polypeptides.
  • the invention relates to the use of the conjugates for making a pharmaceutical product for use in treatment of mammals having circulating antibodies against the parent polypeptide, wherein a conjugate according to the invention has the bioactivity of the parent polypeptide and does not react with such is formulated into an inhalable formulation.
  • circulating antibodies is intended to indicate autoantibodies formed in a mammal in response to having been treated with the parent polypeptide and/or variants thereof different from the conjugates of the dispersion of the invention.
  • Interferon-gamma is a cytokine produced by T-lymphocytes and natural killer cells and exists as a homodimer of two noncovalently bound polypeptide subunits.
  • the mature form of each dimer comprises 143 amino acid residues (shown in SEQ ID NO:l), the precursor form thereof including signal sequence of 166 amino acid residues.
  • Each subunit has two potential N-glycosylation sites (Aggarwal et al., Human Cytokines, Blackwell Scientific Publications, 1992) at positions 25 and 97. Depending on the degree of glycosylation the molecular weight of IFNG in dimer form is 34-50 kDa (Farrar et al., Ann. Rev. Immunol, 1993, 11:571-611).
  • IFNG subunit polypeptides including one comprising a Cys-Tyr-Cys N-terminal amino acid sequence (positions (-3)-(-l) relative to SEQ ID NO:l), one comprising an N-terminal methionine (position -1 relative to SEQ ID NO:l), and various C-terminally truncated forms comprising 127- 134 amino acid residues. It is known that 1-15 amino acid residues may be deleted from the C- terminus without abolishing IFNG activity of the molecule. Furthermore, heterogeneity of the huIFNG C-terminus was described by Pan et al. (Eur. J. Biochem. 166:145-149, 1987).
  • a particular interesting embodiment of the invention relates to a dispersion of particles containing a conjugate comprising at least one non-polypeptide moiety covalently attached to an INFG variant, wherein the amino acid sequence of the INFG variant differs from the amino acid sequence of the corresponding wild-type human INFG shown in SEQ ID NO:l in that at least one amino acid residue comprising an attachment group for said non-polypeptide moiety has been introduced or removed.
  • the non-polypeptide moiety is a sugar moiety, e.g. an O-linked or N-linked sugar moiety
  • the IFNG variant comprises at least one removed and/or at least one introduced in vivo glycosylation site.
  • an in vivo glycosylation site is introduced into a position of the parent IFNG polypeptide occupied by an amino acid residue exposed to the surface of the polypeptide, preferably with more than 25% of the side chain exposed to the solvent, in particular more than 50% exposed to the solvent (these positions are identified in the Examples herein).
  • the N- glycosylation site is introduced in such a way that the N-residue of said site is located in said position.
  • an O-glycosylation site is introduced so that the S or T residue making up such site is located in said position.
  • the in vivo glycosylation site in particular the N residue of the N-glycosylation site or the S or T residue of the O-glycosylation site, is located within the 118 N-terminal amino acid residues of the IFNG polypeptide, more preferably within the 93 N-terminal amino acid residues.
  • the in vivo glycosylation site is introduced into a position wherein only one mutation is required to create the site (i.e. where any other amino acid residues required for creating a functional glycosylation site is already present in the molecule).
  • substitutions that lead to introduction of an additional N- glycosylation site at positions exposed at the surface of the IFNG polypeptide and occupied by amino acid residues having more than 25% of the side chain exposed to the surface include: Q1N + P3S/T, P3N+V5S/T, K6N+A8S/T, E9N+L11S/T, K12S/T,
  • Substitutions that lead to introduction of an additional N-glycosylation site at positions exposed at the surface of the IFNG polypeptide having more than 50% of the side chain exposed to the surface include: P3N+V5S/T, K6N+A8S/T, K12S/T, K13N+F15S/T, G18S/T, D21N+A23S/T, G26N+L28S/T, G31N+L33S/T, K34N+W36S/T, K37N+E39S/T, E38N, E38N+S40S/T, E39N+D41S/T, K55N+F57S/T, K58N+F60S/T, K61S/T, D62N+Q64S/T, Q64N+I66S/T, S65N+Q67S/T, K68N+V70S/T, E71N+I73S/T, E75N+M77S/
  • Substitutions where only one amino acid mutation is required to introduce an N- glycosylation site include K12S/T, G18S/T, G18N, K37S/T, E38N, M45N, I49N, K61S/T, D63N, Q67N, V70N, K80S/T, F82N, N85S/T, K87S/T, K94N, S99N, Q106S/T, El 19N, A124N, K130N and R140N, in particular K12S/T, G18N, G18S/T, K37S/T, E38N, K61S/T, D63N, Q67N, K80S/T, N85S/T, K94N, S99N, Q106S/T, A124N, K130N, and R140N (positions with more than 25% of its site chain exposed to the surface (in a structure without receptor molecule), or more preferably G18N, E38N, D63N, Q67N, K94N
  • existing glycosylation sites may have been removed from the IFNG polypeptide.
  • any of the above listed substitutions to introduce a glycosylation site may be combined with a substitution to remove any of the two natural N-glycosylation sites of huIFNG.
  • the IFNG polypeptide may comprise a substitution of N25 and/or N97, e.g. one of the substitutions N25K C D E and/or N97K/C/D/E, if the conjugate comprises a non-polypeptide polypeptide having the relevant of K, C, D, E as an attachment group.
  • the IFNG polypeptide part of a conjugate may contain a single in vivo glycosylation site per monomer. However, in order to become of a sufficient size to increase functional in vivo half-life it is often desirable that the polypeptide comprises more than one in vivo glycosylation site, in particular 2-7 in vivo glycosylation sites, such as 2, 3, 4, 5, 6 or 7 in vivo glycosylation sites.
  • the IFNG polypeptide may comprise one additional glycosylation site pr monomer, or may comprise two, three, four, five, six, seven or more introduced in vivo glycosylation sites, preferably introduced by one or more substitutions described in any of the above lists.
  • Removal and/or introduction of in vitro glycosylation sites may be achieved as described in the subsequent sections on modification of the IFNG polypeptide to introduce and/or remove polymer attachment sites.
  • Any of the glycosylated IFNG polypeptides disclosed in the present section having introduced and/or removed at least one glycosylation site might further be conjugated to a second non-polypeptide moiety.
  • the second non-polypeptide moiety is a polymer molecule, such as PEG, or any other non-polypeptide moiety.
  • conjugation may be achieved by use of attachment groups already present in the IFNG polypeptide or attachment groups may have been introduced and/or removed, in particular such that a total of 1- 6, in particular 3-4 or 1, 2, 3, 4, 5, or 6 attachment groups are available for conjugation.
  • the number and molecular weight of the non-polypeptide moiety is chosen so as that the total molecular weight added by the non-polypeptide moiety is in the range of 20-40 kDa, in particular about 20 kDa or 30 kDa.
  • the glycosylated IFNG polypeptide may be conjugated to a polymer having cysteine as an attachment group.
  • one or more cysteine residues are inserted into the IFNG polypeptide.
  • the glycosylated IFNG polypeptide may be conjugated to a polymer having lysine as an attachment group.
  • one or more lysine residues of the parent polypeptide may have been removed.
  • a lysine residue may have been introduced, e.g. by any of the substitutions mentioned in said section.
  • the first non-polypeptide moiety is a polymer, e.g.
  • the IFNG polypeptide part of a conjugate may be a glycosylated polypeptide, e.g. using one or both of the natural N- glycosylation sites of huIFNG or an introduced glycosylation site as described in the immediately preceding section.
  • the non-polypeptide moiety is a polymer which has cysteine as an attachment group and at least one cysteine residue is introduced into a position of the IFNG polypeptide that in wild-type human IFNG is occupied by a surface exposed amino acid residue.
  • the cysteine residue is introduced in accordance with the general consideration for introducing and/or removing attachment groups for the non-polypeptide moiety given in the section entitled "Conjugate contained in the dispersion".
  • the IFNG polypeptide may comprise at least one substitution selected from the group consisting of P3C, K6C, N10C, K13C, N16C, D21C, N25C, G26C, G31C, K34C, K37C, E38C, E39C, K55C, K58C, N59C, D62C, Q64C, S65C, K68C, E71C, E75C, N83C, S84C, K86C, K87C, K94C, N97C, S99C, T101C, D102C, L103C and N104C (introduction of a cysteine residue in a position that is occupied by an amino acid residue having more than 50% of its side chain exposed to the surface in a structure with receptor).
  • the substitutions N25C and N97C are of particular interest, and especially N25C+N97C, when the IFNG polypeptide is expressed in a non-glycosylating host cell, such as E. coli, since N25 and N97 constitute part of an inherent glycosylation site of huIFNG.
  • the IFNG polypeptide according to this embodiment may comprise at least one cysteine residue introduced in a position occupied by any of the amino acid residues 121-143 of huEFNG.
  • the IFNG polypeptide of the conjugate comprises a total of 1-8, such as 2-6 Cys residues, e.g. 1-3 Cys residues per monomer.
  • the conjugation between the polypeptide and the polymer may be achieved in any suitable manner, e.g. as described in the section entitled "Conjugation to a polymer molecule", e.g. in using a one step method or in the stepwise manner referred to in said section.
  • the conjugate comprises two or more first non-polypeptide moieties, normally each of these has a molecular weight of 5 or lOkDa.
  • a suitable polymer is VS-PEG.
  • the non-polypeptide is a polymer having lysine as an attachment group and the IFNG polypeptide is modified in that at least one lysine residue is removed, the lysine residue being selected from the group consisting of K6, K12, K13, K34, K37, K43, K55, K58, K61, K68, K74, K80, K86, K87, K88, K94, K108, K125, K128 and K130, the numbering being made relative to SEQ ID NO 2. More preferably, at least one lysine residue selected from the group consisting of K12, K34, K37, K108, K128 and K130 be removed. Thereby, conjugation of this/these residues can be avoided.
  • the lysine residue(s) may be replaced with any other amino acid residue, but is preferably replaced by an arginine or a glutamine.
  • the IFNG polypeptide may be modified to have introduced one or more lysine residues, in particular in a position of huIFNG occupied by a surface exposed amino acid residue.
  • the lysine residue is introduced in accordance with the general consideration for introducing and/or removing attachment groups for the non-polypeptide moiety given in the section entitled "Conjugate contained in the dispersion", in particular in a posi- tion which is occupied by an amino acid residue having at least 25%, such as at least 50% of its side chain exposed to the surface (such positions being identified in the Materials and Methods section herein).
  • at least one lysine residue may be introduced by substitution of any of the amino acid residues 121-143 of SEQ ID NO:l.
  • the IFNG polypeptide may comprise a lysine in at least one position selected from the group consisting of D2, E7, E9, H19, D21, D24, N25, E38, E39, D41, R42, D62, D63, E71, E75, D76, R89, D90, D91, E93, N97, R107, Hill, E112, E119, R129, R131, R137, R139 and R140 of SEQ ID NO 2 (positions occupied by an N, R, D, E or H residue in huIFNG).
  • a lysine in at least one position selected from the group consisting of D2, E7, E9, H19, D21, D24, N25, E38, E39, D41, R42, D62, D63, E71, E75, D76, R89, D90, D91, E93, N97, R107, Hill, E112, E119, R129, R131, R137, R139 and R140 of SEQ
  • the IFNG polypeptide comprises a substitu- tion in one or more of the above positions, in particular in 1-15, such as 1-8 or 2-8, preferably 1-5 or 2-5 positions (removal and/or introduction of lysine residues) per monomer.
  • the IFNG polypeptide may comprise a substitution in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of the above positions.
  • the substitutions N25K and N97K are of particular interest, and especially N25K+N97K, when the IFNG polypeptide is expressed in a non- glycosylating host cell, such as E. coli, since N25 and N97 constitute part of an inherent glycosylation site of huIFNG.
  • the IFNG polypeptide of the conjugate according to this embodiment may comprise at least one of the above substitutions for introduction of a lysine residue in combination with at least one substitution removing a lysine residue as defined above (prefera- bly a substitution to R or Q).
  • the IFNG polypeptide comprises at least one of the following substitutions N25K and N97K in combination with at least one of the substitutions K128R, K128Q, K130R and K130Q.
  • the IFNG polypeptide comprises the substitution N25K+K128R, N25K+K130R, N25K+K128R+K130R, N97K+K128R, N97K+K130R, N97K+K128R+K130R, N25K+N97K+K128R+K130R, N25K+N97K+K128R and N25K+N97K+K130R.
  • non-polypeptide moiety of the conjugate according to this aspect of the invention may be any molecule which, when using the given conjugation method has lysine as an attachment group (such as a sugar moiety, a lipophilic group or an organic derivatizing agent), it is preferred that the non-polypeptide moiety is a polymer molecule.
  • the polymer molecule may be any of the molecules mentioned in the section entitled "Conjugation to a polymer molecule", but is preferably selected from the group consisting of linear or branched polyethylene glycol or polyalkylene oxide.
  • the polymer molecule is SS-PEG, NPC-PEG, aldehyd-PEG, mPEG-SPA, mPEG-SCM or mPEG-BTC from Shearwater Polymers Inc., SC-PEG from Enzon Inc., tresylated mPEG as described in US 5,880,255 or oxycarbonyl- oxy-N-dicarboxyimide-PEG (US 5,122,614).
  • the non-polypeptide moiety has a Mw of about 5 or 10 kDa.
  • the non-polypeptide moiety of the conjugate is a molecule which has an acid group as the attachment group
  • the IFNG polypeptide comprises an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:l in that at least one surface exposed amino acid residue has been substituted with an aspartic acid residue or a glutamic acid residue, preferably in accordance with the general considerations given in the section entitled "Conjugate contained in the dispersion".
  • the Asp or Glu residue may be introduced in a position of the parent IFNG polypeptide occupied by K, R, Q or N. For instance, N25, N97, K125, K128, R129, K130 and/or R131, more preferably N25 and/or N97, most preferably N25+N97, may be substituted with an Asp or Glu residue.
  • one or more Asp or Glu residues may be removed, e.g. from the receptor binding site, in case the non- polypeptide moiety is one that binds to those residues.
  • non-polypeptide moiety of the conjugate according to this aspect of the invention can be any non-polypeptide moiety with such property, it is presently preferred that the non-polypeptide moiety is a polymer molecule or an organic derivatizing agent, in particular a polymer molecule, and the conjugate is prepared, e.g., as described by Sakane and Pardridge, Pharmaceutical Research, Vol. 14, No. 8, 1997, pp 1085-1091.
  • the dispersions of particles comprising the INFG conjugates disclosed herein may be used for treating bacterial and viral infections, cancers or tumors, interstitial pulmonary diseases such as idiopathic pulmonary fibrosis, granulomatous diseases, bone disorders (e.g. a bone metabolism disorder so as malignant osteopetrosis) and autoimmune diseases such rheumatoid arthritis.
  • interstitial pulmonary diseases such as idiopathic pulmonary fibrosis, granulomatous diseases, bone disorders (e.g. a bone metabolism disorder so as malignant osteopetrosis) and autoimmune diseases such rheumatoid arthritis.
  • Interferon ⁇ Human interferon-beta
  • Interferon ⁇ is a regulatory polypeptide with a molecular weight of 22 kDa consisting of 166 amino acid residues. It can be produced by most cells in the body, in particular fibroblasts, in response to viral infection or exposure to other biologies. It binds to a multimeric cell surface receptor, and productive receptor binding results in a cascade of intracellular events leading to the expression of interferon ⁇ inducible genes which in turn produces effects which can be classified as antiviral, antiproliferative and immunomodulatory.
  • a particular interesting embodiment of the invention relates to a dispersion of particles containing a conjugate comprising at least one non-polypeptide moiety covalently attached to an interferon ⁇ variant, wherein the amino acid sequence of the interferon ⁇ variant differs from the amino acid sequence of the corresponding wild-type human interferon ⁇ shown in SEQ ID NO:2 in that at least one amino acid residue comprising an attachment group for said non-polypeptide moiety has been introduced or removed.
  • the non-polypeptide moiety has lysine as an attach- ment group, and thus the interferon ⁇ polypeptide is one that comprises an amino acid sequence that differs from that of wildtype human interferon ⁇ in at least one introduced and/or at least one removed lysine residue. While the non-polypeptide moiety may be any of those binding to a lysine residue, e.g.
  • the ⁇ -amino group thereof such as a polymer molecule, a lipophilic group, an organic derivatizing agent or a carbohydrate moiety, it is preferably any of the polymer molecule mentioned in the section entitled "Conjugation to a polymer molecule", in particular a branched or linear PEG or polyalkylene oxide.
  • the polymer molecule is PEG and the activated molecule to be used for conjugation is SS-PEG, NPC-PEG, aldehyd-PEG, mPEG-SPA, mPEG-SCM, mPEG-BTC from Shearwater Polymers, Inc, SC-PEG from Enzon, Inc., tresylated mPEG as described in US 5,880,255, or oxycarbonyl-oxy-N-dicarboxyimide- PEG (US 5,122,614).
  • the non-polypeptide moiety has a molecular weight of about 5 or 10 kDa.
