WO2001076639A2 - Chemically-modified myelopoietin conjugates - Google Patents

Abstract

The present invention provides a chemically modified Myelopoietin (MPO) prepared by binding a water soluble polymer to the protein. The chemically-modified protein according to the present invention may have a much longer lasting neutrophil-increasing activity than that of the un-modified MPO, enabling reduced dose and scheduling opportunities.

Description

CHEMICALLY-MODIFIED MYELOPOIETIN CONJUGATES

The present application claims priority under Title 35, United States Code, §119 of United States Provisional application Serial No. 60/195,496 filed April 06, 2000.

FIELD OF THE INVENTION

The present invention relates to a chemical modification of myelopoietins (MPOs) , a family of recombinant proteins, which are multifunctional agonists of human interleukin-3 (IL-3) and another hematopoietic growth factor receptor, including but not limited to G-CSF, by which the chemical and/or physiological properties of MPO can be changed. The PEGylated MPOs may have a decreased clearance rate, improved stability, decreased antigenicity, or a combination thereof. The family of MPO proteins is defined as the multifunctional agonists described in US 5,738,849, US 5,858,347, US 6,057,133, US 6,132,991, US 6,022,535, US 6,030,812, WO 95/21197 and WO 95/21256, which are incorporated herein in their entirety. The present invention also relates to processes for the modification of MPO. In addition, the present invention relates to pharmaceutical compositions comprising the modified MPO. A further embodiment is the use of the modified MPO to treat hematopoietic disorders.

BACKGROUND OF THE INVENTION

Myelopoietin may be useful in the treatment of general haematopoietic disorders, including those arising from chemotherapy or from radiation therapy (Mac Vittie, T. J. ; et al . , Exp . Hematol . (1999), 27(10), 1557-1568). MPO may also be useful in bone marrow transplantation, wound healing, burn treatment, and the treatment of parasite, bacterial or viral infection.

It is generally observed that physiologically active proteins administered into a body can show their pharmacological activity only for a short period due to their high clearance rate in the body. Furthermore, the relative hydrophobicity of these proteins may limit their stability.

For the purpose of decreasing the clearance rate, improving stability or abolishing antigenicity of therapeutic proteins, some methods have been proposed wherein the proteins are chemically modified with water-soluble polymers. Chemical modification of this type may block effectively a proteolytic enzyme from physical contact with the protein backbone itself, thus preventing degradation. Chemical attachment may effectively reduce renal clearance. Additional advantages include, under certain circumstances, increasing the stability and circulation time of the therapeutic protein, increasing solubility, and decreasing immunogenicit . A review article describing protein modification and fusion proteins is Francis, Focus on Growth Factors 3 : 4-10 (May 1992) (published by Mediscript, Mountview Court, Friern Barnet Lane, London N20, OLD, UK) .

Poly (alkylene oxide), notably poly (ethylene glycol) (PEG) , is one such chemical moiety, which has been used in the preparation of therapeutic protein products (the verb "pegylate" meaning to attach at least one PEG molecule) . The attachment of poly (ethylene glycol) has been shown to protect against proteolysis, Sada, et al . , J. Fermentation Bioeng_fineering 71: 137-139 (1991), and methods for attachment of certain poly (ethylene glycol) moieties are available. See U.S. Pat. No. 4,179,337, Davis et al . , "Non-Immunogenic Polypeptides," issued Dec. 18, 1979; and U.S. Pat. No. 4,002,531, Royer, "Modifying enzymes with Polyethylene Glycol and Product Produced Thereby," issued Jan. 11, 1977. For a review, see Abuchowski et al . , in Enzymes as Drugs . (J. S. Holcerberg and J. Roberts, eds. pp. 367-383 (1981)).

Other water-soluble polymers have been used, such as copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, poly (vinyl alcohol) , poly(vinyl pyrrolidone) , poly (-1, 3-dioxolane) , poly(- 1, 3 , 6-trioxane) , ethylene/maleic anhydride copolymer, poly- amino acids (either homopolymers or random copolymers) .

A number of examples of pegylated therapeutic proteins have been described. ADAGEN®, a pegylated formulation of adenosine deaminase, is approved for treating severe combined immunodeficiency disease. ONCASPAR®, a pegylated L-asparaginase has been approved for treating hypersensitive ALL patients. Pegylated superoxide dismutase has been in clinical trials for treating head injury. Pegylated α-interferon (U.S. 5,738,846, 5,382,657) has been tested in phase III clinical trials for treating hepatitis with PEG-Intron (pegitron alfa-2b) approved for the treatment of chronic hepatitis C while another molecule, PEGASYS™, still awaits regulatory approval; pegylated glucocerebrosidase and pegylated hemoglobin are reported to have been in preclinical testing. Another example is pegylated IL-6, EF 0 442 724, entitled, "Modified hIL-6, " which discloses poly (ethylene glycol) molecules added to IL-6.

Another specific therapeutic protein, which has been chemically modified, is granulocyte colony stimulating factor, (G-CSF) . G-CSF induces the rapid proliferation and release of neutrophilic granulocytes to the blood stream, and thereby provides therapeutic effect in fighting infection. European patent publication EP 0 401 384, published Dec. 12, 1990, entitled, "Chemically Modified Granulocyte Colony

Stimulating Factor, " describes materials and methods for preparing G-CSF to which poly (ethylene glycol) molecules are attached. Modified G-CSF and analogs thereof are also reported in EP 0 473 268, published Mar. 4, 1992, entitled "Continuous Release

Pharmaceutical Compositions Comprising a Polypeptide Covalently Conjugated To A Water Soluble Polymer, " stating the use of various G-CSF and derivatives covalently conjugated to a water soluble particle polymer, such as poly (ethylene glycol) . A modified polypeptide having human granulocyte colony stimulating factor activity is reported in EP 0 335 423 published Oct. 4, 1989. Provided in U.S. 5,824,784 are methods for N-terminally modifying proteins, including N- terminally chemically modified G-CSF compositions. U.S. 5,824,778 discloses chemically modified G-CSF.

Japanese patent application Hei2 (1990) -30555 discloses chemically modified human IL-3 having decreased antigenicity . The family of MPO proteins is disclosed in US

5,738,849, US 5,858,347, US 6,057,133, US 6,132,991, US 6,022,535, US 6,030,812, WO 95/21197, and WO 95/21256. For poly (ethylene glycol), a variety of means has been used to attach the poly (ethylene glycol) molecules to the protein. Generally, poly (ethylene glycol) molecules are connected to the protein via a reactive group found on the protein.

Amino groups, such as those on lysine residues or at the N-terminus, are convenient for such attachment. For example, Royer (U.S. Pat. No. 4,002,531, above) states that reductive alkylation was used for attachment of poly (ethylene glycol) molecules to an enzyme. EP 0 539 167, published Apr. 28, 1993, Wright, "Peg Imidates and Protein Derivatives Thereof" states that peptides and organic compounds with free amino group (s) are modified with an imidate derivative of PEG or related water-soluble organic polymers. Chamow efc al . , Bioconjugate Chem. 5: 133-140 (1994) report the modification of CD4 immunoadhesin with monomethoxypol (ethylene glycol) aldehyde via reductive alkylation. The authors report that 50% of the CD4-Ig was MePEG-modified under conditions allowing control over the extent of pegylation. Ibid, at page 137. The authors also report that the in vi tro binding capability of the modified CD4-Ig (to the protein gp 120) decreased at a rate correlated to the extent of MePEGylation Ibid. U.S. Pat. No. 4,904,584, Shaw, issued Feb. 27, 1990, relates to the modification of the number of lysine residues in proteins for the attachment of poly (ethylene glycol) molecules via reactive amine groups .

Many methods of attaching a polymer to a protein involve using a moiety to act as a linking group. However, such moieties may be antigenic. A tresyl chloride method involving no linking group is available, but this method may be difficult to use to produce therapeutic products as the use of tresyl chloride may produce toxic by-products. See Francis et al . , In: Stability of protein pharmaceuticals: in vivo pathways of degradation and strategies for protein stabilization (Eds . Ahern. T . and Manning, M. C . ) Plenum, New York, 1991) Also, Delgado et al . , "Coupling of PEG to Protein By Activation With Tresyl Chloride, Applications In Immunoaffinity Cell Preparation", in_ Separations Using Aqueous Phase Systems, Applications In Cell Biology and Biotechnology, Fisher et al . , eds. Plenum Press, New York, N.Y., 1989 pp. 211-213. See also, Rose et al . , Bioconjugate Chemistry 2: 154-159 (1991) which reports the selective attachment of the linker group carbohydrazide to the C-terminal carboxyl group of a protein substrate (insulin) .

The present invention provides chemically modified MPO molecules having decreased clearance rate, increased stability, decreased antigenicity, or combinations thereof .

SUMMARY OF THE INVENTION

The present invention relates to chemically modified MPOs, which have at least one improved chemical or physiological property selected from but not limited to decreased clearance rate, increased stability, and decreased antigenicity. Thus, as described below in more detail, the present invention has a number of aspects relating to chemically modifying MPOs as well as specific modifications using a variety of poly (ethylene glycol) moieties. The present invention also relates to methods of producing the chemically modified MPOs.

The present invention also relates to compositions comprising the chemically modified MPOs. The modified MPO of the present invention may be useful in the treatment of, but not limited to, neutropenia, thrombocytopenia, mobilization of hematopoietic progenitors and stem cells into peripheral blood, bone marrow suppression or hematopoietic deficiencies, and immunodeficiencies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reproduction of the ion-exchange chromatography elution profile of a 30,000 MW PEG-ALD MPO reaction.

FIG. 2 is an SDS-PAGE of 20,000 and 30000 MW PEG-ALD MPO. Lane 1. MW Protein standards; Lane 2. MPO (lOug) ; Lane 3. 20,000 MW PEG-ALD MPO (lOug) ; Lane 4. 30,000 MW PEG-ALD MPO (lOug) .

FIG. 3a is an SEC HPLC profile of recombinant MPO 30,000 NW PEG-ALD RΣ mix, ion exchange purified N- terminally mono-pegylated 30,000 MW PEG-ALD MPO, recombinant MPO 20,000 MW PEG-ALD RX mix, and ion exchange purified N-terminally mono-pegylated 20,000 MW PEG-ALD MPO.

FIG. 3b is an SEC HPLC profile of 10,000 MW branched PEG2-NHS MPO, 20,000 MW branched PEG2-NHS MPO, and 40,000 MW branched PEG2-NHS MPO.

FIG. 4 is reversed phase HPLC profile for 1. MPO; 2. N- terminally mono-PEGylated 20,000 MW PEG-ALD MPO; and 3. N-terminally mono-PEGylated 30,000 MW PEG-ALD MPO. FIG. 5 shows a reversed phase HPLC tryptic maps for MPO, N-terminally mono-PEGylated 30,000 MW PEG-ALD MPO, and N-terminally mono-PEGylated 20,000 MW PEG-ALD MPO.

FIG. 6 illustrates a comparison of response curves for an IL3 receptor agonist, a G-CSF receptor agonist, co- addition of IL3 receptor agonist and a G-CSF receptor agonist, un-PEGylated MPO and mono-PEGylated PEG-ALD MPO in colony forming unit granulocyte/ acrophage (CFU- GM) assay which measures expansion and differentiation of a human bone marrow-derived CD34+ cells.

FIG. 7 compares the in vivo bioactivity of un-PEGylated and N-terminally mono-PEGylated PEG-ALD MPO by illustrating the absolute neutrophil counts (ANC) during a period of 240 hours after a single subcutaneous injection dose in normal rhesus monkeys.

DETAILED DESCRIPTION OF THE INVENTION

Myelopoietin (MPO) proteins are members of a family of recombinant proteins, which are multifunctional agonists of human interleukin-3 (IL-3) and another hematopoietic growth factor. Their recombinant production and methods of use are detailed in US 5,738,849, US 5,858,347, US 6,057,133, US 6,132,991, US 6,022,535, US 6,030,812, WO 95/21197 and WO 95/21256.

Any purified and isolated MPO, which is produced by host cells such as E. coli and animal cells transformed or transfected by using recombinant genetic techniques, may be used in the present invention. Among them, MPO, which is produced by the transformed E. coli, is particularly preferable. Such MPO may be obtained in large quantities with high purity and homogeneity. For example, the above MPO may be prepared according to a method disclosed in US 5,738,849, US 5,858,347, US 6,057,133, US 6,132,991, US 6,022,535, and US 6,030,812. The term "substantially has the following amino acid sequence" means that the above amino acid sequence may include one or more amino-acid changes (deletion, addition, insertion or replacement) as long as such changes will not cause any disadvantageous non-similarity in function to MPO. It is more preferable to use the MPO substantially having an amino acid sequence, in which at least one lysine, aspartic acid, glutamic acid, or unpaired cysteine residue is included. According to the present invention, poly (ethylene glycol) is covalently bound through amino acid residues of MPO. The amino acid residue may be any reactive one(s) having, for example, free amino, carboxyl or sulfhydryl (thiol) groups, to which a terminal reactive group of an activated poly (ethylene glycol) may be bound. The amino acid residues having the free amino groups may include lysine residues and/or N-terminal amino acid residue, those having a free carboxyl group may include aspartic acid, glutamic acid and/or C- terminal amino acid residues, and having a sulfhydryl (thiol) such as cysteine.

In another embodiment, oxine chemistries (Lemieux & Bertozzi Tib Tech 16:506-513, 1998) are used to target N-terminal serine residues. The poly (ethylene glycol) used in the present invention is not restricted to any particular form or molecular weight range. Normally a molecular weight of 500-60,000 is used and preferably of from 1,000-40,000. The poly (ethylene glycol) can also be a branched PEG as described in U.S. 5,932,462, U.S. 5,342,940, U.S. 5,643,575, U.S. 5,919,455, U.S. 6,113,906, and U.S. 5,183,660.

