WO2007075899A2 - Dual agonist compounds and uses thereof - Google Patents

Dual agonist compounds and uses thereof Download PDF

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Publication number
WO2007075899A2
WO2007075899A2 PCT/US2006/048807 US2006048807W WO2007075899A2 WO 2007075899 A2 WO2007075899 A2 WO 2007075899A2 US 2006048807 W US2006048807 W US 2006048807W WO 2007075899 A2 WO2007075899 A2 WO 2007075899A2
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Prior art keywords
compound
dual agonist
polypeptide
amino acid
dual
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PCT/US2006/048807
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French (fr)
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WO2007075899A3 (en
WO2007075899A8 (en
Inventor
Stephen F. Parmley
Madan M. Paidhungat
Stephanie J. Lathrop
Sarah Patrick
Claus M. Krebber
Aaron Kurtzman
Margaret Neighbors
Kim V. Andersen
Steven H. Bass
Sridhar Viswanathan
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Maxygen, Inc.
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Publication of WO2007075899A2 publication Critical patent/WO2007075899A2/en
Publication of WO2007075899A8 publication Critical patent/WO2007075899A8/en
Publication of WO2007075899A3 publication Critical patent/WO2007075899A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/524—Thrombopoietin, i.e. C-MPL ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5403—IL-3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention pertains to compounds having both human IL-3 receptor agonist activity and human thrombopoeitin MpI receptor agonist activity.
  • thrombocytopenia Decreased platelet counts (thrombocytopenia) can be caused by a number of disease processes. Patients undergoing cancer therapies such as chemotherapy and radiation are at risk for developing thrombocytopenia, which is a condition in which platelet counts are reduced to dangerous levels. Platelets are a critical component of hemostasis. Patients with thrombocytopenia are susceptible to complications such as bleeding, prolonged clotting times, and hemorrhaging. These complications can lengthen recovery time and postpone or reduce the efficacy of chemotherapy treatment. Intra- cranial and retroperitoneal bleeding are a major cause of death in hematological cancer patients. In view of these complications, thrombocytopenia can limit the maximum chemotherapy dose that may be administered.
  • Vitamin Bl 2 or folic acid deficiency, leukemia, or myelodysplastic syndromes are associated with a decrease in platelet production.
  • Other diseases associated with a peripheral destruction of platelets can cause thrombocytopenia.
  • Idiopathic thrombocytopenic purpura or ITP is an autoimmune disease in which the platelets are marked as foreign by the immune system and eliminated from the body.
  • Thrombotic thrombocytopenic purpura, hemolytic-uremic syndrome, disseminated intravascular coagulation, paroxysmal nocturnal hemoglobinuria, and antiphospholipid syndrome are also associated with thrombocytopenia.
  • Some commonly used drugs such as heparin may cause heparin induced thrombocytopenia. Chronic infection with hepatitis C or Dengue viruses can also lead to thrombocytopenia.
  • Platelet transfusion has been the primary therapy for reducing the duration and severity of thrombocytopenia after cancer therapy. However, this may be accompanied by complications such as alloimmunization.
  • a number of cytokines that increase platelet counts in normal animals have been evaluated for their efficacy on the duration and severity of thrombocytopenia induced by cancer chemotherapy and/or irradiation exposure in preclinical or clinical studies. These cytokines include interleukin 3 (IL-3), IL-6, and leukemia inhibitory factor. Amelioration of clinically important thrombocytopenia with these cytokines, however, has not yet been achieved. Id.
  • IL-3 interleukin 3
  • IL-6 interleukemia inhibitory factor
  • Interleukin- 1 1 has been approved for the treatment of thrombocytopenia. However, low efficacy combined with fatigue and cardiovascular symptoms are side effects that have been reported with the therapeutic administration of interleukin- 11 limit its use. Tepler, et al. Blood (1996) 87:3607-3614; Isaacs, et al. (1997) J. Clinical Oncology 15:3368- 3377.
  • Platelets produced by megakaryocytes are derived from the differentiation of pluripotent stem cells in the bone marrow or spleen.
  • TPO Thrombopoietin
  • PEG-rHuMGDF pegylated recombinant human megakaryocyte growth and development factor
  • the present invention provides dual agonist compounds that exhibit both IL-3 receptor agonist activity and thrombopoeitin MpI receptor agonist activity, wherein said compound comprises a structure of the following formula: TM 1 -(L') n -TM 2 -(L 2 ) m -IL-3 (I) wherein TM 1 and TM 2 are non-naturally occurring peptides that each independently have a molecular weight of less than about 2,500 daltons and wherein each exhibits thrombopoeitin c-Mpl receptor agonist activity, wherein n and m are each independently 0 or 1 , wherein L 1 is a linker that covalently links the C terminus of TM 1 to the N terminus of TM 2 , wherein L 2 is a linker that covalently links the C terminus of TM 2 to IL-3, wherein the IL-3 is an IL-3 polypeptide that exhibits IL-3 receptor agonist activity, and wherein the dual agonist compound exhibit
  • the present invention further provides dual agonist compounds that exhibit both IL-3 receptor agonist activity and thrombopoeitin c-Mpl receptor agonist activity, wherein said compound comprises a structure of the following formula: TM 1 - (L 2 ) m - IL-3 - (L ⁇ - TM 2 (II) wherein TM 1 and TM 2 are non-naturally occurring peptides that each independently have a molecular weight of less than about 2,500 daltons and wherein each exhibits thrombopoeitin c-Mpl receptor agonist activity, wherein m and p are each independently 0 or 1, wherein L 2 is a linker that covalently links the C-terminus of TM 1 to IL-3, wherein the IL-3 is an IL-3 polypeptide that exhibits IL-3 receptor agonist activity wherein L 3 is a linker that covalently links IL-3 to the N-terminus of TM 2 , and wherein the dual agonist compound exhibits both
  • Figure 1 illustrates the steps in the platelet generation pathway, as well as the roles of cytokines interleukin-6 (IL-6), interleukin-11 (IL-1 1), interleukin-3 (IL-3), stem cell factor (SCF), and TPO in the pathway.
  • IL-6 interleukin-6
  • IL-1 1 interleukin-11
  • IL-3 interleukin-3
  • SCF stem cell factor
  • Figure 2 is a 4323 base pair expression vector (pCKQ3-Dual Agonist) of the present invention comprising a CoIEl origin of replication, a T5 lac promoter, a lad repressor, a lambda to transcriptional terminator, an rmB Tl transcriptional terminator, and a kanamycin resistance gene (KanR).
  • pCKQ3-Dual Agonist a 4323 base pair expression vector (pCKQ3-Dual Agonist) of the present invention comprising a CoIEl origin of replication, a T5 lac promoter, a lad repressor, a lambda to transcriptional terminator, an rmB Tl transcriptional terminator, and a kanamycin resistance gene (KanR).
  • the present invention is directed to novel compounds that exhibit agonist activity with respect to both human interleukin-3 (IL-3) receptor, which is expressed by the human erythroleukemia cell line, TF-I (Deutsche Sammlung von Mikroorganismen und Zellkult ⁇ ren GmbH, DSMZ No. ACC 334) and the human thrombopoietin receptor, c- MpI (SEQ ED NO: 2), as determined by the assays of Examples 3 and 4, respectively.
  • IL-3 human erythroleukemia cell line
  • TF-I Deutsche Sammlung von Mikroorganismen und Zellkult ⁇ ren GmbH, DSMZ No. ACC 33
  • c- MpI SEQ ED NO: 2
  • the human interleukin-3 receptor is a heterodimer of an IL-3 specific alpha chain (SEQ ID NO: 4) and a common beta chain (SEQ ID NO: 5) that is shared with the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5.
  • Human thrombopoietin c-Mpl and IL-3 receptors are two of several receptors implicated in the platelet generation pathway, depicted in Figure 1. This pathway involves several different cytokines, including IL-6, IL-I l , IL-3, stem cell factor (SCF), and thrombopoeitin (TPO).
  • the present invention is directed to dual agonist compounds that exhibit both human IL-3 receptor agonist activity and human thrombopoeitin MpI receptor agonist activity, wherein said compound comprises a structure of the following formula:
  • TM 1 and TM 2 are non-naturally occurring peptides that each independently have a molecular weight of less than about 2,500 daltons and wherein each exhibits thrombopoetin c-Mpl receptor agonist activity, wherein n and m are each independently 0 or 1 , wherein L 1 is a linker that covalently links the C terminus of TM 1 to the N terminus of TM 2 , wherein L 2 is a linker that covalently links the C terminus of TM 2 to IL-3, wherein the IL-3 is an IL-3 polypeptide that exhibits IL-3 receptor agonist activity, and wherein the dual agonist compound exhibits both IL-3 receptor agonist activity and thrombopoeitin c-Mpl receptor agonist activity.
  • the present invention provides dual agonist compounds that exhibit both human IL-3 receptor agonist activity and human thrombopoeitin c-Mpl receptor agonist activity, wherein said compound comprises a structure of the following formula:
  • TM 1 and TM 2 are non-naturally occurring peptides that each independently have a molecular weight of less than about 2,500 daltons and wherein each exhibits thrombopoeitin c-Mpl receptor agonist activity, wherein m and p are each independently 0 or 1 , wherein L 2 is a linker that covalently links the C-terminus of TM 1 to IL-3, wherein the IL-3 is an IL-3 polypeptide that exhibits IL-3 receptor agonist activity wherein L 3 is a linker that covalently links IL-3 to the N-terminus of TM 2 , and wherein the dual agonist compound exhibits both human IL-3 receptor agonist activity and human thrombopoeitin c-Mpl receptor agonist activity.
  • thrombopoeitin MpI receptor agonist activity refers to the ability to induce the proliferation of a murine IL-3-dependent pro-B cell line, Ba/F3- MpI (DSMZ No. ACC 300), that has been transformed to express the human thrombopoeitin MpI receptor (SEQ ID NO:2) as detected in the assay of Example 4.
  • IL-3 receptor agonist activity refers to the ability to induce the proliferation of human erythroleukemia TF-I cells (DSMZ No. ACC 334) which express human IL-3 receptor as detected in the assay of Example 3.
  • polypeptide and peptide refer herein to a polymer of amino acid monomers joined together through amide bonds.
  • peptide refers to a relatively short polymer of up to about 30 amino acid monomer units.
  • TM 1 and TM 2 are thrombopoeitin mimetic peptides that may be the same or different with respect to both sequence and length. Neither TM 1 nor TM 2 are human thrombopoeitin, yet each exhibits human thrombopoeitin MpI receptor agonist activity.
  • TM 1 and TM 2 each have a molecular weight of at least about 800 or 900 daltons, and less than about 2,500 daltons.
  • TM 1 and TM 2 are each independently peptides having a molecular weight of less than about 2,000 daltons, and more typically less than about 1 ,800 daltons.
  • TM 1 and TM 2 are each independently peptides having a molecular weight of less than about 1 ,500 daltons.
  • TM 1 and TM" may each independently have up to about 25 amino acid residues, and in other embodiments.up to about 20 amino acid residues, and sometimes between about 9 and 25 amino acid residues in length, e.g., between about 10 and 20 amino acid residues in length.
  • Suitable peptides for TM 1 and TM 2 include peptides having up to about 10, 1 1, 12, 13, 14, 15, 16, 17, 18, or 19 amino acid residues.
  • Suitable peptides employed in the practice of the present invention typically have at least about 9 amino acid residues.
  • Illustrative TM 1 and TM 2 peptide sequences are provided in SEQ ID NOS: 8-254 and 374-378. [020]
  • Exemplary TM 1 and TM 2 peptides include those having any of the following sequences:
  • X 2 is E, R, H, Q, T, A, G, or S
  • X 7 is R, S, or T
  • X s is A, D, Q, S, or T
  • X 9 is A, C, or W (see, for example, SEQ ID NOS: 89-120 and 374);
  • X 5 is D, E, G 5 or S;
  • X 6 is C, Q, or T;
  • X 7 is G, 1, or V;
  • X s is A, K, L 5 M, S 5 W, or Y;
  • X 9 is A, G, I, K, L, M, Q, R, W, or Y; and
  • X 1 is G, R, S, or T;
  • X 4 is D, E, or Q;
  • X 5 is Q or V;
  • X 6 is I or V;
  • X 7 is C, D, E. G, K, N P, R, or S;
  • X s is A, I, K, L, M, S, W or Y;
  • X 9 is F, H, R, W, or Y;
  • X 10 is F, L 5 M 5 V,
  • X 1 ' is A, C 5 F, H, I, L, M, S, or V (see, for example, SEQ ID NOS: 24-53) ;
  • X 4 is W, T, Q, or C; X 5 is N, G, S, T, or A; X 6 is L, V, or R; X 7 is T, R, K, A. H, S, or N ; X 8 is E, S, D, Q, G, or K; X 9 is F, V, Q ; W, Y, L, or M; X 10 is V, R, K, L, or I; X" is L 5 D, Q 5 A, V, K, I, G, S, or R; X 12 is D, Q, G, R, L, Q, or K; X 13 is T, V, C, D, N, E, G, M, H, or A; X 14 is H, C, T 5 R 5 A, or N; and X 15 is P, A, W, G, T, V, C, L, F, D 5 Y, or R (see, for example, SEQ ID NOS: 54, 59, 61, and 63-76
  • X 3 is G 5 R 5 or S;
  • X 4 is E, M, P, Q, or R;
  • X 5 is H, Q, R, S, or T;
  • X 6 is C, L, P 5 or W- X 7 is A, F 5 K 5 M 5 R, S, or V;
  • X s is E, G 1 M, P, Q. S, or T; and
  • X 10 is L or M (see, for example, SEQ ID NOS: 78-88);
  • X 1 is F, I, L, P, or Y
  • X 2 is E, H, K 5 L, Q, R, S, W, or Y
  • X 5 is R or T
  • X 7 is K or R
  • X 8 is A, S, or V
  • X !o is L, R, or S
  • X n is A or G
  • X 13 is I, L, M, or V (see, for example, SEQ ID NOS: 121-140);
  • X ' is G, S, or Y
  • X 2 is C, L, or P
  • X 4 is F 1 L, or V
  • X 5 is K, P, Q, R, or S
  • X 6 is D, E, H, Q, or Y
  • X 7 is C, F, L, or W
  • X s is I, K, L
  • X 2 is N, Q, R, S, or T;
  • X 3 is C, F, I 1 L, or R;
  • X 4 is A, E, G, H, K, N, Q, R, or S:
  • X 5 is D, E, or Q;
  • X 6 is F, L, V, or Y;
  • X 7 is I, K, L, M, N. R, or V;
  • X s is C, F. I, M, P, T, V, W, or Y;
  • X 9 is A, C, F, G, I, L, Q 1 R, or S (see, for example, SEQ ID NOS: 165- 188);
  • X 1 is C or E
  • X 2 is R or T
  • X 3 is F, G, L, R, or S
  • X 4 is A, G, P, Q, R, S, or T
  • X 5 is E, F, Q, or S
  • X 6 is F or W
  • X 7 is A, K, L 1 or R
  • X 8 is D, E, G, H, K, L, Q, R, or S
  • X 9 is A, C, D, G, or R (see. for example, 189-203);
  • X 2 is A, G, L, M, R, or S
  • X 3 is A, D, E, Q, T, or absent
  • X 5 is L or P
  • X 6 is T, S, or F
  • X 7 is L or V
  • X 8 is R 1 T, or L
  • X 9 is E, P 1 Q, or A
  • X 1 1 is L 1 M 1 or I
  • X 12 is Y 1 L 1 T T,, EE 11 SS 11 oorr aabbsseenntt;
  • X 13 is V, L 1 E, H, F 1 or absent (see, for example, SEQ ID NOS: 205-206 and 209-215);
  • X 2 is D 1 K 1 S 1 T, or V;
  • X 3 is F 1 L, or M;
  • X 4 is A 1 K 1 L. Q 1 R 1 or S;
  • X 5 is D, E 1 or Q;
  • X 6 is F 1 L 1 W, or Y;
  • X 7 is K or L;
  • X 8 is A 1 D, E 1 H 1 L, M 1 Q 1 S, T 1 or V;
  • X 9 is S 1 N 1 F, Y, L 1 H 1 A 1 or P;
  • X 10 is G, H, S 1 or T;
  • X 1 1 is E 1 G 1 L 1 M 1 T, V, or Y;
  • X 12 is A, E 1 G, M 1 N 1 S, V, or Y;
  • X 13 is A, E, L, Q, S, V, W, or Y (see, for example, SEQ ID NOS: 219- 231); and
  • X 2 is L, R, S, or T;
  • X 3 is F or L;
  • X 4 is G, K, L, M, Q, R, or S;
  • X 5 is A, D, E, Q 5 S, or V;
  • X 6 is F or W;
  • X 7 is I, K, L, or V;
  • X s is C, I, K, N, P, T, W, or Y;
  • X 9 is A, D, E, G, L, M, N, S, W, or Y;
  • X 10 is A, E, G, H, L P, Q, R, S, or W;
  • X 1 ' is E, H, I, K, R, or Y (see, for example, SEQ ID NOS: 232-254).
  • Some TM 1 and TM 2 peptides having a sequence of formula (1) have a sequence corresponding to formula (13):
  • X 2 , X 7 , X s , and X 9 are as desc ⁇ bed for formula (1), X 1 1 is A, D, or E and X 12 is
  • X 2 is A, E, R, or S
  • X 7 is R
  • X 9 is W
  • X 1 1 is E or A
  • X 12 is A.
  • TM 1 and TM 2 peptides having the a sequence of formula (2) may have a sequence corresponding to formula (14):
  • X 1 is C, G, E, A, L, or S;
  • X 2 -X 10 are as described for formula (2); and
  • X 11 is C,
  • X 1 is G
  • X 2 is R
  • X 3 is V
  • X 4 is R
  • X 5 is D or E
  • X 6 is Q
  • Some TM 1 and TM 2 peptides having a sequence of formula (4) have a sequence corresponding to formula (15):
  • formula (4) peptides employed in the practice of the present invention have a sequence corresponding to formula (16):
  • X 1 is V, S, G, R, C, N, Y, W, or K
  • X 2 is R, G, Y, O, or S
  • X 3 -X 15 are as described for formulas (4) and (15).
  • Some TM 1 and TM 2 peptides comprising a sequence of formula (5) have a sequence corresponding to formula (17):
  • X 1 is S, R, L, or T
  • X 2 is V, E, or R
  • X 3 -X 10 are as described for sequence
  • X 1 is R or S and X 2 is E.
  • Suitable TM 1 and TM 2 peptides include any of the thrombopoietin mimetic peptides corresponding to SEQ ID NOS: 8-254 and 374-378 in homo- (i.e., TM 1 and TM 2 being identical) or hetero-dimer (i.e., TM 1 and TM 2 being different) format.
  • TM 1 and TM 2 peptides contemplated for use in the invention dual agonist compounds include the specific peptides exemplified herein as well as equivalent peptides that may be somewhat longer or shorter than the peptides specifically recited herein.
  • TM 1 and TM 2 peptides may be readily identified by generating libraries of randomized peptides that are then subjected to affinity enrichment using immobilized human thrombopoeitin MpI receptor (SEQ ID NO: 2). Methods for generating randomized peptides of a desired length followed by identification of those peptides that bind to receptor molecules of interest are well known in the art and are described in references such as U.S. Pat. No. 5,723,286, U.S. Pat. No.