  • the amino acid sequence of the interferon ⁇ polypeptide differs from that of human wild-type interferon ⁇ in at least one removed lysine residue, such as 1- 5 removed lysine residues, in particular 1-4 or 1-3 removed lysine residues.
  • the lysine resi- due(s) to be removed, preferably by replacement, is selected from the group consisting of K19, K33, K45, K52, K99, K105, K108, K115, K123, K134, and K136.
  • the lysine residue(s) may be replaced with any other amino acid residue, but is preferably replaced by an arginine or a glutamine residue in order to give rise to the least structural difference.
  • the polypeptide part may be one, wherein K19, K45, K52 and or K123, preferably K19, K45 and/or K123 has/have been replaced with another any other amino acid residue, preferably arginine or glutamine.
  • the interferon ⁇ polypeptide part of a conjugate comprises a combina- tion of amino acid substitutions selected from the following list: K19R+K45R+K123R; K19Q+K45R+K123R; K19R+K45Q+K123R; K19R+K45R+K123Q; K19Q+K45Q+K123Q; K19Q+K45R+K123Q; K19Q+K45Q+K123Q; K45R+K123R; K45Q+K123Q; K45R+K123Q; K45Q+K123Q; K45R+K123Q; K45R+K123Q; K45R+K123Q; K45R+K123Q; K45R+K123Q; K45R
  • polypeptide part may comprise at least one substitution selected from the group consisting of K33R, K33Q, K52R, K52Q, K99R, K99Q, K105R, K105Q, K108R, K108Q, K115R, K115Q, K134R, K134Q, K136R, and K136Q, e.g.
  • K52R+K134R K99R+K136R; K33R+K105R+K136R; K52R+K108R+K134R; K99R+K115R+K136R; K19R+K33R+K45R+K123R; K19R+K45R+K52R+K123R; K19R+K33R+K45R+K52R+K123R; or K19R+K45R+K52R+K99R+K123R.
  • the amino acid sequence of the interferon ⁇ polypeptide differs from that shown in SEQ ID NO:2 in that a lysine residue has been introduced by substitution of at least one amino acid residue occupying a position that in the parent interferon ⁇ molecule is occupied by a surface exposed amino acid residue, preferably an amino acid residue having at least 25%, such as at least 50% of its side chain exposed to the surface.
  • the amino acid residue to be substituted is selected from the group consisting of N4, F8, L9, Rll, S12, F15, Q16, Q18, L20, W22, Q23, G26, R27, L28, E29, Y30, L32, R35, M36, N37, D39, P41, E42, E43, L47, Q48, Q49, T58, Q64, N65, F67, A68, R71, Q72, D73, S75, S76, G78, N80, E81, 183, E85, N86, A89, N90, Y92, H93, H97, T100, L102, E103, L106, E107, E109, DUO, Fill, R113, G114, L116, M117, L120, H121, R124, G127, R128, L130, H131, E137, Y138, H140, 1145, R147, V148, E149, R152, Y155, F156, N158
  • the amino acid sequence of the interferon ⁇ polypeptide differs from the amino acid sequence shown in SEQ ID NO:2 in that a lysine residue has been intro- prised, by substitution, of at least one amino acid residue occupying a position selected from the group consisting of N4, F8, L9, Rl 1, S12, G26, R27, E29, R35, N37, D39, E42, L47, Q48, Q49, A68, R71, Q72, D73, S75, G78, N80, E85, N86, A89, Y92, H93, DUO, Fill, R113, L116, H121, R124, G127, R128, R147, V148, Y155, N158, R159, G162 and R165, even more preferably selected from the group consisting of N4, Rll, G26, R27, Q48, Q49, R71, D73, S75, N80, E85, A89, Y92, H93, Fill, R
  • the interferon ⁇ polypeptide comprises a substitution to lysine in one or more of the above positions, in particular in 1-15, such as 1-8 or 1-5, and preferably in at least two positions, such as 2-8 or 2-5 positions.
  • amino acid sequence of the interferon ⁇ polypeptide part of a conjugate differs in at least one removed and at least one introduced lysine residue, such as 1-5 or 2-5 removed lysine residues and 1-5 or 2-5 introduced lysine residues. It will be understood that the lysine residues to be removed and introduced preferably are selected from those described in the present section.
  • the total number of conju- gatable lysine residues is preferably in the range of 1-10, such as 2-8 or 3-7.
  • the interferon ⁇ polypeptide part of the conjugate according to this embodiment may comprise at least one of the following substitutions: RllK, Q48K, Q49K, R71K, S75K, N80K, E85K, A89K, H93K, Fll IK, R113K, L116K and Y155K; more preferably RllK, Q49K, R71K, S75K, N80K, E85K, A89K, H93K, Fll IK, R113K, L116K and Y155K, in combination with at least one of the substitutions: K19R/Q K33R/Q K45R/Q, K52R/Q, K99R/Q, K105R/Q, K108R/Q, K115R/Q, K123R/Q, K134R Q, and K136R/Q, wherein R/Q indicates substitution to an R or a Q residue, preferably an R residue.
  • the interferon ⁇ polypeptide comprises at least one of the following substitutions RllK, Q49K, R71K, S75K, N80K, E85K, A89K, H93K, Fll IK, R113K, L116K and Y155K, in par- ticular Q49K, FI 1 IK and/or N80K, in combination with substitution of at least one of K19, K45, K52 and/or K123, preferably to an R or a Q residue.
  • the interferon ⁇ polypeptide comprises at least one of the substitutions Q49K, Fll IK and N80K in combination with at least one of the substitutions mentioned above for removal of a lysine residue.
  • the interferon ⁇ polypeptide may comprise the following substitutions: Y+Z+K19R+K45R+K123R; Y+Z+K19Q+K45R+K123R Y+Z+K19R+K45Q+K123R Y+Z+K19Q+K45Q+K123R Y+Z+K19Q+K45Q+K123Q Y+Z+K19Q+K45R+K123Q Y+Z+K19Q+K45Q+K123Q Y+Z+K45Q+K123Q Y+Z+K45R+K123R Y+Z+K45Q+K123Q Y+Z+K45R+K123R Y+Z+K45Q+K123Q Y+Z+K45R+K123Q Y+Z+K45R+K123Q Y+Z+K45R+K123Q Y+Z+K45R+K123Q Y+Z+K45R+K123Q Y+Z+
  • Y+Z+K19Q+K45Q wherein Y is selected from the group of Q49K, Fll IK, N80K, Q49K+F11 IK, Q49K+N80K, FI 11K+N80K and Q49K+F111K+N80K and Z is absent or comprises at least one substitution selected from the group consisting of K33R, K33Q, K52R, K52Q, K99R, K99Q, K105R, K105Q, K108R, K108Q, K115R, K115Q, K134R, K134Q, K136R, and K136Q.
  • the interferon ⁇ polypeptide comprises the following substitution Y+Z+K19R+K45Q+K123R, wherein Y and Z have the above meaning.
  • the interferon ⁇ polypeptide may comprise one of the following substitutions: K19R+K45R+F111K+K123R; K19R+K45R+Q49K+F111K+K123R; K19R+K45R+Q49K+K123R; K19R+K45R+ F111K; K19R+K45R+Q49K+F11 IK; K19R+Q49K+K123R; K19R+Q49K+F111K+K123R; K45Q+F111K+K123Q; K45R+Q49K+K123R; or K45R+Q49K+F111K+K123R.
  • the interferon ⁇ polypeptide may contain the substitution N80K or C17S+N80K, optionally in combination with one or more of K19R Q; K45R/Q; K52R/Q or K123R/Q.
  • the substitution N80K is of particular interest, when the interferon ⁇ polypeptide is expressed in a non-glycosylating host cell, since N80 constitutes part of an inherent glycosylation site of human interferon ⁇ and conjugation at such site may mimick natural glycosylation.
  • the conjugate according to this aspect comprises at least two first non-polypeptide moieties, such as 2-8 moieties.
  • the invention relates a conjugate exhibiting interferon ⁇ activity and comprising at least one first non-polypeptide conjugated to at least one cysteine residue of an interferon ⁇ polypeptide, the amino acid sequence of which differs from that of wild-type human interferon ⁇ in that at least one cysteine residue has been introduced, prefer- erably by substitution, into a position that in the parent interferon ⁇ molecule is occupied by an amino acid residue that is exposed to the surface of the molecule, preferably one that has at least 25%, such as at least 50% of its side chain exposed to the surface.
  • the amino acid residue is selected from the group consisting of F8, L9, Rll, S12, F15, Q16, Q18, L20, W22, L28, L32, M36, P41, T58, Q64, N65, F67, 183, E85, N86, A89, N90, Y92, H93, H97, T100, L102, E103, L106, M117, L120, H121, R124, G127, R128, L130, H131, H140, 1145, R147, V148, E149, R152, Y155, and F156 of SEQ ID NO:2.
  • the substitution is preferably performed at a position occupied by a threonine or serine residue.
  • position is selected from the group consisting of S2, S12, S13, T58, S74, S75, S76, T77, T82, T100, T112, S118, S119, S139, T144, and T161, more preferably S2, S12, S13, S74, S75, S76, T77, T82, T100, T112, S118, S119, S139, and T144 (side chain surface exposed), still more preferably S2, S12, S75, S76, T82, T100, SI 19 and S139 (at least 25% of its side chain exposed), and even more preferably S12, S75, T82 and T100 (at least 50% of its side chain exposed).
  • the position is selected from the group consisting of S2, S12, S13, S74, S75, S76, S118, S119 and S139, more preferably S2, S12, S13, S74, S75, S76, S118, S119 and S139, even more preferably S2, S12, S75, S76, S119 and S139, and still more preferably S12 and S75.
  • the interferon ⁇ polypeptide part of the conjugate according to this embodiment of the invention comprises the mutation L47C, Q48C, Q49C, D110C, F111C or R113C, in particular only one of these mutations, optionally in combination with the mutation C17S.
  • the interferon ⁇ polypeptide may comprise the substitution C17S+N80C.
  • the first non-polypeptide moiety according to this aspect of the invention may be any molecule which, when using the given conjugation method has cysteine as an attachment group (such as a carbohydrate moiety, a lipophilic group or an organic derivatizing agent), it is preferred that the non-polypeptide moiety is a polymer molecule.
  • the polymer molecule may be any of the molecules mentioned in the section entitled "Conjugation to a polymer molecule", but is preferably selected from the group consisting of linear or branched polyethylene glycol or polyalkylene oxide. Most preferably, the polymer molecule is VS-PEG.
  • the conjugation between the polypeptide and the polymer may be achieved in any suitable manner, e.g. as described in the section entitled "Conjugation to a polymer molecule", e.g. in using a one step method or in the stepwise manner referred to in said section.
  • the interferon ⁇ polypeptide comprises only one conjugatable cysteine residue, this is preferably conjugated to a first non-polypeptide moiety with a molecular weight of at least 20kDa, either directly conjugated or indirectly through a low molecular weight polymer (as disclosed in WO 99/55377).
  • the conjugate comprises two or more first non-polypeptide moieties, normally each of these has a molecular weight of 5 or lOkDa.
  • the invention relates to a conjugate exhibiting interferon ⁇ activity and comprising at least one first non-polypeptide moiety having an acid group as the attachment group, which moiety is conjugated to at least one aspartic acid residue or one glutamic acid residue of an interferon ⁇ polypeptide, the amino acid sequence of which differs from that of wild-type human interferon ⁇ in at least one introduced and/or at least one removed aspartic acid or glutamic acid residue, respectively.
  • the relevant amino acid residue may be introduced in any position occupied by a surface exposed amino acid residue, preferably by an amino acid residue having more than 25% of its side chain surface exposed.
  • At least one amino acid residue occupying a position selected from the group consisting of N4, L5, L6, F8, L9, Q10, Rll, S12, S13, F15, Q16, Q18, K19, L20, W22, Q23, L24, N25, G26, R27, Y30, M36, Q46, Q48, Q49, 166, F67, A68, 169, F70, R71, S75, T82, 183, L87, A89, N90, V91, Y92, H93, Q94, 195, N96, H97, K108, Fill, L116, L120, K123, R124, Y126, G127, R128, L130, H131, Y132, K134, A135, H140, T144, R147, Y155, F156, N158, R159, G162, Y163 and R165 has been substituted with an aspartic acid residue or a glutamic acid residue. More preferably, the position is
  • the conjugate according to this aspect comprises at least two first non-polypeptide moieties.
  • the amino acid sequence of the interferon ⁇ polypeptide differs from that of human wild-type interferon ⁇ in at least one removed aspartic acid or glutamic acid residue, such as 1-5 removed residues, in particular 1-4 or 1-3 removed aspartic acid or glutamic acid residues.
  • the residue(s) to be removed is selected from the group consisting of D34, D39, D54, D73 , D 110, E29, E42, E43, E53, E61, E81, E85, E103, E104, E107, E109, E137 and E149.
  • the aspartic acid or glutamic acid residue(s) may be replaced with any other amino acid residue, but is preferably replaced by an arginine or a glutamine residue.
  • first non-polypeptide moiety can be any non- polypeptide moiety with such property, it is presently preferred that the non-polypeptide moiety is a polymer molecule or an organic derivatizing agent having an acid group as an attachment group, in particular a polymer molecule such as PEG, and the conjugate is prepared, e.g., as described by Sakane and Pardridge, Pharmceutical Research, Vol. 14, No. 8, 1997, pp 1085- 1091.
  • the non-polypeptide moiety has a molecular weight of about 5 or 10 kDa.
  • the conjugate may comprise a second non-polypeptide moiety of a different type as compared to the first non-polypeptide moiety.
  • a sec- ond non- polypeptide moiety is a sugar moiety, in particular an N-linked sugar moiety.
  • the second non-polypeptide moiety may be attached to a natural glycosylation site of human interferon ⁇ , e.g. the N-linked glycosylation site defined by N80, it is normally advantageous to introduce at least one additional glycosylation site in the interferon ⁇ polypeptide.
  • a natural glycosylation site of human interferon ⁇ e.g. the N-linked glycosylation site defined by N80
  • at least one additional glycosylation site in the interferon ⁇ polypeptide is e.g. any of those described in the immediately preceding section.
  • at least one additional glycosylation site is introduced this may be accompanied by removal of an existing glycosylation site as described below.
  • the interferon ⁇ polypeptide may be modified in the number and distribution of attachment groups for the first as well as the second non-polypeptide moiety so as to have e.g. at least one removed attachment group for the first non-polypeptide moiety and at least one introduced attachment group for the second non-polypeptide moiety or vice versa.
  • the interferon ⁇ polypeptide comprises at least two (e.g. 2-5) removed attachment groups for the first non-polypeptide moiety and at least one (e.g. 1-5) introduced attachment groups for the second non-polypeptide moiety or vice versa.
  • the first non-polypeptide moiety is a polymer molecule such as PEG having lysine as an attachment group
  • the second non- polypeptide moiety is an N-linked sugar moiety
  • the conjugate may be one exhibiting interferon ⁇ activity and comprising at least one polymer molecule, preferably PEG, and at least one sugar moiety covalently attached to an interferon ⁇ polypeptide, the amino acid sequence of which differs from that of wild-type human interferon ⁇ in a) at least one introduced and/or at least one removed amino acid residue comprising an attachment group for the polymer molecule; and b) at least one introduced and/or at least one removed in vivo glycosylation site, in particular an N-glycosylation site, provided that when the attachment group for the polymer molecule is a cysteine residue, and the sugar moiety is an N-linked sugar moiety, a cysteine residue is not inserted in such a manner that an N-glycosylation site is destroyed.
  • WO 99/03887 suggests that a cysteine residue can be introduced into the natural N-glycosylation site of interferon ⁇ .
  • the interferon ⁇ polypeptide comprises one of the following sets of mutations: K19R+K45R+Q49N+Q51T+F111N+R113T+K123R; K19R+K45R+Q49N+Q51T+F111N+R113T; or K19R+K45R+Q49N+Q51T+K123R.
  • the conjugate comprises at least one sugar moiety attached to an in vivo glycosylation site, in particular an N-glycosylation site
  • this is either the natural N-glycosylation site of wild-type human interferon ⁇ at position N80, i.e. defined by amino acid residues N80, E81, T82 and 183, or a new in vivo glycosylation site introduced into the interferon ⁇ polypeptide.
  • the in vivo glycosylation site may be an O-glycosylation site, but is preferably an N- glycosylation site.
  • the invention relates to a conjugate exhibiting interferon ⁇ activity and comprising an interferon ⁇ polypeptide, the amino acid sequence of which differs from that of wild-type human interferon ⁇ in at least one introduced glycosylation site, the conjugate further comprising at least one un-PEGylated sugar moiety attached to an introduced glycosylation site.
  • Another embodiment relates to a conjugate exhibiting interferon ⁇ activity and comprising an interferon ⁇ polypeptide, the amino acid sequence of which differs from that of wild-type human interferon ⁇ in that a glycosylation site has been introduced or removed, provided that if only a glycosylation site is removed (and thus that no glycosylation site is introduced) the interferon ⁇ polypeptide does not comprise one or more of the following substutions: N80C, E81C or T82C. The latter substitution is suggested in WO 99/03887.