Pol (alkylene oxides) , notably poly (ethylene glycol) s, are bound to MPO via a terminal reactive group, which may or may not leave a linking moiety (spacer) between the PEG and the protein. In order to form the MPO conjugates of the present invention, polymers such as poly (alkylene oxide) are converted into activated forms, as such term is known to those of ordinary skill in the art. The reactive group, for example, is a terminal reactive group, which mediates a bond between chemical moieties on the protein, such as amino, carboxyl or thiol groups, and poly (ethylene glycol) . Typically, one or both of the terminal polymer hydroxyl end-groups, (i.e. the alpha and omega terminal hydroxyl groups) are converted into reactive functional groups, which allows covalent conjugation. This process is frequently referred to as "activation" and the poly (ethylene glycol) product having the reactive group is hereinafter referred to as "an activated poly (ethylene glycol)". Polymers containing both α and ω linking groups are referred to as "bis-activated poly (alkylene oxides)" and are referred to as "bifunctional" . Polymers containing the same reactive group on and ω terminal hydroxyls are sometimes referred to as "homobifunctional" or "homobis- activated" . Polymers containing different reactive groups on and ω terminal hydroxyls are sometimes referred to as "heterobifunctional" or "heterobis- activated" . Polymers containing a single reactive group are referred to as "mono-activated" polyalkylene oxides or "mono-functional" . Other substantially non- antigenic polymers are similarly "activated" or "functionalized" .

The activated polymers are thus suitable for mediating a bond between chemical moieties on the protein, such as -amino, carboxyl or thiol groups, and poly (ethylene glycol) . Bis-activated polymers can react in this manner with two protein molecules or one protein molecule and a reactive small molecule in another embodiment to effectively form protein polymers or protein-small molecule conjugates through cross linkages. Functional groups capable of reacting with either the amino terminal α-amino group or ε-amino groups of lysines found on the MPO include: carbonates such as the p-nitrophenyl, or succinimidyl; carbonyl imidazole; azlactones; cyclic imide thiones; isocyanates or isothiocyanates and aldehydes. Functional groups capable of reacting with carboxylic acid groups, reactive carbonyl groups and oxidized carbohydrate moieties on MPO include; primary amines; and hydrazine and hydrazide functional groups such as the acyl hydrazides, carbazates, semicarbamates, thiocarbazates, etc. Mercapto groups, if available on the MPO, can also be used as attachment sites for suitably activated polymers with reactive groups such as thiols; maleimides, sulfones, and phenyl glyoxals; see, for example, U.S. Pat. No. 5,093,531, the disclosure of which is hereby incorporated by reference. Other nucleophiles capable of reacting with an electrophilic center include, but are not limited to, for example, hydroxyl, amino, carboxyl, thiol, active methylene and the like.

In one preferred embodiment of the invention secondary amine or amide linkages are formed using the MPO N-terminal amino groups or ε-amino groups of lysine and the activated PEG. In another preferred aspect of the invention, a secondary amine linkage is formed between the N-terminal primary amino group of MPO and single or branched chain PEG aldehyde by reduction with a suitable reducing agent such as NaCNBH₃, NaBH₃, Pyridine Borane etc . as described in Chamow et al . , Bioconjugate Chem. 5: 133-140 (1994) and US Pat. No 5,824,784.

In another preferred embodiment of the invention, polymers activated with amide-forming linkers such as succinimidyl esters, cyclic imide thiones, or the like are used to effect the linkage between the MPO and polymer, see for example, U.S. Pat. No. 5,349,001; U.S. Pat. No. 5,405,877; and Greenwald, et al . , Cri t . Rev. Ther. Drug Carrier Syst . 17:101-161, 2000, which are incorporated herein by reference. One preferred activated poly (ethylene glycol), which may be bound to the free amino groups of MPO includes single or branched chain N-hydroxysuccinylimide poly (ethylene glycol) may be prepared by activating succinic acid esters of poly (ethylene glycol) with N- hydroxysuccinylimide .

Other preferred embodiments of the invention include using other activated polymers to form covalent linkages of the polymer with the MPO via ε-amino or other groups. For example, isocyanate or isothiocyanate forms of terminally activated polymers can be used to form urea or thiourea-based linkages with the lysine amino groups . In another preferred aspect of the invention, carbamate (urethane) linkages are formed with protein amino groups as described in U.S. Pat. Nos. 5,122,614, 5,324,844, and 5,612,640, which are hereby incorporated by reference. Examples include N-succinimidyl carbonate, para-nitrophenyl carbonate, and carbonyl imidazole activated polymers . In another preferred embodiment of this invention, a benzotriazole carbonate derivative of PEG is linked to amino groups on MPO. Another aspect of the invention represents a prodrug or sustained release form of MPO, comprised of a water soluble polymer, such as poly (ethylene glycol), attached to an MPO molecule by a functional linker that can predictably break down by enzymatic or pH directed hydrolysis to release free MPO or other MPO derivative. The prodrug can also be a "double prodrug" (Bundgaard in Advanced Drug Delivery Reviews 3:39-65, 1989) involving the use of a cascade latentiation. In such systems, the hydrolytic reaction involves an initial rate-limiting (slow) enzymatic or pH directed step and a second step involving a rapid non-enzymatic hydrolysis that occurs only after the first has taken place. Such a releasable polymer provides protein conjugates, which are impermanent and could act as a reservoir, that continually discharge MPO. Such functional linkers are described in US 5,614,549; US 5,840,900; US 5,880,131; US 5,965,119; US 6,011,042; US 6,180,095 Bl; Greenwald R.B. et al . , J. Med. Chem. 42 ; 3657-3667 , 1999; Lee, S. et al . , Bioconjugate Chem 12:163-169, 2001; Garman A.J. et al . , FEBS Lett .

223:361-365, 1987; Woghiren C. et al . , Bioconjucate Chem. 4:314-318, 1993; Roberts M.J. et al . , J^". Pharm. Sci . 87 ,-1440-1445, 1998; Zhao X., in Ninth Int . Symp. Recent Adv. Drug Delivery Syst . 199; Greenwald R.B. et al., J^". Med. Chem. 43:475-487, 2000; and Greenwald R.B. Crit. .Rev. Ther. Drug Carrier Syst . 17:101-161, 2000.

Conjugation reactions, referred to as pegylation reactions, were historically carried out in solution with molar excess of polymer and without regard to where the polymer will attach to the protein. Such general techniques, however, have typically been proven inadequate for conjugating bioactive proteins to non- antigenic polymers while retaining sufficient bioactivity. One way to maintain the MPO bioactivity is to substantially avoid the conjugation of those MPO reactive groups associated with the receptor binding site(s) in the polymer coupling process. Another aspect of the present invention is to provide a process of conjugating poly (ethylene glycol) to MPO maintaining high levels of retained activity.

The chemical modification through a covalent bond may be performed under any suitable condition generally adopted in a reaction of a biologically active substance with the activated poly (ethylene glycol) . The conjugation reaction is carried out under relatively mild conditions to avoid inactivating the MPO. Mild conditions include maintaining the pH of the reaction solution in the range of 3 to 10 and the reaction temperatures within the range of from about 0°-37°C. In the cases where the reactive amino acid residues in MPO have free amino groups, the above modification is preferably carried out in a non-limiting list of suitable buffers (pH 3 to 10) , including phosphate, citrate, acetate, succinate or HEPES, for 1-48 hrs at 4° -37°C. In targeting N-terminal amino groups with reagents such as PEG aldehydes pH 4-7 is preferably maintained. The activated pol (ethylene glycol) may be used in 0.05-100 times, preferably 0.05-0.5 times, the molar amount of the number of free amino groups of MPO . On the other hand, where reactive amino acid residues in MPO have the free carboxyl groups , the above modification is preferably carried out in pH from about 3.5 to about 5.5, for example, the modification with poly (oxyethylenediamine) is carried out in the presence of carbodiimide (pH 4-5) for 1-24 hrs at 4°-37°C. The activated poly (ethylene glycol) may be used in 0.05-300 times the molar amount of the number of free carboxyl groups of MPO.

In separate embodiments, the upper limit for the amount of polymer included in the conjugation reactions exceeds about 1:1 to the extent that it is possible to react the activated polymer and MPO without forming a substantial amount of high molecular weight species, i.e. more than about 20% of the conjugates containing more than about one strand of polymer per molecule of MPO. For example, it is contemplated in this aspect of the invention that ratios of up to about 6 : 1 can be employed to form significant amounts of the desired conjugates which can thereafter be isolated from any high molecular weight species .

In another aspect of this invention, bifunctionally activated PEG derivatives may be used to generate polymeric MPO-PEG molecules in which multiple MPO molecules are crosslinked via PEG. Although the reaction conditions described herein can result in significant amounts of unmodified MPO, the unmodified MPO can be readily recycled into future batches for additional conjugation reactions. The processes of the present invention generate surprisingly very little, i.e. less than about 30% and more preferably, less than about 10%, of high molecular weight species and species containing more than one polymer strand per MPO . These reaction conditions are to be contrasted with those typically used for polymeric conjugation reactions wherein the activated polymer is present in several- fold molar excesses with respect to the target. In other aspects of the invention, the polymer is present in amounts of from about 0.1 to about 50 equivalents per equivalent of MPO. In other aspects of the invention, the polymer is present in amounts of from about 1 to about 10 equivalents per equivalent of MPO. The conjugation reactions of the present invention initially provide a reaction mixture or pool containing mono- and di-PEG-MPO conjugates, unreacted MPO, unreacted polymer and usually less than about 20% high molecular weight species. The high molecular weight species include conjugates containing more than one polymer strand and/or polymerized PEG-MPO species. After the unreacted species and high molecular weight species have been removed, compositions containing primarily mono- and di-polymer-MPO conjugates are recovered. Given the fact that the conjugates for the most part include a single polymer strand, the conjugates are substantially homogeneous . These modified MPOs have at least about 5% of the in vi tro biological activity associated with the native or unmodified MPO as measured using standard cell proliferation assays, such as AML, TFl and colony forming unit assays (U.S. Patent 6,030,812 which is incorporated by reference herein) . In preferred aspects of the invention, however, the modified MPOs have about 25% of the in vi tro biological activity, more preferably, the modified MPOs have about 50% of the in vi tro biological activity, more preferably, the modified MPOs have about 75% of the in vi tro biological activity, and most preferably the modified MPOs have equivalent or improved in vi tro biological activity.

The processes of the present invention preferably include rather limited ratios of polymer to MPO. Thus, the MPO conjugates have been found to be predominantly limited to species containing only one strand of polymer. Furthermore, the attachment of the polymer to the MPO reactive groups is substantially less random than when higher molar excesses of polymer linker are used. The unmodified MPO present in the reaction pool, after the conjugation reaction has been quenched, can be recycled into future reactions using ion exchange or size exclusion chromatography or similar separation techniques . A poly (ethylene glycol) -modified MPO, namely chemically modified protein according to the present invention, may be purified from a reaction mixture by conventional methods which are used for purification of proteins, such as dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gel chromatography and electrophoresis. Ion-exchange chromatography is particularly effective in removing unreacted poly (ethylene glycol) and MPO. In a further embodiment of the invention, the mono- and di-polymer- MPO species are isolated from the reaction mixture to remove high molecular weight species, and unmodified MPO. Separation is effected by placing the mixed species in a buffer solution containing from about 0.5- 10 mg/mL of the MPO-polymer conjugates. Suitable solutions have a pH from about 4 to about 10. The solutions preferably contain one or more buffer salts selected from KC1, NaCl, K₂HP0₄, KH₂P0₄, Na₂HP0₄, NaH₂P0 , NaHC0₃, NaB0₄, CH₃C0₂H, and NaOH.

Depending upon the reaction buffer, the MPO polymer conjugate solution may first have to undergo buffer exchange/ultrafiltration to remove any unreacted polymer. For example, the PEG-MPO conjugate solution can be ultrafiltered across a low molecular weight cutoff (10,000 to 30,000 Dalton) membrane to remove most unwanted materials such as unreacted polymer, surfactants, if present, or the like.

The fractionation of the conjugates into a pool containing the desired species is preferably carried out using an ion exchange chromatography medium. Such media are capable of selectively binding PEG-MPO conjugates via differences in charge, which vary in a somewhat predictable fashion. For example, the surface charge of MPO is determined by the number of available charged groups on the surface of the protein. These charged groups typically serve as the point of potential attachment of poly (alkylene oxide) conjugates. Therefore, MPO conjugates will have a different charge from the other species to allow selective isolation.

Strongly polar anion or cation exchange resins such as quaternary amine or sulfopropyl resins, respectively, are used for the method of the present invention. Cation exchange resins are especially preferred. A non-limiting list of included commercially available cation exchange resins suitable for use with the present invention are SP-hitrap®, SP Sepharose HP® and SP Sepharose® fast flow. Other suitable cation exchange resins e.g. S and CM resins can also be used. A non-limiting list of anion exchange resins, including commercially available anion exchange resins, suitable for use with the present invention are Q-hitrap®, Q Sepharose HP®, and Q sepharose® fast flow. Other suitable anion exchange resins, e . g. DEAE resins, can also be used.

For example, the cation exchange resin is preferably packed in a column and equilibrated by conventional means. A buffer having the same pH and osmolality as the polymer conjugated MPO solution is used. The elution buffer preferably contains one or more salts selected from KC1, NaCl, K₂HP0 , KH₂P0₄, Na₂HP0₄, NaH₂P0₄, NaHC0₃, NaB0₄, and (NH )₂C0₃. The conjugate-containing solution is then adsorbed onto the column with unreacted polymer and some high molecular weight species not being retained. At the completion of the loading, a gradient flow of an elution buffer with increasing salt concentrations is applied to the column to elute the desired fraction of polyalkylene oxide- conjugated MPO. The eluted pooled fractions are preferably limited to uniform polymer conjugates after the cation exchange separation step. Any unconjugated MPO species can then be back washed from the column by conventional techniques . If desired, mono and multiply pegylated MPO species can be further separated from each other via additional ion exchange chromatography or size exclusion chromatography.