  • random peptides can be designed to have a defined number of amino acid residues of a specific length.
  • Oligonucleotides encoding random peptides may be prepared which have the codon motif (NNK) x , where N is nucleotide A, C, G, or T in equimolar amounts, K is G or T in equimolar amounts, and x is an integer corresponding to the number of amino acids desired in the peptide.
  • the random peptides may be presented on the surface of a phage particle, as part of a fusion protein comprising either the pill or the pVIII coat protein of a phage fd derivative or as a fusion protein with the Lad peptide fusion protein bound to a plasmid.
  • a fusion protein comprising either the pill or the pVIII coat protein of a phage fd derivative or as a fusion protein with the Lad peptide fusion protein bound to a plasmid.
  • linker moieties When present in dual agonist compounds of formula (I), linker moieties function to separate TM 1 and TM 2 in the case of L 1 , and TM 2 and IL-3 in the case of L 2 .
  • linker moieties When present in dual agonist compounds of formula (II); linker moieties function to separate TM 1 from IL-3 in the case of L 2 , and IL-3 from TM 2 in the case of L 3 .
  • the specific composition of the linker is not critical.
  • the linkers should be of a length that is adequate to link their specific associated substituents in such a way that they assume the correct conformation relative to one another so that they retain agonist activity for their respective receptors.
  • linkers should be a length that allows adequate spacing between the TM 1 and TM * pair and IL-3 in formula (I), and between TM 1 and IL-3 and IL-3 and TM 2 in formula (II), to avoid steric hindrance with the respective receptors.
  • Linker type and length can be readily optimized in the context of the other substituents in the dual agonist compound by using the proliferation assays provided in Examples 3 and 4 to assess agonist activities.
  • L 1 is covalently bound to the C-terminus of TM 1 and the N- terminus of TM 2 , thus covalently linking the two peptides together in a tandem configuration.
  • C-terminus refers to the terminal carboxyl group of a peptide, protein, or polypeptide.
  • N-terminus refers herein to the terminal amino group of a peptide, protein, or polypeptide.
  • L is covalently bound to the C-terminus of TM 2 and an amino acid residue of the IL-3 (i.e., at one of either the N- terminal amino group, the C-terminal carboxyl group, or an ⁇ carbon side chain of any amino acid residue in the IL-3 sequence), thus covalently linking the C-terminus of TM 1 to the IL-3.
  • L 2 is covalently bound to the N-terminal amino group of the IL-3.
  • L 2 is covalently bound to the C-terminus of TM 1 and an amino acid residue of the IL-3 (i.e., one of either the N-terminal amino group, the C-terminal carboxyl group , or an ⁇ carbon side chain of any amino acid residue in the IL-3 sequence), thus covalently linking the C-terminus of TM 1 to the IL-3.
  • L 2 is covalently bound to the N-terminal amino group of the IL-3.
  • L 3 is covalently bound to the N-terminus of TM 2 and an amino acid residue of IL-3 (i.e., at one of either the N- terminal amino group, the C-terminal carboxyl group, or an ⁇ carbon side chain of any of the amino acid residues in the IL-3 sequence), thus covalently linking the IL-3 to the N- terminus of TM 2 .
  • L 3 is covalently bound to the C-terminus of the IL-3.
  • Exemplary linker moieties for either L 1 , L 2 or L 3 include peptides of naturally occurring amino acids, non-natural Iy occurring amino acids, or a combination of both; non-peptidic, non-polymeric aliphatic moieties; and oligonucleotides. Additional L" and L 3 linker moieties include non-peptidic polymeric substituents, polypeptide substituents, and polynucleotide substituents.
  • Dual agonist compounds of the present invention may also have a combination of any two of these different linker types (i.e., peptide; non-peptidic, non-polymeric aliphatic; oligonucleotide; polypeptide; or polynucleotide) such that L 1 and L 2 can be different in the compound of formula (I) and L 2 and L 3 can be different in the compound of formula (II).
  • the difference can be with respect to either linker type or, if the linkers are of the same type, the difference may be with respect to specific structure or sequence.
  • the linkers may be identical.
  • L 1 and L 2 are peptide linkers.
  • both L 1 and L 2 (compound I) and both L 2 and L 3 (compound II) are peptide linkers.
  • L 1 is usually from about 1 to about 40 amino acid residues in length, is often from about 1 to about 20 amino acid residues in length, and typically is from about 1 to about 10 amino acid residues in length, more typically from about 1 to about 6 amino acid residues in length, and often from about 4 to about 6 amino acid residues in length.
  • L 2 is typically from about 40 amino acid residues in length, is often from about 1 to about 20 amino acid residues in length, more typically from about 4 to about 15 amino acid residues in length, and often from about 6 to about 12 amino acid residues in length.
  • L 3 is typically from about 1 to about 40 amino acid residues in length, is often from about 1 to about 20 amino acid residues in length, more typically from about 4 to about 15 amino acid residues in length, and often from about 6 to about 12 amino acid residues in length.
  • the linkers may be a single amino acid residue, such as, for example, a GIy, Ser, Ala, or Thr residue.
  • Peptide linkers employed in the practice of the present invention may be made up of any of the 20 naturally occurring ammo acids (i.e., Ala, Arg, Asn, Asp, Cys, GIn, GIu, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tip, Tyr, and VaI) or any of the non- naturally occurring amino acids known in the art, as well as combinations thereof.
  • Suitable peptide linkers employed in the practice of the present invention include those that have been previously reported in the literature.
  • peptide linkers have also been demonstrated in the production of single-chain antibodies where the variable regions of a light chain (VL) and a heavy chain (V H ) are joined through a peptide linker.
  • VL variable regions of a light chain
  • V H heavy chain
  • a widely used peptide linker is a 15-mer consisting of three repeats of a Gly-Gly-Gly-Gly-Ser amino acid sequence ((Gly. 4 Serb) (SEQ ID NO:3).
  • Phage display technology has been used to diversify and select appropriate linker sequences (Tang, et al. (1996) J.
  • the peptide linker may contain at least 50% glycine residues, and sometimes at least 75% glycine residues.
  • the peptide linker may also be made up of only glycine residues.
  • the linker may contain 1-20 glycine residues, 2-16 glycine residues, 3-15 glycine residues, 4-12 glycine residues or 5-10 glycine residues.
  • the linker may comprise other residues, in particular, residues selected from the group consisting of Ser, Ala and Thr.
  • Peptide linkers employed in invention dual agonist compounds often have only glycine and serine residues in their sequences.
  • the peptide linker may be of the form (Gly 3 Ser) n (SEQ ID NO:363), where n is from 1 to 5, inclusive, or from 1 to 3, inclusive.
  • L 1 may be (Gly 3 Ser) (SEQ ID NO:363) and L 2 may be (Gly 3 Ser) 3 (SEQ ID NO.-364) in compound (I) and L 2 and L 3 may each be (Gly 3 Ser) 3 (SEQ ID NO:364) in compound (II).
  • Other suitable peptide linkers having only glycine and serine residues may be of the form (Gly4Ser) n (SEQ ID NO:364), wherein n is from 1 to 4, inclusive, or from 1 to 3, inclusive.
  • Suitable peptide linkers include those having the amino acid sequence GIy x - Xaa-Gly y -Xaa-Gly Z) wherein each Xaa is independently selected from the group consisting of Ala, VaI, Leu, He, Met, Phe, Trp, Pro, GIy, Ser, Thr, Cys, Tyr, Asn, GIn, Lys, Arg, His, Asp and GIu, and wherein x, y and z are each integers from 1 to 5, inclusive (SEQ ID NO:366). In some embodiments, each Xaa is independently selected from the group consisting of Ser, Ala and Thr.
  • the peptide linker has the amino acid sequence Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly-Gly-Gly-Gly-Gly, wherein each Xaa is independently selected from the group consisting of Ala, VaI, Leu, He, Met, Phe, Trp, Pro, GIy, Ser, Thr, Cys, Tyr, Asn, GIn 1 Lys, Arg, His, Asp and GIu (SEQ ID NO:367).
  • each Xaa is independently selected from the group consisting of Ser, Ala and Thr.
  • Xaa is Ser.
  • Suitable peptide linkers include those comprising at least one proline residue in the amino acid sequence of the peptide linker.
  • Peptide linkers of the present invention also may comprise at least one cysteine residue and/or at least one lysine residue.
  • the peptide linker comprises amino acid residues selected from the group consisting of GIy, Ser, Ala, Thr, Cys, Lys, and Pro.
  • the purpose of introducing an amino acid residue, such as Cys or Lys into the peptide linker is to introduce an attachment group for a non- polypeptide conjugation moiety that can be covalently attached to the amino acid residue.
  • non-polypeptide conjugation moieties may also be incorporated into the peptide linker sequence. Covalent attachment of non-polypeptide conjugation moieties is described in more detail herein below.
  • non-polypeptide conjugation moiety refers to a non-polypeptide polymer, sugar moiety, or non-polymeric lipophilic moiety. Exemplary non-polypeptide conjugation moieties are described in further detail herein below under the heading “Dual Agonist Conjugates.”
  • the amino acid residue to which a non-polypeptide conjugation moiety covalently binds is referred to herein as an "attachment group”.
  • the non-polypeptide conjugation moieties react with specific attachment sites on the attachment groups.
  • the term "attachment site” refers to the specific functional group involved in the conjugation reaction.
  • Additional suitable peptide linkers may be obtained by optimizing the peptide linkers described herein or in the literature by using well known mutagenesis techniques, such as random mutagenesis. For example, libraries of dual agonist compounds in which the TM and IL-3 components remain constant while the type, length and/or amino acid composition of the linker is varied can be prepared and screened using the assays described in Examples 3 and 4. Preferred peptide linkers employed in the practice of the present invention are resistant to proteolysis.
  • Suitable non-peptidic, non-polymeric aliphatic linker moieties include optionally substituted, substantially linear alkylene moieties
  • the non-peptidic, non-polymeric aliphatic linkers are typically derived from an aliphatic compound having at least two functional groups that are capable of reacting with an amino acid (e.g., hydroxyl, carboxyl, amino, and the like).
  • substitution groups include hydroxyl, an alkyl, a lower alkyl (having from 1 to 6 carbon atoms), an alkoxy, a lower alkoxy (having from 1 to 6 carbon atoms), nitro, amino, cyano, and the like.
  • the substantially linear alkylene moiety may optionally have a carbonyl group or heteroatom, such as O, S, or N in its backbone. Usually the backbone heteroatom is oxygen. In some embodiments, the substantially linear alkylene moiety is a lower alkylene having from 1 to 6 carbon atoms.
  • non-peptidic, non-polymeric aliphatic linker moieties include a C6 alkylene, 6-aminocaproic acid, tetra-ethylene glycol, and the like.
  • the molecular weight of the non-peptidic, non-polymeric aliphatic linker moiety is in the range of from about 14 daltons to about 2,000 daltons for L 1 , and more typically from 14 daltons to about 1,000 daltons for L 1 ; and from about 14 daltons to about 3,000 daltons for L 2 or L 3 , more topically from about 14 daltons to about 2,000 daltons for L 2 or L 3 , and usually from about 14 daltons to about 1 ,000 daltons for L 2 or L 3 .
  • Suitable oligonucleotide linkers employed as any one of L 1 , L 2 , or L 3 in the practice of the present invention may be of any composition, and are typically from about 1 to about 20 nucleotides long. L 2 and L 3 may also be a polynucleotide that is up to about 500 nucleotides long.
  • non-peptidic, polymeric linkers When non-peptidic, polymeric linkers are employed as L" and/or L . they typically have an average molecular weight of from about 200 to about 2,000 daltons. Typically, the average molecular weight is from about 300 to about 2,000 daltons.
  • the non-peptidic, polymeric linker is water soluble.
  • Suitable non-peptidic, polymeric linkers include polyalkylene oxides (e.g., polyethylene glycol, polypropylene glycol, and the like), polyvinyl alcohol, polyvinylpyrrolidone, and the like, as well as derivatives and copolymers thereof.
  • Polypeptide linkers that are employed as L 2 and L 3 linkers in the dual agonist compound typically function to increase the circulating serum half-life of the dual agonist compound in vivo and do not by themselves have detectable IL-3 or thrqmbopoeitin MpI receptor agonist activities as determined in the assays of Examples 3 and 4.
  • Suitable polypeptide linkers include the Fc region of the IgG subclass of antibodies, a serum albumin, such as human serum albumin, and the like, as well as variants and fragments thereof. Fusions of proteins with the Fc region of the IgG subclass of antibodies and with serum albumin are described, for example, in U.S. Pat. No. 7,030,226 and U.S. Pat. No.
  • polypeptide linkers employed in the practice of the present invention are resistant to proteolysis.
  • Polypeptide linkers may have an attachment group in their sequences, such as a Cys or Lys, that can be utilized for subsequent attachment of a non-polypeptide conjugation moiety. Glycosylation sites, which are discussed hereinbelow, may also be incorporated into the polypeptide linker sequence.
  • Dual agonist compounds of the present invention having peptide or polypeptide linkers can be readily expressed from a host cell without further processing using the methods described in Example 1. Dual agonist compounds having only peptide or polypeptide linkers are also referred to herein as "dual agonist polypeptides.” When the linker is not a peptide or polypeptide, the dual agonist compound can be ' prepared by separately expressing TM 1 and TM' and IL-3 as described below, then chemically linking each linker substituent to TM 1 , TM 2 , and IL-3. Cross-linking sites may also be incorporated into components of the dual agonist compound to facilitate linking the substituents together.
  • a cassette containing TM 1 , L 1 , TM 2 , L 2 (where L 1 and L 2 may be either peptide or non-peptide in nature) and a C-terminal thiol reactive or amine-reactive group may be synthesized.
  • An IL-3 encoding polynucleotide can be . modified to add a cross-linkable N-terminal or C-terminal residue (e.g., cysteine).
  • the recombinant IL-3 can then be expressed, refolded and cross-linked with the cassette to join all of the substituents together.
  • Both linkers in the dual agonist compounds may be present, or one or both of the linkers in the dual agonist compounds may be absent.
  • IL-3 mature recombinant human IL-3 polypeptide (SEQ ID NO: 7) and muteins thereof that bind to and activate IL-3 receptor as measured by the proliferation of the TF-I cell line (German Resource Centre for Biological Material, DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) No. ACC 334; and described in Kitamura, et al., Blood (1989) 73(2):375-380, which is incorporated herein by reference) in the assay of Example 3.
  • TF-I cell line German Resource Centre for Biological Material, DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) No. ACC 334; and described in Kitamura, et al., Blood (1989) 73(2):375-380, which is incorporated herein by reference
  • the phrase “exhibits IL-3 receptor agonist activity” refers to detectable proliferation of the TF-I cell line in the assay of Example 3.
  • Mature human IL-3 consists of 133 amino acid residues. It has one disulfide bridge and two potential N-glycosylation sites at the asparagines at positions 15 and 70. (Yang, et al. CELL, 47:3 (1986), which is incorporated herein by reference)
  • Suitable IL-3 muteins may have from 1 to 31 amino acid substitutions relative to mature recombinant hu-IL-3 (SEQ ID NO: 7) and/or deletions of from 1 to 14 amino acid residues from the N-terminus and/or 1 to 15 amino acid residues from the C-terminus.
  • IL-3 muteins employed in the practice of the present invention may have from 1 to 25 amino acid substitutions, and some may have from 1 to 15 amino acid substitutions.
  • Other suitable IL-3 muteins may have from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, or 14 amino acid substitutions.
  • IL-3 muteins have been described in the literature.
  • the human sequence for IL-3 was reported by Yang, et al. (Cell 47:3 (1986), which is incorporated herein by reference) as having a serine at position 8 of the mature protein sequence, others have reported the isolation of a proline at position 8 in the mature protein sequence for hJL-3.
  • These apparent allelic forms of hIL-3 may be employed, along with other muteins described in the prior art.
  • WO 88/04691 reports that Alal can be deleted from hIL-3, while still retaining biological activity.
  • This publication also describes other hIL-3 muteins, e.g., Alal Asp + Trpl 3Arg, Alal Asp + Met3Thr, and Alal Asp, Leu9Pro, and Trpl3Arg.
  • the numbering of the above substitutions is with reference to mature recombinant hIL-3 (SEQ ID NO: 7).
  • Additional IL-3 muteins suitable for use in the practice of the present invention are described in U.S. Pat. Nos.
  • IL-3 muteins having deletions at either the N or C terminus are well known in the art. These truncated forms of hIL-3 are suitable for use in the dual agonist compounds of the present invention.
  • U.S. Pat. No. 5,677,149 which is incorporated herein by reference, describes hIL-3 muteins in which amino acid residues 1 to 14 have been deleted from the N terminus.
  • IL-3 mutein comprising residues 15- 1 18 of mature recombinant hIL-3 (SEQ ID NO: 7) was demonstrated by Olins, P.O. et al. ((1995) J. Biol. Chem. 270:23754-23760) which is incorporated herein by reference, to retain IL-3 receptor agonist activity.
  • suitable IL-3 muteins employed in the dual agonist compounds of the present invention may have deletions of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid residues from the N-terminus and/or from 1 10 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 amino acids from the C-terminus.
  • IL-3 muteins employed in the dual agonist compounds of the present invention may have any combination of deletions and substitutions described herein.
  • IL-3 muteins can be readily identified by employing methods for generating variant libraries that are well known in the art. For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides (such as, for example, the polynucleotide encoding mature rhIL-3 (SEQ ID NO: 6), or polynucleotides encoding any of the muteins specifically described herein or in the literature) to generate variant libraries that can be expressed, screened, and assayed using the methods described herein.
  • polynucleotides such as, for example, the polynucleotide encoding mature rhIL-3 (SEQ ID NO: 6), or polynucleotides encoding any of the muteins specifically described herein or in the literature
  • Mutagenesis and directed evolution methods are well known in the art. See, e.g.. Ling, et al., "Approaches to DNA mutagenesis: an overview," Anal, Biochem., 254(2): 157-78 (1997); Dale, et al., “Oligonucleotide-directed random mutagenesis using the phosphorothioate method,” Methods MoI. Biol., 57:369-74 (1996); Smith, “In vitro mutagenesis,” Ann- Rev. Genet..
  • IL-3 muteins suitable for use in the practice of the present invention can be readily identified by assaying candidate IL-3 muteins for IL-3 receptor agonist activity in accordance with the assay described in Example 3.
  • IL-3 muteins employed in the practice of the present invention are those which have had one or more amino acid residues removed (e.g., by deletion or substitution) or introduced (e.g., by insertion or substitution) relative to mature recombinant hIL-3 (SEQ ID NO: 7), to facilitate conjugation of the dual agonist compound to a desired non-polypeptide conjugation moiety.
  • Exemplary non-polypeptide conjugation moieties are described in further detail hereinbelow under the heading "Dual Agonist Conjugates.”
  • attachment groups may also be used to optimize the distribution of the non-polypeptide conjugation moiety along the IL-3 backbone (these attachment sites may also be included in the TM 1 and TM 2 sequences and/or any peptide or polypeptide linkers that might be used in the invention dual agonist compounds). Attachment groups are typically removed or introduced by substitution of amino acid residues in the IL-3 sequence.