  • an in vivo glycosylation site is introduced into a position of the parent interferon ⁇ molecule occupied by an amino acid residue exposed to the surface of the molecule, preferably with more than 25% of the side chain exposed to the solvent, in particular more than 50% exposed to the solvent (these positions are identified in the Methods section herein).
  • the N-glycosylation site is introduced in such a way that the N-residue of said site is located in said position.
  • an O-glycosylation site is introduced so that the S or T residue making up such site is located in said position.
  • the in vivo glycosylation site in particular the N residue of the N-glycosylation site or the S or T residue of the O-glycosylation site, is located within the first 141 amino acid residues of the interferon ⁇ polypeptide, more preferably within the first 116 amino acid residues. Still more preferably, the in vivo glycosylation site is introduced into a position wherein only one mutation is required to create the site (i.e. where any other amino acid residues required for creating a functional glycosylation site is already present in the molecule).
  • Substitutions that lead to introduction of an additional N-glycosylation site at positions exposed at the surface of the interferon ⁇ molecule and occupied by amino acid residues having more than 25% of the side chain exposed to the surface include:
  • Substitutions that lead to introduction of an additional N-glycosylation site at positions exposed at the surface of the interferon ⁇ molecule having more than 50% of the side chain exposed to the surface include:
  • Substitutions that lead to introduction of an N-glycosylation site by only one amino acid substitution include: L6S/T, RUN, D39S/T, Q72N, D73N, S75N, L88S/T, Y92S/T, L98S/T, D110N, L116N, E137N, R159N and L160S/T.
  • a substitution is pre- ferred that is selected from the group consisting of L6S/T, Rl IN, D39S/T, Q72N, D73N, S75N, L88S/T, Y92S/T, L98S/T, Dl ION and LI 16N, more preferably from the group consisting of L6S/T, D39S/T, D73N, S75N, L88S/T, D110N, L116N and E137N; and most preferably selected from the group consisting of L6S/T, D39S/T, D73N, S75N, L88S/T, D110N and L116N.
  • the presently most preferred interferon ⁇ polypeptide according to this aspect in- elude at least one of the following substitutions:
  • amino acid residue in between the N-residue and the S/T residue is different from proline.
  • amino acid residue in between will be that occupying the relevant position in the amino acid sequence shown in SEQ ID NO 2.
  • position 50 is the position in between.
  • the interferon ⁇ polypeptide part of a conjugate may contain a single in vivo glycosylation site.
  • the polypeptide comprises more than one in vivo glycosylation site, in particular 2-7 in vivo glycosylation sites, such as 2, 3, 4, 5, 6 or 7 in vivo glycosylation sites.
  • the interferon ⁇ polypeptide may comprise one additional glycosylation site, or may comprise two, three, four, five, six, seven or more introduced in vivo glycosylation sites, preferably introduced by one or more substitutions described in any of the above lists.
  • the interferon ⁇ polypeptide may comprise a substitution of N80, e.g. one of the substitutions N80K/C D E, when a first non-polypeptide polypeptide is one having one of K, C, D, E as an attachment group.
  • the interferon ⁇ polypeptide may comprise at least one of the following substitutions: S2N+N4T/S, L9N+R11T/S, RUN, S12N+N14T/S, F15N+C17S/T, Q16N+Q18T/S, K19N+L21T/S, Q23N+H25T/S, G26N+L28T/S, R27N+E29T/S, L28N+Y30T/S, D39T/S, K45N+L47T/S, Q46N+Q48T/S, Q48N+F50T/S,
  • the interferon ⁇ polypeptide may comprise the substitution: Q49N+Q51T or F111N+R113T or R71N+D73T, in particular Q49N+Q51T+F111N+R113T or Q49N+Q51T+R71N+D73T+ F111N+ R113T, in combination with N80K/C/D/E.
  • any of the glycosylated variants disclosed in the present section having introduced and/or removed at least one glycosylation site may further be conjugated to a polymer molecule, such as PEG, or any other non-polypeptide moiety.
  • the conjugation may be achieved by use of attachment groups already present in the interferon ⁇ polypeptide or attachment groups may have been introduced and/or removed, in particular such that a total of 1-6, in particular 3-4 or 1, 2, 3, 4, 5, or 6 attachment groups are available for conjugation.
  • the number and molecular weight of the non-polypeptide moiety is chosen so as that the total molecular weight added by the non-polypeptide moiety is in the range of 20-40 kDa, in particular about 20 kDa or 30 kDa.
  • the glycosylated variant may be conjugated to a non-polypeptide moiety via a lysine attachment group, and one or more lysine residues of the parent polypeptide may have been removed, e.g. by any of the substitutions mentioned above, in particular the substitutions K19R+K45R+K123R.
  • a lysine residue may have been introduced, e.g. by any of the substitutions mentioned in said section, in particular the substitution R71K.
  • one specific conjugate is one, which comprises a glycosylated interferon ⁇ polypeptide comprising the mutations Q49N + Q51T + FI 1 IN + Rl 13T + K19R + K45R + K123R or Q49N + Q51T + F111N + R113T + K19R + K45R + K123R + R71K further conjugated to PEG.
  • the glycosylated polypeptide part of said conjugate is favourably produced in CHO cells and PEGylated subsequent to purification using e.g.
  • the glycosylated conjugate according to this embodiment may be PEGylated via a cysteine group as described above (for this purpose the interferon ⁇ polypeptide may, e.g. comprising at least one of the mutations N80C, R71C and C17S), via an acid group as above, or via any other suitable group.
  • the dispersions of particles comprising the interferon ⁇ conjugates disclosed herein may be used for treating most types of viral infections, cancers or tumors (e.g.
  • the dispersion of particles may be used for the treatment of multiple sclerosis (MS), such as any of the generally recognized four types of MS (benign, relapsing remitting MS (RRMS), primary progressive MS (PPMS) and secondary progressive MS (SPMS)) and for monosymptomatic MS), hepatitis, or a herpes infection (the latter treatment optionally being combined with a treatment with IL-10).
  • MS multiple sclerosis
  • RRMS relapsing remitting MS
  • PPMS primary progressive MS
  • SPMS secondary progressive MS
  • FSH Follicle Stimulating Hormone
  • the ⁇ sub- unit is common to the glycoprotein hormone family, which apart from FSH includes chorionic gonadotropin (CG), thyroid stimulating hormone (TSH), and luteinizing hormone (LH), whereas the ⁇ subunit is specific to FSH.
  • the human wild-type ⁇ subunit is a 92 amino acid glycoprotein, the amino acid sequence of which is shown in SEQ ID NO:3. This subunit is re- ferred to herein as hFSH- ⁇ .
  • the human wild-type ⁇ subunit is a 111 amino acid glycoprotein, the amino acid sequence of which is shown in SEQ ID NO:4. This subunit is referred to herein as hFSH- ⁇ .
  • one or both of the FSH subunits may be modified according to the invention.
  • the amino acid sequence of FSH- ⁇ may be modified as described herein, whereas FSH- ⁇ is unmodified, and vice versa.
  • both of FSH- ⁇ and FSH- ⁇ may be modified according to the invention.
  • FSH- ⁇ and/or FSH- ⁇ may be of any origin, it is in particular of mammalian origin, and preferably of human origin. Accordingly, the corresponding wildtype sub- units referred to above are preferably hFSH- ⁇ and hFSH- ⁇ , respectively, with the amino acid sequences shown in SEQ ID NO:3 and 4.
  • An (a) suffix indicates the ⁇ chain, whereas a (b) suffix indicates the ⁇ chain.
  • a particular interesting embodiment of the present invention relates to a dis- persion of particles containing a heterodimeric polypeptide conjugate exhibiting FSH activity, comprising i) a dimeric polypeptide comprising an FSH- ⁇ subunit and an FSH- ⁇ subunit, wherein at least one of said FSH- ⁇ and FSH- ⁇ subunits differs from the corresponding wild- type subunit in that at least one amino acid residue comprising an attachment group for a non- polypeptide moiety has been introduced or removed, and ii) at least one non-polypeptide moiety bound to an attachment group of at least one of said subunits.
  • the wild-type FSH- ⁇ subunit has the amino acid shown in SEQ ID NO:3 and the wild-type FSH- ⁇ subunit has the amino acid shown in SEQ D NO:4.
  • FSH- ⁇ and/or FSH- ⁇ and the corresponding wild-type sequence is that at least one and preferably more, e.g. 1-20, amino acid residues comprising an attachment group for the non- polypeptide moiety have been introduced, by insertion or substitution, into the amino acid sequence.
  • properties such as the molecular weight, shape, size and/or charge of the con- jugate can be optimized.
  • amino acid residues are introduced in positions occupied by an amino acid residue having more than 25%, more preferably more than 50%, such as more than 75% of its side chain exposed at the surface of the molecule.
  • N-glycosylation is important for FSH activity and also that the extent and type of oligosaccharide moiety attached by in vivo glycosylation is important for functional in vivo half -life of the glycosylated FSH. In order to obtain a different, increased glycosylation it is desirable to introduce at least one glycosylation site.
  • the dispersion of particles contain a heterodimeric polypeptide conjugate exhibiting FSH activity comprising a dimeric polypeptide comprising an FSH- ⁇ subunit and an FSH- ⁇ subunit, wherein the amino acid sequence of at least one of the FSH- ⁇ and FSH- ⁇ sub- units differs from that of the corresponding wild-type subunit in that at least one N- glycosylation site has been introduced, and having at least one oligosaccharide moiety bound to an N-glycosylation site of at least one of the subunits.
  • a suitable N-glycosylation site may be introduced by introducing, by substitution or insertion, an asparagine residue in a position occupied by an amino acid residue having more than 25% of its side chain exposed at the surface of the polypeptide, which position does not have a proline residue located in position +1 or +3 therefrom.
  • Ji the amino acid residue located in position +2 is a serine or threonine, no further amino acid substitution is required. However, if this position is occupied by a different amino acid residue, a serine or threonine residue needs to be introduced.
  • a preferred conjugate according to this embodiment is one which comprises a modified FSH- ⁇ subunit having an amino acid residue which differs from that of hFSH- ⁇ in the introduction of at least one N-glycosylation site by means of a mutation selected from the group consisting of P2(a)N+V4(a)S, P2(a)N+V4(a)T, D3(a)N+Q5(a)S, D3(a)N+Q5(a)T, V4(a)N+D6(a)S, V4(a)N+D6(a)S, D6(a)N+P8(a)S, D6(a)N+P8(a)T, E9(a)N+Tll(a)S, E9(a)N, Tll(a)N+Q13(a)S, Tll(a)N+Q13(a)T, L12(a)N+E14(a)S, L12(a)N+E14(a)T,
  • a glycosylation site can be introduced by mutation of a single amino acid residue, i.e. selected from the group consisting of V68(a)S, V68(a)T, E9(a)N, F17(a)N, K44(a)N, R67(a)N, H83(a)N and H90(a)N. More preferably, a glycosylation site is introduced at a position having more than 50% side chain exposure, i.e.
  • glycosylation sites are introduced via mutation of a single amino acid residue selected from the group consisting of E9(a)N, F17(a)N,
  • the FSH- ⁇ part of such conjugates with an altered FSH- ⁇ subunit may be hFSH- ⁇ or any of the modified FSH- ⁇ polypeptides described herein.
  • the conjugate according to this embodiment com- prises a modified FSH- ⁇ having an amino acid residue which differs from that of hFSH- ⁇ in the introduction of at least one N-glycosylation site by a mutation selected from the group consisting of S2(b)N+E4(b)S, S2(b)N+E4(b)T, E4(b)N+T6(b)S, E4(b)N, L5(b)N+N7(b)S, L5(b)N+L7(b)T, T6(b)N+I8(b)S, T6(b)N+I8(b)T, I8(b)N+I10(b)S, I8(b)N+I10(b)T, T9(b)N+All(b)S, T9(b)N+All(b)T, K14(b)N+E16(b)S, K14(b)N+E16(b)T, F19
  • glycosylation sites are introduced by means of mutation of a single amino acid residue selected from the group consisting of E4(b)N, Y58(b)N, L73(b)N, S89(b)N, D90(b)N, D93(b)N, and Y103(b)N.
  • a modified FSH- ⁇ has an amino acid residue which differs from that of hFSH- ⁇ in the introduction of at least one N-glycosylation site by a mutation selected from the group consisting of F19(b)N+I21(b)S, F19(b)N+I21(b)T, Y33(b)N+R35(b)S, Y33(b)N+R35(b)T, A43(b)N+P45(b)S, A43(b)N+P45(b)T, P45(b)N+I47(b)S, P45(b)N+I47(b)T, K46(b)N+Q48(b)S, K46(b)N+Q48(b)T, I47(b)N+K49(b)S, I47(b)N+K49(b)T, K54(b)N+L56(b)S, K54(b)N+L56(b)T, E55(b)N+
  • the FSH- ⁇ part of such conjugates with an altered FSH- ⁇ subunit may be hFSH- ⁇ or any of the modified FSH- ⁇ polypeptides described herein.
  • the FSH- ⁇ and/or FSH- ⁇ polypeptide may further differ from hFSH- ⁇ and/or hFSH- ⁇ in at least one removed, naturally occurring N-glycosylation site.
  • FSH- ⁇ may comprise a substitution of N78(a) and/or T80(a) by any other amino acid residue and/or FSH- ⁇ may comprise a substitution of N7(b), T9(b), N24(b) and/or T26(b) by any other amino acid residue.
  • the N residue is substituted by Q or D, and the T residue by A or G.
  • one or both of the FSH- ⁇ and FSH- ⁇ subunits of the conjugate according to this embodiment may differ from hFSH- ⁇ and hFSH- ⁇ , respectively, in the removal, preferably by substitution, of at least one lysine residue. See the section below on removal of lysine residues for further details.
  • An alternative embodiment of this aspect of the invention is one in which at least one of said FSH- ⁇ and FSH- ⁇ subunits comprises at least one introduced N- or O-glycosylation site at the N-terminal thereof, and wherein the at least one introduced glycosylation site is gly- cosylated; see the discussion of peptide addition below.
  • the respective subunits may comprise one or more of the modifications disclosed elsewhere herein, or one or both of the subunits may be the respective wild-type subunits, but having the at least one introduced terminal glycosylation site.
  • the polypeptide conjugate may be one in which the FSH- ⁇ subunit comprises hFSH- ⁇ having the sequence shown in SEQ ID NO:3, and/or in which the FSH- ⁇ subunit comprises hFSH- ⁇ having the sequence shown in SEQ ID NO:4.
  • both of the subunits correspond to the respective wildtype hFSH subunits, although with either the ⁇ or ⁇ subunit, or both, having an introduced N-terminal glycosylation site.
  • the introduced glycosylation site may be of the type described elsewhere herein; see the discussion of glycosylation under the general discussion of attachment groups above.
  • a non-limiting example of a suitable glycosylation site for introduction at the N-terminal is the sequence Ala-Asn-He-Thr-Nal-Asn-Ue-Thr-Nal, e.g. for insertion of two glycosylation sites upstream of a mature FSH- ⁇ or FSH- ⁇ sequence.
  • each of the polypeptide subunits comprises or consists of or consists essentially of the primary structure
  • X is a peptide addition comprising or contributing to a glycosylation site
  • P is the basic polypeptide subunit to be modified, i.e. FSH- ⁇ or FSH- ⁇ , e.g. a wildtype polypeptide subunit as defined herein or a modified polypeptide having introduced and/or removed glycosylation sites or other attachment sites in the mature part of the polypeptide.
  • the term "comprising a glycosylation site” is intended to mean that a complete glycosylation site is present in the peptide addition, whereas the term “contributing to a glycosylation site” is intended to cover the situation where at least one amino acid residue of an N-glycosylation site is present in the peptide addition while the other amino acid residue of said site is present in the polypeptide P, whereby the glycosylation site can be considered to bridge the peptide addition and the polypeptide.
  • the peptide addition is fused to the N-terminal or C-terminal end of the polypeptide P as reflected in the above shown structure so as to provide an N- or C-terminal elongation of the polypeptide P, preferably at the N-terminal.
  • the peptide addition within the amino acid sequence of the polypeptide P whereby the polypeptide comprises, consists of or consists essentially of the primary structure NH 2 -P x -X-P y - COOH, wherein
  • P x is an N-terminal part of the relevant polypeptide P, Pyis a C-terminal part of said polypeptide P, and X is a peptide addition comprising or contributing to a glycosylation site.
  • P x may be a non-structural N-terminal part of a mature polypeptide P, and P y a structural C-terminal part of said mature polypeptide, or P x may be a structural N-terminal part of a mature polypeptide P, and P y a non-structural C-terminal part of said mature polypeptide.
  • non-structural part is intended to indicate a part of either the C- or N- terminal end of the folded polypeptide subunit that is outside the first structural element, such as an ⁇ -helix or a ⁇ -sheet structure.