Techniques utilizing multiple isocratic steps of increasing concentration can also be used. Multiple isocratic elution steps of increasing concentration will result in the sequential elution of di- and then mono-MPO-polymer conjugates.

The temperature range for elution is between about

4°C and about 25°C. Preferably, elution is carried out at a temperature of from about 6°C to about 22°C. For example, the elution of the PEG-MPO fraction is detected by UN absorbance at 280 nm. Fraction collection may be achieved through simple time elution profiles . A surfactant can be used in the processes of conjugating the poly (ethylene glycol) polymer with the MPO moiety. Suitable surfactants include ionic-type agents such as sodium dodecyl sulfate (SDS) . Other ionic surfactants such as lithium dodecyl sulfate, quaternary ammonium compounds, taurocholic acid, caprylic acid, decane sulfonic acid, etc. can also be used. Non-ionic surfactants can also be used. For example, materials such as poly (oxyethylene) sorbitans (Tweens) , poly (oxyethylene) ethers (Tritons) can be used. See also Neugebauer, A Guide to the Properties and Uses of Detergents in Biology and Biochemistry (1992) Calbiochem Corp. The only limitations on the surfactants used in the processes of the invention are that they are used under conditions and at concentrations that do not cause substantial irreversible denaturation of the MPO and do not completely inhibit polymer conjugation. The surfactants are present in the reaction mixtures in amounts from about 0.01-0.5%; preferably from 0.05-0.5%; and most preferably from about 0.075-0.25%. Mixtures of the surfactants are also contemplated.

It is thought that the surfactants provide a temporary, reversible protecting system during the polymer conjugation process. Surfactants have been shown to be effective in selectively discouraging polymer conjugation while allowing lysine-based or amino terminal-based conjugation to proceed.

The present poly (ethylene glycol) -modified MPO has a more enduring pharmacological effect, which may be possibly attributed to its prolonged half-life in vivo . Furthermore, it is observed that the present poly (ethylene glycol) -modified MPO may accelerate recovery from neutropenia . The present poly (ethylene glycol) -modified MPO may have essentially the same biological activity as an intact MPO and may accordingly be used in the same applications. The poly (ethylene glycol) -modified MPO has an activity for increasing the number of neutrophils, and it is useful therefore in the treatment of general hematopoietic disorders including those arising from chemotherapy or from radiation therapy. It may be also useful in the treatment of infection and in bone marrow transplantation. ^• The modified MPO of the present invention may be useful in the treatment of diseases characterized by decreased levels of either myeloid, erythroid, lymphoid, or megakaryocyte cells of the hematopoietic system or combinations thereof. In addition, they may be used to activate mature myeloid and/or lymphoid cells. Among conditions susceptible to treatment with the polypeptides of the present invention is leukopenia, a reduction in the number of circulating leukocytes (white cells) in the peripheral blood. Leukopenia may be induced by exposure to certain viruses or to radiation. It is often a side effect of various forms of cancer therapy, e.g., exposure to chemotherapeutic drugs, radiation and of infection or hemorrhage. Therapeutic treatment of leukopenia with these modified MPO of the present invention may avoid undesirable side effects caused by treatment with presently available drugs .

The modified MPO of the present invention may be useful in the treatment or prevention of neutropenia and, for example, in the treatment of such conditions as aplastic anemia, cyclic neutropenia, idiopathic neutropenia, Chediak-Higashi syndrome, systemic lupus erythematosus (SLE) , leukemia, myelodysplastic syndrome and myelofibrosis .

The modified MPO of the present invention may be useful in the treatment or prevention of thrombocytopenia. Currently the only therapies for thrombocytopenia are platelet transfusions, which are costly and carry the significant risks of infection (HIV, HBV) and alloimmunization, and IL-11 (Neumega™) , which is approved for certain thrombocytopenia. The modified MPO may alleviate or diminish the need for platelet transfusions. Severe thrombocytopenia may result from genetic defects such as Fanconi ' s Anemia, Wiscott-Aldrich, or May-Hegglin syndromes. Acquired thrombocytopenia may result from auto- or allo- antibodies as in Immune Thrombocytopenia Purpura, Systemic Lupus Erythematosis, hemolytic anemia, or fetal maternal incompatibility. In addition, splenomegaly, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, infection, or prosthetic heart valves may result in thrombocytopenia. Severe thrombocytopenia may also result from chemotherapy and/or radiation therapy or cancer. Thrombocytopenia may also result from marrow invasion by carcinoma, lymphoma, leukemia, or fibrosis.

The modified MPO- of the present invention may be useful in the mobilization of hematopoietic progenitors and stem cells into peripheral blood. Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation. Hematopoietic growth factors including G-CSF and GM-CSF have been shown to enhance the number of circulating progenitors and stem cells in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of phereses required. The modified MPO may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation .

Another projected clinical use of growth factors has been in the in vi tro activation of hematopoietic progenitors and stem cells for gene therapy. In order to have the gene of interest incorporated into the genome of the hematopoietic progenitor or stem cell one needs to stimulate cell division and DNA replication. Hematopoietic stem cells cycle at a very low frequency, which means that growth factors may be useful to promote gene transduction and thereby enhance the clinical prospects for gene therapy. Many drugs may cause bone marrow suppression or hematopoietic deficiencies. Examples of such drugs are AZT, DDI, alkylating agents and anti-metabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin, gancyclovir, daunomycin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, analgesics such as aminopyrine and dipyrone, anti- convulsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics. The modified MPO of the present invention may be useful in preventing or treating the bone marrow suppression or hematopoietic deficiencies, which often occur in patients treated with these drugs. Hematopoietic deficiencies may also occur because of viral, microbial, or parasitic infections and as a result of treatment for renal disease or renal failure, e . g. , dialysis. The modified MPO of the present invention may be useful in treating such hematopoietic deficiency.

The treatment of hematopoietic deficiency may include administration of a pharmaceutical composition containing the modified MPO to a patient. The modified MPO of the present invention may also be useful for the activation and amplification of hematopoietic precursor cells by treating these cells in vi tro with the modified MPO of the present invention prior to injecting the cells into a patient.

Various immunodeficiencies e. g. , in T and/or B lymphocytes, or immune disorders, e . g. , rheumatoid arthritis, may also be beneficially affected by treatment with the modified MPO of the present invention. Immunodeficiencies may be the result of viral infections e . g. HTLV-I, HTLV-II, HTLV-III, severe exposure to radiation, cancer therapy or the result of other medical treatment. The modified MPO of the present invention may also be employed, alone or in combination with other hematopoietins, in the treatment of other blood cell deficiencies, including thrombocytopenia (platelet deficiency), or anemia. Other uses for these novel polypeptides are in the treatment of patients recovering from bone marrow transplants in vivo and ex vivo, and in the development of monoclonal and polyclonal antibodies generated by standard methods for diagnostic or therapeutic use. The present poly (ethylene glycol) -modified MPO may be formulated into pharmaceuticals containing also a pharmaceutically acceptable diluent, an agent for preparing an isotonic solution, a pH-conditioner and the like in order to administer them into a patient. The above pharmaceuticals may be administered subcutaneously, intramuscularly, intravenously, or orally, depending on a purpose of treatment. A dose may be also based on the kind and condition of the disorder of a patient to be treated, being normally between 0.1 mg and 50 mg by injection and between 0.1 mg and 5 g in an oral administration for an adult

The polymeric substances included are also preferably water-soluble at room temperature. A non- limiting list of such polymers include poly (alkylene oxide) homopolymers such as poly (ethylene glycol) or poly (propylene glycols), poly (oxyethylenated polyols), copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.

As an alternative to PEG-based polymers, effectively non-antigenic materials such as dextran, poly(vinyl pyrrolidones) , poly (acrylamides) , poly(vinyl alcohols) , carbohydrate-based polymers, and the like can be used. Indeed, the activation of α- and co- terminal groups of these polymeric substances can be effected in fashions similar to that used to convert poly (alkylene oxides) and thus will be apparent to those of ordinary skill. Those of ordinary skill in the art will realize that the foregoing list is merely illustrative and that all polymer materials having the qualities described herein are contemplated. For purposes of the present invention, "effectively non- antigenic" means all materials understood in the art as being nontoxic and not eliciting an appreciable immunogenic response in mammals .

Definitions

The following is a list of abbreviations and the corresponding meanings as used interchangeably herein: g gram (s) mg milligram(s) ml or mL milliliter (s) RT room temperature

PEG poly (ethylene glycol)

The complete content of all publications, patents, and patent applications cited in this disclosure are herein incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference . Although the foregoing invention has been described in some detail by way of illustration and example for the purposes of clarity of understanding, it will be readily apparent to one skilled in the art in light of the teachings of this invention that changes and modifications can be made without departing from the spirit and scope of the present invention. The following examples are provided for exemplification purposes only and are not intended to limit the scope of the invention, which has been described in broad terms above .

In the following examples, the MPO polypeptide is that of residues 2-322 of SEQ ID NO: 224. It is understood that other members of the MPO family of polypeptides could also be pegylated in a similar manner as exemplified in the subsequent examples .

EXAMPLE 1 Straight Chain 30,000 MW PEG-ALD MPO

M-PEG-Aldehyde 5,000, 20,000 and 30,000 MW

This example demonstrates a method for generation of substantially homogeneous preparations of N-terminally monopegylated MPO by reductive alkylation. Methoxy- linear PEG-propionaldehyde reagent of approximately 30,000 MW (Shearwater Polymers Inc.) was selectively coupled via reductive amination to the N-terminus of MPO by taking advantage of the difference in the relative pK_a value of the primary amine at the N- terminus versus pK_a values of primary amines at the ε- amino position of lysine residues .

MPO protein dissolved at 4.5 mg/mL in 10-20 mM sodium acetate, pH 4.5, was reacted with Methoxy-PEG- propionaldehyde (M-PEG-ALD) by addition of solid M-PEG- ALD to yield a relative PEG:Myelopoietin molar ratio of 6.5:1. Reactions were catalyzed by addition of stock 1M NaCNBH₄ dissolved in H₂0 to a final concentration of

20 mM. Reactions were carried out at 4°C for 18-96 hours . Reactions were stopped by lowering the pH to 4.0 with 0.1 N acetic acid or by adding a 5X molar excess of Tris HCl.

EXAMPLE 2

Straight Chain 20,000 MW PEG-ALD MPO

Methoxy-linear 20,000 MW PEG-propionaldehyde reagent (Shearwater Polymers Inc.) was coupled to the N- terminus of MPO using the procedure described for Example 1.

EXAMPLE 3

Straight chain 5,000 MW PEG-ALD MPO

Methoxy-linear 5,000 MW PEG-propionaldehyde reagent

(Fluka) was coupled to the N-terminus of MPO using the procedure described for Example 1.

EXAMPLE 4 Branched chain 40,000 MW PEG2-ALD MPO

Methoxy-branched 40,000 MW PEG-propionaldehyde (PEG2- ALD) reagent (Shearwater Polymers Inc.) was coupled to the N-terminus of MPO using the procedure described for Example 1.

EXAMPLE 5

Straight chain 20,000 MW PEG-SPA MPO

PEG-SPA 5,000 and 20,000 MW

This example demonstrates a method for generation of substantially homogeneous preparations of monopegylated Myelopoietin (MPO) using N-hydroxysuccinimidyl (NHS) active esters. MPO protein stock solution dissolved at 4.5 mg/mL in 10-20 mM sodium acetate, pH 4.5 was titrated to pH 7.2 by addition of 0.25 M HEPES buffer. The solution was then reacted with Methoxy-PEG- succinimidyl propionate (SPA-PEG) by addition of solid SPA-PEG to yield a relative PEG:Myelopoietin molar ratio of 6.5:1. Reactions were carried out at 4°C for 1 hour. Reactions were stopped by lowering the pH to 4.0 with 0.1 N acetic acid or by adding a 5X molar excess of Tris HCl.

EXAMPLE 6

Straight chain 3,400 MW Biotin-PEG-NHS MPO

Biotin-PEG-NHS 3,400 MW

3,400 MW Biotin-PEG-C0₂-NHS reagent (Shearwater Polymers Inc.) was coupled to MPO using the procedure described for Example 5.

EXAMPLE 7

Branched 10 , 000 MW PEG2-NHS MPO

PEG2-NHS 10,000, 20,000 and 40,000 MW

10 , 000 MW branched PEG2-NHS ( Shearwater Polymers Inc . ) was coupled to MPO using the procedure described for Example 5 .

EXAMPLE 8

Branched 20,000 MW PEG2-NHS MPO

20,000 MW branched PEG2-SPA (Shearwater Polymers Inc.) was coupled to MPO using the procedure described for Example 5.

EXAMPLE 9

Branched 40,000 MW PEG2-NHS MPO

40,000 MW branched PEG2-NHS (Shearwater Polymers Inc.) was coupled to MPO using the procedure described for Example 5.

EXAMPLE 10

Straight chain 20 , 000 MW PEG-BTC MPO

20,000 MW PEG-BTC (Shearwater Polymers Inc.) was coupled to MPO using the procedure described for Example 4. This example demonstrates a method for generation of substantially homogeneous preparations of pegylated Myelopoietin (MPO) using benzotriazole carbonate derivatives of PEG.

EXAMPLE 11

Straight chain 5,000 MW PEG-SS MPO

PEG-SS 5,000MW

5,000 MW succinimidyl succinate-PEG (PEG-SS) (Shearwater Polymers Inc . ) was coupled to MPO using the procedure described for Example 5. This example demonstrates a method for generation of substantially homogeneous preparations of monopegylated Myelopoietin (MPO) using a hydrolyzable linkage.