  • attachment groups in any of the IL-3, TM 1 , TM", peptide or polypeptide linker sequences may facilitate a more efficient, specific and/or extensive conjugation of a desired non-polypeptide conjugation moiety during a subsequent conjugation reaction.
  • the attachment group to be removed or introduced is selected based on the nature of the non-polypeptide conjugation moiety to be attached.
  • the attachment group may be cysteine, lysine, the N-terminal amino acid residue (i.e., via the terminal ⁇ -amino group), aspartic acid, glutamic acid, histidine, or arginine, or any combination of two or more thereof.
  • Lysine and cysteine are the typical attachment groups which are introduced or removed in the context of adding or removing attachment sites for conjugation to a non-polypeptide polymer in the IL-3 component of the dual agonist compounds.
  • IL-3 muteins having one or more introduced cysteine residues have been reported in U.S. Pat. No. 5,166,322, which is incorporated herein by reference, as retaining the biological activity of hIL-3.
  • the introduced cysteine residues provide attachment sites for sulfhydryl reactive compounds such as, for example, a polyalkylene glycol (including polyethylene glycol, polypropylene glycol, and the like) and derivatives thereof, dextran, colominic acids and other carbohydrate-based polymers, biotin, and the like.
  • sulfhydryl reactive compound refers to a compound that reacts and forms a covalent attachment to the sulfhydryl group (-SH) of a cysteine residue.
  • Sulfhydryl reactive compounds include non-polypeptide polymers such as, for example, polyalkylene glycols that have been activated by the addition of a sulfhydryl group, thiol, triflate, tresylate, aziridine or oxirane, s-pyridyl, or malemeide.
  • Specific IL-3 muteins having an introduced cysteine residue that are suitable for use in the practice of the present invention include those having cysteine substitutions or additions introduced within positions 1-14, inclusive, of the mature form of hIL-3, as described in U.S. Pat. No. 5,166,322, which is incorporated herein by reference.
  • IL-3 muteins include those having one or more of the following cysteine substitutions and substitution combinations relative to the mature form of recombinant hIL-3 (SEQ ID NO: 7): M3C, KlOC, T6C, S8C, S12C, M19C, KlOOC, T6C + KlOC, L9C + KlOC, T6C + S8C, T6C + S8C + KlOC, or S8C + L9C + KlOC.
  • IL-3 muteins employed in the dual agonist compounds of the present invention may also have a cysteine residue inserted after the C-terminus (e.g., 134C).
  • cysteine substitutions and insertions are described in U.S. Pat. No. 5,166,322, which is incorporated herein by reference. Cysteine residues may also be substituted in any one or more of the 8 C-terminal amino acid positions (i.e.,- 125C, 126C, 127C, 128C, 129C, 130C, 131C, 132C, and/or 133C).
  • Cysteine attachment sites may also be removed from mature recombinant hIL-3 (SEQ ID NO: 7) or mutein thereof by substituting another amino acid for the cysteine residues at position 16, 84, or both (with amino acid position numbering corresponding to SEQ ID NO: 7), or by deleting one or both of these cysteine residues.
  • Suitable IL-3 muteins include those having one or more lysine residues introduced or having one or more lysine residues removed from mature recombinant hlL- 3 (SEQ ID NO: 7) or mutein thereof.
  • the ⁇ amino groups of lysine provide convenient attachment sites for non-polypeptide polymers and other desirable non-polypeptide conjugation moieties having amine reactivity.
  • the lysine residues are thus attachment groups for amine reactive non-polypeptide conjugation moieties.
  • amine-reactive refers herein to a compound that reacts with and forms a covalent attachment to the amine group (-NH 2 ) of a lysine residue.
  • IL-3 muteins employed in the practice of the present invention may have one or more of the following substitutions relative to mature recombinant hIL-3 (SEQ ID NO: 7): R54K, R55K, R63K, R94K, R108K, R109K. and any combination of two or more substitutions thereof, all of which introduce an attachment site for an amine reactive non-polypeptide conjugation moiety; and KlOR, K28R, K66R, K79R, KlOOR, Kl 1OR, Kl 16R, and any combination of two or more substitutions thereof which function to remove an attachment site for an amine reactive non-polypeptide conjugation moiety.
  • Dual agonist compounds include those having an IL-3 mutein component (for example, an IL-3 polypeptide having an K- ⁇ R or an R->K substitution), where the IL-3 receptor activity (as measured by the assay of Example 3) is at least the same as or greater than the corresponding dual agonist compound having an IL-3 component that is mature recombinant hIL-3 (SEQ ID NO: 7) instead of an IL-3 mutein.
  • Suitable IL-3 muteins include those having an attachment site introduced or removed within the first 14 amino acid residues or last 8 amino acid residues of mature recombinant hIL-3 (SEQ ID NO: 7).
  • the lysine at position 10 in mature recombinant hIL-3 may be substituted with another amino acid (i.e., Ala, Arg, Asn, Asp, Cys, GIn, GIu, GIy, His, He, Leu, Met, Pbe, Pro, Ser, Thr, Tip, Tyr, or VaI) or deleted to remove it as an attachment site, or may be substituted with a cysteine residue to substitute a lysine attachment group for introduce a cysteine attachment group, Lysine or cysteine may be substituted for any of the other amino acid residues in these regions, i.e., AlalLys/Cys, Pro2Lys/Cys, Met3Lys/Cys, Thr4Lys/Cys, Gln5Lys/Cys, Thr6Lys/Cys, Thr7Lys/Cys, Ser8Lys/Cy
  • the attachment group is typically an in vivo glycosylation site, such as, for example, an N-glycosylation or an O-glycosylation site.
  • N-glycosylation site refers to the sequence N-X-S/T/C, wherein X is any amino acid residue except proline, N is asparagine and S/T/C is either serine, threonine or cysteine, and typically, serine or threonine, and preferably threonine.
  • S/T/C is either serine, threonine or cysteine, and typically, serine or threonine, and preferably threonine.
  • removal or introduction of an attachment group refers to an alteration in one, two, or all three of the amino acid residues that constitute an N-glycosylation site in order to introduce or remove a functional N-glycosylation site.
  • hIL-3 has two natural N-glycosylation asparagine attachment sites, N15 and N70.
  • IL-3 muteins suitable for use in the practice of the present invention include those having these attachment sites removed by substituting the asparagines at these positions for another amino acid, or alternatively, introducing a substitution at S 17 and/or S72 with an amino acid that is not threonine or cysteine.
  • suitable IL-3 muteins having introduced N-glycosylation sites that may be employed in dual agonist compounds of the present invention include those having a substitution selected from the following: P2N, T4N, Q5N, L9N, KlON, I20S/T, I23N, L40S/T, N41 S/T, E43S/T, L53S/T, R54S/T, A64S/T, V65N, I74N, L87N, F107S/T, Kl ION, Ll 15N, Q122S/T, Q124N, Q125N, T127N, or any combination of two or more thereof, where position numbering is based on correspondence with mature recombinant hIL-3 (SEQ ID NO: 7).
  • the ⁇ one or more introduced glycosylation sites are made by making one or more of the following substitutions: I20S/T, L40S/T, N41S/T, E43S/T, L53S/T, R54S/T, A64S/T, F107S/T, Q122S/T, or any combination of two or more thereof.
  • the substitution is I20T, L40T, N42T, E43T, L53T, R54T, A64T, F107T, Q122T, or any combination of two or more thereof.
  • one or more of the following substitutions is made: I20S/T, N41 S/T, E43S/T, L53S/T, Q122S/T, or any combination of two or more thereof.
  • the substitution is usually I20T, N41T, E43T, L53T, and/or Q122T.
  • two residues "downstream" from an existing S/T can be made to introduce a glycosylation site: P2N, T4N, Q5N. L9N.
  • the substitution is P2N, T4N, Q5N, L9N, KlON, L87N, Q124N, Q125N, and/or T127N. More typically, the substitution is P2N, T4N, Q5N, L9N, KlON, L87N, Q124N, Q125N, and/or Tl 27N.
  • Glycosylated dual agonist compounds comprising the IL-3 muteins described above typically have sugar moieties attached to the asparagine residues at any one or more of positions 2, 4, 5, 9, 10, 18, 23, 38, 39, 41 , 51, 52, 62, 65, 74, 87, 105, 110, 115, 120, 124, 125, and/or 127, respectively (with amino acid position numbering corresponding to that of SEQ ID NO: 7).
  • the introduced N-glycosylation site is not within about 20 amino acid residues from the C-te ⁇ ninal end of the IL-3 (e.g., an IL-3 with a substitution selected from I20S/T, L40S/T, N41S/T, E43S/T, L53S/T, R54S/T, or A64S/T). Usually, it is not within about 30, 40, and in some embodiments, 50 amino acid residues from the C-terminal end.
  • Desirable substitutions that create the third position of an N- glycosylation site in the IL-3 component include I20S/T, L40S/T, N41 S/T, E43S/T, L53S/T, R54S/T, and A64S/T, usually I20S/T, N41 S/T, E43S/T, and L53S/T, and more typically, I20S/T, N41 S/T, E43S/T, and L53S/T.
  • Desirable substitutions that introduce an asparagine to create an N-glycosylation site in the IL-3 component include P2N, T4N, Q5N, L9N, Kl ON, I23N, V65N, I74N, T4N, Q5N, L9N, or KlON.
  • Preferable substitutions of either type include I20S/T, N41S/T, E43S/T, L53S/T, P2N, T4N, Q5N, L9N, KlON, and particularly I20T, N41T, E43T, and L53T.
  • Illustrative TM'-L'-TM 2 combinations are provided as SEQ ID NOS: 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, and 308 (encoded by polynucleotides corresponding to SEQ ID NOS: 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281 , 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, and 307, respectively).
  • exemplary dual agonist compounds include those in which TM 1 and TM 2 each have a molecular weight of at least about 900 daltons and less than about 1 ,800 daltons, the linkers (either L 1 and L 2 for compound (T) or L 2 and L 3 for compound (H)) are both present and are peptide linkers, and the IL-3 is mature recombinant hIL-3 (SEQ ID NO: 7) or an IL-3 mutein having from 1 to 15 amino acid substitutions, and/or a deletion of 1 to 14 amino acid residues from the N-terminus, and/or a deletion of 1 to 8 amino acid residues from the C-terminus.
  • the substitutions in the IL-3 are those which either introduce or remove an attachment site for a non-polypeptide conjugation moiety.
  • Specific dual agonist compounds are illustrated in SEQ ID NOS: 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, and 362.
  • the present invention provides a method for identifying a dual agonist compound that exhibits a desired dual agonist activity, said method comprising: providing a library of peptide dimers having the structure
  • P 1 and P 2 are each independently non-naturally occurring peptides having a molecular weight of less than about 2,500 daltons, wherein L 1 is a linker that covalently links the C terminus of P 1 to the N terminus of P 2 , screening the library of peptide dimers to identify one or more peptide dimers having a desired thrombopoeitin c-Mpl receptor agonist activity, providing a library of IL-3 fusion compounds comprising one or more of the identified dimers of peptides fused to an IL-3 polypeptide, wherein each IL-3 fusion compound has the structure p i _ L' -P 2 - (L 2 ) m — IL-3 wherein m is 0 or 1 , wherein P 1 -L 1 -P 2 is the one or more identified dimers or peptides, wherein L 2 is a linker that covalently links the C terminus of TM 2 to
  • P 1 and P 2 are random peptides, which are described in more detail hereinabove.
  • P 1 and P may also be mutated variants of one or more random peptides previously selected by panning against immobilized human thromobopoeitin MpI receptor in a prior step.
  • P 1 and P 2 typically each exhibit thrombopoeitin c-Mpl receptor agonist activity in accordance with the assay of Example 4.
  • the molecular weights and typical lengths of P 1 and P 2 are the same as described above for TM 1 and TM 2 .
  • P 1 and P 2 may be the same or different in both sequence and length.
  • Suitable L 1 and L 2 linkers are the same as described for the dual agonist compounds described hereinabove.
  • linkers L 1 and L 2 may be the same or different from library member to library member.
  • Suitable IL-3 polypeptides are the same as described above for the dual agonist compounds.
  • the library of peptide dimers is screened using the proliferation assay of Example 4 to identify one or more peptide dimers having a desired level of thrombopoeitin c-Mpl receptor agonist activity. Those dimers having a desired level of thrombopoetin c-Mpl receptor agonist activity are subsequently employed in the construction of IL-3 fusion compounds using, for example, the methods described in Example 1 or other suitable methods. [077] With respect to the IL-3 fusion compound library, the sequence of the IL-3 polypeptide may differ among the IL-3 fusion compound library members.
  • the library of IL-3 fusion compounds can be screened using the proliferation assays of both Example 3 and Example 4 to identify a dual agonist compound that has the desired combination of thrombopoeitin c-Mpl receptor agonist activity and IL-3 receptor activity.
  • Dual agonist compounds of the present invention typically exhibit agonist activities with respect to either thrombopoeitin c-Mpl receptor or hJL-3 receptor that are from about 0.1 to about 5 or more fold more potent than that observed for native ligands recombinant human thrombopoeitin (SEQ ID NO: 373, encoded by the polynucleotide of SEQ ID NO: 372) and mature recombinant IL-3 (SEQ ID NO: 7) in the assays of
  • Examples 4 and 3 respectively (i.e., an EC 50 in either assay that is from about 10 times to about 0.2 or less times the ECJO with respect to either recombinant human thrombopoietin (SEQ ID NO: 373) or mature rhIL-3 (SEQ ID NO: 7) in their respective assays). It may be desirable in certain circumstances to utilize a dual agonist compound that is less potent than either or both rh-TPO (SEQ ID NO: 373) and/or mature rhIL-3 (SEQ ID NO: 7) with respect to agonizing the TPO c-Mpl receptor or hIL-3 receptor, respectively. For example, lower potency dual agonist molecules may be more suitable for use in an extended release composition or in the form of a dual agonist conjugate, which is discussed in more detail hereinbelow.
  • Dual agonist compounds of the present invention stimulate the proliferation of hematopoietic bone marrow stem cells in the assay of Example 8. These cells have both the human TPO c-Mpl and human IL-3 receptors. Typically, dual agonist compounds of the present invention stimulate the proliferation of more hematopoeitic stem cells in this assay than are stimulated by each individual growth factor, rhTPO (SEQ ID NO: 373) and mature rhIL-3 (SEQ ID NO: 7), as measured individually on an equimolar basis.
  • dual agonist compounds of the present invention stimulate the proliferation of more hematopoeitic stem cells in the assay of Example 8 than the sum of stem cells stimulated by each individual growth factor, rhTPO (SEQ ID NO: 373) and mature rhlL- 3 (SEQ ID NO: 7), as measured individually on an equimolar basis in the assay of Example 8.
  • rhTPO SEQ ID NO: 373
  • rhlL- 3 SEQ ID NO: 7
  • the phrase, "synergistic dual agonist activity” refers to a compound that stimulates the proliferation of more stem cells in the assay of Example 8 than the sum of combined individual effects of rhTPO (SEQ ID NO: 373) and mature rhIL-3 (SEQ ID NO: 7), i.e., as measured individually, in the assay of Example 8, where all are compared on an equimolar basis.
  • the present invention provides polynucleotides encoding invention dual agonist polypeptides.
  • Exemplary dual agonist polynucleotides include those corresponding to SEQ ID NOS: 309, 311 , 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, and 361.
  • polynucleotides encode the dual agonist polypeptides corresponding to SEQ ID NOS: 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, and 362 respectively.
  • Table I is a Codon Table that provides the synonymous codons for each amino acid.
  • the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine.
  • the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence. Table I: Codon Table
  • Dual agonist polypeptides of the present invention may be codon optimized for expression in a particular host organism by modifying the polynucleotides to conform with the optimum codon usage of the desired host organism.
  • Polynucleotides of the present invention can be prepared using methods that are well known in the art. Typically, oligonucleotides of up to about 50 to 120 bases are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form essentially any desired continuous sequence. Dual agonist polynucleotides of the present invention can be prepared by chemical synthesis using, for example, the classical phosphoramidite method described by Beaucage, et al. (1981) Tetrahedron Letters. 22:1859-69, or the method described by Matthes, et al. (1984) EMBO J,, 3:801-05, both of which are incorporated herein by reference.
  • oligonucleotides are synthesized, purified, annealed, ligated and cloned in appropriate vectors.
  • essentially any oligonucleotide can be custom ordered from any of a variety of commercial sources, such as, for example.
  • the Midland Certified Reagent Company Modland, TX
  • the Great American Gene Company Ramona, CA
  • ExpressGen Inc. Choicago, IL
  • Polynucleotides may also be synthesized by well-known techniques as described in, for example, Carruthers, et al., Cold Spring Harbor Syrnp. Quant.
  • Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by generating the complementary strand in a polymerase chain reaction using DNA polymerase with an appropriate primer sequence.
  • General texts which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other relevant topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, vol.
  • RNA polymerase mediated techniques e.g., NASBA
  • RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase as described in Ausebel, Sambrook, and Berger, supra.
  • the present invention also includes recombinant constructs comprising one or more of the dual agonist polynucleotides, which are described above.
  • nucleic acid construct refers herein to a nucleic acid, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature.
  • nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a dual agonist encoding polynucleotide sequence of the present invention.
  • the present invention also provides an expression vector comprising a dual agonist polynucleotide of the present invention operably linked to a promoter.
  • expression vector refers herein to a DNA molecule, linear or circular that comprises a segment encoding a dual agonist polypeptide of the invention, which is operably linked to additional segments that provide for its transcription.
  • expression includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • Example 1 provides a description of how to make expression constructs for expression of dual agonist polypeptides of the present invention.
  • Nucleic acid constructs of the present invention typically include a control sequence, such as a promoter.
  • control sequences refers herein to all of the components that are necessary or advantageous for the expression of a dual agonist polypeptide of the present invention.
  • Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide.
  • control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator.
  • the control sequences include a promoter and a transcriptional and a translational stop signals.
  • the control sequences may be provided with additional sequences that introduce specific restriction sites, which facilitate ligation of the control sequences with the coding region of the nucleotide sequence encoding the polypeptide.
  • operably linked refers herein to a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polypeptide.
  • coding sequence refers to a polynucleotide sequence that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon.
  • the coding sequence typically includes a DNA, cDNA, and/or recombinant polynucleotide sequence.
  • Dual agonist polynucleotides of the present invention can be incorporated into any one of a variety of expression vectors that are well known in the art. Suitable vectors include chromosomal, nonchromosomal, and synthetic DNA sequences.
  • Exemplary vectors include a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a plasmid, such as, for example, a bacterial plasmid or a yeast plasmid, a cosmid, a phage, vectors derived from viral DNA, such as, for example, vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses, and the like, as well as vectors derived from combinations of plasmids and phage DNA. Any vector that transduces genetic material into a cell, and if replication is desired, which is replicable and viable in the relevant host can be used.
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • plasmid such as, for example, a bacterial plasmid or a yeast plasmid, a cosmid, a phage
  • a polynucleotide of the invention When incorporated into an expression vector, a polynucleotide of the invention is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis, such as, for example, T5 promoter.
  • promoter known to control expression of genes in prokaryotic or eukaryotic cells or their viruses and which can be used in some embodiments of the invention include SV40 promoter, E. coli lac or trp promoter, phage lambda PL promoter, tac promoter, T7 promoter, and the like.