  • the non-structural part can easily be identified in a three- dimensional structure or model of the polypeptide. If no structure or model is available, a non- structural part typically comprises or consists of the first or last 1-20 amino acid residues, such as 1-10 amino acid residues of the amino acid sequence constituting the mature form of the polypeptide.
  • the peptide addition can be inserted into a loop structure of the polypeptide P and thereby elongate the loop.
  • the peptide addition X can be any stretch of amino acid residues ranging from a single amino acid residue to a mature protein.
  • each peptide addition will normally comprise up to about 50 amino acid residues, such as 2-30 or 3-20 amino acid residues.
  • the peptide addition may be designed by a site- specific or random approach.
  • N- or C-terminal extensions of the FSH sequence that comprise peptide sequences that are part of naturally occurring human proteins.
  • Non-limiting examples of such peptide sequences include the sequence NSTQNATA, which corresponds to positions 231 to 238 of the human calcium activated channel 2 precursor (to add two N-glycosylation sites to FSH), or the sequence ANLTNRNLTRNVTV, which corresponds to positions 538 to 551 of the human G protein coupled receptor 64 (to add three N-glycosylation sites to FSH).
  • each peptide addition X comprises 1-10 glycosylation sites.
  • the peptide addition X may thus comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 glycosylation sites. It is well known that a frequently occurring consequence of modifying an amino acid sequence of, e.g., a human protein is that new epitopes are created by such modification. Non-polypeptide moieties may be used to shield any new epitopes created by the peptide addition, and therefore it is desirable that sufficient glycosylation sites (or attachment groups for another non-polypeptide moiety, e.g. a polymer such as PEG) are present to enable shielding of all epitopes introduced into the sequence. This is e.g.
  • the peptide addition X comprises at least one gly- cosylation site within a stretch of 30 contiguous amino acid residues, preferably as at least one glycosylation sites within 20 amino acid residues, more preferably at least one attachment group within 10 amino acid residues, in particular 1-3 attachment groups within a stretch of 10 contiguous amino acid residues in the peptide addition X.
  • the glycosylation site of the peptide addition is an in vivo glycosylation site, preferably an N-glycosylation site.
  • the peptide addition X may have the structure X 1 -N-X 2 -T/S/C-Z, wherein X .
  • X 2 is any amino acid residue different from P
  • Z is absent or is a peptide comprising at least one amino acid residue.
  • Xy may absent
  • X 2 may be an amino acid residue selected from the group consisting of I, A, G, V and S (all relatively small amino acid residues)
  • Z may comprise at least 1 amino acid residue.
  • Z can e.g. be a peptide comprising up to 50 amino acid residues and e.g. up to 10 glycosylation sites.
  • X may comprise at least one amino acid residue, e.g. 1-50 amino acid residues with 1-10 glycosylation sites, X may be an amino acid residue selected from the group consisting of I, A, G, V and S, and Z may be absent.
  • peptide additions for use in the present invention are ANITVNITV, NDTVNF ⁇ and NITVNITV.
  • the peptide addition can comprise one or more of these peptide sequences, i.e. at least two of said sequences either directly linked together or separated by one or more amino acid residues, or can contain two or more copies of any of these peptide sequence. It will be understood that the above specific sequences are given for illustrative purposes and thus do not constitute an exhaustive list of peptide sequences of use in the present invention.
  • the peptide addition X has an N residue in position -2 or -1
  • the polypeptide P or P x has a T or an S residue in position +1 or +2, respectively, the resi- due numbering being made relative to the N-terminal amino acid residue of P or P x , whereby an N-glycosylation site is formed.
  • the polypeptide may have a T or S residue in position 2, preferably a T residue
  • the peptide addition is AN or comprises AN as the C- terminal amino acid residues.
  • the het- erodimeric polypeptide may comprise one or more introduced O-glycosylation sites, for example the amino acid sequence AATPAP, which has been found to be an efficient signal sequence for O-glycosylation in vivo (Asada et al. (1999) Glycoconj. J. 16(7):321-6).
  • the AATPAP se- quence for O-glycosylation is preferably introduced by way of insertion at the N- and/or C- terminus of the FSH- ⁇ and/or FSH- ⁇ subunit.
  • the polypeptide in order to prepare a conjugate according to this aspect, the polypeptide must be expressed in a glycosylating host cell capable of attaching oligosaccha- ride moieties at the glycosylation site(s) in vivo or alternatively subjected to in vitro glycosylation.
  • glycosylating host cells are given in the section further below entitled “Coupling to an oligosaccharide moiety”.
  • the conjugate according to the aspect of the invention described in the present section may contain additional non-polypeptide moieties different from O-linked or N-linked oligosaccharide moieties, in particular a polymer molecule such as PEG as described herein conjugated to one or more attachment groups present in the polypeptide part of the conjugate.
  • a polymer molecule such as PEG as described herein conjugated to one or more attachment groups present in the polypeptide part of the conjugate.
  • a lysine residue or any other amino acid residue comprising an attachment group for the polymer molecule in question
  • any of the amino acid changes specified in this section can be combined with any of the amino acid changes specified in the other sections herein disclosing specific amino acid changes.
  • the conjugate is one wherein the amino acid residue comprising an attachment group for the non-polypeptide moiety is a lysine residue and the non-polypeptide moiety is any molecule which has lysine as an attachment group.
  • the non-polypeptide moiety may be a polymer molecule, in particular any of the molecules mentioned in the section entitled "Conjugation to a polymer molecule", and preferably selected from the group consisting of linear or branched polyethylene glycol and polyalkylene oxide.
  • the polymer molecule is mPEG-SPA or oxycarbonyl-oxy-N- dicarboxyimide PEG (US 5,122,614).
  • the FSH- ⁇ and/or FSH- ⁇ having introduced and/or removed at least one lysine may advantageously be in vivo glycosylated, e.g. using naturally occurring glycosylation sites present in the relevant FSH polypeptide.
  • the conjugate is one wherein the amino acid sequence of FSH- ⁇ and/or FSH ⁇ differs from that of FSH- ⁇ and/or FSH- ⁇ in that an N-glycosylation site has been introduced and/or removed.
  • Such introduced/removed sites may be any of those described above.
  • the conjugate is one which comprises a modified FSH- ⁇ having an amino acid residue which differs from that of hFSH- ⁇ in the removal of at least one lysine residue selected from the group consisting of K44(a), K45(a), K51(a), K63(a), K75(a), and K91(a), in particular at least one amino acid residue selected from of the group consisting of K44(a), K45(a), K63(a), K75(a), and K91(a) (these residues having more than 25% of their side chain exposed to the surface), and preferably from the group consisting of K45(a), K63(a), K75(a), and K91(a) (these residues having more than 50% of their side chain exposed to the surface).
  • K44(a), K45(a), K51(a), K63(a), K75(a), and K91(a in particular at least one amino acid residue selected from of the group consisting of K44(a), K45(a), K63(a), K75(a
  • the conjugate is one which comprises a modified FSH- ⁇ having an amino acid residue which differs, from that of hFSH- ⁇ in the removal of at least one lysine residue selected from the group consisting of K14(b), K40(b), K46(b), K49(b), K54(b), K86(b), and Kl 10(b), in particular at least one amino acid residue selected from of the group consisting of K14(b), K40(b), K46(b), K49(b), K54(b), K86(b), and Kl 10(b) (these residues having more than 25% of their side chain exposed to the surface), and preferably from the group consisting of K46(b), K54(b), K86(b), and Kl 10(b) (these residues having more than 50% of their side chain exposed to the surface).
  • the FSH- ⁇ part of this conjugate may be hFSH- ⁇ or any of the modified FSH- ⁇ polypeptides described herein.
  • the conjugate is one which comprises a modified FSH- ⁇ and a modified FSH- ⁇ , each of which differ from the corresponding hFSH subunit in the re- moval of at least one of the above identified lysine residues.
  • the conjugate may be one wherein the modified FSH- ⁇ and modified FSH- ⁇ subunit differ from the corresponding hFSH subunit in at least one of K45(a), K63(a), K75(a), and K91(a) and at least one of K46(b), K54(b), K86(b), and Kl 10(b).
  • lysine residues are preferably achieved by substi- tution by any other amino acid residue, in particular by an arginine or a glutamine residue.
  • lysine residue in hFSH in particular in a position occupied by an amino acid residue having a side chain which is more than 25% surface exposed and which is not part of a cystine or located at a receptor binding site.
  • the conjugate is one which comprises a modified FSH- ⁇ having an amino acid residue which differs from that of hFSH- ⁇ in the introduction of at least one lysine residue in a position selected from the group consisting of Al(a), P2(a), D3(a), V4(a), Q5(a), D6(a), P8(a), E9(a), Tll(a), L12(a), Q13(a), E14(a), P16(a), F17(a), Q20(a), P21(a), G22(a), A23(a), P24(a), L26(a), M29(a), F33(a), R42(a), S43(a), T46(a), L48(a), V49(a), Q50(a), N52(a), V61(a), S64(a), Y65(a), N66(a), R67(a), V68(a), T69(a), M71(a), G72(a), G73(
  • the FSH- ⁇ part of this conjugate may be hFSH- ⁇ or any of the modified FSH- ⁇ polypeptides described herein.
  • the conjugate is one which comprises a modified FSH- ⁇ having an amino acid residue which differs from that of hFSH- ⁇ in the introduction of at least one lysine residue in a position selected from the group consisting of Nl(b), S2(b), E4(b), L5(b), T6(b), N7(b), 18(b), T9(b), E15(b), E16(b), R18(b), F19(b), 121(b), S22(b), N24(b), Y31(b), Y33(b), R35(b), D36(b), L37(b), Y39(b), D41(b), P42(b), A43(b), R44(b), P45(b), 147(b), E55(b), L56(b), V57(b), Y58(b), E59(
  • the conjugate is one which comprises a modified FSH- ⁇ and a modified FSH- ⁇ , each of which differ from the corresponding hFSH subunit in the introduction of a lysine residue, preferably by substitution, in at least one of the above identified positions.
  • the conjugate may be one wherein the modified FSH- ⁇ and modified FSH- ⁇ subunit differ from the corresponding hFSH subunit in that a lysine residue has been introduced in at least one of R42(a) and R67(a), and at least one of R18(b), R35(b), R44(b), R62(b), and R97(b), and more preferably in R67(a), and at least one of R18(b), R44(b), R62(b), R97(b).
  • the conjugate may comprise at least one introduced lysine residue, in particular any of those described above, and at least one removed lysine residue, in particular any of those described above.
  • the conjugate comprises a modified FSH- ⁇ and/or a modified FSH- ⁇ which differs from the corresponding hFSH- ⁇ / ⁇ in at least one introduced and at least one removed lysine residue, wherein the lysine residue is introduced by substitution of an amino acid residue selected from the group consisting of R42(a) and R67(a), R18(b), R35(b), R44(b), R62(b), and R97(b), and more preferably from the group consisting of R67(a), R18(b), R44(b), R62(b), and R97(b) and removal of a lysine residue selected from the group consisting of K45(a), K63(a), K75(a), K91(a) K46(b), K54(b), K86(b), and Kl 10(b), the removal preferably being achieved by substitution by any other amino acid residue, in particular by an arginine residue.
  • the lysine residue is introduced by substitution of an amino acid residue selected from the group
  • amino acid residues comprising other attachment groups may be introduced into and/or removed from FSH- ⁇ and/or FSH- ⁇ , using the same approach as that illustrated above by lysine residues.
  • one or more amino acid residues comprising an acid group glutamic acid or aspartic acid
  • asparagine, tyrosine or cysteine may be introduced into positions which in hFSH are oc- cupied by amino acid residues having surface exposed side chains (i.e. the positions mentioned above as being of interest for introduction of lysine residues), or removed.
  • introduction or removal of such amino acid residues is preferably performed by substitution.
  • Asp is substituted by Asn, Glu by Gin, Tyr by Phe, and Cys by Ser.
  • Another possi- bility is introduction and/or removal of a histidine, e.g. by substitution with arginine.
  • the dispersions of particles comprising the FSH conjugates disclosed herein may be used for treatment of infertility or diseases associated with insufficient endogenous production of FSH.
  • Interleukin 10 (IL-10)
  • E -10 was initially described as an activity in the supematants of activated T- helper type 2 (Th2) clones that could inhibit the production of cytokines, especially interferon gamma (IFN-gamma) by T-helper type 1 (Thl) clones (J Exp Med 1989; 170:2081-2095).
  • Viral homologues of IL-10 have been detected in the genomes of Epstein-Barr virus (EBV) and equine herpesvirus 2 (Science 1990; 248:1230-1234) (Proc Natl Acad Sci USA 1991; 88:1172- 1176) (Virus Genes 1993; 7:111-116).
  • IL-10 has also been described under the names cytokine synthesis inhibitory factor (CSIF), mast cell growth factor TE (MCGF-DI) and B-cell derived T-cell growth factor (B- TCGF).
  • CIF cytokine synthesis inhibitory factor
  • MGF-DI mast cell growth factor TE
  • B-TCGF B-cell derived T-cell growth factor
  • Mature human IL-10 (ML-IO) consists of 160 amino acid residues, is biologically active as a homodimer and is derived from a precursor consisting of 178 amino acid residues. The mature human IL-10 is shown herein as SEQ ID NO: 5. The DNA sequences encoding these proteins have been reported (Human Cytokines, Handbook of Basic and Clinical Research, Volume JJ, Blackwell Science, Eds. Aggarwal and Gutterman, 1996, pp. 19-42). The same publication discloses the IL-10 receptor, its amino acid sequence and underlying DNA sequences, a bioassay for IL-10, methods of pur
  • H-10 has two intramolecular disulphide bonds and an un-occupied glycosylation site.
  • the three-dimensional structure of IL-10 has been reported (Biochemistry 1995; 34:12118- 12125) (Structure 1995; 3:591-601) (Protein Sci. 1996 5:1955-1962).
  • IL-10 has been suggested as an anti-inflammatory agent for treatment of inflammatory bowel disease, rheumatoid arthritis, uveitis, etc. It has also been suggested in connection with transplantation, immunodeficiencies and parasitic infections.
  • a particular interesting embodiment relates to a dispersion of particles containing a conjugate comprising at least one non-polypeptide moiety covalently attached to an IL-10 variant, wherein the amino acid sequence of the IL-10 variant differs from the amino acid sequence of the corresponding wild-type human IL-10 shown in SEQ ID NO: 5 in that at least one amino acid residue comprising an attachment group for said non-polypeptide moiety has been introduced or removed.
  • the conjugate is one, wherein the amino acid residue comprising an attachment group for the non-polypeptide moiety is a lysine residue and the non- polypeptide moiety is any molecule which has lysine as an attachment group.
  • the non-polypeptide moiety is a polymer molecule, in particular any of the molecules mentioned in the section entitled "Conjugation to a polymer molecule", and preferably selected from the group consisting of linear or branched polyethylene glycol or polyalkylene oxide.
  • the polymer molecule is PEG and the activated molecule to be used for conjugation is SS-PEG, NPC-PEG, aldehyd-PEG, mPEG-SPA, mPEG-SCM, mPEG-BTC from Shearwater Polymers, Inc, SC-PEG from Enzon, Inc., tresylated mPEG as described in US 5,880,255, or oxycarbonyl-oxy-N-dicarboxyimide-PEG (US 5,122,614).
  • the non-polypeptide moiety has a molecular weight of about 5 or 10 kDa.
  • the polypeptide variant having introduced and/or removed at least one lysine is preferably in vivo glycosylated, e.g. using naturally-occurring glycosylation sites present in the polypeptide.
  • the conjugate is one, wherein the amino acid sequence of the polypeptide variant differs from that of SEQ ID NO:5 in that an N- glycosylation site has been introduced and/or removed.
  • lysine residue(s) ML- 10 contains thirteen lysine residues one or more of which may be located in the receptor-binding domain. In order to avoid conjugation to one or more of these lysine residues (since this may inactivate or severely reduce the activity of the resulting conjugate) it may be desirable to remove at least one lysine residue.
  • the conjugate according to this embodiment comprises a polypeptide comprising an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO: 5 in the removal of at least one lysine residue selected from the group consisting of K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138 and K157, in particular selected from the group consisting of K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, and K157 (having more than 25% of their side chains surface exposed, even more preferably from the group consisting of K34, K49, K57, K88, K99, K117, K119, and K130 (having more than 50% of their side-chain surface exposed), and ii) a non-polypeptide moiety, which has a lysine residue as an attachment group.
  • the removal is preferably achieved by substitution with any other amino acid residue, in particular an arginine or a glutamine residue.
  • lysine residues in hIL-10, in particular in a position which in hIL-10 is occupied by an amino acid residue having a side chain which is more than 25% surface exposed and not part of a cystine or located at a receptor binding site.
  • amino acid residues are identified in the Examples section hereinafter or form part of the state of the art.