EXAMPLE 12

Straight chain 20 , 000 MW PEG-HZ MPO

OCH₂CONHNH₂

PEG-Hydrazide 20,000 MW

This example demonstrates a method for generation of substantially homogeneous preparations of pegylated Myelopoietin (MPO) using 20,000 MW methoxy-PEG- hydrazide, PEG-HZ (Shearwater Polymers Inc.). MPO protein stock solution dissolved at 4-8 mg/mL in 250 mM MES, pH 4 - 5. The solution was then reacted with PEG- HZ by addition of solid to yield a relative PEG:Myelopoietin molar ratio of 6.5 - 26:1 reactions were catalyzed with carbodiimide (EDC, EOAC) at a final concentration of 2mM. Reactions were carried out at 4°C for 2 hours. Reactions were stopped by lowering the pH to 4 with 0.1 N acetic acid.

EXAMPLES 13

Multi-pegylated species

Modified MPOs having two or more PEGs (multi-pegylated) attached were also obtained from Examples 1-12 and were separated from the mono-pegylated using either anion or cation exchange chromatography. EXAMPLE 14

Purification of Pegylated MPO

Pegylated MPO species were purified from the reaction mixture to >95% (SEC analysis) using either a single anion or cation exchange chromatography step (FIG 1) . While the present example shows the purification of 2OK PEG-ALD MPO or 3OK PEG-ALD MPO it is understood that similar purification methods could be used for other MPO molecules exemplified or disclosed herein.

Anion exchange chromatography

Anion exchange chromatography was carried out on a 5 mL Hitrap Q column (Pharmacia Biotech) equilibrated in 50 mM Tris pH 9.0 (Buffer A) . The reaction mixture was diluted 5 fold with buffer A and loaded onto the column at a flow rate of 5 mL/min. Next, the column was washed with 5 column volumes of buffer A, followed by elution of the pegylated-MPO with a linear gradient of 0 to 20% buffer B (50 mM Tris pH 9.0, 1 M NaCl) in 20 column volumes. The eluant was monitored at 280 nm and 2 mL fractions were collected. Fractions containing monopegylated-MPO were pooled.

Cation Exchange Chromatography

Cation exchange chromatography was carried out on an SP Sepharose high performance column (Pharmacia XK 26/20, 70 ml bed volume) equilibrated in 10 mM sodium acetate pH 4.5 (Buffer C) . The reaction mixture was diluted 10X with buffer C and loaded onto the column at a flow rate of 5 mL/min. Next the column was washed with 5 column volumes of buffer C, followed by 5 column volumes of 12% buffer D (10 mM acetate pH 4.5, 1 M NaCl) . Subsequently, the PEG-MPO species were eluted from the column with a linear gradient of 12 to 27% buffer D in 20 column volumes. The eluant was monitored at 280 nm and 10 mL fractions were collected. Fractions were pooled according to extent of pegylation (mono, di, tri etc.), exchanged into 10 mM acetate pH 4.5 buffer and concentrated to 1-5 mg/mL in a stirred cell fitted with an Amicon YM10 membrane. Protein concentration of pool was determined by A280 nm using an extinction coefficient of 0.71. Total yield of monopegylated MPO from this process was 10 to 50%.

EXAMPLE 15

Biochemical Characterization

The purified pegylated MPO pools were characterized by SDS-PAGE (FIG 2), Size Exclusion Chromatography (FIG 3a & 3b), RP HPLC (FIG 4), Tryptic mapping (FIG 5), and Sedimentation Analysis (Table 1) .

SDS PAGE

SDS PAGE was carried out on 1 mm thick 12% reducing Novex Tris glycine gels .and stained using a Novex Colloidal Coomassie ™ G-250 staining kit (FIG 2).

Size Exclusion High Performance Liquid Chromatography (SEC-HPLC)

Analytical SEC-HPLC was carried out using a Pharmacia Superdex 200 HR 10/30 column in 50 mM Tris pH 7.5, 150 mM NaCl at a flow rate of 0.4 mL/minute. PEG/protein elution was followed using a triple detector system including UV monitor (220nm) , differential refractometer (RI) , and light scattering (LS) detector. FIG 3a shows a single peak corresponding to N- terminally monopegylated MPO and a single peak corresponding to MPO.

N-terminal Sequence and Peptide Mapping

Following purification, Pegylated MPO(ie. 2OK PEG-ALD MPO or 3OK PEG-ALD MPO) was buffer-exchanged into a dilution buffer comprised of lOmM Tris, pH 7.5/1X Modified Dulbecco's phosphate-buffered saline (MD-PBS) to a concentration of >2mg/ml using centrifugal concentration (Microsep™ Filtron, 10K Fast-Flux) . N- terminal sequence analysis was determined (PE-

Biosystems Model 494 Procise) and sample aliquots were digested overnight at 37°C using trypsin (Promega, V511C, from porcine) at 1:50 E:S. Digestions were quenched with 1M HCl and tryptic maps were then effected using a Vydac C-18 column eluted at l.Oml/min flow-rate using an acetonitrile gradient of ~0.33%/minute in 0.1% TFA. N-terminal sequence analysis and MALDI-TOF MS (PerSeptive Biosystems Voyager-DE™RP BioSpectrometry Workstation) were used to identify the disulfide-linked N-terminal fragment (s) in the tryptic profile of control MPO.

N-terminal sequence analysis for both 2OK PEG-ALD MPO and 3 OK PEG-ALD MPO revealed N-terminal heterogeneity, suggesting the presence of a combination of free N- terminal alanine as well as preview sequence coming from the N-alkylated (PEGylated) sample component. Differences in relative Edman degradation efficiency for free protein versus alkylated proteins, however, preclude absolute quantitation. As such, tryptic map profiling (FIG 5) was utilized to indicate that -90% of the pegylated MPO (both 20,000 and 30,000 PEG-ALD MPO species) was pegylated on the -NH₂ group of the N- terminal alanine residue when judged by the relative decrease in intensity of the N-terminal disulfide- linked peptide (s) (peak between 105 and 120) compared to a tryptic map of control MPO. Since HPLC-SEC data had previously indicated that both pegylated-MPO species were quantitatively pegylated, it is likely that the other 10% of the pegylated MPO samples are pegylated elsewhere in the molecule, i.e. most likely on one or more of the ε-NH₂ groups of internal lysine residues, noting that there were only subtle differences observed for the other, non-N-terminal peaks in the fingerprint (s) as developed. MALDI-MS and N-terminal sequence analysis were used to positively identify the N-terminal fragment (s) in the map of control MPO, but were somewhat less definitive as tools for the identification of the more hydrophobic difference peaks eluting from the maps of the PEGylated MPO samples presumably due to the complex nature of these fragments .

-Reversed Phase HPLC (RP HPLC)

RP HPLC was carried out on a Phenomenex Jupiter Cis column (4.6 X 250 mm, 5μm particle size) at a temperature of 50°C. Samples were loaded onto the column equilibrated in 40% acetonitrile, 0.1% TFA at 1 mL/min. The column was washed with 3 mL 58% acetonitrile. Subsequently, the protein was eluted with a gradient from 58 to 63% acetonitrile over 27 minutes. The monopegylated MPO species eluted as a single peak (FIG 4) .

Sedimentation analysis

The hydrodynamic radii of the two PEGylated MPO molecules (2OK PEG-ALD MPO and the 3OK PEG-ALD MPO) were determined using analytical ultracentrifugation technology. In sedimentation velocity experiments, one can measure the sedimentation coefficient ^,λs" and the diffusional coefficient "D" . From D, one can calculate the hydrodynamic radius (Rh) using the Stokes-Einstein Equation. Using the s/D ratio, one can calculate the molecular weight. TABLE 1 shows data from sedimentation velocity experiments carried out on MPO, N-terminally mono-PEGylated 20,000 MW PEG MPO, and N-terminally mono-PEGylated 30,000 MW PEG MPO.

TABLE 1

Sedimentation Velocity Results Hydrodynamic Radii and Molecular Weights

EXAMPLE 16

BAF 3fG-CSFR Cell proliferation assay

Mouse BaF 3 cell line transfected with genes encoding the human G-CSF (mBaF 3/hG-CSFR) receptor were used to examine hG-CSF agonist activity. mBaF3 /hG-CSFR cells were seeded at 2.5 X IO⁴ cells/well in 96 well microtiter plates containing serial dilution of cytokines. Cells were pulsed at T₅₆ hours with [methyl- ³H] -thymidine at 0.5 mCi per well for 18 hours. Plates were harvested onto glass fiber filter mats, and the incorporated radioactivity was measured by scintillation spectroscopy. The assay medium for the cell lines consisted of IMDM supplemented with bovine serum albumin (BSA, 500 μg/ml, Boehringer Mannheim), human transferrin (100 μg/ml, Sigma), a lipid substitute consisting of 2.5 mg of phosphatidyl choline/ml of BSA and 50 mM 2-mercaptoethanol. N- terminally pegylated MPO was active in this assay.

TABLE 2 compares the in vitro IL-3 receptor and G-CSF receptor agonist bioactivities of mono and di- PEG-MPO species with un-PEGylated MPO.

TABLE 2

Relative Potency MPO species AML TF-1 BAF3/G-CSFR

MPO 1.0 1.0 1.0

PEG-ALD 5K PEG MPO 0.124 0.144 0.775 20KPEGMPO 0.088 0.113 0.955 30KPEGMPO 0.133 0.149 0.870 di5KPEGMPO 0.093 0.105 1.509 di30K PEG MPO 0.051 0.092 1.502

PEG-NHS

20KPEGMPO 0.268 0.600 1.696 di20KPEGMPO 0.123 0.278 3.192 tetra5KPEGMPO 0.086 0.090 4.216 tetra5K NHS-SS PEG MPO 0.179 0.226 3.962 lOK-Branch PEG MPO 0.297 0.632 0.980

20K-BranchPEGMPO 0.281 0.550 1.505

40K-BranchPEGMPO 0.337 0.613 1.654 dilOK-Branch PEG MPO 0.097 0.147 1.105

PEG-BTC

20KPEGMPO 0.321 0.925 4.686

PEG-Hvdrazide 20KPEGMPO 0.149 0.250 1.717 di20KPEGMPO 0.129 0.165 1.757 EXAMPLE 17

TF-1 proliferation assay

Human TF-1 cells, which express the hIL3 receptor were used to identify hIL-3 receptor agonist activity. Human TF-1 cells were seeded at 1.25 X IO⁴ cells/well in 96 well microtiter plates containing serial dilutions of cytokines. Cells were pulsed at T₇₂ hours with [methyl- ³H] -thy idine at 0.5 mCi per well for 6 hours. Plates were harvested onto glass fiber filter mats, and the incorporated radioactivity was measured by scintillation spectroscopy. The assay medium for the cell lines consisted of IMDM supplemented with bovine serum albumin (BSA, 500 μg/ml, Boehringer Mannheim) , human transferrin (100 μg/ml, Sigma) , a lipid substitute consisting of 2.5 μg of phosphatidyl choline/ml of BSA and 50 mM 2-mercaptoethanol. N- terminally pegylated MPO was active in this assay. TABLE 2 compares the in vi tro IL-3 receptor and G-CSF receptor agonist bioactivities of assorted PEG-MPO species with un-PEGylated MPO.

EXAMPLE 18

AML proliferation assay

Human AML cells, which express the hIL3 and hG-CSF receptors were also used to measure MPO in vi tro receptor agonist activity. Human AML cells were seeded at 2.5 X IO⁴ cells/well in 96 well microtiter plates containing a serial dilution of cytokines. Cells were pulsed at T₇₂ hours with [methyl-³H] -thymidine at 0.5 mCi per well for 24 hours. Plates were harvested onto glass fiber filter mats, and the incorporated radioactivity was measured by scintillation spectroscopy. The assay medium for the cell lines consisted of IMDM supplemented with bovine serum albumin (BSA, 500 μg/ml, Boehringer Mannheim) , human transferrin (100 μg/ml, Sigma) , a lipid substitute consisting of 2.5 μg of phosphatidyl choline/ml of BSA and 50 mM 2-mercaptoethanol. N-terminally pegylated MPO was active in this assay. TABLE 2 compares the in vitro IL-3 receptor and G-CSF receptor agonist bioactivities of assorted PEG-MPO species with un- PEGylated MPO.

EXAMPLE 19

CD34⁺ cell proliferation assays

Further assessment of the in vi tro biological activity of N-terminally pegylated MPO (PEG-ALD MPO) was carried out in bone marrow CD34⁺ cell proliferation bioassays. Fresh bone marrow aspirates were obtained through a collaboration with the St. Louis University Medical School . Mononuclear cell fractions were recovered following density gradient centrifugation with Ficoll- Hypaque. CD34⁺ (stem and progenitor) cells were subsequently isolated by positive selection using the Isolex 50 stem cell reagent kit (Baxter Healthcare Corporation, Deerfield, IL) . This procedure yields an enriched cellular product where >90% of the cells express the CD34+ cell surface antigen. For proliferation assays, the CD34⁺ cells were incubated overnight at 4°C in X-VIVO 10 media supplemented with 1% Human Serum Albumin. Following this incubation, the cells were washed, resuspended in X-VIVO 10 with 1% HSA, counted for viability and plated at 1 X IO³ cells/well in 96 well microtiter plates containing a serial dilution of cytokines. Concentrations of the respective receptor agonists in co-addition experiments were equimolar at the indicated concentration value. After 6 days, cells were pulsed with [methyl-³H] - thymidine at 0.5 mCi per well for 7 hr, harvested onto glass fiber filter mats, and the incorporated radioactivity was measured by scintillation spectroscopy. The N-terminally pegylated MPO yielded proliferative responses slightly less than unmodified MPO yet greater or equal to the responses from co- administration of IL-3 and G-CSF.