  • An expression vector optionally contains a ribosome binding site for translation initiation, and a transcription terminator, such as Pin II.
  • the vector also optionally includes appropriate sequences for amplifying expression, such as, for example, an enhancer.
  • the expression vectors of the present invention optionally contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Suitable marker genes include those coding for resistance to the antibiotic spectinomycin or streptomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycinn resistance.
  • Additional selectable marker genes include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance in E. coli.
  • Vectors of the present invention can be employed to transform an appropriate host to permit the host to express a dual agonist polypeptide, or the separate components of specific dual agonist compounds (e.g., TM 1 , TM 2 , TM'-L'-TM 2 (where L 1 is a peptide linker), IL-3, TM 2 -L 2 -IL-3 (where L 2 is a peptide or polypeptide linker), and the like).
  • appropriate expression hosts include bacterial cells, such as E. coli, B. subtilis, and Streptomyces. In bacterial systems, a number of expression vectors may be selected, such as, for example, multifunctional E. coli cloning and expression vectors.
  • Dual agonist polypeptides of the invention can also be fused, for example, in- frame to nucleic acids encoding a secretion/localization sequence, to target polypeptide expression to a desired cellular compartment, membrane, or organelle of a cell, or to direct polypeptide secretion to the periplasmic space or into the cell culture media.
  • sequences are known to those of skill in the art, and include secretion leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, endoplasmic reticulum (ER) retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization/anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.
  • the present invention also relates to engineered host cells that are transduced (transformed or transfected) with a vector or construct of the invention, as well as the production of polypeptides of the invention by recombinant techniques.
  • the term "host cell” refers to any cell type which is susceptible to transformation with a nucleic acid construct of the present invention. Therefore, the present invention includes host cells composing any polynucleotide of the present invention that is described hereinabove. Typically, the polynucleotide is operably connected to one or more promoters and/or enhancers that provide for expression of the polynucleotide of the host cell.
  • the host cell can be a eukaryotic cell, such as a mammalian cell, a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell (e.g., E. coli, Bacillus sp., and the like).
  • a prokaryotic cell such as a bacterial cell (e.g., E. coli, Bacillus sp., and the like).
  • Introduction of the nucleic acid construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation, gene ir vaccine gun, injection, or other common techniques (see, e.g., Davis, L., Dibner, M., and Battey, I.
  • a host cell strain is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "pre” or a "prepro” form of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as E.
  • coli Bacillus sp., yeast or mammalian cells such as CHO, HeLa, BHK, MDCK, HEK 293, WI38, and the like, have specific cellular machinery and characteristic mechanisms for such post- translational activities and may be chosen to ensure the correct modification and processing of the introduced foreign protein.
  • yeast or mammalian cells such as CHO, HeLa, BHK, MDCK, HEK 293, WI38, and the like, have specific cellular machinery and characteristic mechanisms for such post- translational activities and may be chosen to ensure the correct modification and processing of the introduced foreign protein.
  • Stable expression can be used for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express a polypeptide of the present invention are transduced using expression vectors which contain viral on gins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • Host cells transformed with a polynucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the polypeptide produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides encoding polypeptides of the invention can be designed to include signal sequences which direct secretion of mature polypeptides through a prokaryotic or eukaryotic cell membrane.
  • the present invention provides a method of making a dual agonist polypeptide having both IL-3 receptor agonist activity and thrombopoeitin MpI receptor agonist activity, said method comprising: culturing a host cell transformed with a dual agonist polynucleotide of the present invention under conditions suitable for expression of the encoded dual agonist polypeptide; and recovering the dual agonist polypeptide from the culture medium or from the transformed and cultured host cells.
  • the selected promoter is induced by appropriate means, such as, for example, by temperature shift or chemical induction, and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well known to those skilled in the art.
  • many references are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archebacterial origin.
  • Dual agonist polypeptides of the present invention can be recovered/isolated and optionally purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or solvent precipitation (such as, for example, by using a solvent like ethanol, acetone, and the like), acid extraction, ion (anion or cation) exchange chromatography, high performance liquid chromatography (HPLC), phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography.
  • ammonium sulfate or solvent precipitation such as, for example, by using a solvent like ethanol, acetone, and the like
  • acid extraction ion (anion or cation) exchange chromatography
  • HPLC high performance liquid chromatography
  • phosphocellulose chromatography hydrophobic interaction chromatography
  • affinity chromatography affinity chromatography
  • hydroxylapatite chromatography hydroxylapati
  • the present invention provides a method of producing a dual agonist polypeptide having both IL-3 receptor agonist activity and thrombopoeitin MpI receptor agonist activity, said method comprising: culturing a host cell transformed with a dual agonist polynucleotide of the present invention under conditions suitable for expression of a dual agonist polypeptide; recovering inclusion bodies comprising the encoded dual agonist polypeptide from the transformed and cultured host cells; solubilizing the recovered inclusion bodies comprising the encoded dual agonist polypeptide with a solubilizing agent; purifying the solubilized encoded dual agonist polypeptide; allowing the encoded dual agonist polypeptide to refold; and purifying the refolded dual agonist polypeptide.
  • Inclusion bodies are typically solubilized in solvent, such as, for example urea. Refolding can be accomplished by incubating solubilized dual agonist polypeptide in a solution of urea This process is illustrated in Example 2D-E The refolded dual agonist polypeptides of the present invention may be purified as described above and in Example 2E.
  • the present invention provides dual agonist conjugates comprising a dual agonist compound of the present invention covalently bound to at least one non-polypeptide conjugation moiety, either directly or indirectly via a linker moiety.
  • dual agonist conjugates have a dual agonist compound covalently bound to one or more of a non- polypeptide polymer, a sugar moiety, a non-polypeptide, non-polymeric lipophilic moiety, or any combination of two or more thereof.
  • non-polypeptide polymer refers herein to a water soluble polymer that may be a natural or synthetic polymer (homopolymer, copolymer, terpolymer, and the like), that is not a peptide, polypeptide, or protein.
  • the term "sugar moiety” refers to a carbohydrate molecule attached by an in vivo or in vitro glycosylation process, such as an N- or O-glycosylation process.
  • Non-polypeptide conjugation moieties are typically selected to alter specific attributes of the dual agonist compounds, such as, for example, in vivo serum half life or functional in vivo half life, stability, immunogenicity, and the like.
  • in vivo serum half-life refers to the time at which 50% of the compound of interest circulates in the bloodstream of a non-human mammal such as a rat, mouse, rabbit, or monkey.
  • the term "serum” is used herein to refer to its normal meaning, i.e., as blood plasma without fibrinogen and other clotting factors.
  • the term "functional in vivo half-life” refers herein to the time at which 50% of the biological activity of the compound of interest is still present in the body or target organ, or the time at which the activity of the compound of interest is 50% of the initial value.
  • the functional in vivo half-life may be determined in a non-human mammal, such as a rat, mouse, rabbit, dog, or monkey. Methods for determining both in vivo serum half-life and functional in vivo half-life are well known in the art.
  • dual agonist compounds and conjugates thereof may be administered to a non-human mammal, and blood samples collected at fixed time intervals.
  • the blood samples may be analyzed for levels of both platelets and dual agonist compound by a standard blood count and quantitative IL-3 ELISA, respectively.
  • the half life can be determined from a plot of dual agonist concentration versus time.
  • Dual agonist conjugates of the present invention typically exhibit greater functional in vivo half-life and/or greater serum half-life as compared to the corresponding non-conjugated dual agonist compound.
  • These dual agonist conjugates usually have a non-polypeptide polymer or a sugar moiety conjugated to the dual agonist compound.
  • each non-polypeptide polymer and/or sugar moiety may be conjugated either directly or indirectly via a linker, to the dual agonist compound.
  • the non-polypeptide conjugation moiety is typically bound, either directly or indirectly via a linker, to an attachment group in the dual agonist compound.
  • the non-polypeptide conjugation moiety may be bound to them, either directly or indirectly via a linker, via functional groups that are the same as the attachment sites described herein.
  • the term "greater” as it is used in connection with the functional in vivo half-life or serum half-life is used herein to indicate that the relevant half-life of the dual agonist conjugate is statistically significantly greater than a reference molecule, such as the corresponding non-conjugated dual agonist compound or a component thereof, when determined under comparable conditions.
  • dual agonist conjugates include those which have a functional in vivo half-life or a serum half-life that is greater than that of the corresponding non-conjugated dual agonist compound.
  • Dual agonist conjugates include those where the ratio between the funcational in vivo half-life (or serum half-life) of the conjugate and that of the corresponding non- conjugated dual agonist compound is at least 1.25, at least 1.5, at least 1.75, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8.
  • the dual agonist conjugate may exhibit greater bioavailability than the corresponding non-conjugated dual agonist compound.
  • An indication of bioavailability is provided by the "Area Under the Curve when administered subcutaneously" parameter or "AUCsc".
  • AUCsc or "Area Under the Curve when administered subcutaneously” is used in its normal meaning to refer to the area under the activity-in-serum vs. time curve, where the dual agonist conjugate has been administered subcutaneously to an experimental animal. Once the experimental activity time points have been determined, the AUCsc may conveniently be calculated by a computer program, such as GraphPad Prism 3.01, GraphPad Software Inc., San Diego, CA.
  • the term “greater” as it is used in connection with AUCsc is used to indicate that the Area Under the Curve for a dual agonist conjugate, when administered subcutaneously, is statistically significantly greater than the corresponding non- conjugated dual agonist or component thereof, when determined under comparable conditions.
  • Exemplary dual agonist conjugates include those in which the ratio between the AUCsc of the conjugate and the AUCsc of the corresponding non-conjugated dual agonist compound is at least 1.25, at least, 1.5, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 when administered under comparable conditions to the same species of experimental animal (e.g., rat, monkey, and the like).
  • the ratio between the AUCsc of the conjugate and the AUCsc of the corresponding non-conjugated dual agonist compound is at least 1.25, at least, 1.5, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 when administered under comparable conditions to the same species of experimental animal (e.g., rat, monkey, and the like).
  • Certain dual agonist conjugates of the present invention exhibit greater activity after a longer duration post-administration than the corresponding non-conjugated dual agonist compound. These dual agonist conjugates exhibit a Tmax that is greater than the Tmax for the corresponding non-conjugated dual agonist compound.
  • Tmax refers to the time point in the activity-in-serum vs. time curve where the highest activity in serum is observed.
  • the ratio of Tmax for this dual agonist conjugates to the Tmax of the corresponding non-conjugated dual agonist compound is at least 1.2, at least 1.4, at least 1.6, at least 1.8, at least 2, at least 2.5, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, when administered subcutaneously, in particular when administered subcutaneously in an experimental animal such as a rat or a monkey.
  • Polymers suitable for use in the practice of the present invention may be branched (i.e., having two or more linear polymer chains linked together by a linker group) or linear and typically have an average molecular weight in the range of from about 300 to about 100,000 daltons and typically from about 1,000 daltons to about 50,000 daltons, or from about 2,000 to about 30,000 or 20,000, or 10,000 daltons, and in some embodiments from about 1,000 daltons to about 5,000 daltons.
  • the polymer molecule will typically have an average molecular weight of about 2,000 daltons, 5,000 daltons, 10,000 daltons, 10,000 daltons, 12,000 daltons, 15,000 daltons, 20,000 daltons, 30,000 daltons, 40,000 daltons, or 50,000 daltons.
  • Exemplary polymers that are suitable for use in the conjugates of the present invention include polyalkylene oxides (PAO), such as a polyalkylene glycol (PAG) that may, for example, be a polyethylene glycol (PEG), a monomethoxypolyethylene glycol (mPEG), a polypropylene glycol (PPG), a branched polyethylene glycol having two or more polyethylene glycol chains linked together by a linker group (a linker, such as, for example, lysine, glycerol, and the like), polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone), polyethylene-co-maleic acid anhydride, a dextran (such as, for example, carboxymethyl dextran), and other like polymers.
  • PAO polyalkylene oxides
  • PEG polyalkylene glycol
  • PEG polyethylene glycol
  • mPEG monomethoxypolyethylene glycol
  • PPG polypropylene glycol
  • Polyalkylene glycol-derived polymers are typically employed in the conjugates of the present invention because they are generally biocompatible, non-toxic, non-antigenic, non-irnmunogenic, water soluble, and are easily excreted from living organisms.
  • Polyethylene glycol (PEG) in particular is favored because it has only a few reactive groups that are capable of cross-linking to other compounds, such as polysacchararides (e.g., dextran).
  • Monofunctional PEG such as, for example, a monomethoxypolyethylene glycol (mPEG), is particularly suitable for use in the conjugates of the present invention because its coupling chemistry is relatively simple. There is only one reactive group available for conjugating with the dual agonist compound.
  • the resulting population of dual agonist conjugates is more homogeneous with respect to having one PEG conjugated to only one attachment site.
  • the reaction of the polymer molecules with the dual agonist compound is also easier to control.
  • At least one terminal hydroxyl group of the polymer molecule is typically provided in activated form, i.e., derivatized with functional groups that are reactive with the target attachment group in the dual agonist compound.
  • Exemplary reactive functional groups include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl propnonate (SPA), succinimidyl carboxymethylate (SCM), benzot ⁇ azole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)).
  • HZ hydrazide
  • SUC succinate
  • SS succinimidyl succinate
  • SSA succinimidyl succinamide
  • SPA succinimidyl propnonate
  • SCM succinimidyl carboxymethylate
  • BTC benzot ⁇ azole carbonate
  • NHS N-hydroxysuccinimide
  • NPC nitrophenylcarbonate
  • TAS tresylate
  • the polymer molecules can be activated by conventional methods known in the art, such as those disclosed in WO 90/13540, which is incorporated herein by reference.
  • activated linear and branched polymer molecules that are suitable for use in the dual agonist conjugates of the present invention are described in the Nektar 2005-2006 Advanced Pegylation Catalogue, which is incorporated herein by reference.
  • activated polyethylene glycol polymers include the following linear PEGs: NHS-PEG (e.g., SPA-PEG, SSPA- PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PET, SG-PEG, and SCM-PET), and NOR- PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES- PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as mPEG2-NHS, disclosed in the Nektar 2005-2006 "Advanced Pegylation" Catalogue (which is incorporated herein by reference) and those disclosed in U.S.
  • NHS-PEG e.g., SPA-PEG, SSPA- PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PET, SG-PEG, and
  • activated polyethylene glycol polymers include the following PEGs: NHS-PEG (e.g., SUNBRIGHT ME-020(-050, -100, -20O)-CS (CH 3 O(CH 2 CHO) n -CO-CH 2 CH 2 -COO-NHS); SUNBRIGHT MEGC-20 (-5O)-HS and SUNBRIGHT MEGC-10(-20, -3O)-TS (CH 3 O(CH 2 CH 2 O) n -CO-CH 2 CH 2 CH 2 -COO- NHS); SUNBRIGHT ME-020 (-05O)-AS (CH 3 O(CH 2 CH 2 O) n -CH 2 -COO-NHS); SUNBRIGHT ME-050HS (CH 3 O(CH 2 CH 2 O) n -(CH 2 ) S -COO-NHS)); Aldehyde PEG (e.g., SUNBRIGHT ME-050 (-100, -200, -30O)-AL (CH 3 O
  • activated PEG polymers particularly preferred for coupling to cysteine residues include the following linear PEGs: Vinyl sulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG 9VS-mPEG); maleimide-PEG (MAL-PEG, as well as other maleimide PEGs described herein) preferably maleimide-mPEG (MAL-mPEG, as well as other maleimide mPEGs described herein) and orthopyridyl disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG).
  • PEG or mPEG polymers will have an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 12,000 daltons, about 20,000 daltons, about 30,000 daltons, or about 40,000 daltons.
  • the process of conjugating an activated polyethylene glycol to the dual agonist compound is referred to herein as "PEGylation”.
  • Covalent coupling of a polyethylene glycol moiety to the dual agonist compound can be targeted to a specific attachment site by selection of the appropriate activated polyethylene glycol and reaction conditions. These are well known in the art and are described in more detail hereinbelow.
  • the conjugation may be achieved in one step or in a stepwise manner using known methods, such as those described in WO 99/55377, which is incorporated herein by reference.
  • the polypeptide is usually treated with a reducing agent, such as dithiothreitol (DDT) prior to PEGylation.
  • DDT dithiothreitol
  • the reducing agent is subsequently removed by any conventional method, such as by desalting.
  • Conjugation of PEG to a cysteine residue typically takes place in a suitable buffer at pH 6-9 at temperatures varying from 4°C to 25°C for periods up to about 16 hours.
  • PEGylation of lysines often employs PEG moieties activated with active esters, such as, for example, N-hydroxysuccinimidyl (NHS) ester (e.g., mPEG-N- hydroxylsuccinimide (e.g., mPEG-NHS or mPEG2-NHS), esters such as PEG succinimidyl propionate (e.g., mPEG-SPA) or PEG succinimidyl butanoate (e.g., mPEG- SBA), and the like, including other activated PEG NHS esters described herein).
  • active esters such as, for example, N-hydroxysuccinimidyl (NHS) ester (e.g., mPEG-N- hydroxylsuccinimide (e.g., mPEG-NHS or mPEG2-NHS), esters such as PEG succinimidyl propionate (e.g., mPEG-
  • One or more PEG moieties can be attached to a protein within 30 minutes at pH 8-9.5 at room temperature if about equimolar amounts of PEG and protein are mixed. A molar ratio of PEG to protein amino groups of 1-5 to 1 will usually suffice. Increasing pH increases the rate of reaction, while lowering pH reduces the rate of reaction. These highly reactive active esters can couple at physiological pH, but less reactive derivatives typically require higher pH. Low temperatures may also be employed if a labile protein is being used. Under low temperature conditions, a longer reaction time may be used. [133] Covalent attachment of a PEG moiety to the N-terminal amino group of a polypeptide is referred to herein as "N-terminal PEGylation".
  • N-terminal PEGylation is facilitated by the difference between the pKa values of the ⁇ -amino group of the N- terminal amino acid (-7.6 to 8.0) and the ⁇ -amino group of lysine ( ⁇ 10).
  • PEGylation of the N-terminal amino group often employs PEG-aldehydes, which are more selective for amines and thus are less likely to react with the imidazole group of histidine; in addition, PEG reagents used for lysine conjugation may also be used for conjugation of the N- terminal amine.
  • Conjugation of a PEG-aldehyde to the N-terminal amino group typically takes place in a suitable buffer (such as, 100 mM sodium acetate or 100 ⁇ iM sodium bisphosphate buffer with 20 mM sodium cyan ⁇ borohydride) at pH ⁇ 5.0 overnight at temperatures varying from about 4°C to 25°C.
  • a suitable buffer such as, 100 mM sodium acetate or 100 ⁇ iM sodium bisphosphate buffer with 20 mM sodium cyan ⁇ borohydride
  • a PEG moiety may be conjugated to any one or more components of the dual agonist compound, i.e., TM 1 , L 3 , TM 2 , L 2 , and/or the IL-3.
  • the TM 1 , L 1 , TM 2 , L 2 , and IL-3 components may be designed to have one or more attachment groups (or functionality) that are suitable for reaction with the desired PEG moiety. Suitable attachment groups are described hereinabove.