  • the conjugate is one, which comprises a non-polypeptide moiety having lysine as attachment group and a polypeptide comprising an amino acid sequence that differs from that shown in SEQ ID NO: 5 in the introduction of at least one lysine residue in a position selected from the group consisting of SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sl l, T13, H14, P16, G17, N18, P20, N21, R24, D25, R27, D28, S31, R32, T35, Q38, M39, Q42, L43, D44, N45, L46, E50, S51, E54, G58, S66, Q70, E74, E75, P78, Q79, N82, Q83, D84, P85, D86, A89, H90, N92, S93, E96, N97, TlOO, R102, L103, R106, R107, H109,
  • the conjugate comprises at least one introduced lysine residue, in particular any of those described above, and at least one removed lysine residue, in particular any of those described above.
  • the conjugate contains a polypeptide comprising an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO: 5 in that at least one lysine residue has been introduced, by substitution, in a position selected from the group consisting of SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sll, T13, H14, P16, G17, N18, P20, N21, R24, D25, R27, D28, S31, R32, T35, Q38, M39, Q42, L43, D44, N45, L46, E50, S51, E54, G58, S66, Q70, E74, E75, P78, Q79, N82, Q83, D84, P85, D86, A89, H90, N92, S93, E
  • amino acid residues comprising other attachment groups may be introduced and or removed from hJL-10 with the amino acid sequence shown in SEQ ID NO: 5, using the same approach as that illustrated above with lysine residues.
  • amino acid residues comprising an acid group glutamic acid and aspartic acid
  • asparagine, tyrosine and cystein may be intro- prised into positions which in hIL-10 are occupied by amino acid residues having surface exposed side chains (i.e.
  • conjugates according to this aspect include a non-polypeptide moiety reactive with an aspartic acid or glutamic acid residue and a polypeptide variant that
  • - comprises an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO: 5 in the introduction of at least one aspartic acid residue in a position selected from the group consisting of SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sll, T13, H14, P16, G17, N18, P20, N21, R24, D25, R27, D28, S31, R32, T35, Q38, M39, Q42, L43, D44, N45, L46, E50, S51, E54, G58, S66, Q70, E74, E75, P78, Q79, N82, Q83, D84, P85, D86, A89, H90, N92, S93, E96, N97, TlOO, R102, L103, R106, R107, H109, R110, PI 13, E122, Q123, N126, A127, N129, Q132, E133, D144, 1145, N148, E
  • - comprises an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO: 5 in the introduction of at least one glutamic acid residue selected from the group consisting of introduction of at least one glutamic acid residue in a position selected from the group consisting of SI, P2, G3, Q4, G5, T6, Q7, S8, E9, N10, Sll, T13, H14, P16, G17, N18, P20, N21, R24, D25, R27, D28, S31, R32, T35, Q38, M39, Q42, L43, D44, N45, L46, E50, S51, E54, G58, S66, Q70, E74, E75, P78, Q79, N82, Q83, D84, P85, D86, A89, H90, N92, S93, E96, N97, TlOO, R102, L103, R106, R107, H109, R110, PI 13, E122, Q123, N126, A127, N129
  • the invention relates to a conjugate comprising a glycosylated polypeptide exhibiting IL-10 activity, which comprises an amino acid sequence that differs from that shown in SEQ ID NO: 5 in that at least one non-naturally occurring glycosylation site has been introduced into the amino acid sequence.
  • a suitable N-glycosylation site may be introduced by introducing, preferably by substitution, an asparagine residue in a position occupied by an amino acid residue having more than 25% of its side chain exposed at the surface of the polypeptide, which position does not have a proline residue located in position +1 and +3 therefrom. If the amino acid residue located in position +2 is a serine or threonine, no further amino acid substitution need to be made. However, if this position is occupied by a different amino acid residue a serine or threonine residue need to be introduced.
  • the glycosylation site(s) is/are introduced by way of a substitution selected from the group consisting of P2N+Q4S, P2N+Q4T, G3N+G5S, G3N+G5T, Q4N+T6S, Q4N, G5N+Q7S, G5N+Q7T, T6N, T6N+S8T, Q7N+E9S, Q7N+E9T, S8N+N10S, S8N+N10T, E9N, E9N+S11T, S11N+T13S, SUN, H14N+P16S, H14N+P16T, P16N+N18S, P16N+N18T, P20S, P20T, P20N+M22S, P20N+M22T, L23S, L23T, R24N+L26S, R24N+L26T, D25N+R27S, D25N+R27T, R27
  • the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 introduced glycosylation sites.
  • glycosylated polypeptide comprises at least one sugar moiety, but may contain more, e.g. 1-10 sugar moieties (depending on the number of glycosyla- tion sites and the extent to which they are used).
  • the polypeptide variant may have an amino acid sequence which differs from that of SEQ ID NO: 5 in at least one removed, naturally occurring N- glycosylation site.
  • the conjugate comprises a polypeptide comprising an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO: 5 in the re- moval, preferably by substitution, of at least one of the amino acid residues selected from the group consisting of N116 and S118.
  • N116 may be substituted with any other amino acid residue, and SI 18 with any other amino acid residue than T.
  • the N residue is substituted with Q or D, and the T residue with A or G.
  • the amino acid sequence of polypeptide i) having at least one of the above mentioned N-glycosylation site modifications may differ from that of SEQ ID NO: 5 in the removal of at least one lysine residue as identified above.
  • the polypeptide variant in order to prepare a conjugate according to this aspect the polypeptide variant must be expressed in a glycosylating host cell capable of attaching sugar moieties at the glycosylation site(s) or alternatively subjected to in vitro glycosylation.
  • glycosylating host cells are given in the section further below entitled "Coupling to a sugar moiety”.
  • the conjugate according to the aspect of the invention described in the present section may contain additional non-polypeptide moieties, in particular a polymer molecule, as described in the present application, conjugated to one or more, optionally introduced attachment groups present in the polypeptide part of the conjugate.
  • the dispersions of particles comprising the IL-10 conjugates disclosed herein may be used for treatment of diseases, in particular inflammatory diseases, such as rheumatoid arthritis or Crohn's disease, and in connection with transplantation, immunodeficiencies and parasitic infections.
  • diseases in particular inflammatory diseases, such as rheumatoid arthritis or Crohn's disease, and in connection with transplantation, immunodeficiencies and parasitic infections.
  • G-CSF Granulocyte Colony-Stimulating Factor
  • CSF colony-stimulating factors
  • G-CSF colony-stimulating factor
  • inter- leukins inter- leukins
  • the principal biological effect of G-CSF in vivo is to stimulate the growth and development of certain white blood cells known as neutrophilic granulocytes or neutrophils (Welte et al, PNAS-USA 82:1526-1530, 1985, Souza et al, Science, 232:61-65, 1986). When released into the blood stream, neutrophilic granulocytes function to fight bacterial infection.
  • hG-CSF human G-CSF
  • SEQ ID NO:6 The amino acid sequence of human G-CSF
  • hG-CSF is a monomeric protein that dimer- izes the G-CSF receptor by formation of a 2:2 complex of 2 G-CSF molecules and 2 receptors (Horan et al. (1996), Biochemistry 35(15): 4886-96).
  • Aritomi et al. Nature 401:713-717, 1999 has described the X-ray structure of a complex between hG-CSF and the BN-BC domains of the G-CSF receptor.
  • hG-CSF residues are part of the receptor binding interfaces: G4, P5, A6, S7, S8, L9, P10, Qll, S12, L15, K16, E19, Q20, L108, D109, D112, T115, T116, Q119, E122, E123, and L124.
  • Expression of rhG-CSF in Escherichia coli, Saccharomyces cerevisiae and mammalian cells has been reported (Souza et al, Science 232:61-65, 1986, Nagata et al, Nature 319: 415-418, 1986, Robinson and Wittrup, Biotechnol. Prog. 11:171-177, 1985).
  • Recombinant human G-CSF is generally used for treating various forms of leukopenia.
  • commercial preparations of rhG-CSF are available under the names filgrastim (Gran® and Neupogen®), lenograstim (Neutrogin® and Granocyte®) and nartogras- tim (Neu-up®).
  • Gran® and Neupogen® are non-glycosylated and produced in recombinant E. coli cells.
  • Neutrogin® and Granocyte® are glycosylated and produced in recombinant CHO cells and Neu-up® is non-glycosylated with five amino acids substituted at the N-terminal region of intact rhG-CSF produced in recombinant E. coli cells.
  • a particular interesting embodiment of the invention relates to a dispersion of particles containing a conjugate comprising at least one non-polypeptide moiety covalently attached to a G-CSF variant, wherein the amino acid sequence of the G-CSF variant differs from the amino acid sequence of the corresponding wild-type human G-CSF polypeptide shown in SEQ ID NO:6 in that at least one amino acid residue comprising an attachment group for said non-polypeptide moiety has been introduced or removed.
  • one difference between the amino acid sequence of the polypeptide and the amino acid sequence shown in SEQ ID NO:6 is that at least one and often more, e.g.
  • amino acid residues comprising an attachment group for the non-polypeptide moiety has been introduced, preferably by substitution, into the amino acid sequence.
  • the polypeptide part is altered in the content of the specific amino acid residues to which the non-polypeptide moiety of choice binds, whereby a more efficient, specific and/or extensive conjugation is achieved.
  • the clearance of the conjugate is typically significantly reduced, due to the altered shape, size and/or charge of the molecule achieved by the conjugation.
  • one difference between the amino acid sequence of the polypeptide and the amino acid sequence shown in SEQ ID NO: 6 is that at least one and preferably more, e.g. 1-15, amino acid residues comprising an attachment group for the non-polypeptide moiety has/have been removed, preferably by substitution, from the amino acid sequence.
  • the term "functional site" is intended to indicate one or more amino acid residues which is/are essential for or otherwise involved in the function or performance of hG-CSF. Such amino acid residues are a part of the functional site.
  • the functional site may be determined by methods known in the art and is preferably identified by analysis of a structure of the polypeptide complexed to a relevant receptor, such as the hG-CSF receptor (See Aritomi et al. Nature 401:713-717, 1999).
  • the amino acid sequence of the polypeptide differs from the amino acid sequence shown in SEQ ID NO: 6 in that a) at least one specified amino acid residue comprising an attachment group for the non-polypeptide moiety and present in the amino acid sequence shown in SEQ ID NO: 6 has been removed, preferably by substitution, and b) at least one specified amino acid residue comprising an attachment group for the non-polypeptide moiety has been introduced into the amino acid sequence, preferably by substitution, the specified amino acid residues being any of those described in the subsequent sections herein.
  • This embodiment is considered of particular interest in that it is possible to specifically design the polypeptide so as to obtain an optimal conjugation to the non-polypeptide moiety of choice.
  • the conjugate will in general comprise a sufficient number and type of non- polypeptide moieties to provide the conjugate with an increased functional in vivo half-life and/or serum half-life as compared to hG-CSF, e.g. filgrastim, lenograstim or nartograstim, and preferably as compared to rhG-CSF comprising a single N-terminally attached 20 kDa PEG moiety.
  • the increased functional in vivo half -life is conveniently determined as described in the Materials and Methods section herein.
  • the conjugate may comprise at least one non-conjugated, conjugatable attachment group for the non-polypeptide moiety.
  • conjugatable attachment group is intended to indicate an attachment group that is located in a position of the polypeptide where it is accessible for conjugation, and that but for special precautions is conjugated to the relevant non-polypeptide moiety when subjected to conjugation.
  • attachment group may be part of an amino acid residue involved in or otherwise essential for the polypeptide to exert its activity.
  • a convenient way to avoid conjugation of an otherwise conjugatable attachment group is to shield the attachment group by means of a helper molecule, e.g. as described in the section entitled "Blocking of the functional site”.
  • the number of non-conjugated, conjugatable attachment groups depends on the specific G-SCF polypeptide and the location of the conjugatable attachment groups.
  • the polypep- tide conjugate comprises one or two non-conjugated, conjugatable attachment groups, and at least one, and preferably two or more conjugated attachment groups.
  • the conjugate contains: i) a polypeptide exhibiting G-CSF activity, comprising an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:6 in at least one substitution selected from the group consisting of T1K, P2K, L3K, G4K, P5K, A6K, S7K, S8K, L9K, P10K, Q11K, S12K, F13K, L14K, L15K, E19K, Q20K, V21K, Q25K, G26K, D27K, A29K, A30K, E33K, A37K, T38K, Y39K, L41K, H43K, P44K, E45K, E46K, V48K, L49K, L50K, H52K, S53K, L54K, I56K, P57K, P60K, L61K, S62K, S63K, P65K, S66K, Q67K, A68K, L69
  • hG-CSF contains four lysine residues, of which K16 is located in the receptor- binding domain and the others are located in positions 23, 34 and 40, respectively, all relatively close to the receptor-binding domain.
  • K16 is located in the receptor- binding domain
  • the others are located in positions 23, 34 and 40, respectively, all relatively close to the receptor-binding domain.
  • the invention relates to a polypeptide conjugate as defined above, wherein at least one of the amino acid residues selected from the group consisting of K16, K23, K34 and K40 has been deleted or substituted with another amino acid residue.
  • at least K16 is substituted with another amino acid residue.
  • prefe ⁇ ed amino acid substitutions include one or more of
  • Q70K, Q90K, T105K, Q120K and T133K such as two, three or four of these substitutions, for example: Q70K+Q90K, Q70K+T105K, Q70K+120K, Q70K+T133K, Q90K+T105K, Q90K+Q120K, Q90K+T133K, T105K+Q120K, T105K+T133K, Q120K+T133K, Q70K+Q90K+T105K, Q70K+Q90K+Q120K, Q70K+Q90K+T133K, Q70K+T105K+T120K, Q70K+T105K+T133K, Q70K+Q120K+T133K, Q70K+Q120K+T133K, Q09K+T105K+Q120K, Q90K+T105K+T133K, Q90K+Q120K+T133K, T105K+T120K+T133K, Q90K+T105K+Q
  • the polypeptide of the conjugate according to the second of the embodiments described in the present section preferably comprises at least one, such as one, two, three or four, of the substitutions selected from the group consisting of K16R, K16Q, K23R, K23Q, K34R, K34Q, K40R and K40Q, more preferably at least one of the substitutions K16R and K23R, whereby conjugation of these residues can be avoided.
  • the polypeptide comprises at least one substitution selected from the group consisting of K16R+K23R, K16R+K34R, K16R+K40R, K23R+K34R, K23R+K40R, K34R+K40R, K16R+K23R+K34R, K16R+K23R+K40R, K23R+K34R+K40R, K16R+K34R+K40R and K16R+K23R+K34R+K40R.
  • substitutions are likely to give rise to the least structural difference compared to the native or parent polypeptide.
  • the polypeptide conjugate may also include one or more substitutions selected from R22K, R146K, R147K, R166K and R169K.
  • non-polypeptide moiety of the conjugate according to this aspect of the invention may be any molecule which, when using the given conjugation method has lysine as an attachment group such as a carbohydrate moiety, it is preferred that the non-polypeptide moiety is a polymer molecule.
  • the polymer molecule may be any of the molecules mentioned in the section entitled "Conjugation to a polymer molecule", but is preferably selected from the group consisting of linear or branched polyethylene glycol or another polyalkylene oxide.
  • Pre- ferred polymer molecules are e.g. mPEG-SPA from Shearwater Polymers, Inc. or oxycarbonyl- oxy-N-dicarboxyimide PEG (US 5,122,614).
  • any of the amino acid changes, in particular substitutions, specified in this section can be combined with any of the amino acid changes, preferera- bly substitutions, specified in the other sections herein disclosing specific amino acid modifica- tions, including introduction and/or removal of glycosylation sites.
  • the conjugate contains: i) a polypeptide exhibiting G-CSF activity, which comprises an amino acid sequence that differs from the amino acid sequence of hG-CSF shown in SEQ ID NO: 6 in at least one substitution selected from the group consisting of TIC, P2C, L3C, G4C, P5C, A6C, S7C, S8C, L9C, P10C, QllC, S12C, F13C, L14C, L15C, E19C, Q20C, V21C, R22C, Q25C, G26C, D27C, A29C, A30C, E33C, A37C, T38C, Y39C, L41C, H43C, P44C, E45C, E46C, V48C, L49C, L50C, H52C, S53C, L54C, I56C, P57C, P60C, L61C, S62C, S63C, P65C, S66C, Q67
  • the receptor-binding domain of hG-CSF contains a cysteine residue in position 17 which does not take part in a cystine and which may advantageously be removed in order to avoid conjugation of a non-polypeptide moiety to said cysteine.
  • the conjugate contains: i) a polypeptide exhibiting G-CSF activity, which comprises an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO: 6 in at least one substitution selected from the group consisting of TIC, P2C, L3C, G4C, P5C, A6C, S7C, S8C, L9C, P10C, QllC, S12C, F13C, L14C, L15C, E19C, Q20C, N21C, R22C, Q25C, G26C, D27C, A29C, A30C, E33C, A37C, T38C, Y39C, L41C, H43C, P44C
  • substitutions according to this aspect of the invention are substitutions of arginine with cysteine, for example one or more of R146C, R147C, R166C and R169C.