EXAMPLE 20

CFU-GM clonogenic assays

Expansion of hematopoietic progenitors was demonstrated using human bone marrow-derived CD34+ cells in a colony forming unit granulocyte/macrophage (CFU-GM) assay, where clonogenic progenitors divide and differentiate in a semi-solid media in response to growth factors. CD34⁺ cells (isolated as described in example 19) were seeded in 35 mm tissue culture plates (10,000 cells/dish) in MethoCult H4230 (StemCell Technologies, Vancouver, BC) containing 0.9% Methylcellulose in IMDM, 30% FBS, 1% BSA, lxlO^~M 2-mercaptoethanol and 2mM L- glutamine . Cultures were incubated with growth factors for 10-12 days at 37°C in humidified air containing 5% C0₂. The concentrations of the respective receptor agonists in co-addition experiments are equimolar each at the indicated concentration value. Hematopoietic colonies (>50 cells) were counted using an inverted microscope. FIG 6 shows that the unmodified MPO molecule induces differentiation and expansion of hematopoietic progenitor cells into colony forming unit granulocyte/macrophage cells (CFU-GM) greater or equal to the responses from co-administration of hIL-3 and G- CSF.

Example 21

Normal Rhesus Monkey studies

The pharmacology of N-terminally pegylated MPO was assessed in normal Rhesus monkey (Macaca mulatta) studies. Rhesus monkeys (~5 Kg) were acclimated for 2 weeks during which the baseline blood data were collected. Monkeys were given a single dose of PEG-MPO or control MPO by subcutaneous (SC) , or intravenous (IV) injection. Monkeys were observed for clinical pharmacokinetics (PK) and pharmacodynamics (PD) parameters up to 50 days post dosing. Blood samples were taken at regular intervals and hematological analyses were performed. Samples were dosed SC at 20 and 200 mg/kg. IV dosing studies were carried out at 10 mg/kg. Blood samples were taken at regular intervals and hematological analyses were performed. Plasma samples were collected before dosing and at 0.5, 1, 2, 4, 6, 8, 14, 23.5, 47.5, 71.5, and 95.5 hr. after dosing.

Example 22

Normal Rhesus Pharmacokinetics MPO protein concentration levels in rhesus plasma for PK analysis were determined using a sandwich ELISA. 96-well microtiter plates were coated with 150-mL/well affinity-purified goat-anti-G-CSF polyclonal diluted to lmg/mL in 100 mM NaHC0₃, pH 8.2. Plates were incubated overnight at room temperature in a humidified chamber and blocked for one hour at 37°C with phosphate buffered saline, containing 3% bovine serum albumin (BSA) and 0.05% Poly (oxyethylene) -Sorbitan Monolaurate (Tween 20), pH 7.4. Plates were washed four times with 150 mM NaCl containing 0.05% Tween 20 (wash buffer) . Plasma PK samples were initially diluted in assay buffer (PBS, 0.1% BSA, 0.01% Tween 20 pH 7.4), added to the plate and serially dlited 1:2 in an assay matrix of untreated Rhesus pooled plasma. The plasma concentration of the matrix and the samples were matched by percentage . Plates were incubated for 2.5 hours at 37°C in a humidified chamber, then washed 4 times. Wells were washed four times. Affinity purified goat~anti-hIL3 receptor agonist polyclonal antibody was diluted 1:5000 in assay buffer and 150 mg/mL well were added to each plate. Plates were incubated for 1.5 hours at 37°C in a humidified chamber. Wells were emptied and each well was again washed four times with wash buffer. Each well then received 150 mL of TMB peroxidase substrate solution. Plates were incubated at room temperature for 10 minutes and read at a test wavelength of 650 nm on a microtiter plate reader (Molecular Devices Corporation) . Concentrations of immuno-reactive MPO in unknown PK samples were calculated from a standard curve using a four-parameter curve-fitting program supplied by Molecular Devices. Plasma concentration data and pharmacokinetic parameters, which were derived from non-compartmental analysis, are shown in TABLE 3. The results indicate that the plasma residence time of the N-terminally pegylated MPO is greatly protracted when compared to the unmodified MPO. Pegylation dramatically increases the drug exposure as AUC increases -7-9 fold and -10-20 fold for 20K and 30K PEG-MPO, respectively, compared to the unmodified MPO; and clearance rates decreased by up to 10 and 20 fold for 2OK and 3OK MPO, respectively, at the higher dose tested. MPO exposure increases in greater-than- proportional manner with dose, which indicates a nonlinear PK saturation of some "early" clearance mechanism. The IV profile suggests multi-phasic elimination distribution into a "deeper" compartment. Absolute bioavailability of PEG-MPO appears to be approximately 50% (data not shown) .

TABLE 3 compares the in vivo pharmacokinetics of un- PEGylated and N-terminally mono-PEGylated 20,000 MW and 30,000 MW PEG-ALD MPO after a single subcutaneous dose in normal Rhesus monkeys .

TABLE 3

Plasma Concentration Data and Pharmacokinetic Parameters following a single subcutaneous dose of MPO, 20,000 MW OR 30,000 PEG-ALD MPO

^a ng protein/mL plasma (n=2) ^b Pharmacokinetic parameters derived from one compartmental analysis

NS - no sample

C_nax - maximum concentration in plasma

T_{m x} - time at which C_max is achieved AUC (0-24 hr) - Area under the curve from 0 to 24 hours

AUC all - total area under the curve

AUC (I) - AUC infinity

Tι_ast - final time point at which sample is detected

Tj ₂ - terminal half life CL - clearance rate

F - Bioavailability - fraction of dose reaching systemic circulation V_d - volume distribution EXAMPLE 23

Normal Rhesus Pharmacodynamics

Normal rhesus plasma samples were also analyzed for MPO pharmacological responses . Leukocytes were counted in a Technicon HIE Hematology Analyzer (Technicon Instruments Corp.). FIG 7 shows profiles for absolute neutrophil count (ANC) following SC dosing at 200 μg/kg as described above. A dramatic protraction in the WBC and ANC response is observed with pegylated MPO. Also observed was a nearly identical 5-fold increase in WBC counts (C_maχ) after a single SC administration of either MPO or pegylated MPO (PEG-ALD MPO) at 200 μg/kg to normal rhesus monkeys was driven by a 8-fold increase in neutrophil numbers within 8 hrs. Elevated total WBC and ANC levels returned to pretreatment levels within 3 or 4 days after dosing with MPO at 200 μg/kg. Both the 20,000 PEG MPO and 30,000 MW pegylated MPO maintained elevated levels for over 160 hours with the latter yielding a remarkable increase (-6 fold) in total ANC at 80-100 hours compared to unmodified MPO. These data show good correlation with increases in the pharmacokinetic profile described above.

EXAMPLE 27

Myelosuppressed rhesus (efficacy) studies

Male rhesus monkeys were housed in individual stainless steel cages in conventional holding rooms in animal facilities accredited by the American Association for Accreditation of Laboratory Care. Following a prehabituation period, monkeys were unilaterally irradiated in Lucite restraining chairs with 250 kVp x- radiation at 13 cGy/min in the post-anterior position, rotated 180° at the mid-dose (300 cGy) to the anterior- posterior position for completion of the total 600 cGy midline tissue exposure and randomly assigned to a treatment protocol utilizing pegylated MPO or control autologous serum (AS) . Pegylated MPO (3 OK PEG-ALD MPO) was subcutaneously administered according to the following two protocols: a) as two doses [200 μg/kg, n=4] given one day and seven days following TBI, b) as two doses [200 μg/kg, n=4] given one day and four days following TBI, or c) as a single dose (600 μg/kg, n=5) given one day following TBI. The irradiation controls (n=7) received 0.1% autologous serum (AS), daily for 18 days. Complete blood counts were monitored for up to 70 days following irradiation and the durations of neutropenia [absolute neutrophil count (ANC) < 500/μL] and thrombocytopenia (platelets (PLT) < 20,000/μL) were assessed. All animals received clinical support, which consisted of antibiotics, fresh irradiated whole blood, and fluids as needed. An antibiotic regimen was initiated prophylactically when the white blood cell count (WBC) was <1000/μL and continued daily until the WBC was >1000/μL for three consecutive days. Fresh, irradiated (1500 cGy Co-60) whole blood (approximately 30 mLs/transfusion) from a random donor pool was administered when the PLT count was < 20,000/μL and the hematocrit was <18%. Hematologic evaluations were determined using peripheral blood drawn from the saphenous vein in order to assay complete blood (Sysmex K-4500) and differential counts (Wright-Giemsa Stain) . TABLE 4 shows the efficacy of N-terminally mono- PEGylated 30,000 MW PEG MPO in a Rhesus monkey model of myelosuppression. Clinically relevant indicators such as days where ANC's fell below 500/ml , ANC nadir, days to recovery, and days on antibiotics were measured for N-terminally mono-pegylated MPO using three dosing schedules .

One or more Key indicators in this model for clinical efficacy improve at all dosing schedules for pegylated MPO when compared to daily administration of un-Pegylated-MPO as a comparator.

TABLE4

Example 28

Normal Rat Pharmacokinetics and Pharmacodynamics (PK/PD)

The pharmacokinetic and pharmacodynamic properties of PEGylated MPO molecules were assessed in normal rats. Male Sprague Dawley rats in groups of 5 were given a single subcutaneous injection of PEGylated MPO or control MPO at 500ug/kg. Blood samples were taken at regular intervals and analyzed for plasma protein concentrations and pharmacological responses as described in Examples 22 and 23.

TABLE 5

PK PD

MPO cone, AUC (ng /mL plasma) (Neutrophils hours

/nL plasma)

24hr 48hr 72hr

30K PEG ALD 634 326 13 2026 MPO

20K PEG ALD 468 242 1 2299 MPO

5K PEG ALD 503 2 0 1866 MPO di5KPEG ALD 970 158 7 2156 MPO

40K Branch 1539 518 19 2268

PEG2-NHS

MPO

20K Branch 301 72 7 2093

PEG2-NHS

MPO

20K PEG-SPA 711 139 2 1761 MPO di20K PEG - 735 360 33 2329 SPA MPO

10K Branch 982 157 0 1384 PEG NHS MPO dilOK Branch 945 247 10 1490

PEG2-NHS

MPO tetra 20K PEG- 572 192 10 2316 BTC MPO

20K PEG-HZ 2155 709 91 2569 MPO di20K PEG-HZ 499 289 47 3010 MPO tetra5K PEG-SS 881 40 0 1838 MPO tetra5K PEG- 710 137 11 2272 SPA MPO di30K PEG- 504 193 28 2275 ALD MPO

MPO 14 1 0 693

Claims

WHAT IS CLAIMED IS:

1. A Myelopoietin conjugate having at least one water-soluble polymer molecule covalently attached to at least one amino acid residue of a biologically active Myelopoietin polypeptide.

2. The Myelopoietin conjugate of claim 1 wherein said Myelopoietin polypeptide comprises a sequence of the formula selected from the group consisting of;

R1-L-R2, R2-L-R1, R3.-R2, R2-L-R1, Met-Ala-Rι-L-R₂ , Met~Ala-R2-L-Rι_/ Met-Ala-Rι-R2, Met-Ala-R2~Ri, Met- R1-L-R2, Met-R2-L-Rι, Met-Rχ-R2, Met-R2~Rl, Ala-Rη.- L-R2, Ala-R2-L-Rl, Ala-Rχ-R2 and Ala-R2~Rl;

wherein R± is a modified human interleukin-3 (hlL- 3) amino acid sequence of SEQ ID NO:l;

wherein

Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gin, or Arg;

Xaa at position 18 is Asn, His, Leu, lie, Phe, Arg, or Gin;

Xaa at position 19 is Met, Phe, lie, Arg, Gly, Ala, or Cys;

Xaa at position 20 is lie, Cys, Gin, Glu, Arg, Pro, or Ala; Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gin, Asn, Thr, Ser or Val;

Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gin, Leu, Val or Gly;

Xaa at position 23 is lie, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;

Xaa at position 24 is lie, Gly, Val, Arg, Ser, Phe, or Leu;

Xaa at position 25 is Thr, His, Gly, Gin, Arg, Pro, or Ala;

Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;

Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 28 is Lys, Arg, Leu, Gin, Gly, Pro, Val or Trp;

Xaa at position 29 is Gin, Asn, Leu, Pro, Arg, or Val;

Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gin, Ser, Leu, or Lys; Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gin; Xaa at position 32 is Leu, Val, Arg, Gin, Asn, Gly, Ala, or Glu; Xaa at position 33 is Pro, Leu, Gin, Ala, Thr, or Glu; Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gin,

Thr, Arg, Ala, Phe, lie or Met; Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gin, or Val; Xaa at position 36 is Asp, Leu, or Val; Xaa at position 37 is Phe, Ser, Pro, Trp, or lie; Xaa at position 38 is Asn, or Ala;

Xaa at position 40 is Leu, Trp, or Arg;

Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;

Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr,

Leu, Val, Glu, Phe, Tyr, lie, Met or Ala; Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala,

Cys, Gin, Arg, Thr, Gly or Ser; Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met,

Trp, Glu, Asn, Gin, Ala or Pro; Xaa at position 45 is Gin, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, lie, Glu or His; Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn,

Gin, Lys, His, Ala, Tyr, lie, Val or Gly; Xaa at position 47 is lie, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 48 is Leu, Ser, Cys, Arg, lie, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;

Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn,

Ser, Ala, lie, Val, His, Phe, Met or Gin; Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys,

Ser, or Met; Xaa at position 54 is Arg, Asp, lie, Ser, Val, Thr, Gin,

Asn, Lys, His, Ala or Leu; Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;

Xaa at position 56 is Pro, Gly, Cys, Ser, Gin, Glu, Arg,

His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 57 is Asn or Gly;

Xaa at position 58 is Leu, Ser, Asp Arg, Gin, Val, or Cys;

Xaa at position 59 is Glu, Tyr, His Leu, Pro, or Arg;