  • At least one or more PEG moieties is conjugated to the IL-3 component of the dual agonist conjugate at one or more of the following attachment groups in the IL-3 (with reference to the amino acid position number corresponding to that of mature recombinant huIL-3 (SEQ ID NO: 7): KlO, C 16, K28, K66, K79, C84, KlOO, Kl 10, Kl 16, the ⁇ amino group on the N-terminal amino acid residue of IL-3 if that group is not bound to a linker moiety, and any combination of two or more thereof.
  • dual agonist conjugates of the present invention comprise a PEG moiety bound to a lysine attachment group in the IL-3 component selected from KlO, K28, K66, K79, KlOO, Kl 10, Kl 16, or any combination of two or more thereof.
  • dual agonist conjugates of the present invention comprise a PEG moiety bound to K28.
  • dual agonist conjugates of the present invention comprise a PEG moiety bound to KlO, K28, and K66 or KlO and K28.
  • dual agonist conjugates of the present invention comprise a PEG moiety bound to Kl O 5 K28, K66, K79, Kl 00, Kl 10, and Kl 16.
  • the dual agonist conjugate may have an IL-3 mutein component that has a substitution selected from Kl OR, K28R, K66R, K79R, Kl 0OR, Kl 1OR, Kl 16R or any combination of two or more substitutions thereof (where numbering is with reference to mature recombinant hIL-3 (SEQ ID NO: 7).
  • Dual agonist conjugates typically contain an IL-3 mutein with a substitution that is a substitution selected from KlOR or K28R.
  • reactive PEG attachment sites may be introduced into the IL-3 component of the dual agonist component as described above, for example in paragraphs 057-064.
  • Dual agonist conjugates typically have one or more polyethylene glycol moieties attached to a lysine residue in the IL-3 moiety of the dual agonist conjugate.
  • the polyethylene glycol moiety may be linear or branched, and typically has an average molecular weight of about 20,000 to about 40,000 daltons, usually about 30,000 to about 40,000 daltons, and often about 40,000 daltons.
  • cysteine pegylation methods are utilized, dual agonist conjugates of the present invention typically comprise a PEG moiety bound to the IL-3 component at the following attachment groups: C 16, C84, or both. PEG moieties may be bound to any combination of the aforedescribed attachment groups, as well as any introduced attachment groups described herein.
  • the polyethylene glycol moiety may be linear or branched, and typically has an average molecular weight of about 20,000 to about 40,000 daltons, usually about 30,000 to about 40,000 daltons, and often about 40,000 daltons.
  • dual agonist conjugates of the present invention also include conjugates having one or more sugar moieties (i.e., carbohydrate molecules) bound to the dual agonist conjugate. These conjugates are also referred to herein as "glycosylated” dual agonist compounds.
  • the glycosylation sites on the dual agonist compound are typically either an N- or O-glycosylation site.
  • N-glycosylation site refers to the sequence N-X-S/T/C", wherein X is any amino acid residue except proline, N is asparagines and S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine.
  • An "O-glycosylation site” refers herein to the —OH group of a serine or threonine residue.
  • Dual agonist compounds may be glycosylated by either an in vitro or in vivo process.
  • the carbohydrate(s) may be attached to the following attachment groups: a) arginine and histidine, as described in Lundblad and Noyes, "Chemical Reagents for Protein Modification", CRC Press me. Boca Raton, FI, which is incorporated herein by reference b) free carboxyl groups (e.g.
  • TGases transglutaminases
  • Dual agonist polypeptides of the present invention may be glycosylated in vivo by introducing a polynucleotide encoding a dual agonist polypeptide having one or more N- or O-glycosylation sites into a glycosylating eukaryotic expression host cell.
  • the glycosylation site may be one of the natural glycosylation sites in mature recombinant ML-3, SEQ ID NO: 7 (i.e., Nl 5 and N70, with amino acid position numbering corresponding to SEQ ID NO: 7), or a glycosylation site that is introduced into any of the components of the dual agonist polypeptide (i.e., TM 1 , TM 2 , L 1 , L 2 , L 3 , or the IL-3).
  • Substitutions in mature recombinant hIL-3 (SEQ ID NO: 7) that are useful for introducing one or more glycosylation sites into the IL-3 component of the dual agonist compound are described in more detail in the discussion of IL-3 muteins hereinabove.
  • the glycosylating eukaryotic expression host cell may be selected from a fungal cell (e.g., a filamentous fungal cell or a yeast cell), an insect cell , a mammalian cells, a plant cell, or any other glycosylating eukaryotic expression host cell known in the art.
  • dual agonist conjugates of the present invention comprise a sugar moiety bound to one or both of the natural glycosylation sites in mature recombinant hlL- 3 (SEQ ID NO: 7) or a mutein thereof which retains the two natural glycosylation sites, i.e., Nl 5 and N70 (with amino acid position numbering corresponding to SEQ ID NO: 7).
  • dual agonist conjugates of the present invention comprise one or more sugar moieties bound to one or more introduced glycosylation sites in the IL-3, TM 1 , L 1 , TM 2 , or combination of any two or more thereof.
  • These conjugates may further comprise a sugar moiety bound to one or both of the natural glycosylation (Nl 5 and N70) sites in mature recombinant hIL-3 (SEQ ID NO: 7).
  • sugar moieties When one or more sugar moieties is bound to an introduced glycosylation site in the IL-3, it is typically bound to an asparagine residue at one or more of the following amino acid positions corresponding to SEQ ID NO: 7: P2N, T4N, Q5N, L9N, KlON, Nl 8, 123N > N38, N39, N41, N51, N52, N62, N63, N72, L87N, N105, Kl ION, Ll 15N 5 N120, Q124N, Q125N, T127N, and any combination or two or more thereof (with substitutions shown relative to SEQ ID NO: 7).
  • IL-3 muteins having introduced glycosylation sites are described in more detail hereinabove.
  • Dual agonist conjugates of the present invention may also comprise a combination of one or more non-polypeptide polymers (such as, for example, PEG, mPEG, and the like) and one or more sugar moieties bound to the dual agonist compound.
  • Non-polypeptide lipophilic moieties that are suitable for conjugation to the dual agonist compounds of the present invention include a natural compound such as a saturated or an unsaturated fatty acid, a fatty acid diketone, a te ' rpene, a prostaglandin, a vitamin, a carotenoid or a steroid, a phospholipid, or alternatively, a synthetic compound, such as a linear or branched aliphatic, aryl, alkaryl acid (e.g., carboxylic, sulphonic, and the like), alcohol, amine, and the like.
  • Conjugation to non-polypeptide lipophilic moieties may take place at any one of the following exemplary attachment sites: the N- terminus or the C-terminus of the dual agonist compound, the hydroxyl groups of the amino acid residues Ser, Thr or Tyr, the ⁇ -amino group of Lys, the SH group of Cys or the carboxyl group of Asp and GIu.
  • the dual agonist compound and the non-polypeptide lipophilic moiety may be conjugated to each other either directly or indirectly via a linker in accordance with methods known in the art, such as those described in Bodanszky, "Peptide Synthesis", John Wiley, New York (1976) and WO 96/12505, both of which are incorporated herein by reference.
  • Non-polypeptide conjugation moieties may be bound, either directly or indirectly via a linker moiety, to any one or more components of the dual agonist compound, i.e., TM 1 , L 1 , TM 2 , L 2 , or IL-3.
  • Linker moieties that are suitable for conjugating the non- polypeptide conjugation moieties indirectly to the dual agonist compound include any of the linker types described hereinabove for L 1 and L 2 .
  • Other suitable linkers will be readily apparent to the skilled person.
  • the non-polypeptide polymer may be conjugated to the dual agonist compound via a cyanuric chloride linker as described in Abuchowski et al., (1977), J. Biol.
  • dual agonist conjugates for example, PEGylated with a branched or a linear PEG moiety of from 20 kDa to about 40 KDa
  • dual agonist conjugates may induce the production of at least about 2 times more platelets in a mammal (non-human or human) than that observed for the corresponding non-conjugated form of the dual agonist measured 6 days after intravenous administration of a single equivalent dose of the dual agonist conjugate or the non-conjugated form of the dual agonist, respectively.
  • Suitable dual agonist conjugates include those described hereinabove, and in particular the dual agonist conjugates having a dual agonist of formula I, and TMP 1 and TMP 2 moieties of sequence formula (1) or sequence formula (13), and PEGylated with a branched or a linear PEG moiety, for example a PEG having a molecular weight of from 20 kDa to about 40 kDa.
  • the foregoing dual agonist conjugates of the present invention may induce the production of at least about 2.5 and 3 times more platelets in a mammal, than that observed for the corresponding non-conjugated form of the dual agonist measured 6 days after intravenous administration of a single equivalent dose of the dual agonist conjugate or the non-conjugated form of the dual agonist, respectively.
  • Exemplary mammals include a rat (using the assay of Example 9 and a single dose of 100 ⁇ g/kg), a mouse, a monkey, a human, and the like.
  • the inventors have also discovered that dual agonist conjugates having one or more PEG moieties are more stable to proteolysis.
  • Biotinylated PEGylated dual agonist compounds (i.e., dual agonist conjugates) were incubated with rat plasma at 37 0 C overnight and the samples were analyzed by western blot probed with avidin-horse radish peroxidase to detect peptides cleaved from the molecules by proteases in the rat plasma. The results suggested that the bands detected as likely degradation products were less than 1% compared to the full length protein. Accordingly, the present invention provides dual agonist conjugates that are more resistant to proteolytic degradation in vivo as compared to the corresponding non-conjugated dual agonist compound. [150] Dual agonist conjugates may exhibit reduced immunogenicity as compared to hTPO.
  • the reduction may be on the order of at least 10%, 25%, 50%, or at least 75% as compared to hTPO.
  • Irnmunogenicity may be determined by use of any suitable method known in the art.
  • the term "reduced immunogenicity" is intended to indicate that the dual agonist compound or conjugate of the present invention gives rise to a measurably lower immune response than that of a reference molecule (such as, for example, hTPO, hIL3, or a fragment or variant thereof), as determined under comparable conditions.
  • Reduced antibody reactivity e.g., reduced reactivity towards antibodies present in serum from patients treated with, for example, hTPO or a fragment thereof, or hIL3 or a fragment thereof
  • Dual agonist conjugates of the present invention may exhibit reduced or no neutralization when contacted with antibodies or antisera isolated from patients treated with hTPO or a fragment thereof.
  • Neutralization may be evidenced by a decrease in biological activity (such as, receptor binding activity or agonist activity) of a test molecule when an antibody or antisera raised against a control molecule is incubated with the test molecule.
  • the test molecule is the conjugate of the invention and the control molecule is the hTPO or fragment thereof.
  • Reduced neutralization may be expressed as the percent difference between the decrease in biological activity of the test molecule and the decrease in biological activity of the control molecule and assayed under comparable conditions.
  • a conjugate of the invention may exhibit at least 25% reduced neutralization, such as at least 50% reduced neutralization, e.g., at least 75% reduced neutralization, for example at least 90% reduced neutralization, e.g., at least 95% reduced neutralization, relative to the neutralization observed when the hTPO or fragment thereof is contacted with antibodies or antisera isolated from patients treated with hTPO or a fragment thereof.
  • a polypeptide comprising the sequence of hTPO or a fragment thereof may exhibit reduced or no neutralization when contacted with antibodies or antisera isolated from patients treated with a conjugate of the invention, relative to the neutralization observed when contacted with antibodies or antisera isolated from patients treated with hTPO or a fragment thereof.
  • a polypeptide comprising the sequence of hTPO or a fragment thereof may exhibit at least 25% reduced neutralization, such as at least 50% reduced neutralization, e.g., at least 75% reduced neutralization, when contacted with antibodies or antisera isolated from patients treated with a conjugate of the invention, relative to the neutralization observed when the TPO or fragment thereof is contacted with antibodies or antisera isolated from patients treated with a conjugate of the invention, relative to the neutralization observed when the hTPO or fragment thereof is contacted with antibodies or antisera isolated from patients treated with hTPO or a fragment thereof.
  • the present invention also provides dual agonist derivatives comprising a dual agonist compound modified by an organic derivatizing agent.
  • the organic derivatizing agent is employed to alter an amino acid residue in the dual agonist compound.
  • Such derivatizing agents and methods are well known in the art.
  • cysteinyl residues most commonly are reacted with oc-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ -(4-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2- pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7- nitrobenzo-2-oxa-l,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Suitable reagents for derivatizing ⁇ -amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase- catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, and ninhydrin.
  • arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as wen as the arginine guanidino group.
  • the present invention provides a composition comprising a dual agonist compound of the present invention or conjugate thereof as described hereinabove, together with a pharmaceutically acceptable carrier or excipient.
  • Suitable pharmaceutically acceptable excipients include processing agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl- ⁇ - cyclodextrin, polyvinylpyrrolidone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof.
  • Other suitable pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences", 18 th edition, A.R. Gennaro, Ed., Mack Pub. Co.
  • compositions containing an invention dual agonist compound or conjugate thereof may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion.
  • Liquid carriers contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils and fats, and the like, as well as mixtures of any two or more thereof.
  • the liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, and the like.
  • Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols.
  • Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like.
  • the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like.
  • Compositions of the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.
  • the dual agonist compounds and conjugates of the present invention may be administered orally, parenterally, sublingually, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
  • Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoretic devices.
  • transdermal administration such as transdermal patches or iontophoretic devices.
  • parenteral as used herein includes subcutaneous injections, intravenous administration, intramuscular administration, intrasternal injections, transdermal or transmucosal administration, or infusion techniques.
  • Injectable preparations may be formulated using standard methods and materials known in the art, such as, for example, suitable dispersing, wetting, and suspension agents.
  • the sterile injectable preparation may also be a solvent, for example, as a solution in 1,3- propanediol.
  • the acceptable vehicles and solvents that may be employed are water. Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch.
  • Such dosage forms may also comprise additional substances other than inert diluents, such as, for example, lubricating agents (e.g., magnesium stearate), and the like.
  • the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
  • Liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in lipid form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. Typical lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic.
  • the present invention provides a method for stimulating platelet production in a human or non-human animal subject (e.g., a patient in need thereof), said method comprising administering to a subject an amount of dual agonist compound or conjugate thereof (or composition comprising such compound or conjugate) effective to stimulate platelet production in the subject.
  • the present invention also provides a method for stimulating cell proliferation comprising administering to the cell an amount of invention dual agonist compound, conjugate or composition thereof effective to induce proliferation of the cell.
  • Typical cells include platelets and/or megakaryocytes.
  • a composition of the invention may stimulate megakaryocyte production by inducing proliferation of early and late platelet progenitor cells and maturation of megakaryocytes to produce platelets.
  • a composition of the invention may increase the reservoir of platelet precursors and protect megakaryocytes from chemotherapy induced apoptosis.
  • compositions are useful in a number of therapeutic and/or prophylactic applications, primary of which is the ability to enhance platelet production in a subject, for example to prevent or reduce the duration of thrombocytopenia in a subject undergoing chemotherapy, without the risk of the subject generating neutralizing antibodies, for example, to endogenous hTPO.
  • Use of a composition of the invention may reduce or eliminate the need for platelet transfusions, in instances such as prior to, concurrent with or following myelosuppressive chemotherapy.
  • Thrombocytopenia is a blood disorder characterized by an abnormally low number of platelets in the bloodstream.
  • a normal platelet count in human blood is usually at least about 150,000 cells per microliter ( ⁇ l) of blood, with a range generally between about 150,000 and 350,000 platelets/ ⁇ l of blood.
  • the platelet number drops below 150,000/ ⁇ l, such as below 100,000/ ⁇ l, the patient is considered to be ' thrombocytopenic.
  • Patients with thrombocytopenia are predisposed to a risk of hemorrhage which increases with decreasing platelet levels.
  • Treatment for thrombocytopenia may be initiated when platelet counts are, for example, below 50,000/ ⁇ l, below 40,000/ ⁇ l, or below 30,000/ ⁇ l, such as in the range of 50,000/ ⁇ l to 20,000/ ⁇ l, e.g., in the range of 30,000/ ⁇ l - 20,000/ ⁇ l. At platelet levels below 10,000/ ⁇ l, spontaneous, and sometimes fatal, hemorrhage can occur.
  • Thrombocytopenia may occur as a result of decreased platelet production in the subject, increased platelet destruction in the subject, and/or other factors associated with platelet loss, and may be associated with a variety of diseases, disorders, treatments or other factors.
  • thrombocytopenia may arise from decreased platelet production, which, for example, may be associated with chemotherapy or radiation therapy, may be associated with intake of drugs or other substances (e.g., alcohol, thiazide diuretics), may be associated with a congenital or inherited disorder (such as May Hegglin Anomaly, Bernard Soulier syndrome, Alport syndrome), or may result from an acquired condition, such as a viral infection (for example, chicken pox, mumps, rubella) or bone marrow infiltration (e.g., associated with cancer).
  • a viral infection for example, chicken pox, mumps, rubella
  • bone marrow infiltration e.g., associated with cancer
  • Thrombocytopenia may occur as a result of increased platelet destruction.
  • platelet destruction may be associated with immune-related disorders (such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus), may be drug induced (e.g., heparin, depakote, quinine), may be associated with HIV, thrombotic thrombocytopenic purpura, hemolytic uraemic syndrome, disseminated intravascular coagulation, may be associated with physical trauma (e.g., cardiopulmonary bypass, artificial heart valve), or certain infections (such as mononucleosis, cytomegalovirus).
  • Thrombocytopenia may occur as a result of platelet loss associated with, for example, large volume transfusion or splenomegaly (e.g., cirrhosis of the liver, Gaucher's disease).
  • the invention includes a method of increasing the number of platelets in a subject suffering from thrombocytopenia or at risk for thrombocytopenia.
  • the method comprises administering an effective amount of a composition comprising a dual agonist polypeptide or conjugate thereof to the subject (e.g., a mammalian host) such that an increased number of platelets are produced in the subject compared to the number of platelets present in the subject prior to such administration.
  • the subject in need of such treatment may be suffering from thrombocytopenia associated with, for example, chemotherapy for treatment of cancer, AIDS, and the like, or radiation therapy, may be undergoing a bone marrow transplant procedure, or may be suffering from a disorder characterized by low platelet numbers, such as idiopathic thrombocytopenic purpura or thrombotic thrombocytopenic purpura.
  • a patient with a myeloid or nonmyeloid malignancy who is at risk for thrombocytopenia may be administered a composition of the invention prior to, concurrent with, or following the chemotherapy regimen.
  • a patient undergoing a bone marrow transplant procedure may be administered a composition of the invention prior to, concurrent with, or following the procedure.
  • composition of the invention may be administered prophylactically, for example, to a patient prior to or concurrent with the start of a course of chemotherapy or radiation therapy or prior to a procedure (such as, bone marrow transplant), to prevent or reduce the duration of thrombocytopenia known or suspected to be associated with such' therapy or procedure.
  • the composition of the invention may be administered therapeutically to a patient exhibiting a low platelet count (such as, a thrombocytopenic patient) to increase the number of platelets in the patient.