  • the invention relates to a conjugate containing i) a polypeptide exhibiting G-CSF activity, which comprises an amino acid sequence that differs from the amino acid sequence shown in SEQ ID ⁇ O:6 in at least one substitution selected from the group consisting of T1D, P2D, L3D, G4D, P5D, A6D, S7D, S8D, L9D, P10D, QllD, S12D, F13D, L14D, L15D, K16D, Q20D, V21D, R22D, K23D, Q25D, G26D, A29D, A30D, K34D, A37D, T38D, Y39D, K40D, L41D, H43D, P44D, V48D, L49D, L50D, H52D, S53D, L54D, I56D, P57D, P60D, L61D, S62D, S63D, P65D, S66D, Q
  • a non-polypeptide moiety having an aspartic acid or a glutamic acid residue as an attachment group examples include Q67D E, Q70D/E, Q77D/E, Q86D E, Q90D/E, Q120D/E, Q131D/E, Q134D/E, Q145D/E and Q173D/E.
  • the polypeptide of the conjugate according to any of the above aspects may comprise removal, preferably by substitution, of at least one of the amino acid residues selected from the group consisting of D27, D104, D109, D112, E19, E33, E45, E46, E93, E98, E122, E123, and E163.
  • the substitution may be for any other amino acid residue, in particular for an asparagine or a glutamine residue, whereby conjugation of these residues can be avoided.
  • the polypeptide may comprise at least one of the following substitutions: D27 ⁇ , D104N, D109N, D112N, E19Q, E33Q, E45Q, E46Q, E93Q, E98Q, E122Q, E123Q and E163Q.
  • the amino acid substitution in one or more of the above positions may in addition be combined with at least one of the following substitutions: D109N, D112N, E19Q, E122Q and E123Q. Substitution with any of these amino acid residues is likely to give rise to the least structural difference.
  • non-polypeptide moiety of the conjugate according to this aspect of the invention which has an acid group as an attachment group
  • the non-polypeptide moiety is a polymer molecule or an organic derivatizing agent, in particular a polymer molecule, and the conjugate is prepared e.g. as described by Sakane and Pardridge, Pharmaceutical Research, Vol. 14, No. 8, 1997, pp 1085-1091.
  • the conjugate may contain further carbohydrate moieties as a consequence of the polypeptide being expressed in a glycosylating host cell and glycosylation achieved at the glycosylation sites or introduced gly- cosylation site(s).
  • the conjugate contains a glycosylated polypeptide exhibiting G-CSF activity, which comprises an amino acid sequence that differs from that shown in SEQ ID NO: 6 in that at least one non-naturally occurring glycosylation site has been introduced into the amino acid sequence by way of at least one substitution selected from the group consisting of L3N+P5S/T, P5N, A6N, S8N+P10S/T, P10N, Ql 1N+F13S/T, S12N+L14S/T, F13N+L15S/T, L14N+K16S/T, K16N+L18S/T, E19N+V21S/T, Q20N+R22S/T, V21N+K23S/T, R22N+I24S/T, K23N+Q25S/T, Q25N+D27S/T, G26N+G28S/T, D27N+A29S/T, A29N+L31S/
  • the polypeptide in order to prepare a conjugate according to this aspect the polypeptide must be expressed in a glycosylating host cell capable of attaching oligosaccha- ride moieties at the glycosylation site(s) or alternatively subjected to in vitro glycosylation.
  • glycosylating host cells are given in the section further below entitled “Coupling to an oligosaccharide moiety”.
  • the conjugate according to this aspect contains a polypeptide exhibiting G-CSF activity, which comprises an amino acid sequence that differs from that shown in SEQ ID NO:6 in at least one substitution selected from the group consisting of P5N, A6N, P10N, P60N, L61N, L78N, F113N and E162N, in particular from the group consisting of P5N, A6N, P10N, P60N, L61N, FI 13N and E162N, such as from the group consisting of P60N, L61N, F113N and E162N.
  • the conjugate according to this aspect comprises a polypeptide ex- hibiting G-CSF activity, which comprises an amino acid sequence that differs from that shown in SEQ ID NO: 6 in at least one substitution selected from the group consisting of D27N+A29S, D27N+A29T, D104N+L106S, D104N+L106T, D109N+A111S, D109N+A111T, Dl 12N+A114S and Dl 12N+A114T, more preferably from the group consisting of D27N+A29S, D27N+A29T, D104N+L106S, D104N+L106T, D112N+A114S and D112N+A114T, such as from the group consisting of D27N+A29S, D27N+A29T, D104N+L106S and D104N+L106T.
  • a polypeptide ex- hibiting G-CSF activity which comprises an amino acid sequence that differs from that shown in SEQ ID NO
  • the conjugate according to the aspect of the invention described in the present section may contain additional non-polypeptide moieties, in particular a polymer molecule, as described in the present application, conjugated to one or more attachment groups present in the polypeptide part of the conjugate.
  • the polypeptide conjugate may be characterized as being a conjugate exhibiting G-CSF activity and comprising a polypeptide with an amino acid sequence that differs in at least one amino acid residue from the amino acid sequence shown in SEQ ID NO: 6 and having at least one non-polypeptide moiety attached to an attachment group of the polypeptide, the polypeptide conjugate further fulfilling at least one of the following criteria (A)-(D):
  • the dispersions of particles comprising the G-CSF conjugates disclosed herein may be used for treatment of diseases, in particular prevention of infection in cancer patients undergoing certain types of chemotherapy, radiation therapy and bone marrow transplantations, mobilisation of progenitor cells for collection in peripheral blood progenitor cell transplantations, treatment of severe chronic or relative leukopenia, treatment of patients with acute myeloid leukaemia, treatment of AIDS or other immunodeficiency diseases, and for antifungal therapy, in particular for treatment of systemic or invasive candidiasis
  • proteins suitable to be modified for the purposes described herein include proteins such as a plasma protein, e.g. a factor from the fibrinolytic system such as pro-urokmase, urokinase, tissue plasminogen activator, plasminogen activator inhibitor 1 (PAI-1) or plasminogen activator inhibitor 2 (PAI- 2), the Von Willebrand factor, or an ⁇ -1 -proteinase inhibitor, ii) a cytokine, e.g.
  • a plasma protein e.g. a factor from the fibrinolytic system such as pro-urokmase, urokinase, tissue plasminogen activator, plasminogen activator inhibitor 1 (PAI-1) or plasminogen activator inhibitor 2 (PAI- 2), the Von Willebrand factor, or an ⁇ -1 -proteinase inhibitor, ii) a cytokine, e.g.
  • an interleukin such as interleukin (IL) 1, IL-2, IL-4, IL-5, IL-6, IL-9, IL-11, IL-12, JL-13, IL-15, JL-16, IL-17, IL-18, or IL-lRa
  • a colony-stimulating factor such as GM-CSF, stem cell factor (SCF), a mem- ber of the tumor necrosis factor family (e.g TNF- ⁇ , lymphotoxin- ⁇ , lymphotoxin- ⁇ , FasL, CD40L, CD30L, CD27L, Ox40L, 4-1BBL, RANKL, TRAIL, TWEAK, LIGHT, TRANCE, APRIL, THANK or TALL-1), iii) a growth factor, e.g platelet-derived growth factor (PDGF), transforming growth factor ⁇ (TGF- ⁇ ), transforming growth factor ⁇ (TGF- ⁇ ), epidermal growth factor (EGF), vascular endothelial
  • aprotinin or CI-2A v) an enzyme, e.g. superoxide dismutase, catalase, uricase, bilirubin oxidase, trypsin, papain, asparaginase, arginase, arginine deiminase, adenosin deaminase, ribo- nuclease, alkaline phosphatase, ⁇ -glucuronidase, purine nucleoside phosphorylase or batroxo- bin, vi) an opioid, e.g. endorphins, enkephalins or non-natural opioids, vii) a hormone or neu- ropeptide, e.g.
  • an enzyme e.g. superoxide dismutase, catalase, uricase, bilirubin oxidase, trypsin, papain, asparaginase, arginase, arginine deiminase,
  • glucagons insulin, calcitonin, glucagons, adrenocorticotropic hormone (ACTH), soma- tostatin, gastrins, cholecystokinins, parathyroid hormone, luteinizing hormone, gonadotropin- releasing hormone, chorionic gonadotropin, corticotropin-releasing factor, vasopressin, oxyto- cin, antidiuretic hormones, thyroid-stimulating hormone, thyrotropin-releasing hormone, re- laxin, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), prolactin, neuropep- tide Y, peptide YY, pancreatic polypeptide, leptin, orexin, CART (cocaine and amphetamine regulated transcript), a CART-related peptide, melanocortins (melanocyte-stimulating hor- mones),
  • SEQ ID NO:l shows the amino acid sequence of wild-type human interferon gamma.
  • SEQ ID NO: 2 shows the amino acid sequence of wild-type human interferon beta.
  • SEQ ID NO: 3 shows the amino acid sequence of the ⁇ subunit of wild-type human follicle stimulating hormone (FSH).
  • SEQ ID NO:4 shows the amino acid sequence of the ⁇ subunit of wild-type hu- man follicle stimulating hormone (FSH).
  • SEQ ID NO: 5 shows the amino acid sequence of wild-type human interleukin 10 (D -10).
  • SEQ ID NO:6 shows the amino acid sequence of wild-type granulocyte colony- stimulating factor (G-CSF).
  • ISRE Biter- feron Stimulated Response Element
  • HeLa cells are co-transfected with ISRE-Luc and pCDNA 3.1/hygro and foci (cell clones) are created by selection in DMEM media containing Hygromycin B. Cell clones are screened for luciferase activity in the presence or absence of IFNG. Those clones showing the highest ratio of stimulated to unstimulated luciferase activity are used in further assays.
  • radioactive labelled IFNG can be used to study the subcutaneous absorption and local distribution of IFNG.
  • Croos and Roberts J. Pharm., 1993, vol 45, p.606-609 have done studies of 125 EFNG in anaesthetized female Spraque-Dawley rats. After administration subcutaneous administration, blood and tissue samples were collected and the amount of IFNG was determined by gamma-counting.
  • ASA Accessible Surface Area
  • ASA accessible surface area
  • the fractional ASA of the side chain atoms is computed by division of the sum of the ASA of the atoms in the side chain with a value representing the ASA of the side chain at- oms of that residue type in an extended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton (1991) J.Mol.Biol.: 220,507-530.
  • the CA atom is regarded as a part of the side chain of Glycine residues but not for the remaining residues.
  • the following table are used as standard 100% ASA for the side chain:
  • Residues not detected in the structure are defined as having 100% exposure as they are thought to reside in flexible regions.
  • the distance between atoms was determined using molecular graphics software e.g. rnsightH v. 98.0, MSI INC.
  • the receptor-binding site is defined as comprising of all residues having their accessible surface area changed upon receptor binding. This is determined by at least two ASA calculations; one on the isolated ligand(s) in the ligand(s)/receptor(s) complex and one on the complete ligand(s)/receptor(s) complex.
  • the X-ray structure used was of an IFNG homo-dimer in complex with two mole- cules of a soluble form of the IFNG receptor having a third molecule of the IFNG receptor in the structure not making interactions with the IFNG homodimer reported by Thiel et.al. Struc- ture 8:927-936 (2000).
  • the structure consists of the IFNG homodimer wherein the two molecules are labeled A and B.
  • the M0 is removed from the structure in all the calculations of this example.
  • the structure of the two IFNG monomers has very weak electron density after residue 120 and residues were only modeled until residue T126. Therefore, residues S121-T126 were removed from the structure prior to the calculations in this example.
  • the two receptor fragments labeled C and D make direct interactions with the IFNG homodimer and a third receptor molecule labeled E makes no contact with the IFNG homodimer and are not included in these calculations.
  • Residues not determined in the structure are treated as fully surface exposed, i.e. residues S121, P122, A123, A124, K125, T126, G127, K128, R129, K130, R131, S132, Q133, M134, L135, F136, R137, G138, R139, R140, A141, S142, Q143.
  • residues S121, P122, A123, A124, K125, T126, G127, K128, R129, K130, R131, S132, Q133, M134, L135, F136, R137, G138, R139, R140, A141, S142, Q143 are also consti- tute separate targets for introduction of attachment groups in accordance with the present invention (or may be viewed as belonging to the group of surface exposed amino acid residues, e.g. having more than 25% or more than 50% exposed side chains).
  • Receptor binding site Performing ASA calculations as described above results in the following residues of the IFNG molecule having reduced ASA in at least one of the monomers in the complex as compared to the calculation on the isolated dimer: Ql, D2, Y4, V5, E9, K12, G18, H19, S20, D21, V22, A23, D24, N25, G26, T27, L30, K34, K37, K108, Hill, E112, 1114, Q115, A118, E119.
  • the resulting solution is sti ⁇ ed in a ice bath for more than 4 hours and then stands overnight in a refrigerator. Approx. 3 ml of the supernatant, after removing the resulting precipitates by centrifugation, is applied to a column (1.2 x 45 cm) packed with Sephadex G-75 equilibrated with a 25 mM ammonium acetate buffer solution (pH 6.0) for elution, yielding approx. 5 ml of a main fraction. Reduced form glutathione is added to the fraction to a concentration of 10 mM, yielding a solu- tion containing rhuIFNG modified with polyethylene glycol imidoester.
  • JFNG activity ⁇ nzyme-Immunoassay method: EIA method
  • antiviral activity of the resulting solution show the values of 3.4xl0 4 ng rhuIFNG/ml and 2.31xl0 4 IU/ml, respectively.
  • Part of the supernatant described above is passed through a column (0.9 x 50 cm) packed with Sephadex G-75 equilibrated with 0.1M acetic acid, yielding a protein fraction. After lyophiliza- tion, the protein fraction is hydrolyzed with 6N HCl at 110 degrees Celcius in a sealed degassed tube for 24 hours and followed by amino acid analysis.
  • nucleotide sequence encoding IFNG is modified to encode IFNG with the substitution N25K.
  • the variant is purified and modified as in the above example. Said modification is carried by standard DNA manipulation procedures well known to the man of average skill in the art.
  • the resulting PEG-IFNG-N25K conjugate has an additional PEG-molecule attached as compared with the conjugate prepared in the above example.
  • PEG-IFNG-N25K conjugate co ⁇ esponding It is desirable to deliver doses of PEG-IFNG-N25K conjugate co ⁇ esponding to injection of 1-5 microgram/kg rhuIFNG.
  • a preparation of PEG-IFNG-N25K having a concentration of 1 mg conjugate/mL is made and nebulized using an Acorn ⁇ , Ultravent or AERx de- vice.
  • the volume to be nebulized is varied from 0.05 mL to 2 mL, the optimal dose to be determined in accordance with the indication of interest and individual patient characteristics such as weight and age.
  • the dispersion of particles is inhaled by the patient in need thereof and rele- vant clinical end points for the indication in question are monitored in accordance with well- known clinical dose finding and efficacy methods.
  • ISRE Interferon Stimulated Response Element
  • HeLa cells are co-transfected with ISRE-Luc and pCDNA 3.1/hygro and foci (cell clones) are created by selection in DMEM media containing Hygromycin B. Cell clones are screened for luciferase activity in the presence or absence of interferon ⁇ . Those clones showing the highest ratio of stimulated to unstimulated luciferase activity are used in further assays. To screen variants, 15,000 cells/well are seeded in 96 well culture plates and incubated overnight in DMEM media. The next day variants as well as a known standard are added to the cells in various concentrations.
  • the plates are incubated for 6 hours at 37°C in a 5% CO 2 air atmosphere LucLite substrate (Packard Bioscience, Groningen The Netherlands ) is subsequently added to each well. Plates are sealed and luminescence measured on a TopCount luminometer (Packard) in SPC (single photon counting) mode. Each individual plate contains wells incubated with interferon ⁇ as a stimulated control and other wells containing normal media as an unstimulated control. The ratio between stimulated and unstimulated luciferase activity serves as an internal standard for both variant activity and experiment-to-experiment variation.
  • the ⁇ -Rl gene is activated by interferon ⁇ but not by other interferons.
  • the tran- sciption of ⁇ -Rl thus serves as a second marker of interferon ⁇ activation and is used to ensure that muteins retain interferon ⁇ activity.
  • a 300 bp promoter fragment of ⁇ -Rl shown to drive interferon sensitive transcription was isolated by PCR from human genomic DNA and inserted into the pGL3 basic vector (Promega). The resulting ⁇ -Rl.
  • 'luciferase gene is used in assays similar to the primary assay described above. In astrocytoma cells, the resulting ⁇ -Rl:luciferase gene has been described to show 250 fold higher sensitivity to interferon ⁇ than to interferon ⁇ (Rani et al. op cit).
  • the concentration of IFN- ⁇ is quantitated by use of a commercial sandwich immunoassay (PBL Biomedical Laboratories, New Brunswick, NJ, USA).
  • the kit is based on an ELISA with monoclonal mouse anti-IFN- ⁇ antibodies for catching and detection of IFN- ⁇ in test samples.
  • the detecting antibody is conjugated to biotin.