Xaa at position 60 is Ala, Ser, Pro Tyr, Asn, or Thr; Xaa at position 61 is Phe, Asn, Glu Pro, Lys, Arg, or Ser;

Xaa at position 62 is Asn, His, Val Arg, Pro, Thr, Asp, or lie;

Xaa at position 63 is Arg, Tyr, Trp Lys, Ser, His, Pro, or Val;

Xaa at position 64 is Ala, Asn, Pro Ser, or Lys;

Xaa at position 65 is Val, Thr, Pro His, Leu, Phe, or Ser; Xaa at position 66 is Lys, lie, Arg Val, Asn, Glu, or Ser;

Xaa at position 67 is Ser, Ala, Phe Val, Gly, Asn, lie,

Pro, or His;

Xaa at position 68 is Leu, Val, Trp Ser, lie, Phe, Thr, or His;

Xaa at position 69 is Gin, Ala, Pro Thr, Glu, Arg, Trp, Gly, or Leu;

Xaa at position 70 is Asn, Leu, Val Trp, Pro, or Ala;

Xaa at position 71 is Ala, Met, Leu Pro, Arg, Glu, Thr,

Gin, Trp, or Asn;

Xaa at position 72 is Ser, Glu, Met Ala, His, Asn, Arg, or Asp; Xaa at position 73 is Ala, Glu, Asp Leu, Ser, Gly, Thr, or Arg;

Xaa at position 74 is lie. Met, Thr Pro, Arg, Gly, Ala;

Xaa at position 75 is Glu, Lys, Gly Asp, Pro, Trp, Arg,

Ser, Gin, or Leu;

Xaa at position 76 is Ser, Val, Ala Asn, Trp, Glu, Pro, Gly, or Asp;

Xaa at position 77 is lie, Ser, Arg Thr, or Leu;

Xaa at position 78 is Leu, Ala, Ser Glu, Phe, Gly, or Arg;

Xaa at position 79 is Lys, Thr, Asn Met, Arg, lie, Gly, or Asp;

Xaa at position 80 is Asn, Trp, Val Gly, Thr, Leu, Glu, or Arg; Xaa at position 81 is Leu, Gin, Gly Ala, Trp, Arg, Val, or Lys;

Xaa at position 82 is Leu, Gin, Lys Trp, Arg, Asp, Glu,

Asn, His, Thr, Ser, Ala, Tyr, Phe, lie. Met or Val;

Xaa at position 83 is Pro, Ala, Thr Trp, Arg, or Met;

Xaa at position 84 is Cys, Glu, Gly Arg, Met, or Val; Xaa at position 85 is Leu, Asn, Val or Gin;

Xaa at position 86 is Pro, Cys, Arg Ala, or Lys;

Xaa at position 87 is Leu, Ser, Trp or Gly; Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;

Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His,

Asn, or Ser; Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, lie, or Met; Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala,

Gly, lie or Leu; Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 94 is Arg, lie, Ser, Glu, Leu, Val, Gin, Lys, His, Ala, or Pro;

Xaa at position 95 is His, Gin, Pro, Arg, Val, Leu, Gly,

Thr, Asn, Lys, Ser, Ala, Trp, Phe, lie, or Tyr; Xaa at position 96 is Pro, Lys, Tyr, Gly, lie, or Thr; Xaa at position 97 is lie, Val, Lys, Ala, or Asn; Xaa at position 98 is His, lie, Asn, Leu, Asp, Ala, Thr,

Glu, Gin, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 99 is lie, Leu, Arg, Asp, Val, Pro, Gin,

Gly, Ser, Phe, or His; Xaa at position 100 is Lys, Tyr, Leu, His, Arg, lie, Ser, Gin, or Pro;

Xaa at position 101 is Asp;

Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 103 is Asp, or Ser;

Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gin, Lys, Ala, Phe, or Gly;

Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gin,

Tyr, Leu, Lys, lie, Asp, or His; Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, lie, Gly, or Pro; Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, lie, Gin, His, Ser, Ala or Pro;

Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gin,

His, Glu, Ser, Ala, or Trp; Xaa at position 111 is Leu, lie, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gin, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr,

Asp, Lys, Leu, lie, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;

Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His,

Thr, Trp, or Met;

Xaa at position 116 is Lys; Xaa at position 117 is Thr, Ser, Asn, lie, Trp, Lys, or Pro;

Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;

Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;

Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gin;

Xaa at position 121 is Ala, Ser, lie, Asn, Pro, Lys, Asp, or Gly; Xaa at position 122 is Gin, Ser, Met, Trp, Arg, Phe, Pro,

His, lie, Tyr, or Cys;

Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu,-

wherein from 1 to about 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 ; and wherein from 1 to 14 amino acids are optionally deleted from the N- terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus of said sequence of SEQ ID NO : 1 ;

R2 is a factor selected from the group consisting of: a colony stimulating factor, a cytokine, a lymphokine, an interleukin, and a hematopoietic growth factor; and

L is a linker capable of linking Ri to 2.

3. The Myelopoietin conjugate of claim 2 wherein in said Myelopoietin polypeptide said factor is selected from the group consisting of;

GM-CSF, G-CSF, G-CSF Ser¹⁷, c-mpl ligand (TPO) ,

MGDF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL- 3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3/flk2 ligand, and stem cell factor (SCF) .

4. The Myelopoietin conjugate of claim 1 wherein in said biologically active Myelopoietin polypeptide ]_ is selected from the group consisting of:

SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, residues 2-113 of SEQ ID NO: 25, residues 3-113 of SEQ ID NO -.25, SEQ ID NO: 26, residues 2-113 of SEQ ID NO:26, residues 3-113 of SEQ ID NO.-26, SEQ ID NO: 27, residues 2-113 of SEQ ID NO: 27, residues 3-113 of SEQ ID NO: 27, SEQ ID NO: 28, residues 2-113 of SEQ ID NO: 28, residues 3-113 of SEQ ID NO: 28, SEQ ID NO: 29, residues 2-113 of SEQ ID NO: 29, residues 3-113 of SEQ ID NO: 29, SEQ ID NO: 30, residues 2-113 of SEQ ID NO:30, residues 3-113 of SEQ ID NO:30, SEQ ID NO:31, residues 2-113 of SEQ ID NO:31, residues 3-113 of SEQ ID NO: 31, SEQ ID NO: 32, residues 2-113 of SEQ ID NO: 32, residues 3-113 of SEQ ID NO: 32, SEQ ID NO: 33, residues 2-113 of SEQ ID NO: 33, residues 3-113 of SEQ ID NO: 33, SEQ ID NO: 34, residues 2-113 of SEQ ID NO: 34, residues 3-113 of SEQ ID NO:34, SEQ ID NO:35, residues 2-113 of SEQ ID NO: 35, residues 3-113 of SEQ ID NO: 35, SEQ ID NO: 36, residues 2-113 of SEQ ID NO: 36, residues 3-113 of SEQ ID NO: 36, SEQ ID NO: 37, residues 2-113 of SEQ ID NO: 37, residues 3-113 of SEQ ID NO: 37, SEQ ID NO: 38, residues 2-113 of SEQ ID NO: 38, residues 3-113 of SEQ ID NO: 38, SEQ ID NO: 39, residues 2-113 of SEQ ID NO: 39, residues 3-113 of SEQ ID NO: 39, SEQ ID NO: 40, residues 2-113 of SEQ ID NO: 40, residues 3-113 of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, residues 2-113 of SEQ ID NO: 43, residues 3-113 of SEQ ID NO: 43, SEQ ID NO: 44, residues 2-113 of SEQ ID NO: 45, residues 3-113 of SEQ ID NO: 45, SEQ ID NO: 48, residues 2-113 of SEQ ID NO: 48, residues 3-113 of SEQ ID NO: 48, SEQ ID NO: 49, residues 2-113 of SEQ ID NO: 49, residues 3-113 of SEQ ID NO: 49, SEQ ID NO: 53 through SEQ ID NO: 120, residues 2-113 of SEQ ID NO: 53 through SEQ ID NO: 120, residues 3-113 of SEQ ID NO: 53 through SEQ ID NO: 120.

5. The Myelopoietin conjugate of claim 1 where said biologically active Myelopoietin polypeptide is selected from the group consisting of:

SEQ ID NO:121-124, 134, 135, 146, 147, 152, 158, 159, residues 2-307 of SEQ ID NO:121-124, 134, 135, 146, 147, 152, 158, 159, residues 3-307 of SEQ ID NO:121-124, 134, 135, 146, 147, 152, 158, 159, SEQ ID NO:125-127, 136, 153, residues 2-244 of SEQ ID NO:125-127, 136, 153, residues 3-244 of SEQ ID NO: 125-127, 136, 153, SEQ ID NO.-128-130, 138, 149, 150, 154, 156, 157, residues 2-322 of SEQ ID NO: 128-130, 138, 149, 150, 154, 156, 157, residues 3-322 of SEQ ID NO: 128-130, 138, 149, 150, 154, 156, 157, SEQ ID NO:131-133, 137, 141, 155, residues 2-259 of SEQ ID NO:131-133, 137, 141, 155, residues 3-259 of SEQ ID NO: 131-133, 137, 141, 155, SEQ ID NO:139, 151, residues 2-349 of SEQ ID NO:139, 151, residues 3-349 of SEQ ID NO: 139, 151, SEQ ID NO: 141, residues 2-301 of SEQ ID NO: 141, residues 3-301 of SEQ ID NO: 141, SEQ ID NO: 143, residues 2-335 of SEQ ID NO: 143, residues 3-335 of SEQ ID NO: 143, SEQ ID NO: 144, residues 2-274 of SEQ ID NO: 144, residues 3-274 of SEQ ID NO: 144, SEQ ID NO: 145, residues 2-317 of SEQ ID NO: 145, residues 3-317 of SEQ ID NO: 145, SEQ ID NO: 148, residues 2-337 of SEQ ID NO: 148, residues 3-337 of SEQ ID NO: 148, SEQ ID NO -.165-167, residues 2-286 of SEQ ID NO:165-167, residues 3-286 of SEQ ID N0:165-167, SEQ ID NO: 168, residues 2-290 of SEQ ID NO: 168, residues 3-290 of SEQ ID NO: 168, SEQ ID NO: 178, SEQ ID NO: 181, SEQ ID NO:184, SEQ ID NO:187, SEQ ID NO:190, SEQ ID NO:193, SEQ ID NO: 196, SEQ ID NO: 199, SEQ ID NO: 202, SEQ ID NO: 205, SEQ ID NO:208, SEQ ID NO:211, SEQ ID NO:214, SEQ ID NO:217, SEQ ID NO:220, SEQ ID NO:223, SEQ ID NO:226, SEQ ID NO:229, SEQ ID NO:232, SEQ ID NO:235, SEQ ID NO:238, SEQ ID NO: 241, SEQ ID NO: 244, SEQ ID NO: 247, SEQ ID NO:250, SEQ ID NO:253, SEQ ID NO:256, SEQ ID NO:259, SEQ ID NO: 262, SEQ ID NO: 265, SEQ ID NO: 268, SEQ ID NO: 271, SEQ ID NO:274, SEQ ID NO:277, SEQ ID NO:280, SEQ ID NO:283, SEQ ID NO:286, SEQ ID NO-.289, SEQ ID NO:292, SEQ ID NO:295, SEQ ID NO:298, SEQ ID NO:301, SEQ ID NO:304, SEQ ID NO :307, SEQ ID NO: 310, SEQ ID NO: 313, SEQ ID