  • a low platelet count e.g., less than 100,000 platelets/ ⁇ l of blood, such as less than 50,000 platelets/ ⁇ l of blood, e.g., less than 30,000 platelets/ ⁇ l of blood, for example, between 20,000-30,000 platelets/ ⁇ l/blood
  • a low platelet count e.g., less than 100,000 platelets/ ⁇ l of blood, such as less than 50,000 platelets/ ⁇ l of blood, e.g., less than 30,000 platelets/ ⁇ l of blood, for example, between 20,000-30,000 platelets/ ⁇ l/blood
  • 100,000 platelets/ ⁇ l of blood such as to at least about 150,000 platelets/ ⁇ l of blood, e.g., to between about 150,000 and 350,000 platelets/ ⁇ l of blood.
  • Effective amounts of compounds, conjugates, and compositions of the invention generally include any amount sufficient to detectably stimulate platelet stimulation which can be assessed by a complete blood count (CBC) from whole blood drawn from the subject in question.
  • CBC complete blood count
  • Successful treatment of a subject in accordance with the invention may result in the inducement of a reduction or alleviation of symptoms in a subject afflicted with thrombocytopenia, chemotherapy-induced, idiopathic pupura (ITP), liver disease, bone marrow failure, myelodysplastic syndrome (MDS), and other similar disorders which are characterized by platelet deficiency.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the disorder. The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.
  • a therapeutically effective dose will generally be from about 0.1 ⁇ g/kg/day to about 10 ⁇ g/kg/day of a dual agonist compound or conjugate of the present invention, which may be administered in one or multiple doses.
  • the compounds and conjugates of the invention can be administered as the sole active pharmaceutical agent, they may also be used in combination with one or more other agents used in the treatment of other disorders, as well as thrombocytopenia.
  • the present invention provides combining a dual agonist compound or conjugate thereof, with a cytokine (such as, for example, stem cell factor (SCF), leukemia inhibitory factor (LIF), interferon- ⁇ , consensus interferon, IFN- ⁇ , IFN- ⁇ , an interleukin (e.g..
  • a cytokine such as, for example, stem cell factor (SCF), leukemia inhibitory factor (LIF), interferon- ⁇ , consensus interferon, IFN- ⁇ , IFN- ⁇ , an interleukin (e.g..
  • SCF stem cell factor
  • LIF leukemia inhibitory factor
  • interferon- ⁇ consensus interferon
  • IFN- ⁇ IFN- ⁇
  • IFN- ⁇ interleukin
  • IL-7, IL-8, IL-9, IL-IO, and the like and/or a hematopoietic factor (such as, for example, erythropoietin (EPO), granulocyte colony stimulating factor (G- CSF), and the like) and/or an angiopoeitin (such as, for example, angiopoeitin-1 (Ang-1), angiopoeitin-2 (Ang-2), and the like, the human angiopoetin-like polypeptide, and the like), and/or other therapeutically active agent in a pharmaceutical composition that is administered in accordance with the methods provided herein.
  • EPO erythropoietin
  • G- CSF granulocyte colony stimulating factor
  • an angiopoeitin such as, for example, angiopoeitin-1 (Ang-1), angiopoeitin-2 (Ang-2), and the like, the human angiopoetin-like polypeptide, and the like
  • the compounds and conjugates of the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower dosages. Dosage levels of the active compounds and conjugates in the compositions of the invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease, and the response of the patient.
  • the combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • Variants were designed with different amino acid substitutions within TM ' and TM 2 . Once the amino acid sequences were selected, DNA sequences encoding each TM 1 -L 1 -TM 2 component were designed using codons optimal for E. coli and mammalian expression. In addition, codons were selected at each position in order to avoid repeat sequences in TM 1 versus TM 2 . For example:
  • ATC GAA GGT CCA ACG CTT ACT CAG TGG TTG GCA GCC CGT GCA GGC GGTGGC TCCATT GAG GGC CCT ACC CTT AGT CAA TGG TTG GCC GCA CGT GCA (SEQ ID NO: 263);
  • ATC AGC GGT CCA ACG CTT CGT CAG TGG TTG GAA GCC CGT GCA GGC GGTGGC TCCATT GCG GGC CCTACC CTT CGT CAATGG TTG GAG GCA CGT GCA (SEQ IDNO: 275).
  • TM'-L'-TM 2 peptide sequences are provided as SEQ ID NOS: 264 and 276, respectively.
  • the first underlined section is TM 1
  • the sequence in italics is the linker, L 1
  • the second underlined sequence is TM 2 .
  • TM'-L ⁇ TM 2 Further exemplary nucleic acids encoding TM'-L ⁇ TM 2 are illustrated in SEQ ID NOS: 255, 257, 259, 261, 265, 267, 269, 271, 273, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, and 307.
  • the corresponding TM'-L ⁇ TM 2 encoded sequences are provided as SEQ ID NOS: 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, and 308, respectively.
  • Mature recombinant human IL-3 (SEQ ID NO: 7) was back translated using codon biases for E. coli and mammalian cells. The gene was designed and optimized based on E. coli codon biases and was predicted to be acceptable for mammalian expression as well. Overlapping 50-mers were assembled using PCR, and the full-length gene was rescued using terminal primers.
  • IL-3 gene get cct atg ace caa ace ace tec ttg aaa acg tec tgg gtc aat tgt tec aat atg ate gac gag ate ate act cac ttg aaa caa cct cct ttg cca ctt ctt gac ttc aat aac ttg aac ggt gag gac caa gat att ttg atg gag aat aat ttg cgt cgt cca aat ttg gaa get ttc aat cgt get gtc aaaagt ctt cag aac gcc tea gcc att gaa tct ate ctt aag aac ctt ctt cct tgtttt act
  • bridging oligos were designed to overlap with one end of the TM 1 -L 1 -TM 2 peptide sequence and one end of the IL-3 gene (for example, linking the 3' end of the TM '-L 1 -TM 2 peptide sequence to the 5' end of the IL-3 sequence).
  • the bridging oligos can also encode for an "in-frame" second linker "L 2 " comprising neutral amino acids such as Glycine, Serine, and Threonine to create a linker between the TM 1 - L'-TM2 peptide and IL-3 of lengths such as 6 or 12 amino acids residues (for example: GGGSGG (SEQ ID NO: 368) or GGGSGGGSGGGS (SEQ ID NO: 364).
  • L 2 comprising neutral amino acids such as Glycine, Serine, and Threonine to create a linker between the TM 1 - L'-TM2 peptide and IL-3 of lengths such as 6 or 12 amino acids residues (for example: GGGSGG (SEQ ID NO: 368) or GGGSGGGSGGGS (SEQ ID NO: 364).
  • Illustrative dual agonist polynucleotides are provided as SEQ ID NOS: 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, and 361.
  • dual agonist polynucleotides encode the dual agonist polypeptides corresponding to SEQ ID NOS-/310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348-, 350, 352, 354, 356, 358, 360, and 362 respectively.
  • the assembled nucleic acids encoding the dual agonist polypeptides were cloned into the pCR4 vector (Invitrogen, Carlsbad, CA) using a TOPO Cloning® kit and sequenced.
  • nucleic acids were subsequently subcloned into vector pCDNA3.1 (+) (Invitrogen, Carlsbad, CA) for protein expression in mammalian cells or subcloned into vector pCKQ3 ( Figure 2) for E. coli expression.
  • Mammalian expression constructs were made carrying these assembled genes under the transcriptional control of the eukaryotic CMV promoter and carrying an amino terminal TPA signal peptide for secretion.
  • Expression in the bacterial expression vector was under the transcriptional control of a T5 lac promoter and was restricted to the cytoplasm to favor production of inclusion bodies. Both mammalian and bacterial expression constructs were made with and without amino- terminal 6HIS tags for purification.
  • the Chinese hamster ovary cell line CHO-Kl (ATCC Accession No. CCL-61) was maintained in DMEM/F12 (GIBCO brand, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (GIBCO brand, Invitrogen, Carlsbad, CA) 3 1% penicillin/streptomycin/glutamine (PSG) (GIBCO brand, Invitrogen, Carlsbad, CA).
  • FBS fetal bovine serum
  • PSG penicillin/streptomycin/glutamine
  • E. coli strain W3110 E. coli Genetic Stock Center, New Haven, CT
  • plasmids encoding dual agonist polypetides as described in Example 1C.
  • Liquid cultures were inoculated from glycerol stocks into shake flasks containing 2XYT medium (EMD, Gibbstown, NJ) with 50 ⁇ g/ml kanamycin (Sigma, St. Louis, MO) and 0.5 % glucose (Sigma, St. Louis, MO). The culture was grown overnight at 37 0 C, shaking at 250 RPM.
  • the culture was then diluted 1 :3.5 into 2XYT medium with 50 ⁇ g/ml kanamycin and 2 mM PTG (Calbiochem, La Jolla, CA) for a four hour induction at 37 0 C and 250 JAPM.
  • the cells were harvested by centrifugation and resuspended in 7 ml PBS (GIBCO brand, Invitrogen, Carlsbad, CA) per pellet.
  • the cells were lysed using a French Press (Thermo Electron Corporation, Waltham, MA). Three passes were done at 1500 psi.
  • the inclusion bodies were harvested by centrifugation and washed twice with 1 % Triton X-IOO and once with water for irrigation (WFI).
  • inclusion bodies were solubilized at 40 mg inclusion body/ml 8M urea (JT Baker, Phillipsburg, NJ) in 1/2X PBS and incubated for one hour at room temperature. Insoluble material was removed by centrifugation.
  • the solubilized inclusion bodies were prepared for cation exchange purification by 1 :4 dilution with equilibration buffer, pH adjustment to 6.5 and filtration through a 0.2 ⁇ filter.
  • the material was loaded at ⁇ 7 AU/ml gel onto a SP Sepharose HP column (GE Healthcare, Piscataway, NJ) equilibrated with 25 mM sodium phosphate (Sigma, St. Louis, MO), 8 M urea, pH 6.5.
  • the protein was eluted using a 0 - 80 mM NaCl (Sigma, St. Louis, MO) 5 20 column volume gradient. [192] The eluate pool was refolded overnight at 0.1 mg/ml in 3 M urea with 1:10 oxidizingrreducing glutathione (Sigma, St. Louis, MO) (0.1:1 mM) atpH 8. The refold mixture was then adjusted to pH 6.5 and filtered using a 0.2 ⁇ filter. This material was loaded onto a SP Sepharose HP column equilibrated with 25 mM sodium phosphate, pH 6.5. The protein was eluted with a 0 - 150 mM NaCl, 30 column volume gradient.
  • the eluate pool was either concentrated to between 0.4 and 0.7 mg/ml and dialyzed against PBS, pH 6.5 or dialyzed against 50 mM sodium borate (Sigma, St. Louis, MO), pH 9 for a subsequent PEGylation reaction.
  • the TF-I cells were maintained in growth media A [RPMI 1640 (GIBCO brand, Invitrogen, Carlsbad, CA), 10% fetal bovine serum (FBS) (GIBCO brand, Invitrogen, Carlsbad, CA), 1 ng/ml GM-CSF (R&D Systems, Minneapolis, MN), 1 ng/ml penicillin/streptomycin (PS) (GIBCO brand, Invitrogen, Carlsbad, CA)] in a humidified incubator at 37 0 C and 5% CO 2 .
  • Ba/F3-Mpl Cells a murine IL-3 dependent pro-B cell line, was engineered to express human c-Mpl (SEQ ID NO: 2) in accordance with the protocol described in Abe, M., et al. (2002) Leukemia 16:1500-1506, which is incorporated herein by reference.
  • Ba/F3 was obtained from DSMZ 5 accession no.
  • ACC 300 and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 10 ng/ml penicillin/streptomycin (P/S) and 10 ng/ml recombinant mouse IL-3 (rmIL-3, R&D Systems, Minneapolis, MN)-
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • rmIL-3 recombinant mouse IL-3
  • cDNA encoding a c-Mpl was amplified by RT-PCR using MpI primers (5' TCAAG GCTGC TGCCA ATAGC TTAGT GG; SEQ ID NO: 368) and (g ( ATGCC CTCCT GGGCC CTCTT CATGG; SEQ ID NO: 369).
  • MpI primers 5' TCAAG GCTGC TGCCA ATAGC TTAGT GG; SEQ ID NO: 368) and (g ( ATGCC CTCCT GGGCC CTCTT CATGG; SEQ ID NO: 369).
  • the PCR fragment was cloned into the pCR4 TOPO vector (Invitrogen, Carlsbad, CA) and sequenced.
  • the cDNA encoding c-Mpl was subsequently subcloned into pCDNA3.1(+) (Invitrogen, Carlsbad, CA) along with an upstream Kozak sequence for optimal transcription.
  • Ba/F3 cells were transfected by electroporation withl5 ⁇ g of the pcDNA3.1+c-Mpl construct that had been linearized with Mfe I.
  • Transfected cells were selected in selection media B [RPMI 1640 (GIBCO brand, Invitrogen, Carlsbad, CA), 10% fetal bovine serum (FBS) (GIBCO brand, Invitrogen, Carlsbad, CA), 1 ng/ml purified, recombinant human TPO (rhTPO; PeproTech, Inc. Rocky Hill, NJ), 1 ng/ml penicillin/streptomycin (PS) (GIBCO brand, Invitrogen, Carlsbad, CA)and 1 mg/ml G418].
  • selection media B [RPMI 1640 (GIBCO brand, Invitrogen, Carlsbad, CA), 10% fetal bovine serum (FBS) (GIBCO brand, Invitrogen, Carlsbad, CA), 1 ng/ml purified, recombinant
  • a pool of cells resistant to G418 was sorted for single cells using flow-cytometric cell sorting. Individual clones were selected for robust growth in 1 ng/ml rhTPO (SEQ ID NO: 373) (PeproTech, Inc. Rocky Hill, NJ) and 1 mg/ml G418 (GIBCO brand, Invitrogen, Carlsbad, CA)]. Stable Ba/F3-M ⁇ l cell clones were evaluated based on their responsiveness to rhTPO (SEQ ID NO: 373) in the cell proliferation assay described below in part B.
  • SEQ ID NO: 373 PeproTech, Inc. Rocky Hill, NJ
  • G418 G418
  • Stable Ba/F3-M ⁇ l cell clones were evaluated based on their responsiveness to rhTPO (SEQ ID NO: 373) in the cell proliferation assay described below in part B.
  • Ba/F3-Mpl cells were maintained in growth media B [RPMI 1640 (GIBCO brand, Invitrogen, Carlsbad, CA) 5 10% fetal bovine serum (FBS) (GIBCO brand, Invitrogen, Carlsbad, CA), 1 ⁇ g/ml rhTPO (SEQ ID NO: 373) (PeproTech, Inc. Rocky Hill, NJ), 1 mg/ml G418 (GIBCO brand, Invitrogen, Carlsbad, CA), and 1 ng/ml penicillin/streptomycin (PS) (GIBCO brand, Invitrogen, Carlsbad, CA)].
  • RPMI 1640 GBCO brand, Invitrogen, Carlsbad, CA
  • FBS fetal bovine serum
  • 1 ⁇ g/ml rhTPO SEQ ID NO: 373
  • PS penicillin/streptomycin
  • EXAMPLE 5 PEGylation of Dual Agonist Polypeptides [196]
  • the dual agonist protein was concentrated to 4 mg/ml in 50 mM sodium borate, pH 9.
  • the protein was mixed with a 3X molar excess of 4OK branched PEG-NHS reagent (GL2-400GS2, NOF Corporation, Tokyo, Japan).
  • the reaction was incubated at room temperature for 1.5 hours.
  • the PEGylation mixture was buffer-exchanged into 50 mM sodium acetate (Sigma, St. Louis, MO), pH 5.5, using a PD-IO desalting column (GE Healthcare, Piscataway, NJ).
  • Size exclusion analysis was used to detect aggregates in the unPEGylated sample and unPEGylated or multiPEGylated species in the PEGylated sample. Five micrograms of protein was injected onto a TSK-GeI G3000SWXL size exclusion column (Tosoh Bioscience LLC 5 Montgomeryville, PA), which is run in PBS 5 pH 6.5 at 1 ml/minute.
  • the mobile phases used were, buffer A: 5% acetonitrile in 0.05% TFA/H2O and buffer B: 80% acetonitrile in 0.04% TFATH 2 O.
  • the peptides were resolved using a gradient fromlO-60% B over 45 min at the flow rate of 0.15 mL/min on an Agilent HP 1100.
  • the peptide identities were confirmed by on-line measurement of their mass using an ABI Q-TOF mass spectrometer.
  • the degree of PEGylation at each PEGylation site was calculated based on the ratio of chromatographic peak areas of peptides from the PEGylated vs unPEGylated sample.
  • Dual agonist polypeptides having the sequences of SEQ ID NOS: 318 and 33O 5 were prepared in accordance with the methods of Examples -1 and 2 using encoding • polynucleotide sequences corresponding to SEQ ID NOS: 317 and 329.
  • IL-3 receptor agonist activity and human thrombopoeitin MpI receptor agonist activity were assessed in accordance with the assays provided in Examples 3 and 4 and the data is provided in Table 3 below.
  • RhIL-3 (SEQ ID NO: 7) from CHO 115 pM [+/- 30 pM] rhTPO (Peprotech, SEQ ID NO: 373) — . 5O pM [+/- 3.7 pM]
  • the TF-I human erythroleukemia cell line proliferates in response to several growth factors, including human IL-3 and human TPO (Drexler H.G. and Quentmeier H. (1996) Leukemia 10(9): 1405- 1421, both incorporated herein by reference) and can be used to measure the bioactivity of both of these cytokines.
  • Dual agonist polypeptides isolated and purified from mammalian expression or E. coli expression were tested in the TF-I proliferation assay described in Example 3, above.
  • TF-I cells proliferated in the presence of dual agonist polypeptides and the hIL-3 control (SEQ ID NO: 7), but not in the presence of conditioned media controls.
  • TF-I cells proliferate in response to both hIL-3 and hTPO, the TF- 1 proliferative activity of the polypeptides of the invention could arise from signaling either through the hIL-3 receptor or through the hTPO c-Mpl receptor, or, through both of these receptors.
  • differential cytokine sensitivity and neutralization data suggest that the TF-I proliferative activity of the polypeptides of the invention is largely due to IL-3 receptor activation.
  • TF-I cells are approximately 3 log units more sensitive to IL-3 than to TPO.
  • Neutralizing antibodies directed against the alpha chain of the IL-3 receptor mAb rhIL-3 sRa, R&D Cat.
  • 203-IL reduced the TF- 1 cell proliferative activity of dual agonist polypeptides by 2 log units, suggesting that the TF-I cell proliferative activity of the polypeptides of the invention is primarily due to signaling through the IL-3 receptor.
  • BaF3-mpl cell line is a specific biological indicator for TPO agonism because the original naive BaF3 cell line from mouse (Abe, M., et al. 2002 Leukemia 16: 1500-1506, which is incorporated herein by reference ) does not proliferate in response to TPO or TPO mimetic peptides. Although the BaF3 cell line responds to species-specific IL-3 from mouse, the response to human IL-3 is at least 3 log units lower.
  • the cell line proliferates in response to human TPO or TPO mimetic peptides at sensitivities 3-4 log units more potent than the proliferative responses induced from human IL-3. Therefore, the BaF3-mpl proliferative response induced by dual agonist polypeptides is due to the TPO mimetic portion of the molecule and not due to human IL-3 portion of the molecule.