  • Tests samples and recombinant human IFN- ⁇ standard are added in 0.1 mL in concentrations from 10-0.25 ng/mL to microtiter plates, precoated with catching antibody. The plates are incubated at RT for 1 hr. Samples and standard are diluted in kit dilution buffer.
  • the plates are washed in the kit buffer and incubated with the biotinylated detecting antibody in 0.1 mL for 1 hr at RT. After another wash the streptavidin- horseradishperoxidase conjugate is added in 0.1 mL and incubated for 1 hr at RT.
  • TMB Tetramethylbenzidine
  • the receptor binding capability of a polypeptide or conjugate can be determined using the assay described in WO 95/25170 entitled "Analysis Of _FN- ⁇ (Phe 101 ) For Receptor Binding"(which is based on Daudi or A549 cells). Soluble domains of IFNARl and IFNAR2 can be obtained essentially as described by Arduini et al, Protein Science, 1999, vol. 8, 1867- 1877 or as described in Example 9 of WO 01/15736. Alternatively, the receptor binding capability is determined using a crosslinking agent such as disuccinimidyl suberate (DSS) available from Pierce, Rockford, IL, USA as follows:
  • DSS disuccinimidyl suberate
  • the polypeptide or conjugate is incubated with soluble IFNAR-2 receptor in the presence or absence of DSS in accordance with the manufacturer's instructions. Samples are separated by SDS-PAGE, and a western blot using anti-interferon ⁇ or anti-IFNAR2 antibodies is performed.
  • the presence of a functional interferon ⁇ polypeptide/conjugate: receptor interaction is apparent by an increase in the molecular size of receptor and interferon ⁇ in the presence of DSS.
  • a crosslinking assay using a polypeptide or conjugate and both receptor subunits IFNAR-1 and IFNAR-2) can establish Interferon receptor 1 binding ability.
  • IFNAR-1 binds only after an interferon ⁇ : IFNAR-2 complex is formed (Mogensen et al., Journal of Interferon and Cytokine Research, 19:1069- 1098, 1999).
  • Reduced immunogenicity of a conjugate or polypeptide is determined by use of an ELISA method measuring the immunoreactivity of the conjugate or polypeptide relative to a reference molecule or preparation.
  • the reference molecule or preparation is normally a recom- binant human interferon ⁇ preparation such as Avonex, Rebif or Betaseron, or another recombinant human interferon ⁇ preparation produced by a method equivalent to the way these products are made.
  • the ELISA method is based on antibodies from patients treated with one of these recombinant interferon ⁇ preparations. The immunogenicity is considered to be reduced when the conjugate or polypeptide has a statistically significant lower response in the assay than the reference molecule or preparation.
  • Another method of determining immunogenicity is by use of sera from patients treated with interferon beta (i.e. any commercial interferon ⁇ product) in an analogous manner to that described by Ross et al. J. Clin Invest. 95, 1974-78, 1995.
  • interferon beta i.e. any commercial interferon ⁇ product
  • reduced immunogenicity results in reduced inhibition of a conjugate by patient sera compared to a wt IFN-beta reference molecule.
  • a less immunogenic conjugate is expected to bind to patient IgG to a lesser extent than reference IFN-beta molecules.
  • the reference and conjugate molecules are added in a concentration that produces approximately 80% virus protection in the antiviral neutralisation bioassay.
  • the IFN- ⁇ proteins are mixed with patient sera in various dilutions (starting at 1:20).
  • the antiviral bioassay is performed using A549 cells (CCL 185, American tissue culture collection) and Encephalomyocarditis (EMC) virus (VR-129B, American tissue culture collection).
  • the cells are seeded in 96 well tissue culture plates at a concentration of 10,000 cells/well and incubated at 37°C in a 5% CO 2 air atmosphere.
  • a polypeptide or conjugate is added in concentrations from 100-0.0001 IU/mL in a total of lOO ⁇ l DMEM medium containing fetal calf serum and antibiotics.
  • the medium is removed and 0.1 mL fresh medium containing EMC virus is added to each well.
  • the EMC virus is added in a concentration that causes 100% cell death in IFN- ⁇ free cell cultures after 24 hours.
  • the antiviral effect of the polypeptide or conjugate is measured using the WST-1 assay.
  • 0.01 mL WST-1 WST-1 cell proliferation agent, Roche Diagnostics GmbH, Mannheim, Germany
  • the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells results in the formation of formazan that is quantified by measuring the absorbance at 450 nm.
  • ISRE Interferon Stimulated Response Element
  • interferon ⁇ neutralising effect of anti-interferon ⁇ sera are analysed using the ISRE-Luciferase activity assay.
  • Sera from interferon ⁇ treated patients or from immunised animals are used. Sera are added either in a fixed concentration (dilution 1:20-1:500 (pt sera) or 20-600 ng/mL (animal sera)) or in five-fold serial dilutions of sera starting at 1/20 (pt sera) or 600 ng/mL (animal sera). Interferon ⁇ is added either in five fold-dilutions starting at 25.000 IU/mL or in a fixed concentration (0.1- 10 IU/mL) in a total volume of 80 ⁇ l DMEM medium + 10% FCS. The sera are incubated for 1 hr. at 37°C with IFN- ⁇ .
  • the samples are then transfe ⁇ ed to 96 well tissue culture plates containing HeLa cells transfected with ISRE-Luc grown from 24 firs before (15,000 cells/well) in DMEM media.
  • the cultures are incubated for 6 hours at 37°C in a 5% CO 2 air atmosphere.
  • LucLite substrate (Packard Bioscience, Groningen, The Netherlands) is subsequently added to each well. Plates are sealed and luminescence measured on a TopCount luminometer (Packard) in SPC (single photon counting) mode.
  • Measurement of biological half -life can be carried out in a number of ways described in the literature.
  • One method is described by Munafo et al (European Journal of Neurology 1998, vol 5 No2 p 187-193), who used an ELISA method to detect serum levels of interferon ⁇ after subcutaneous and intramuscular administration of interferon ⁇ .
  • the rapid decrease of interferon ⁇ serum concentrations after i.v. administration has made it important to evaluate biological responses to interferon ⁇ treatment.
  • the conjugates of the present invention will have prolonged serum half lifes also after i.v. administration making it possible to measure by e.g. an ELISA method or by the primary screening assay.
  • Different pharmacodynamic markers e.g.
  • Assays to assess the biological effects of interferon ⁇ such as antiviral, antiprolif- erative and immunomodulatory effects (as described in e.g. Annals of Neurology 1995 vol 37 No 1 p 7-15) can be used together with the primary and secondary screening assays described herein to evaluate the biological efficacy of the conjugate in comparison to wild type interferon ⁇ .
  • EAE experimental autoimmune encephalomyelitis
  • TMEV Theiler's murine encephalomyelitis virus
  • ASA Accessible Surface Area
  • ASA accessible surface area
  • This method typically uses a probe- size of 1.4 A and defines the Accessible Surface Area (ASA) as the area formed by the centre of the probe. Prior to this calculation all water molecules and all hydrogen atoms are removed from the coordinate set, as are other atoms not directly related to the protein.
  • Alternative pro- grams are available for computing ASA, e.g. the program Whatlf G.Vriend, J. Mol. Graph.
  • the fractional ASA of the side chain atoms is computed by division of the sum of the ASA of the atoms in the side chain with a value representing the ASA of the side chain atoms of that residue type in an extended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton (1991) J.Mol.Biol.220,507-530.
  • the CA atom is regarded as a part of the side chain of Glycine residues but not for the remaining residues.
  • the following table indicates the 100% ASA standard for the side chain: Ala 69.23 A 2
  • HCG HCG for which two published structures are available: Wu, H., Lustbader, J. W., Liu, Y., Canfield, R. E., Hendrickson, W. A.: Structure 2 pp. 545 (1994) and Lapthom, A. J., Harris, D. C, Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan, F. J., Isaacs, N. W.: Nature 369 pp. 455 (1994), both including the ⁇ chain of HCG.
  • the ⁇ chain of hFSH is 32 per- cent identical to the amino acid sequence of the structural part of the ⁇ chain of HCG (see the sequence alignment of Figure 1).
  • ASA Accessible Surface Area
  • ASA accessible surface area
  • This method typically uses a probe-size of 1.4A and defines the Accessible Surface Area (ASA) as the area formed by the center of the probe. Prior to this calculation all water molecules and all hydrogen atoms should be removed from the coordinate set, as should other atoms not directly related to the protein.
  • the fractional ASA of the side chain atoms is computed by division of the sum of the ASA of the atoms in the side chain with a value representing the ASA of the side chain atoms of that residue type in an extended Ala-x-Ala tripeptide, see Hubbard, Campbell & Thornton (1991) J. Mol. Biol. 220,507-530.
  • the CA atom is regarded as being a part of the side chain of glycine residues but not other residues.
  • the following values are used as standard 100% ASA for the side chain:
  • CHO FSH-R / CRE-luc cells were seeded in white 96 well culture plates at a density of about 15,000 cells/well.
  • the cells were in 100 Dl DMEM/F- 12 (without phenol red) with 1.25% FBS. After incubation overnight (at 37°C, 5% CO 2 ), 25 ⁇ l of sample or standard diluted in DMEM F-12 (without phenol red) with 10% FBS was added to each well. The plates were further incubated for 3 hrs, followed by addition of 125 ⁇ l LucLite substrate (Packard Bioscience). Subsequently, plates were sealed and luminescence was meas- ured on a TopCount luminometer (Packard) in SPC (single photon counting) mode.
  • TopCount luminometer Packard
  • DRG FSH EIA DRG Instruments GmbH, Marburg, Germany
  • DRG FSH EIA is a solid phase immunosorbent assay (ELISA) based on the sandwich principle.
  • the microtiter wells are coated with a monoclonal antibody directed towards a unique antigenic site on the FSH- ⁇ subunit.
  • An aliquot of FSH-containing sample (diluted in H 2 0 with 0.1% BSA) and an anti-FSH antiserum conjugated with horseradish peroxidase are added to the coated wells. After incubation, unbound conjugate is washed off with water.
  • the amount of bound peroxidase is proportional to the concentration of FSH in the sample.
  • the intensity of colour developed upon addition of substrate solution is proportional to the concentration of FSH in the sample.
  • ASA Accessible Surface Area
  • ASA accessible surface area
  • This method typically uses a probe- size of 1.4A and defines the Accessible Surface Area (ASA) as the area formed by the centre of the probe. Prior to this calculation all water molecules and all hydrogen atoms should be removed from the coordinate set, as should other atoms not directly related to the protein.
  • the fractional ASA of the side chain atoms is computed by division of the sum of the ASA of the atoms in the side chain with a value representing the ASA of the side chain atoms of that residue type in an extended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton (1991) J.Mol.Biol.220,507-530.
  • the CA atom is regarded as a part of the side chain of Glycine residues but not for the remaining residues.
  • the following table are used as standard 100% ASA for the side chain:
  • Val 114.14 A 2 Residues not detected in the structure are defined as having 100% exposure as they are thought to reside in flexible regions.
  • JL-10 can be measured by its cytokine inhibitory activity on activated peripteral blood mononuclear cells (PBMCs) (Science 1990; 250:830-832).
  • PBMCs activated peripteral blood mononuclear cells
  • the receptors may be purified extracellular receptor domains, receptors bound to purified cellular plasma membranes, or whole cells - the cellular sources being either cell lines that inherently express IL-10 receptors or cells transfected with cDNAs encoding the receptors.
  • the ability of rhIL-10 or variants thereof to compete for the binding sites with native IL-10 is analyzed by incubating with a labeled IL-10-analog for instance biotinylated ML- 10 or radioiodinated ML- 10.
  • the extracellular domains of the hIL-10 receptor can optionally be coupled to Fc and immobilized in 96 well plates. RhIL-10 or variants thereof are subsequently added and the binding of these detected using either specific anti-ML-10 antibodies or biotinylated or radioiodinated ML- 10.
  • An important aspect of the invention is the prolonged biological half-life that is obtained by the conjugation of the polypeptide to the polymer moiety.
  • the rapid decrease of ML- 10 serum concentrations has made it important to evaluate biological responses to ML- 10 treatment.
  • the conjugates of the present invention have prolonged serum half lifes also after i.v. administration making it possible to measure by e.g. an ELISA method or by the primary screening assay. Measurement of in vivo biological half -life can be carried out in a number of ways as described in the literature.
  • the molecular weight of conjugated or unconjugated ML-10 or variants thereof is determined by either SDS-PAGE, gel filtration, matrix assisted laser desorption mass spectrometry or equilibrium centrifugation
  • ASA Accessible Surface Area
  • a 3D ensemble of 10 structures determined by NMR spectroscopy is available from the Protein Data Bank (PDB) (www.rcsb.org/pdb/).
  • PDB Protein Data Bank
  • This information can be entered into the computer program Access (B. Lee and F.M. Richards, J. Mol. Biol. 55: 379-400 (1971)) version 2 ( ⁇ 1983 Yale University) and used to compute the accessible surface area (ASA) of the individual atoms in the structure.
  • This method typically uses a probe size of 1.4A and defines the Accessible Surface Area (ASA) as the area formed by the centre of the probe. Prior to this calculation all water molecules and all hydrogen atoms should be removed from the coordinate set as should other atoms not directly related to the protein.
  • the fractional ASA of the side chain atoms is computed by division of the sum of the ASA of the atoms in the side chain with a value representing the ASA of the side chain atoms of that residue type in an extended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton (1991) J.Mol.Biol.220, 507-530.
  • the CA atom is regarded as a part of the side chain of glycine residues but not for the remaining residues.
  • the values in the following table are used as standard 100% ASA for the side chain: .19
  • Residues not detected in the structure are defined as having 100% exposure as they are thought to reside in flexible regions.
  • the distance between atoms is most easily determined using molecular graphics software, e.g. InsightJJ® v. 98.0, MSI INC.
  • amino acid residues to be modified are preferably those whose side chains are surface exposed, in particular those with more than about 25% of the side chain exposed at the surface of the molecule, and more preferably those with more than 50% side chain exposure.
  • residues located in receptor interfaces are preferably excluded so as to avoid or at least minimize possible interference with receptor binding or activation.
  • residues that are less than lOA from the nearest Lys (Glu, Asp) CB-CB (CA for Gly) should also be excluded.
  • preferred positions for modification are in particular those that have a hydro- pMlic and/or charged residue, i.e. Asp, Asn, Glu, Gin, Arg, His, Tyr, Ser and Thr, positions that have an arginine residue being especially prefe ⁇ ed.
  • the molecular graphics program Insightll® v.98.0 was used to determine residues having their CB atom (CA in the case of glycine) at a distance of more than 15 A from the nearest amine group, defined as the NZ atoms of lysine and the N atom of the N-terminal residue Tl.
  • the following list includes the residues that fulfill this criteria in at least one of the 10 NMR structures.
  • G4 P5, A6, S7, S8, L9, P10, Qll, L14, L15, L18, V21, R22, Q25, G26, D27, G28, A29, Q32, L35, C36, T38, Y39, C42, H43, P44, E45, E46, L47, V48, L49, L50, G51, H52, S53, L54, G55, 156, P57, W58, A59, P60, L61, S62, S63, C64, P65, S66, Q67, A68, L69, Q70, L71, A72, G73, C74, L75, S76, Q77, L78, H79, S80, G81, L82, F83, L84, Y85, Q86, G87, L88, L89, Q90, A91, L92, E93, G94, 195, S96, P97, E98, L99, G100, PlOl, T102, L103,
  • the Insight ⁇ ® v.98.0 program was similarly used to determine residues having their CB atom (CA atom in the case of glycine) at a distance of more than lOA from the nearest acidic group, defined as the CG atoms of aspartic acid, the CD atoms of glutamic acid and the C atom of the C-terminal residue P174.
  • the following list includes the residues that fulfill this criteria in at least one of the 10 NMR structures.
  • the molecular weight of conjugated or non-conjugated hG-CSF or variants thereof is determined by either SDS-PAGE, gel filtration, matrix assisted laser desorption mass spectrome- try or equilibrium centrifugation. Determination of polypeptide concentration
  • the concentration of a polypeptide can be measured using optical density measurements at 280 nm, an enzyme-linked immunoadsorption assay (ELISA), a radio- immunoassay (RIA), or other such immunodetection techniques well known in the art.
  • ELISA enzyme-linked immunoadsorption assay
  • RIA radio- immunoassay
  • Fur- thermore the polypeptide concentration in a sample can be measured with the Biacore® instrument using a Biacore® chip coated with an antibody specific for the polypeptide.
  • Such an antibody can be coupled covalently to the Biacore® chip by various chemistries.
  • the antibody can be bound non-covalently e.g. by means of an antibody specific for the Fc portion of the anti-polypeptide antibody.
  • the Fc specific antibody is first coupled covalently to the chip.
  • the anti-polypeptide antibody is then flowed over the chip and is bound by the first antibody in a directed fashion.
  • biotinylated antibodies can be immobilised using a streptavidin coated surface (e.g.
  • Biacore Sensor Chip S A® Real- Time Analysis of Biomolecular Interactions, Nagata and Handa (Eds.), 2000, Springer Verlag, Tokyo; Biacore 2000 Instrument Handbook, 1999, Biacore AB.