NO:316, SEQ ID NO:319, SEQ ID NO:322, SEQ ID NO:325, SEQ ID NO:328, SEQ ID NO:331, SEQ ID NO:334, SEQ ID NO:337, SEQ ID NO: 340, SEQ ID NO: 343, SEQ ID NO: 346, SEQ ID NO:349, SEQ ID NO:352, SEQ ID NO:355, SEQ ID NO:358, SEQ ID NO:361, SEQ ID NO:364, SEQ ID NO:367, SEQ ID NO:370, SEQ ID NO: 373, SEQ ID NO: 376, SEQ ID NO: 379, SEQ ID NO:382, residues 2-307 of SEQ ID NO:178, residues 2-307 of SEQ ID NO: 181, residues 2-307 of SEQ ID NO: 184, residues 2-307 of SEQ ID NO: 187, residues 2-307 of SEQ ID NO: 190, residues 2-307 of SEQ ID NO: 193, residues 2- 307 of SEQ ID NO: 196, residues 2-307 of SEQ ID NO: 199, residues 2-307 of SEQ ID NO:202, residues 2-307 of SEQ ID NO: 205, residues 2-307 of SEQ ID NO: 208, residues 2- 307 of SEQ ID NO:211, residues 2-307 of SEQ ID NO:214, residues 2-307 of SEQ ID NO: 217, residues 2-307 of SEQ ID NO:220, residues 2-307 of SEQ ID NO:223, residues 2- 307 of SEQ ID NO:226, residues 2-307 of SEQ ID NO:229, residues 2-307 of SEQ ID NO: 232, residues 2-307 of SEQ ID NO: 235, residues 2-307 of SEQ ID NO: 238, residues 2- 307 of SEQ ID NO:241, residues 2-307 of SEQ ID NO:244, residues 2-307 of SEQ ID NO: 247, residues 2-307 of SEQ ID NO: 250, residues 2-307 of SEQ ID NO: 253, residues 2- 307 of SEQ ID NO:256, residues 2-307 of SEQ ID NO.-259, residues 2-307 of SEQ ID NO: 262, residues 2-307 of SEQ ID NO:265, residues 2-307 of SEQ ID NO:268, residues 2- 307 of SEQ ID NO:271, residues 2-307 of SEQ ID NO:274, residues 2-307 of SEQ ID NO:277, residues 2-307 of SEQ ID NO: 280, residues 2-307 of SEQ ID NO: 283, residues 2- 307 of SEQ ID NO:286, residues 2-307 of SEQ ID NO:289, residues 2-307 of SEQ ID NO: 292, residues 2-307 of SEQ ID NO: 295, residues 2-307 of SEQ ID NO: 298, residues 2- 307 of SEQ ID NO:301, residues 2-307 of SEQ ID NO:304, residues 2-307 of SEQ ID NO:307, residues 2-307 of SEQ ID NO: 310, residues 2-307 of SEQ ID NO: 313, residues 2- 307 of SEQ ID NO:316, residues 2-307 of SEQ ID NO:319, residues 2-307 of SEQ ID NO: 322, residues 2-307 of SEQ ID NO: 325, residues 2-307 of SEQ ID NO: 328, residues 2- 307 of SEQ ID NO:331, residues 2-307 of SEQ ID NO:334, residues 2-307 of SEQ ID NO: 337, residues 2-307 of SEQ ID NO: 340, residues 2-307 of SEQ ID NO: 343, residues 2- 307 of SEQ ID NO:346, residues 2-307 of SEQ ID NO:349, residues 2-307 of SEQ ID NO: 352, residues 2-307 of SEQ ID NO.-355, residues 2-307 of SEQ ID NO:358, residues 2- 307 of SEQ ID NO: 361, residues 2-307 of SEQ ID NO: 364, residues 2-307 of SEQ ID NO: 367, residues 2-307 of SEQ ID NO:370, residues 2-307 of SEQ ID NO:373, residues 2- 307 of SEQ ID NO:376, residues 2-307 of SEQ ID NO:379, residues 2-307 of SEQ ID NO: 382, residues 3-307 of SEQ ID NO:178, residues 3-307 of SEQ ID NO:181, residues 3- 307 of SEQ ID NO: 184, residues 3-307 of SEQ ID NO: 187, residues 3-307 of SEQ ID NO: 190, residues 3-307 of SEQ ID NO:193, residues 3-307 of SEQ ID NO:196, residues 3- 307 of SEQ ID NO:199, residues 3-307 of SEQ ID NO:202, residues 3-307 of SEQ ID NO: 205, residues 3-307 of SEQ ID NO:208, residues 3-307 of SEQ ID NO:211, residues 3- 307 of SEQ ID NO:214, residues 3-307 of SEQ ID NO.-217, residues 3-307 of SEQ ID NO: 220, residues 3-307 of SEQ ID NO:223, residues 3-307 of SEQ ID NO:226, residues 3- 307 of SEQ ID NO:229, residues 3-307 of SEQ ID NO:232, residues 3-307 of SEQ ID NO: 235, residues 3-307 of SEQ ID NO: 238, residues 3-307 of SEQ ID NO: 241, residues 3- 307 of SEQ ID NO: 244, residues 3-307 of SEQ ID NO: 247, residues 3-307 of SEQ ID NO -.250, residues 3-307 of SEQ ID NO:253, residues 3-307 of SEQ ID NO:256, residues 3- 307 of SEQ ID NO:259, residues 3-307 of SEQ ID NO:262, residues 3-307 of SEQ ID NO: 265, residues 3-307 of SEQ ID NO: 268, residues 3-307 of SEQ ID NO: 271, residues 3- 307 of SEQ ID NO: 274, residues 3-307 of SEQ ID NO: 277, residues 3-307 of SEQ ID NO: 280, residues 3-307 of SEQ ID NO: 283, residues 3-307 of SEQ ID NO: 286, residues 3- 307 of SEQ ID NO: 289, residues 3-307 of SEQ ID NO: 292, residues 3-307 of SEQ ID NO:295, residues 3-307 of SEQ ID NO-.298, residues 3-307 of SEQ ID NO:301, residues 3- 307 of SEQ ID NO: 304, residues 3-307 of SEQ ID NO: 307, residues 3-307 of SEQ ID NO: 310, residues 3-307 of SEQ ID NO:313, residues 3-307 of SEQ ID NO:316, residues 3- 307 of SEQ ID NO:319, residues 3-307 of SEQ ID NO:322, residues 3-307 of SEQ ID NO: 325, residues 3-307 of SEQ ID NO:328, residues 3-307 of SEQ ID NO:331, residues 3- 307 of SEQ ID NO-.334, residues 3-307 of SEQ ID NO:337, residues 3-307 of SEQ ID NO: 340, residues 3-307 of SEQ ID NO: 343, residues 3-307 of SEQ ID NO: 346, residues 3- 307 of SEQ ID NO: 349, residues 3-307 of SEQ ID NO: 352, residues 3-307 of SEQ ID NO: 355, residues 3-307 of SEQ ID NO:358, residues 3-307 of SEQ ID NO:361, residues 3- 307 of SEQ ID NO: 364, residues 3-307 of SEQ ID NO -.367, residues 3-307 of SEQ ID NO:370, residues 3-307 of SEQ ID NO: 373, residues 3-307 of SEQ ID NO: 376, residues 3- 307 of SEQ ID NO:379, residues 3-307 of SEQ ID NO:382, SEQ ID NO: 179, SEQ ID NO: 182, SEQ ID NO: 185, SEQ ID NO :188, SEQ ID NO: 191, SEQ ID NO: 194, SEQ ID NO: 197, SEQ ID NO:200, SEQ ID NO:203, SEQ ID NO:206, SEQ ID NO:209, SEQ ID NO :212, SEQ ID NO: 215, SEQ ID NO: 218, SEQ ID NO:221, SEQ ID NO:224, SEQ ID NO:227, SEQ ID NO:230, SEQ ID NO: 233, SEQ ID NO: 236, SEQ ID NO: 239, SEQ ID NO: 242, SEQ ID NO: 245, SEQ ID NO: 248, SEQ ID NO: 251, SEQ ID NO.-254, SEQ ID NO:257, SEQ ID NO:260, SEQ ID NO:263, SEQ ID NO:266, SEQ ID NO:269, SEQ ID NO:272, SEQ ID NO:275, SEQ ID NO:278, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO: 287, SEQ ID NO: 290, SEQ ID NO: 293, SEQ ID NO: 296, SEQ ID NO: 299, SEQ ID NO: 302, SEQ ID NO: 305, SEQ ID NO: 308, SEQ ID NO:311, SEQ ID NO:314, SEQ ID NO:317, SEQ ID NO: 320, SEQ ID NO: 323, SEQ ID NO: 326, SEQ ID NO: 329, SEQ ID NO: 332, SEQ ID NO: 335, SEQ ID NO: 338, SEQ ID NO: 341, SEQ ID NO:344, SEQ ID NO:347, SEQ ID NO:350, SEQ ID

NO:353, SEQ ID NO:356, SEQ ID NO:359, SEQ ID NO:362, SEQ ID NO: 365, SEQ ID NO: 368, SEQ ID NO: 371, SEQ ID NO: 374, SEQ ID NO: 377, SEQ ID NO: 380, SEQ ID NO: 383, residues 2- 322 of SEQ ID NO: 179, residues 2-322 of SEQ ID NO: 182, residues 2-322 of SEQ ID NO: 185, residues 2-322 of SEQ ID NO: 188, residues 2-322 of SEQ ID NO: 191, residues 2- 322 of SEQ ID NO: 194, residues 2-322 of SEQ ID NO: 197, residues 2-322 of SEQ ID NO: 200, residues 2-322 of SEQ ID NO: 203, residues 2-322 of SEQ ID NO: 206, residues 2- 322 of SEQ ID NO:209, residues 2-322 of SEQ ID NO-.212, residues 2-322 of SEQ ID NO: 215, residues 2-322 of SEQ ID NO: 218, residues 2-322 of SEQ ID NO: 221, residues 2- 322 of SEQ ID NO: 224, residues 2-322 of SEQ ID NO: 227, residues 2-322 of SEQ ID NO.-230, residues 2-322 of SEQ ID NO: 233, residues 2-322 of SEQ ID NO: 236, residues 2- 322 of SEQ ID NO: 239, residues 2-322 of SEQ ID NO: 242, residues 2-322 of SEQ ID NO: 245, residues 2-322 of SEQ ID NO:248, residues 2-322 of SEQ ID NO:251, residues 2- 322 of SEQ ID NO-.254, residues 2-322 of SEQ ID NO:257, residues 2-322 of SEQ ID NO:260, residues 2-322 of SEQ ID NO: 263, residues 2-322 of SEQ ID NO: 266, residues 2- 322 of SEQ ID NO: 269, residues 2-322 of SEQ ID NO: 272, residues 2-322 of SEQ ID NO: 275, residues 2-322 of SEQ ID NO :278, residues 2-322 of SEQ ID NO: 281, residues 2- 322 of SEQ ID NO: 284, residues 2-322 of SEQ ID NO: 287, residues 2-322 of SEQ ID NO: 290, residues 2-322 of SEQ ID NO: 293, residues 2-322 of SEQ ID NO: 296, residues 2- 322 of SEQ ID NO: 299, residues 2-322 of SEQ ID NO: 302, residues 2-322 of SEQ ID NO: 305, residues 2-322 of SEQ ID NO:308, residues 2-322 of SEQ ID NO:311, residues 2- 322 of SEQ ID NO: 314, residues 2-322 of SEQ ID NO: 317, residues 2-322 of SEQ ID NO: 320, residues 2-322 of SEQ ID NO:323, residues 2-322 of SEQ ID NO:326, residues 2- 322 of SEQ ID NO: 329, residues 2-322 of SEQ ID NO: 332, residues 2-322 of SEQ ID NO: 335, residues 2-322 of SEQ ID NO: 338, residues 2-322 of SEQ ID NO: 341, residues 2- 322 of SEQ ID NO: 344, residues 2-322 of SEQ ID NO: 347, residues 2-322 of SEQ ID NO: 350, residues 2-322 of SEQ ID NO:353, residues 2-322 of SEQ ID NO:356, residues 2- 322 of SEQ ID NO: 359, residues 2-322 of SEQ ID NO: 362, residues 2-322 of SEQ ID NO: 365, residues 2-322 of SEQ ID NO:368, residues 2-322 of SEQ ID NO: 371, residues 2- 322 of SEQ ID NO: 374, residues 2-322 of SEQ ID NO: 377, residues 2-322 of SEQ ID NO: 380, residues 2-322 of SEQ ID NO: 383, residues 3-322 of SEQ ID NO: 179, residues 3- 322 of SEQ ID NO: 182, residues 3-322 of SEQ ID NO: 185, residues 3-322 of SEQ ID NO: 188, residues 3-322 of SEQ ID NO: 191, residues 3-322 of SEQ ID NO: 194, residues 3- 322 of SEQ ID NO: 197, residues 3-322 of SEQ ID NO: 200, residues 3-322 of SEQ ID NO: 203, residues 3-322 of SEQ ID NO: 206, residues 3-322 of SEQ ID NO: 209, residues 3- 322 of SEQ ID NO: 212, residues 3-322 of SEQ ID NO: 215, residues 3-322 of SEQ ID NO: 218, residues 3-322 of SEQ ID NO: 221, residues 3-322 of SEQ ID NO: 224, residues 3- 322 of SEQ ID NO: 227, residues 3-322 of SEQ ID NO: 230, residues 3-322 of SEQ ID NO: 233, residues 3-322 of SEQ ID NO.-236, residues 3-322 of SEQ ID NO.-239, residues 3- 322 of SEQ ID NO: 242, residues 3-322 of SEQ ID NO: 245, residues 3-322 of SEQ ID NO: 248, residues 3-322 of SEQ ID NO:251, residues 3-322 of SEQ ID NO:254, residues 3- 322 of SEQ ID NO: 257, residues 3-322 of SEQ ID NO: 260, residues 3-322 of SEQ ID NO: 263, residues 3-322.of SEQ ID NO:266, residues 3-322 of SEQ ID NO:269, residues 3- 322 of SEQ ID NO:272, residues 3-322 of SEQ ID NO:275, residues 3-322 of SEQ ID NO: 278, residues 3-322 of SEQ ID NO:281, residues 3-322 of SEQ ID NO:284, residues 3- 322 of SEQ ID NO: 287, residues 3-322 of SEQ ID NO: 290, residues 3-322 of SEQ ID NO -.293, residues 3-322 of SEQ ID NO: 296, residues 3-322 of SEQ ID NO: 299, residues 3- 322 of SEQ ID NO:302, residues 3-322 of SEQ ID NO:305, residues 3-322 of SEQ ID NO.-308, residues 3-322 of SEQ ID NO:311, residues 3-322 of SEQ ID NO:314, residues 3- 322 of SEQ ID NO: 317, residues 3-322 of SEQ ID NO: 320, residues 3-322 of SEQ ID NO: 323, residues 3-322 of SEQ ID NO: 326, residues 3-322 of SEQ ID NO: 329, residues 3- 322 of SEQ ID NO: 332, residues 3-322 of SEQ ID NO: 335, residues 3-322 of SEQ ID NO: 338, residues 3-322 of SEQ ID NO -.341, residues 3-322 of SEQ ID NO: 344, residues 3- 322 of SEQ ID NO:347, residues 3-322 of SEQ ID NO:350, residues 3-322 of SEQ ID NO: 353, residues 3-322 of SEQ ID NO: 356, residues 3-322 of SEQ ID NO -.359, residues 3- 322 of SEQ ID NO:362, residues 3-322 of SEQ ID NO:365, residues 3-322 of SEQ ID NO:368, residues 3-322 of SEQ ID NO: 371, residues 3-322 of SEQ ID NO: 374, residues 3- 322 of SEQ ID NO:377, residues 3-322 of SEQ ID NO:380, residues 3-322 of SEQ ID NO: 383, SEQ ID NO: 180, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 198, SEQ ID NO: 201, SEQ ID NO: 204, SEQ ID NO: 207, SEQ ID NO: 210, SEQ ID NO: 213, SEQ ID NO:^'216, SEQ ID NO: 219, SEQ ID NO: 222, SEQ ID NO: 225, SEQ ID NO: 228, SEQ ID NO: 231, SEQ ID NO: 234, SEQ ID NO: 237, SEQ ID NO: 240, SEQ ID NO: 243, SEQ ID NO: 246, SEQ ID NO: 249, SEQ ID NO: 252, SEQ ID NO: 255, SEQ ID NO: 258, SEQ ID N0.261, SEQ ID NO:264, SEQ ID NO:267, SEQ ID NO:270, SEQ ID NO: 273, SEQ ID NO: 276, SEQ ID NO: 279, SEQ ID