  • [206J Human or rhesus CD34+ hematopoietic bone marrow stem cells were treated as in section B for 10-14 days. Maturation to megakaryocytes was measured by staining for CD41 surface expression (a megakaryocyte-specific marker) and analyzed using FACS. Cells were pre-treated with purified human IgG (10 ⁇ L of 1 mg/mL/10 6 cells for 15 minutes at room temperature in order to block Fc-mediated interactions).
  • Dual agonist polypeptides exhibited agonist activities on human hematopoietic stem cells with respect to both human TPO c-Mpl and H-3 receptors that was greater than that observed for each single growth factor (human thrombopoietin or IL-3) or the theoretical additive effects of both human IL-3 and thrombopoietin in the bone marrow stem cell assay. Dual agonist polypeptides exhibited activities on human hematopoietic stem cells that were at least as potent as that observed with the equal molar mixture of human IL-3 and thrombopoietin.
  • Dual agonist polypeptides stimulated more stem cells to proliferate than equal concentrations of the single growth factors human thrombopoietin or IL-3. Dual agonist polypeptides were more potent than equal concentrations of human thrombopoietin or IL-3 at stimulating stem cells to mature into the megakaryocyte lineage as evidenced by more cells detected by FACS as expressing the cell surface marker CD41.
  • the IL-3 ELISA is a standard sandwich ELISA containing mouse monoclonal anti-human-IL-3 coating and detection antibodies.
  • the coating antibody is unconjugated and the detection antibody is conjugated with biotin.
  • High sensitivity of detection of the complex is achieved by exploiting the affinity of biotin to a streptavidin- horseradish peroxidase conjugate.
  • the quantity of active compound in each plasma sample may be measured by a semi-quantitative method using the TF-I cell and BaF3- mpl cell assays of Example 3 and 4.
  • Pharmacokinetic parameters such are the area under the curve (AUC) and half life (t ⁇ / ,) can be calculated from concentration-time profiles (concentration of dual agonist or dual agonist conjugate vs. time) observed by IL-3 ELISA. The platelet counts are plotted as a function of time. . . [211] While preferred embodiments of the invention have been illustrated and described, it will be readily appreciated that various changes can be made therein without departing from the spirit and scope of the invention. S

Abstract

The invention provides dual agonist compounds that exhibit agonist activity with . respect to both human thrombopoeitin c-Mpl receptor and human IL-3 receptor.. The invention provides dual agonist compounds that include dual agonist polypeptides, and also provides related nucleic acids, conjugates, vectors, cells, and methods of producing and using the dual agonist compounds.

Description

DUAL AGONIST COMPOUNDS AND USES THEREOF
[01] This application claims the benefit under 35 U.S.C. § 119(e) of USSN 60/752,924, filed December 21, 2005, and USSN 60/831,087, filed July 14, 2006, both of which are incorporated herein by reference.
FIELD OF THE INVENTION
[02] The present invention pertains to compounds having both human IL-3 receptor agonist activity and human thrombopoeitin MpI receptor agonist activity.
BACKGROUND OF THE INVENTION
[03] Decreased platelet counts (thrombocytopenia) can be caused by a number of disease processes. Patients undergoing cancer therapies such as chemotherapy and radiation are at risk for developing thrombocytopenia, which is a condition in which platelet counts are reduced to dangerous levels. Platelets are a critical component of hemostasis. Patients with thrombocytopenia are susceptible to complications such as bleeding, prolonged clotting times, and hemorrhaging. These complications can lengthen recovery time and postpone or reduce the efficacy of chemotherapy treatment. Intra- cranial and retroperitoneal bleeding are a major cause of death in hematological cancer patients. In view of these complications, thrombocytopenia can limit the maximum chemotherapy dose that may be administered. Akahori, et al., Stem Cells (1996) 14:678- 689. [04] Vitamin Bl 2 or folic acid deficiency, leukemia, or myelodysplastic syndromes are associated with a decrease in platelet production. Other diseases associated with a peripheral destruction of platelets can cause thrombocytopenia. Idiopathic thrombocytopenic purpura or ITP is an autoimmune disease in which the platelets are marked as foreign by the immune system and eliminated from the body. Thrombotic thrombocytopenic purpura, hemolytic-uremic syndrome, disseminated intravascular coagulation, paroxysmal nocturnal hemoglobinuria, and antiphospholipid syndrome are also associated with thrombocytopenia. Some commonly used drugs such as heparin may cause heparin induced thrombocytopenia. Chronic infection with hepatitis C or Dengue viruses can also lead to thrombocytopenia.
[05] Platelet transfusion has been the primary therapy for reducing the duration and severity of thrombocytopenia after cancer therapy. However, this may be accompanied by complications such as alloimmunization. A number of cytokines that increase platelet counts in normal animals have been evaluated for their efficacy on the duration and severity of thrombocytopenia induced by cancer chemotherapy and/or irradiation exposure in preclinical or clinical studies. These cytokines include interleukin 3 (IL-3), IL-6, and leukemia inhibitory factor. Amelioration of clinically important thrombocytopenia with these cytokines, however, has not yet been achieved. Id.
Interleukin- 1 1 has been approved for the treatment of thrombocytopenia. However, low efficacy combined with fatigue and cardiovascular symptoms are side effects that have been reported with the therapeutic administration of interleukin- 11 limit its use. Tepler, et al. Blood (1996) 87:3607-3614; Isaacs, et al. (1997) J. Clinical Oncology 15:3368- 3377.
[06] Platelets produced by megakaryocytes are derived from the differentiation of pluripotent stem cells in the bone marrow or spleen. Thrombopoietin (TPO) has been described as the hematopoietic factor that primarily regulates megakaryocytopoiesis and platelet production. In vivo administration of either glycosylated TPO or pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a pegylated truncated molecule related to TPO, was reported to increase circulating platelets. Shibuya, et al. Blood (1998) 91(l):37-45; Lok, et al., Nature (1994) 369-565. Marginal efficacy and notable side effects were reported in association with the use of these compounds. Vadhan-Raj, et al. J, Clin. Oncol. (2003) 21(16):3158-67; Schiffer et al.Blood (2000) 95(8):2530-5.
[07] Accordingly, a need exists for an alternative therapy for the treatment of thrombocytopenia. SUMMARY OF THE INVENTION
[08] The present invention provides dual agonist compounds that exhibit both IL-3 receptor agonist activity and thrombopoeitin MpI receptor agonist activity, wherein said compound comprises a structure of the following formula: TM1-(L')n-TM2-(L2)m-IL-3 (I) wherein TM1 and TM2 are non-naturally occurring peptides that each independently have a molecular weight of less than about 2,500 daltons and wherein each exhibits thrombopoeitin c-Mpl receptor agonist activity, wherein n and m are each independently 0 or 1 , wherein L1 is a linker that covalently links the C terminus of TM1 to the N terminus of TM2, wherein L2 is a linker that covalently links the C terminus of TM2 to IL-3, wherein the IL-3 is an IL-3 polypeptide that exhibits IL-3 receptor agonist activity, and wherein the dual agonist compound exhibits both IL-3 receptor agonist activity and thrombopoeitin c-Mpl receptor agonist activity.
[09] The present invention further provides dual agonist compounds that exhibit both IL-3 receptor agonist activity and thrombopoeitin c-Mpl receptor agonist activity, wherein said compound comprises a structure of the following formula: TM1 - (L2)m - IL-3 - (L\ - TM2 (II) wherein TM1 and TM2 are non-naturally occurring peptides that each independently have a molecular weight of less than about 2,500 daltons and wherein each exhibits thrombopoeitin c-Mpl receptor agonist activity, wherein m and p are each independently 0 or 1, wherein L2 is a linker that covalently links the C-terminus of TM1 to IL-3, wherein the IL-3 is an IL-3 polypeptide that exhibits IL-3 receptor agonist activity wherein L3 is a linker that covalently links IL-3 to the N-terminus of TM2, and wherein the dual agonist compound exhibits both IL-3 receptor agonist activity and human thrombopoeitin c-Mpl receptor agonist activity. [010] Also provided are dual agonist conjugates, dual agonist polynucleotides, vectors, and host cells, as well as related compositions and methods thereof. BRIEF DESCRIPTION OF THE FIGURES
[Oil] Figure 1 illustrates the steps in the platelet generation pathway, as well as the roles of cytokines interleukin-6 (IL-6), interleukin-11 (IL-1 1), interleukin-3 (IL-3), stem cell factor (SCF), and TPO in the pathway.
[012] Figure 2 is a 4323 base pair expression vector (pCKQ3-Dual Agonist) of the present invention comprising a CoIEl origin of replication, a T5 lac promoter, a lad repressor, a lambda to transcriptional terminator, an rmB Tl transcriptional terminator, and a kanamycin resistance gene (KanR).
DETAILED DESCRIPTION OF THE INVENTION
Dual Agonist Compounds
[013] The present invention is directed to novel compounds that exhibit agonist activity with respect to both human interleukin-3 (IL-3) receptor, which is expressed by the human erythroleukemia cell line, TF-I (Deutsche Sammlung von Mikroorganismen und Zellkultυren GmbH, DSMZ No. ACC 334) and the human thrombopoietin receptor, c- MpI (SEQ ED NO: 2), as determined by the assays of Examples 3 and 4, respectively. The human interleukin-3 receptor is a heterodimer of an IL-3 specific alpha chain (SEQ ID NO: 4) and a common beta chain (SEQ ID NO: 5) that is shared with the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5. Human thrombopoietin c-Mpl and IL-3 receptors are two of several receptors implicated in the platelet generation pathway, depicted in Figure 1. This pathway involves several different cytokines, including IL-6, IL-I l , IL-3, stem cell factor (SCF), and thrombopoeitin (TPO). [014] More specifically, the present invention is directed to dual agonist compounds that exhibit both human IL-3 receptor agonist activity and human thrombopoeitin MpI receptor agonist activity, wherein said compound comprises a structure of the following formula:
TM1-(L1)n-TM2-(L2)m-IL-3 (I) wherein TM1 and TM2 are non-naturally occurring peptides that each independently have a molecular weight of less than about 2,500 daltons and wherein each exhibits thrombopoetin c-Mpl receptor agonist activity, wherein n and m are each independently 0 or 1 , wherein L1 is a linker that covalently links the C terminus of TM1 to the N terminus of TM2, wherein L2 is a linker that covalently links the C terminus of TM2 to IL-3, wherein the IL-3 is an IL-3 polypeptide that exhibits IL-3 receptor agonist activity, and wherein the dual agonist compound exhibits both IL-3 receptor agonist activity and thrombopoeitin c-Mpl receptor agonist activity.
[015] In another embodiment, the present invention provides dual agonist compounds that exhibit both human IL-3 receptor agonist activity and human thrombopoeitin c-Mpl receptor agonist activity, wherein said compound comprises a structure of the following formula:
TM1 - (L2)m - IL-3 - (L3)p - TM2 (II) wherein TM1 and TM2 are non-naturally occurring peptides that each independently have a molecular weight of less than about 2,500 daltons and wherein each exhibits thrombopoeitin c-Mpl receptor agonist activity, wherein m and p are each independently 0 or 1 , wherein L2 is a linker that covalently links the C-terminus of TM1 to IL-3, wherein the IL-3 is an IL-3 polypeptide that exhibits IL-3 receptor agonist activity wherein L3 is a linker that covalently links IL-3 to the N-terminus of TM2, and wherein the dual agonist compound exhibits both human IL-3 receptor agonist activity and human thrombopoeitin c-Mpl receptor agonist activity.
[016] The terms "thrombopoeitin MpI receptor agonist activity",. "human thrombopoeitin MpI receptor agonist activity", "TPO MpI receptor agonist activity", and "human TPO MpI receptor agonist activity" are all used interchangeably herein to refer to the ability to induce the proliferation of a murine IL-3-dependent pro-B cell line, Ba/F3- MpI (DSMZ No. ACC 300), that has been transformed to express the human thrombopoeitin MpI receptor (SEQ ID NO:2) as detected in the assay of Example 4. The terms "IL-3 receptor agonist activity, " "interleukin-3 receptor agonist activity," "human IL-3 receptor agonist activity," and "human interleukin-3 receptor agonist activity" are used interchangeably herein to refer to the ability to induce the proliferation of human erythroleukemia TF-I cells (DSMZ No. ACC 334) which express human IL-3 receptor as detected in the assay of Example 3.
[017] The terms "polypeptide" and "peptide" refer herein to a polymer of amino acid monomers joined together through amide bonds. The term "peptide" refers to a relatively short polymer of up to about 30 amino acid monomer units. [018] TM1 and TM2 are thrombopoeitin mimetic peptides that may be the same or different with respect to both sequence and length. Neither TM1 nor TM2 are human thrombopoeitin, yet each exhibits human thrombopoeitin MpI receptor agonist activity. [019] Peptides employed in the practice of the present invention as TM1 and TM2 each have a molecular weight of at least about 800 or 900 daltons, and less than about 2,500 daltons. Typically, TM1 and TM2 are each independently peptides having a molecular weight of less than about 2,000 daltons, and more typically less than about 1 ,800 daltons. In some embodiments, TM1 and TM2 are each independently peptides having a molecular weight of less than about 1 ,500 daltons. TM1 and TM" may each independently have up to about 25 amino acid residues, and in other embodiments.up to about 20 amino acid residues, and sometimes between about 9 and 25 amino acid residues in length, e.g., between about 10 and 20 amino acid residues in length. Suitable peptides for TM1 and TM2 include peptides having up to about 10, 1 1, 12, 13, 14, 15, 16, 17, 18, or 19 amino acid residues. Suitable peptides employed in the practice of the present invention typically have at least about 9 amino acid residues. Illustrative TM1 and TM2 peptide sequences are provided in SEQ ID NOS: 8-254 and 374-378. [020] Exemplary TM1 and TM2 peptides include those having any of the following sequences:
I _ χ2 _ G - P - T - L - X7 - X8 - X9 (1)
wherein X2 is E, R, H, Q, T, A, G, or S; X7 is R, S, or T; Xs is A, D, Q, S, or T; and X9 is A, C, or W (see, for example, SEQ ID NOS: 89-120 and 374); X2~X3-X4-X5-Xδ-X7-Xs-X9-X10 (2) wherein X2 is D, G, K, R, S, or T; X3 is L or V; X4 is K, R, or W; X5 is D, E, G5 or S; X6 is C, Q, or T; X7 is G, 1, or V; Xs is A, K, L5 M, S5 W, or Y; X9 is A, G, I, K, L, M, Q, R, W, or Y; and X10 is A, C, G, H, L, S, W, or Y (see, for example, SEQ ID NOS: S-23);
χ> -_v-R-X4-X5-X6-X7-Xs-X9-X10~X" (3)
wherein X1 is G, R, S, or T; X4 is D, E, or Q; X5 is Q or V; X6 is I or V; X7 is C, D, E. G, K, N P, R, or S; Xs is A, I, K, L, M, S, W or Y; X9 is F, H, R, W, or Y; X10 is F, L5 M5 V,
W, or Y; and X1 ' is A, C5 F, H, I, L, M, S, or V (see, for example, SEQ ID NOS: 24-53) ;
X< - X* _ X6 _ X7 _ χ8 _ χ9 _ χl0 _ χl 1 _ χ12 _ χ13 _ χH.χ15 (4)
wherein X4 is W, T, Q, or C; X5 is N, G, S, T, or A; X6 is L, V, or R; X7 is T, R, K, A. H, S, or N ; X8 is E, S, D, Q, G, or K; X9 is F, V, Q; W, Y, L, or M; X10 is V, R, K, L, or I; X" is L5 D, Q5 A, V, K, I, G, S, or R; X12 is D, Q, G, R, L, Q, or K; X13 is T, V, C, D, N, E, G, M, H, or A; X14 is H, C, T5 R5 A, or N; and X15 is P, A, W, G, T, V, C, L, F, D5 Y, or R (see, for example, SEQ ID NOS: 54, 59, 61, and 63-76);
X3-X4-X5-X6-X7-X8-W-X10 (5)
wherein X3 is G5 R5 or S; X4 is E, M, P, Q, or R; X5 is H, Q, R, S, or T; X6 is C, L, P5 or W- X7 is A, F5 K5 M5 R, S, or V; Xs is E, G1 M, P, Q. S, or T; and X10 is L or M (see, for example, SEQ ID NOS: 78-88);
χ'_χ2_G-C-X5-L-X7-X8-W-X10-X1!-G-X13-C (6)
wherein X1 is F, I, L, P, or Y; X2 is E, H, K5 L, Q, R, S, W, or Y; X5 is R or T; X7 is K or R; X8 is A, S, or V; X!o is L, R, or S; Xn is A or G; and X13 is I, L, M, or V (see, for example, SEQ ID NOS: 121-140); X'-X2-T-X4-X5-Xδ-X7-X8 (7) wherein X ' is G, S, or Y; X2 is C, L, or P; X4 is F1 L, or V; X5 is K, P, Q, R, or S; X6 is D, E, H, Q, or Y; X7 is C, F, L, or W; and Xs is I, K, L1 M. R, or V (see, for example SEQ ID NOS: 141-164);
C - X2 - X3 - X4 - X5 - X6 - X7 - X8 - X9 - C (S)
wherein X2 is N, Q, R, S, or T; X3 is C, F, I1 L, or R; X4 is A, E, G, H, K, N, Q, R, or S: X5 is D, E, or Q; X6 is F, L, V, or Y; X7 is I, K, L, M, N. R, or V; Xs is C, F. I, M, P, T, V, W, or Y; and X9 is A, C, F, G, I, L, Q1 R, or S (see, for example, SEQ ID NOS: 165- 188);
X1 - X2 - X3 - X4 - X5 - X6 - X7 - X8 - X9 (9)
wherein X1 is C or E; X2 is R or T; X3 is F, G, L, R, or S; X4 is A, G, P, Q, R, S, or T; X5 is E, F, Q, or S; X6 is F or W; X7 is A, K, L1 or R; X8 is D, E, G, H, K, L, Q, R, or S; and X9 is A, C, D, G, or R (see. for example, 189-203);
C-X2-X3-G-X5-X6- X7-Xs-X9-W-X' '-Xl2-X13-C (10)
wherein X2 is A, G, L, M, R, or S; X3 is A, D, E, Q, T, or absent; X5 is L or P ; X6 is T, S, or F; X7 is L or V; X8 is R1 T, or L; X9 is E, P1 Q, or A; X1 1 is L1 M1 or I; X12 is Y1 L1 T T,, EE11 SS11 oorr aabbsseenntt;; aanndd ? X13 is V, L1 E, H, F1 or absent (see, for example, SEQ ID NOS: 205-206 and 209-215);
C-x2-x3-x4-x5-x6-x7-x8-x9-x10-x1'-x12-x13-C (11)
wherein X2 is D1 K1 S1 T, or V; X3 is F1 L, or M; X4 is A1 K1 L. Q1 R1 or S; X5 is D, E1 or Q; X6 is F1 L1 W, or Y; X7 is K or L; X8 is A1 D, E1 H1 L, M1 Q1 S, T1 or V; X9 is S1 N1 F, Y, L1 H1 A1 or P; X10 is G, H, S1 or T; X1 1 is E1 G1 L1 M1 T, V, or Y; X12 is A, E1 G, M1 N1 S, V, or Y; and X13 is A, E, L, Q, S, V, W, or Y (see, for example, SEQ ID NOS: 219- 231); and
C - X2 - X3 - X4 - Xs - X6 - X7 - X8 - X9 - X10 - X11 - C (12)
wherein X2 is L, R, S, or T; X3 is F or L; X4 is G, K, L, M, Q, R, or S; X5 is A, D, E, Q5 S, or V; X6 is F or W; X7 is I, K, L, or V; Xs is C, I, K, N, P, T, W, or Y; X9 is A, D, E, G, L, M, N, S, W, or Y; X10 is A, E, G, H, L P, Q, R, S, or W; and X1 ' is E, H, I, K, R, or Y (see, for example, SEQ ID NOS: 232-254). [021] TM1 and TM2 peptides comprising a sequence of formula (1) typically have X2 = A, E, R, or S; X7 = R, and X9 = W. Some TM1 and TM2 peptides having a sequence of formula (1) have a sequence corresponding to formula (13):
I - X2 - G - P - T - L - X7 - Xs - X9 - L - X1 ' - X12 - R - A (13)
wherein X2, X7, Xs, and X9 are as descπbed for formula (1), X1 1 is A, D, or E and X12 is
A or E. Typically, X2 is A, E, R, or S; X7 is R; X9 is W; X1 1 is E or A; and X12 is A.