  • the polypeptide will bind to the coated antibody and the increase in mass can be measured.
  • a standard curve can be established and subsequently the concentration of the polypeptide in the sample can be determined.
  • the sensor chip is regenerated by a suitable eluent (e.g. a low pH buffer) that removes the bound ana- lyte.
  • the applied antibodies will be monoclonal antibodies raised against the wild type polypeptide.
  • Introduction of mutations or other manipulations of the wild type polypeptide may alter the recognition by such antibodies.
  • manipulations that give rise to an increased molecular weight of the polypeptide will result in an increased plasmon resonance signal. Consequently, it is necessary to establish a standard curve for every molecule to be tested.
  • NFS-60 Proliferation of the murine cell line NFS-60 (obtained from Dr. J. Bile, St. Jude Children' s Research Hospital, Tennessee, USA) is dependent on the presence of active G-SCF in the growth medium.
  • the in vitro biological activity of hG-CSF and variants thereof can be determined by measuring the number of dividing NFS-60 cells after addition of a G-CSF sample to the growth medium followed by incubation over a fixed period of time. NFS-60 cells are maintained in Iscoves DME Medium containing 10% w/w FBS (fetal bovine serum), 1% w/w Pen/Strep, 10 ⁇ g per litre hG-CSF and 2 mM Glutamax.
  • FBS fetal bovine serum
  • Pen/Strep 10 ⁇ g per litre hG-CSF
  • 2 mM Glutamax 2 mM Glutamax.
  • cells Prior to sample addition, cells are washed twice in growth medium without hG-CSF and diluted to a concentration of 2.2 x 10 5 cells per ml. 100 ⁇ l of the cell suspension is added to each well of a 96 well microtiter plate (Coming).
  • Samples containing conjugated or non-conjugated G-CSF or variants thereof are diluted to concentrations between l.lxlO "6 M and l.lxlO "13 M in the growth medium.
  • 10 ⁇ l of each sample is added to 3 wells containing NFS-60 cells.
  • a control consisting of 10 ⁇ l of mammalian growth medium is added to 8 wells on each microtiter plate.
  • the cells are incubated for 48 hours (37°C, 5% CO 2 ) and the number of dividing cells in each well is quantified using the WST-1 cell proliferation agent (Roche Diagnostics GmbH, Mannheim, Germany). 0.01 ml WST-1 is added to the wells followed by incubation for 150 min.
  • dose-response curves for each conjugated and non- conjugated G-CSF molecule or variants thereof are calculated, after which the EC50 value for each molecule can be determined.
  • This value is equal to the amount of active G-CSF protein that is necessary to obtain 50% of the maximum proliferation activity of non-conjugated human G-CSF.
  • the EC50 value is a direct measurement of the in vitro activity of the given protein.
  • the murine hematopoietic cell line BaF3 is transfected with a plasmid carrying the human G-CSF receptor and the promoter of the transcription regulator, fos, in front of the luciferase reporter gene.
  • a number of intracellular reactions lead to stimulation of fos expression, and consequently to expression of luciferase.
  • This stimulation is monitored by the Steady-GloTM Luciferase Assay System (Promega, Cat. No. E2510) whereby the in vitro activity of the G-CSF sample may be quantified.
  • BaF3/hGCSF-R pfos-lux cells are maintained at 37°C in a humidified 5% CO 2 atmosphere in complete culture media (RPMI-1640/HEPES (Gibco/BRL, Cat. No. 22400), 10% FBS (HyClone, characterized), lx Penicillin/Streptomycin (Gibco/BRL, Cat. No. 15140-122), lx L-Glutamine (Gibco/BRL, Cat. No. 25030-081), 10% WEHI-3 conditioned media (source of muIL-3), and grown to a density of 5 x 10 5 cells/mL (confluent). The cells are reseeded at about 2 x 10 4 cells/mL every 2-3 days.
  • log-phase cells are resuspended at 2 x 10 5 cells/mL in starving media (DMEM/F-12 (Gibco/BRL, Cat. No. 11039), 1% BSA (Sigma, Cat. No. A3675), lx Penicillin/Streptomycin (Gibco/BRL, Cat. No. 15140-122), lx L-Glutamine (Gibco/BRL, Cat. No. 25030-081), 0.1% WEHI-3 conditioned media) and starved for 20 hours. The cells are washed twice with PBS and tested for viability using Trypan Blue viability staining.
  • the cells are resuspended in assay media (RPMI-1640 (phenol-red free, Gibco/BRL, Cat. No. 11835), 25 mM HEPES, 1% BSA (Sigma, Cat. No. A3675), lx Penicillin/Streptomycin (Gibco/BRL, Cat. No. 15140-122), lx L-Glutamine (Gibco/BRL, Cat. No. 25030-081) at 4 x 10 6 cells/mL, and 50 ⁇ L are aliquotted into each well of a 96-well microtiter plate (Coming).
  • Samples containing conjugated or non-conjugated G-CSF or variants thereof are diluted to concentrations between l.lxlO "7 M and l.lxlO "12 M in the assay medium.
  • 50 ⁇ l of each sample is added to 3 wells containing BaF3/hGCSF-R/pfos-lux cells.
  • a negative control consisting of 50 ⁇ l of medium is added to 8 wells on each microtiter plate. The plates are mixed gently and in- cubated for 2 hours at 37°C.
  • the luciferase activity is measured by following the Promega
  • Steady-GloTM protocol Promega Steady-GloTM Luciferase Assay System, Cat. No. E2510
  • 100 ⁇ L of substrate is added per well followed by gentle mixing.
  • Luminescence is measured on a TopCount luminometer (Packard) in SPC (single photon counting) mode.
  • dose-response curves for each conjugated and non- conjugated G-CSF molecule or variants thereof are calculated, after which the EC50 value for each molecule can be determined.
  • Binding of rhG-CSF or variants thereof to the hG-CSF receptor is studied using standard binding assays.
  • the receptors may be purified extracellular receptor domains, receptors bound to purified cellular plasma membranes, or whole cells - the cellular sources being either cell lines that inherently express G-CSF receptors (e.g. NFS-60) or cells transfected with cDNAs encoding the receptors.
  • G-CSF receptors e.g. NFS-60
  • the ability of rhG-CSF or variants thereof to compete for the binding sites with native G-CSF is analyzed by incubating with a labeled G- CSF-analog, for instance biotinylated hG-CSF or radioiodinated hG-CSF.
  • hG-CSF receptor can optionally be coupled to Fc and immobilized in 96 well plates. RhG-CSF or variants thereof are subsequently added and the binding of these is detected using either specific anti-hG-CSF antibodies or biotinylated or radioiodinated hG-CSF.
  • An important aspect of the invention is the prolonged biological half-life that is obtained by construction of a hG-CSF with or without conjugation of the polypeptide to the polymer moiety.
  • the rapid decrease of hG-CSF serum concentrations has made it important to evaluate biological responses to treatment with conjugated and non-conjugated hG-CSF and variants thereof.
  • the conjugated and non-conjugated hG-CSF and variants thereof of the present invention have prolonged serum half -lives also after i.v. administration, making it possible to measure by e.g. an ELISA method or by the primary screening assay. Measurement of in vivo biological half-life was carried out as described below. Male Sprague Dawley rats (7 weeks old) were used.
  • the weights of the animals were measured (280-310 gram per animal). 100 ⁇ g per kg body weight of the non-conjugated and conjugated hG-CSF samples were each injected intravenously into the tail vein of three rats. At 1 minute, 30 minutes, 1, 2, 4, 6, and 24 hours after the injection, 500 ⁇ l of blood was withdrawn from the eyes of each rat while under CO 2 - anaesthesia. The blood samples were stored at room temperature for V ⁇ hours followed by isolation of serum by centrifugation (4°C, 18000xg for 5 minutes). The serum samples were stored at -80°C until the day of analysis.
  • the amount of active G-CSF in the serum samples was quantified by the G-CSF in vitro activity assays (see primary assay 1 and 2) after thawing the samples on ice.
  • Another example of an assay for the measurement of in vivo half-life of G-CSF or variants thereof is described in US 5,824,778, the content of which is hereby incorporated by reference. Measurement of the in vivo biological activity of conjugated and non-conjugated hG-CSF and variants thereof
  • the rats On the day of arrival the rats are randomly allocated into groups of 6. The animals are acclimatised for a period of 7 days wherein individuals in poor condition or at extreme weights are rejected.
  • the weight range of the rats at the start of the acclimatization period is 250-270g.
  • the blood samples are analyzed for the following haematological parameters: Haemoglobin, red blood cell count, haematocrit, mean cell volume, mean cell haemo- globin concentration, mean cell haemoglobin, white blood cell count, differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes).
  • haematological parameters Haemoglobin, red blood cell count, haematocrit, mean cell volume, mean cell haemo- globin concentration, mean cell haemoglobin, white blood cell count, differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes).
  • the strength of the binding between a receptor and ligand can be measured using an enzyme-linked immunoadsorption assay (ELISA), a radio-immunoassay (RIA), or other such immunodetection techniques well known in the art.
  • ELISA enzyme-linked immunoadsorption assay
  • RIA radio-immunoassay
  • the ligand-receptor binding interaction may also be measured with the Biacore® instrument, which exploits plasmon resonance detection (Zhou et al., Biochemistry, 1993, 32, 8193-98; Faegerstram and O'Shannessy, 1993, In Handbook of Affinity Chromatography, 229-52, Marcel Dekker, Inc., NY).
  • the Biacore® technology allows one to bind receptor to a gold surface and to flow ligand over it.
  • the reduced immunogenicity of a conjugate can be determined by use of an ELISA method measuring the immunoreactivity of the conjugate relative to a reference mole- cule or preparation.
  • the reference molecule or preparation is normally a recombinant human G- CSF preparation such as Neupogen® or another recombinant human G-CSF preparation, e.g. an N-terminally PEGylated rhG-CSF molecule as described in US 5,824,784.
  • the ELISA method is based on antibodies from patients treated with one of these recombinant G-CSF preparations.
  • the immunogenicity is considered to be reduced when the conjugate has a statistically signifi- cant lower response in the assay than the reference molecule or preparation.
  • hG-CSF conjugates by anti-G-CSF sera is analyzed using the G-CSF bioassay described above.
  • Sera from patients treated with the G-CSF reference molecule or from immunized animals are used.
  • Sera are added either in a fixed concentration (dilution 1:20-1:500 (pt sera) or 20-1000 ng/ l (animal sera)) or in five-fold serial dilutions of sera starting at 1:20 (pt sera) or 1000 ng/ml (animal sera).
  • HG-CSF conjugate is added either in seven fold-dilutions starting at 10 nM or in a fixed concentration (1-100 pM) in a total volume of 80 ⁇ l DMEM medium + 10% FCS.
  • the sera are incubated for 1 hr. at 37°C with hG-CSF conjugate.
  • the samples (0.01 ml) are then transfe ⁇ ed to 96 well tissue culture plates containing NFS-60 cells in 0.1 ml DMEM media.
  • the cultures are incubated for 48 hours at 37°C in a 5% CO 2 air atmosphere.
  • 0.01 ml WST-1 WST-1 cell proliferation agent, Roche Diagnostics GmbH, Mannheim, Germany
  • the cleavage of the tetrazolium salt WST-1 by mitochondrial dehy- drogenases in viable cells results in the formation of formazan that is quantified by measuring the absorbance at 450 nm.
  • FI fold inhibition
  • aqueous solution of both a conjugate as described herein and a reference polypeptide where the latter is either the co ⁇ esponding wild-type human polypeptide or the co ⁇ esponding polypeptide variant, at an equal and appropriate concentration (i.e. within the range of 0.01 to 1.0 mg/ml) in a buffer solution of relevant pH and without addition of further stabilizers.
  • the relevant pH is chosen within the range of 2 pH values from the known pi value for the wild-type human polypeptide in question (i.e. pH values within ["pI-2" to "pI+2"].
  • a volume of 0.5 ml of the solution is shaken in a closed container of at least 2 ml volume (e.g. a 2 ml Eppendorf tube), using a high speed mixer for a period of 5 minutes, at room temperature, or ii) A volume of 0.5 ml of the solution is rotated in a closed container of at least 2 ml volume (e.g.
  • a volume of 0.5 ml of the solution is kept unstirred for 24 hours at a temperature, which is 5-10°C below the melting temperature for the conjugate as determined by DSC using a suitable scanning rate, or iv) A volume of 0.5 ml of the solution is processed 3 times through a freeze-thaw cyclus within a period of Iweek. A freezing temperature of -20°C and a thaw temperature of 20°C is used.
  • the degree of aggregation within each pair of solutions processed under the conditions mentioned above, is then analyzed by: a) SEC-HPLC technique with UV detection, or b) SEC-HPLC technique with Multi Angeled Light Scattering (MALS) detection, after which the "area under the curve" for the monomer is determined (AUC m0n omer >C onjugate), and the sum of the "area under the curve” for dimer and higher aggregates is determined (AUC a ggr e- g a t e,conjugate)- Similar values are determined for the reference polypeptide (AUC mon omer ) ref and AUCaggregate.ref)- The relative amount of aggregated conjugate (a CO njugate) is determined as AUC ag- gregate,conjugat_/(AUC a gg re gate,conjugate+ AUC m onomer,conjugate)- U a Similar Way, the relative amount OI aggregated reference polypeptide (a re f
  • the percentage reduction in the tendency to form aggregates is then calculated as ((A re _ - A co nj u at e VAre f ) x 100%, wherein A ref is the absorbance readout for the reference polypeptide and A CO nj u gat e is the absorbance readout for the conjugate.

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Abstract

La présente invention concerne de nouvelles dispersions d'un conjugué contenant une variante polypeptidique et un groupe caractéristique non polypeptidique, et la délivrance pulmonaire de quantités thérapeutiquement efficaces de cette dispersion pour le traitement de maladies. L'invention concerne également une dispersion de particules contenant un conjugué comprenant au moins un groupe caractéristique non polypeptidique fixé de façon covalente à une variante polypeptidique. La séquence d'acides aminés de cette variante polypeptidique diffère de celle du polypeptide humain de type sauvage correspondant en ce qu'au moins un résidu d'acide aminé comprenant un groupe de fixation de ce groupe caractéristique non polypeptidique a été introduit et/ou retiré. Des exemples des polypeptides humains de type sauvage contiennent des cytokines, des facteurs de croissance, des hormones et des activateurs plasminogènes.
PCT/DK2001/000182 2000-03-17 2001-03-16 Dispersions de conjugues polypeptidiques WO2001068141A2 (fr)

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AU42310/01A AU4231001A (en) 2000-03-17 2001-03-16 Dispersions of polypeptide conjugates

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DKPA200000447 2000-03-17
DKPA200000447 2000-03-17
US19084400P 2000-03-20 2000-03-20
US60/190,844 2000-03-20
PCT/DK2000/000471 WO2001015736A2 (fr) 1999-08-27 2000-08-25 Nouvelles molecules analogues a l'interferon beta
DKPCT/DK00/00471 2000-08-25
DKPCT/DK00/00631 2000-11-13
PCT/DK2000/000631 WO2001036001A2 (fr) 1999-11-12 2000-11-13 Conjugues d'interferon gamma

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2795734A1 (fr) * 1999-07-02 2001-01-05 Hoffmann La Roche Conjugues d'erythropoietine,compositons les comprenant et procede pour leur preparation
WO2004044006A1 (fr) * 2002-11-14 2004-05-27 Maxygen, Inc. Conjugues de l'interleukine-10 et de polymeres
US6838076B2 (en) 2001-05-21 2005-01-04 Nektar Therapeutics Pulmonary administration of chemically modified insulin
CN110582515A (zh) * 2018-11-12 2019-12-17 天境生物科技(上海)有限公司 包含cd47抗体和细胞因子的融合蛋白

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
No Search *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2795734A1 (fr) * 1999-07-02 2001-01-05 Hoffmann La Roche Conjugues d'erythropoietine,compositons les comprenant et procede pour leur preparation
EP1064951A3 (fr) * 1999-07-02 2002-03-20 F. Hoffmann-La Roche Ag Dérivés de l' Erythropoietine
US6583272B1 (en) 1999-07-02 2003-06-24 Hoffmann-La Roche Inc. Erythropoietin conjugates
EP1839676A3 (fr) * 1999-07-02 2008-09-03 F. Hoffmann-La Roche AG Dérivés dýerythropoiétine
US6838076B2 (en) 2001-05-21 2005-01-04 Nektar Therapeutics Pulmonary administration of chemically modified insulin
US6890518B2 (en) 2001-05-21 2005-05-10 Nektar Therapeutics Compositions of chemically modified insulin
WO2004044006A1 (fr) * 2002-11-14 2004-05-27 Maxygen, Inc. Conjugues de l'interleukine-10 et de polymeres
CN110582515A (zh) * 2018-11-12 2019-12-17 天境生物科技(上海)有限公司 包含cd47抗体和细胞因子的融合蛋白

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