NO:282, SEQ ID NO:285, SEQ ID NO:288, SEQ ID NO: 291, SEQ ID NO: 294, SEQ ID NO: 297, SEQ ID NO: 300, SEQ ID NO: 303, SEQ ID NO: 306, SEQ ID NO: 309, SEQ ID NO: 312, SEQ ID NO: 315, SEQ ID NO: 318, SEQ ID NO: 321, SEQ ID NO: 324, SEQ ID NO:327, SEQ ID NO:330, SEQ ID NO:333, SEQ ID NO-.336, SEQ ID NO: 339, SEQ ID NO: 342, SEQ ID NO: 345, SEQ ID NO: 348, SEQ ID NO: 351, SEQ ID NO: 354, SEQ ID NO: 357, SEQ ID NO:360, SEQ ID NO:363, SEQ ID NO:366, SEQ ID NO:369, SEQ ID NO: 372, SEQ ID NO: 375, SEQ ID NO: 378, SEQ ID NO: 381, SEQ ID NO: 384, residues 2-349 of SEQ ID NO: 179, residues 2-349 of SEQ ID NO: 182, residues 2-349 of SEQ ID NO: 185, residues 2-349 of SEQ ID NO: 188, residues 2- 349 of SEQ ID NO: 191, residues 2-349 of SEQ ID NO: 194, residues 2-349 of SEQ ID NO: 197, residues 2-349 of SEQ ID NO:200, residues 2-349 of SEQ ID NO:203, residues 2- 349 of SEQ ID NO: 206, residues 2-349 of SEQ ID NO: 209, residues 2-349 of SEQ ID NO: 212, residues 2-349 of SEQ ID NO: 215, residues 2-349 of SEQ ID NO: 218, residues 2- 349 of SEQ ID NO:221, residues 2-349 of SEQ ID NO:224, residues 2-349 of SEQ ID NO: 227, residues 2-349 of SEQ ID NO: 230, residues 2-349 of SEQ ID NO: 233, residues 2- 349 of SEQ ID NO: 236, residues 2-349 of SEQ ID NO: 239, residues 2-349 of SEQ ID NO: 242, residues 2-349 of SEQ ID NO: 245, residues 2-349 of SEQ ID NO: 248, residues 2- 349 of SEQ ID NO:251, residues 2-349 of SEQ ID NO:254, residues 2-349 of SEQ ID NO: 257, residues 2-349 of SEQ ID NO: 260, residues 2-349 of SEQ ID NO:263, residues 2- 349 of SEQ ID NO: 266, residues 2-349 of SEQ ID NO: 269, residues 2-349 of SEQ ID NO: 272, residues 2-349 of SEQ ID NO: 275, residues 2-349 of SEQ ID NO: 278, residues 2- 349 of SEQ ID NO: 281, residues 2-349 of SEQ ID NO: 284, residues 2-349 of SEQ ID NO-.287, residues 2-349 of SEQ ID NO: 290, residues 2-349 of SEQ ID NO: 293, residues 2- 349 of SEQ ID NO: 296, residues 2-349 of SEQ ID NO: 299, residues 2-349 of SEQ ID NO: 302, residues 2-349 of SEQ ID NO: 305, residues 2-349 of SEQ ID NO: 308, residues 2- 349 of SEQ ID NO: 311, residues 2-349 of SEQ ID NO: 314, residues 2-349 of SEQ ID NO: 317, residues 2-349 of SEQ ID NO: 320, residues 2-349 of SEQ ID NO: 323, residues 2- 349 of SEQ ID NO: 326, residues 2-349 of SEQ ID NO: 329, residues 2-349 of SEQ ID NO: 332, residues 2-349 of SEQ ID NO: 335, residues 2-349 of SEQ ID NO: 338, residues 2- 349 of SEQ ID NO:341, residues 2-349 of SEQ ID NO:344, residues 2-349 of SEQ ID NO: 347, residues 2-349 of SEQ ID NO: 350, residues 2-349 of SEQ ID NO: 353, residues 2- 349 of SEQ ID NO: 356, residues 2-349 of SEQ ID NO: 359, residues 2-349 of SEQ ID NO:362, residues 2-349 of SEQ ID NO: 365, residues 2-349 of SEQ ID NO: 368, residues 2- 349 of SEQ ID NO: 371, residues 2-349 of SEQ ID NO: 374, residues 2-349 of SEQ ID NO: 377, residues 2-349 of SEQ ID NO: 380, residues 2-349 of SEQ ID NO: 383, residues 3- 349 of SEQ ID NO: 179, residues 2-349 of SEQ ID NO: 182,. residues 2-349 of SEQ ID NO: 185, residues 2-349 of SEQ ID NO: 188, residues 2-349 of SEQ ID NO: 191, residues 2- 349 of SEQ ID NO: 194, residues 2-349 of SEQ ID NO: 197, residues 2-349 of SEQ ID NO: 200, residues 2-349 of SEQ ID NO: 203, residues 2-349 of SEQ ID NO: 206, residues 2- 349 of SEQ ID NO: 209, residues 2-349 of SEQ ID NO: 212, residues 2-349 of SEQ ID NO: 215, residues 2-349 of SEQ ID NO: 218, residues 2-349 of SEQ ID NO: 221, residues 2- 349 of SEQ ID NO: 224, residues 2-349 of SEQ ID NO: 227, residues 2-349 of SEQ ID NO: 230, residues 2-349 of SEQ ID NO: 233, residues 2-349 of SEQ ID NO: 236, residues 2- 349 of SEQ ID NO: 239, residues 2-349 of SEQ ID NO: 242, residues 2-349 of SEQ ID NO: 245, residues 2-349 of SEQ ID NO: 248, residues 2-349 of SEQ ID NO: 251, residues 2- 349 of SEQ ID NO: 254, residues 2-349 of SEQ ID NO: 257, residues 2-349 of SEQ ID NO: 260, residues 2-349 of SEQ ID NO: 263, residues 2-349 of SEQ ID NO: 266, residues 2- 349 of SEQ ID NO:269, residues 2-349 of SEQ ID NO:272, residues 2-349 of SEQ ID NO: 275, residues 2-349 of SEQ ID NO:278, residues 2-349 of SEQ ID NO:281, residues 2- 349 of SEQ ID NO:284, residues 2-349 of SEQ ID NO:287, residues 2-349 of SEQ ID NO: 290, residues 2-349 of SEQ ID NO: 293, residues 2-349 of SEQ ID NO: 296, residues 2- 349 of SEQ ID NO: 299, residues 2-349 of SEQ ID NO: 302, residues 2-349 of SEQ ID NO: 305, residues 2-349 of SEQ ID NO:308, residues 2-349 of SEQ ID NO:311, residues 2- 349 of SEQ ID NO: 314, residues 2-349 of SEQ ID NO: 317, residues 2-349 of SEQ ID NO: 320, residues 2-349 of SEQ ID NO: 323, residues 2-349 of SEQ ID NO: 326, residues 2- 349 of SEQ ID NO: 329, residues 2-349 of SEQ ID NO: 332, residues 2-349 of SEQ ID NO: 335, residues 2-349 of SEQ ID NO: 338, residues 2-349 of SEQ ID NO: 341, residues 2- 349 of SEQ ID NO:344, residues 2-349 of SEQ ID NO:347, residues 2-349 of SEQ ID NO: 350, residues 2-349 of SEQ ID NO:353, residues 2-349 of SEQ ID NO:356, residues 2- 349 of SEQ ID NO: 359, residues 2-349 of SEQ ID NO: 362, residues 2-349 of SEQ ID NO: 365, residues 2-349 of SEQ ID NO: 368, residues 2-349 of SEQ ID NO: 371, residues 2- 349 of SEQ ID NO: 374, residues 2-349 of SEQ ID NO: 377, residues 2-349 of SEQ ID NO: 380, and residues 2-349 of SEQ ID NO: 383.

6. The Myelopoietin conjugate of claim 1 wherein said Myelopoietin polypeptide is selected from the group consisting of: SEQ ID NO: 224, residues 2-322 of SEQ ID NO: 224, and residues 3-322 of SEQ ID NO: 224.

7. . The Myelopoietin conjugate of claim 1, 2, 3, 4, 5 or 6 wherein said polymer is a poly (ethylene oxide) molecule.

8. The Myelopoietin conjugate of claim 7 wherein said poly (ethylene oxide) molecule is a poly (ethylene glycol) molecule.

9. The Myelopoietin conjugate of claim 8 wherein the poly (ethylene glycol) is attached at an amino acid residue having a free amino, carboxyl or sulfhydryl group (s).

10. The Myelopoietin conjugate of claim 9 wherein said poly (ethylene glycol) is conjugated through a activated poly (ethylene glycol) .

11. The Myelopoietin conjugate of claim 10 wherein said activated poly (ethylene glycol) is selected from the group consisting of, para-nitrophenyl, succinimidyl, carbonyl imidazole, azlactones, cyclic imide thiones, isocyanates, isothiocyanates, aldehydes, primary amines, hydrazine, acyl hydrazides, carbazates, semicarbamates, thiocarbazates, thiols, maleimides, sulfones, and phenyl glyoxals.

12. The Myelopoietin conjugate of claim 11 wherein said activated poly (ethylene glycol) is selected from the group consisting of, succinimidyl, carbonyl imidazole, aldehydes, acyl hydrazides, carbazates, semicarbamates, and maleimides.

13. The Myelopoietin conjugate of claim 9 wherein said poly (ethylene glycol) has a molecular weight of between about 0.5 kDa and about 100 kDa.

14. The Myelopoietin conjugate of claim 9 wherein said poly (ethylene glycol) has a molecular weight of between about 3. kDa and about 40 kDa .

15. The Myelopoietin conjugate of claim 9 wherein said poly (ethylene glycol) is a branched polymer.

16. The Myelopoietin conjugate of claim 15 wherein branched poly (ethylene glycol) polymer has a molecular weight of between about 10 kDa and about 40 kDa.

17. The Myelopoietin conjugate of claim 9 wherein said poly (ethylene glycol) is a bifunctional polymer.

18. The Myelopoietin conjugate of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 wherein said Myelopoietin conjugate is a prodrug.

19. A composition comprising the Myelopoietin of claim 1, 2, 3, 4, 5, 6, 7, 8, or 9 and at least one pharmaceutically acceptable carrier.

20. A composition comprising the Myelopoietin of claim 10 and at least one pharmaceutically acceptable carrier.

21. A composition comprising the Myelopoietin of claim 11, 12, 13, 14, 15, 16, 17, or 18 and at least one pharmaceutically acceptable carrier.

22. A method of treating a patient having a hematopoietic disorder comprising administering to said patient a therapeutically effective amount of the Myelopoietin conjugate of claim 1, 2, 3, 4, 5, 6, 7, 8, or 9.

23. A method of treating a patient having a hematopoietic disorder comprising administering to said patient a therapeutically effective amount of the Myelopoietin conjugate of claim 10.

24. A method of treating a patient having a hematopoietic disorder comprising administering to said patient a therapeutically effective amount of the Myelopoietin conjugate of claim 11, 12, 13, 14, 15, 16, 17, or 18.

25. The method of claim 22 wherein said hematopoietic disorder is neutropenia, leukopenia, thrombocytopenia, or anemia.

26. The method of claim 23 wherein said hematopoietic disorder is neutropenia, leukopenia, thrombocytopenia, or anemia.

27. The method of claim 24 wherein said hematopoietic disorder is neutropenia, leukopenia, thrombocytopenia, or anemia.

28. The method of claim 22 wherein said hematopoietic disorder is the result of chemotherapy, radiation therapy, or bone marrow suppressive drugs.

29. The method of claim 23 wherein said hematopoietic disorder is the result of chemotherapy, radiation therapy, or bone marrow suppressive drugs.

30. The method of claim 24 wherein said hematopoietic disorder is the result of chemotherapy, radiation therapy, or bone marrow suppressive drugs.

31. A method of treating a patient recovering and/or suffering from a bone marrow transplant, burn, wound, parasite, bacterial or viral infection comprising administering to said patient a therapeutically effective amount of the Myelopoietin conjugate of claim

I, 2, 3, 4, 5-, 6, 7, 8, or 9.

32. A method of treating a patient recovering and/or suffering from a bone marrow transplant, burn, wound, parasite, bacterial or viral infection comprising administering to said patient a therapeutically effective amount of the Myelopoietin conjugate of claim 10.

33. A method of treating a patient recovering and/or suffering from a bone marrow transplant, burn, wound, parasite, bacterial or viral infection comprising administering to said patient a therapeutically effective amount of the Myelopoietin conjugate of claim

II, 12, 13, 14, 15, 16, 17, or 18.

34. A method of mobilizing hematopoietic progenitors and stem cells into peripheral blood comprising administering to said patient a therapeutically effective amount of the Myelopoietin conjugate of claim 1, 2, 3, 4, 5, 6, 7, 8, or 9.

35. A method of mobilizing hematopoietic progenitors and stem cells into peripheral blood comprising administering to said patient a therapeutically effective amount of the Myelopoietin conjugate of claim 10.

36. A method of mobilizing hematopoietic progenitors and stem cells into peripheral blood comprising administering to said patient a therapeutically effective amount of the Myelopoietin conjugate of claim 11, 12, 13, 14, 15, 16, 17, or 18.