[022] TM1 and TM2 peptides comprising formula (2) typically have X2 = R, S, or G; X3
= V; X4 = R; X5 = D or E; X6 = Z; X7 = V or I; X8 = A or M; X9 = A or L; and X10 = W, L, or S.
[023] TM1 and TM2 peptides having the a sequence of formula (2) may have a sequence corresponding to formula (14):
X1 - X2 -X3 -X4 - X5 - X6 - X7 - X8 -X9 - X10 - X1 ' (14)
wherein X1 is C, G, E, A, L, or S; X2-X10 are as described for formula (2); and X11 is C,
L, M, or H. Typically, X1 is G; X2 is R; S, or G; X3 is V; X4 is R; X5 is D or E; X6 is Q;
X7 is V or I; X8 is A or M; X9 is A or L; X10 is W, L, or S; and X" is L.
[024] TM1 and TM2 peptides comprising a sequence of formula (3) typically have X1 = S; X4 = E or D; X5 = Q; X6 = V; X7 = V, F, M, A, Y, W, or I; X8 = A or L; X10 = L; and
X" = A, S, or C. [025] TM1 and TM2 peptides comprising a sequence of formula (4) typically have X4 = C; X5 = S or T; X6 = L or R; X7 = R or A; Xs = E, D, or Q; X9 = W; X10 = L, V, or K; X11 = L, A, Q or G; X12 = G or R; and X14 = C. Some TM1 and TM2 peptides having a sequence of formula (4) have a sequence corresponding to formula (15):
χ3_χ^_χ5_χό_χ7_χS_χ9_χ]0_χπ_χl2_χl3_χl4_χl5 (J5)
wherein X3 is Q, R, or G and X4-X15 are as described for formula (4). Typically, X3 is G. [026J In some embodiments, formula (4) peptides employed in the practice of the present invention have a sequence corresponding to formula (16):
X' _χ2_χ3_X^_χ5_χ6_χ7_χ8_χP_χl0_χM_χ.2_χ.3_χ14_χl5 (j6)
wherein X1 is V, S, G, R, C, N, Y, W, or K, X2 is R, G, Y, O, or S, and X3-X15 are as described for formulas (4) and (15).
[027] TM1 and TM2 peptides comprising a sequence of formula (5) typically have X3 = G; X4 = P; X5 = T; X6 = L; X7 = R; and X10 = L. Some TM1 and TM2 peptides comprising a sequence of formula (5) have a sequence corresponding to formula (17):
XI-X2-X3-X4-X5-X6-X7-XS-W-X10 (17)
wherein X1 is S, R, L, or T, X2 is V, E, or R, and X3 -X10 are as described for sequence
(5). Typically, X1 is R or S and X2 is E.
[028] TM1 and TM2 peptides comprising a sequence of formula (6) typically have X1 = L; X2 = Q; or K; X5 = T; X7 = R; X8 = A; X10 = A; and X13 = M or I. TM1 and TM2 peptides comprising a sequence of formula (7) typically have X1 = G; X2 = P or C; X4 = L; Xs = R; X6 = Q or E; X7 = W; and Xs = L or V. TM1 and TM2 peptides comprising sequence (8) typically have X2 = S or T; X3 = L or R; X4 = S, E, A, or R; X6 = L or F; X7 = R or L; Xs = R or A; and X9 = G. TM1 and TM2 peptides comprising a sequence of formula (9) typically have X1 = C; X2 = T; X3 = R or L; X4 = G, T, R, or Q; X5 = E or Q; X6 = W; X7 = L or K; and X9 = G. TM1 and TM2 peptides comprising a sequence of
IU formula (10) typically have X2 = A or R; X5 = P; X6 = T; X7 = L; X8 = R or T; X9 = E or ■ Q; and X1 1 = L. TM1 and TM2 peptides comprising a sequence of formula (1 1) typically have X2 = T or S; X3 = L; X4 = R, S, or Q; X5 = E; X6 = F; X7 - L; and X10 = G. TM1 and TM2 peptides comprising a sequence of formula (12) typically have X2 = T; X3 = L; X4 = R; X5 = E or Q; X6 = W; X7 = L; X9 = G or D; X10 = G; and X1 ' = T.
[029) Suitable TM1 and TM2 peptides include any of the thrombopoietin mimetic peptides corresponding to SEQ ID NOS: 8-254 and 374-378 in homo- (i.e., TM1 and TM2 being identical) or hetero-dimer (i.e., TM1 and TM2 being different) format. TM1 and TM2 peptides contemplated for use in the invention dual agonist compounds include the specific peptides exemplified herein as well as equivalent peptides that may be somewhat longer or shorter than the peptides specifically recited herein. For example, the skilled artisan could readily make peptides having from about 1 to about 15 or more amino acid residues added to, or removed from either end of the disclosed peptides using standard techniques known in the art. [030] Other suitable TM1 and TM2 peptides may be readily identified by generating libraries of randomized peptides that are then subjected to affinity enrichment using immobilized human thrombopoeitin MpI receptor (SEQ ID NO: 2). Methods for generating randomized peptides of a desired length followed by identification of those peptides that bind to receptor molecules of interest are well known in the art and are described in references such as U.S. Pat. No. 5,723,286, U.S. Pat. No. 5,770,358, U.S. Pat. No. 5,639,603, U.S. Pat. No. 5,432,018, U.S. Pat. No. 5,270,170, U.S. Pat. No. 6,251,864, all of which are incorporated herein by reference.
[031] For example, random peptides can be designed to have a defined number of amino acid residues of a specific length. Oligonucleotides encoding random peptides may be prepared which have the codon motif (NNK)x, where N is nucleotide A, C, G, or T in equimolar amounts, K is G or T in equimolar amounts, and x is an integer corresponding to the number of amino acids desired in the peptide. The random peptides may be presented on the surface of a phage particle, as part of a fusion protein comprising either the pill or the pVIII coat protein of a phage fd derivative or as a fusion protein with the Lad peptide fusion protein bound to a plasmid. By panning the phage or plasmid against immobilized human thrombopoietin MpI receptor (SEQ ID NO: 2, which is encoded by the polynucleotide sequence of SEQ ID NO: 1), peptides that bind the receptor can be enriched. The encoding nucleic acids may then be isolated from the phage and the sequence of the desired peptide can be readily deduced. [032] When present in dual agonist compounds of formula (I), linker moieties function to separate TM1 and TM2 in the case of L1, and TM2 and IL-3 in the case of L2. When present in dual agonist compounds of formula (II); linker moieties function to separate TM1 from IL-3 in the case of L2, and IL-3 from TM2 in the case of L3. The specific composition of the linker is not critical. The linkers should be of a length that is adequate to link their specific associated substituents in such a way that they assume the correct conformation relative to one another so that they retain agonist activity for their respective receptors. Furthermore the linkers should be a length that allows adequate spacing between the TM1 and TM* pair and IL-3 in formula (I), and between TM1 and IL-3 and IL-3 and TM2 in formula (II), to avoid steric hindrance with the respective receptors. Linker type and length can be readily optimized in the context of the other substituents in the dual agonist compound by using the proliferation assays provided in Examples 3 and 4 to assess agonist activities.
[033] In compound (I), L1 is covalently bound to the C-terminus of TM1 and the N- terminus of TM2, thus covalently linking the two peptides together in a tandem configuration. As used herein, the term "C-terminus" refers to the terminal carboxyl group of a peptide, protein, or polypeptide. The term "N-terminus" refers herein to the terminal amino group of a peptide, protein, or polypeptide. L" is covalently bound to the C-terminus of TM2 and an amino acid residue of the IL-3 (i.e., at one of either the N- terminal amino group, the C-terminal carboxyl group, or an α carbon side chain of any amino acid residue in the IL-3 sequence), thus covalently linking the C-terminus of TM1 to the IL-3. Typically, L2 is covalently bound to the N-terminal amino group of the IL-3. [034] In compound (II), L2 is covalently bound to the C-terminus of TM1 and an amino acid residue of the IL-3 (i.e., one of either the N-terminal amino group, the C-terminal carboxyl group , or an α carbon side chain of any amino acid residue in the IL-3 sequence), thus covalently linking the C-terminus of TM1 to the IL-3. Typically, L2 is covalently bound to the N-terminal amino group of the IL-3. L3 is covalently bound to the N-terminus of TM2 and an amino acid residue of IL-3 (i.e., at one of either the N- terminal amino group, the C-terminal carboxyl group, or an α carbon side chain of any of the amino acid residues in the IL-3 sequence), thus covalently linking the IL-3 to the N- terminus of TM2. Typically. L3 is covalently bound to the C-terminus of the IL-3. [035] Exemplary linker moieties for either L1 , L2 or L3 include peptides of naturally occurring amino acids, non-natural Iy occurring amino acids, or a combination of both; non-peptidic, non-polymeric aliphatic moieties; and oligonucleotides. Additional L" and L3 linker moieties include non-peptidic polymeric substituents, polypeptide substituents, and polynucleotide substituents. [036] Dual agonist compounds of the present invention may also have a combination of any two of these different linker types (i.e., peptide; non-peptidic, non-polymeric aliphatic; oligonucleotide; polypeptide; or polynucleotide) such that L1 and L2 can be different in the compound of formula (I) and L2 and L3 can be different in the compound of formula (II). The difference can be with respect to either linker type or, if the linkers are of the same type, the difference may be with respect to specific structure or sequence. In some embodiments, the linkers may be identical.
[037] Typically, at least one of L1 and L2 (compound Y) and at least one of L2 and L3 (compound II) is a peptide linker. Usually, both L1 and L2 (compound I) and both L2 and L3 (compound II) are peptide linkers. [038] When n is 1 in compound (I) and L1 is a peptide linker, L1 is usually from about 1 to about 40 amino acid residues in length, is often from about 1 to about 20 amino acid residues in length, and typically is from about 1 to about 10 amino acid residues in length, more typically from about 1 to about 6 amino acid residues in length, and often from about 4 to about 6 amino acid residues in length. When m is 1 in compound (I) or compound (II) and L2 is a peptide linker, L2 is typically from about 40 amino acid residues in length, is often from about 1 to about 20 amino acid residues in length, more typically from about 4 to about 15 amino acid residues in length, and often from about 6 to about 12 amino acid residues in length. Similarly, when p is 1 in compound (II) and L3 is a peptide linker, L3 is typically from about 1 to about 40 amino acid residues in length, is often from about 1 to about 20 amino acid residues in length, more typically from about 4 to about 15 amino acid residues in length, and often from about 6 to about 12 amino acid residues in length. In some embodiments, the linkers may be a single amino acid residue, such as, for example, a GIy, Ser, Ala, or Thr residue. [039] Peptide linkers employed in the practice of the present invention may be made up of any of the 20 naturally occurring ammo acids (i.e., Ala, Arg, Asn, Asp, Cys, GIn, GIu, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tip, Tyr, and VaI) or any of the non- naturally occurring amino acids known in the art, as well as combinations thereof. Suitable peptide linkers employed in the practice of the present invention include those that have been previously reported in the literature. For example, use of peptide linkers made up of either naturally occurring amino acid residues or non-naturally occurring amino acid residues to connect polypeptides into novel fusion polypeptides is well known and has been reported in the following references, each of which is incorporated herein by reference: Hallewell, et al. (1989) J. Biol. Chem. 264:5260-5268; Alfthan, et al. (1995) Protein Ene. 8:725-731 ; Robinson & Suer (1996) Biochemistry 35:109-1 16; Khandekar, et al. (1997) J. Biol. Chem., 272:32190-32197; Fares, et al. (1998) Endocrinology 139:2459-2464; Smallshaw et al. (\ 999) Protein Eng. 12:623-630; and U.S. Pat. No. 5,856,456. The use of peptide linkers has also been demonstrated in the production of single-chain antibodies where the variable regions of a light chain (VL) and a heavy chain (VH) are joined through a peptide linker. A widely used peptide linker is a 15-mer consisting of three repeats of a Gly-Gly-Gly-Gly-Ser amino acid sequence ((Gly.4 Serb) (SEQ ID NO:3). Phage display technology has been used to diversify and select appropriate linker sequences (Tang, et al. (1996) J. Biol. Chem. 271 :15682-15686; Hennecke, et al. (1998) Protein Eng. 11 :405-410, both of which are incorporated herein by reference). [040] In some embodiments, the peptide linker may contain at least 50% glycine residues, and sometimes at least 75% glycine residues. The peptide linker may also be made up of only glycine residues. For example, the linker may contain 1-20 glycine residues, 2-16 glycine residues, 3-15 glycine residues, 4-12 glycine residues or 5-10 glycine residues. In addition to the glycine residues, the linker may comprise other residues, in particular, residues selected from the group consisting of Ser, Ala and Thr. Peptide linkers employed in invention dual agonist compounds often have only glycine and serine residues in their sequences. For example, the peptide linker may be of the form (Gly3Ser)n (SEQ ID NO:363), where n is from 1 to 5, inclusive, or from 1 to 3, inclusive. To illustrate, L1 may be (Gly3Ser) (SEQ ID NO:363) and L2 may be (Gly3Ser)3 (SEQ ID NO.-364) in compound (I) and L2 and L3 may each be (Gly3Ser)3 (SEQ ID NO:364) in compound (II). Other suitable peptide linkers having only glycine and serine residues may be of the form (Gly4Ser)n (SEQ ID NO:364), wherein n is from 1 to 4, inclusive, or from 1 to 3, inclusive.
(041] Other suitable peptide linkers include those having the amino acid sequence GIyx- Xaa-Glyy-Xaa-GlyZ) wherein each Xaa is independently selected from the group consisting of Ala, VaI, Leu, He, Met, Phe, Trp, Pro, GIy, Ser, Thr, Cys, Tyr, Asn, GIn, Lys, Arg, His, Asp and GIu, and wherein x, y and z are each integers from 1 to 5, inclusive (SEQ ID NO:366). In some embodiments, each Xaa is independently selected from the group consisting of Ser, Ala and Thr. More particularly, the peptide linker has the amino acid sequence Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly, wherein each Xaa is independently selected from the group consisting of Ala, VaI, Leu, He, Met, Phe, Trp, Pro, GIy, Ser, Thr, Cys, Tyr, Asn, GIn1 Lys, Arg, His, Asp and GIu (SEQ ID NO:367). In some embodiments, each Xaa is independently selected from the group consisting of Ser, Ala and Thr. Typically, Xaa is Ser.
[042] Suitable peptide linkers include those comprising at least one proline residue in the amino acid sequence of the peptide linker. Peptide linkers of the present invention also may comprise at least one cysteine residue and/or at least one lysine residue. Thus, in some embodiments of the present invention the peptide linker comprises amino acid residues selected from the group consisting of GIy, Ser, Ala, Thr, Cys, Lys, and Pro. [043] In some embodiments, the purpose of introducing an amino acid residue, such as Cys or Lys into the peptide linker is to introduce an attachment group for a non- polypeptide conjugation moiety that can be covalently attached to the amino acid residue. Glycosylation sites, which are discussed in more detail below, may also be incorporated into the peptide linker sequence. Covalent attachment of non-polypeptide conjugation moieties is described in more detail herein below. As used herein, the term "non- polypeptide conjugation moiety" refers to a non-polypeptide polymer, sugar moiety, or non-polymeric lipophilic moiety. Exemplary non-polypeptide conjugation moieties are described in further detail herein below under the heading "Dual Agonist Conjugates." The amino acid residue to which a non-polypeptide conjugation moiety covalently binds is referred to herein as an "attachment group". The non-polypeptide conjugation moieties react with specific attachment sites on the attachment groups. The term "attachment site" refers to the specific functional group involved in the conjugation reaction. [044] Additional suitable peptide linkers may be obtained by optimizing the peptide linkers described herein or in the literature by using well known mutagenesis techniques, such as random mutagenesis. For example, libraries of dual agonist compounds in which the TM and IL-3 components remain constant while the type, length and/or amino acid composition of the linker is varied can be prepared and screened using the assays described in Examples 3 and 4. Preferred peptide linkers employed in the practice of the present invention are resistant to proteolysis.
[045] Suitable non-peptidic, non-polymeric aliphatic linker moieties include optionally substituted, substantially linear alkylene moieties The non-peptidic, non-polymeric aliphatic linkers are typically derived from an aliphatic compound having at least two functional groups that are capable of reacting with an amino acid (e.g., hydroxyl, carboxyl, amino, and the like).
[046] The term "optionally substituted" refers herein to the replacement of hydrogen with a monovalent radical. Suitable substitution groups include hydroxyl, an alkyl, a lower alkyl (having from 1 to 6 carbon atoms), an alkoxy, a lower alkoxy (having from 1 to 6 carbon atoms), nitro, amino, cyano, and the like. The substantially linear alkylene moiety may optionally have a carbonyl group or heteroatom, such as O, S, or N in its backbone. Usually the backbone heteroatom is oxygen. In some embodiments, the substantially linear alkylene moiety is a lower alkylene having from 1 to 6 carbon atoms. Exemplary non-peptidic, non-polymeric aliphatic linker moieties include a C6 alkylene, 6-aminocaproic acid, tetra-ethylene glycol, and the like. Typically, the molecular weight of the non-peptidic, non-polymeric aliphatic linker moiety is in the range of from about 14 daltons to about 2,000 daltons for L1, and more typically from 14 daltons to about 1,000 daltons for L1; and from about 14 daltons to about 3,000 daltons for L2 or L3, more topically from about 14 daltons to about 2,000 daltons for L2 or L3, and usually from about 14 daltons to about 1 ,000 daltons for L2 or L3. [047] Suitable oligonucleotide linkers employed as any one of L1, L2, or L3 in the practice of the present invention may be of any composition, and are typically from about 1 to about 20 nucleotides long. L2 and L3 may also be a polynucleotide that is up to about 500 nucleotides long. [048] When non-peptidic, polymeric linkers are employed as L" and/or L . they typically have an average molecular weight of from about 200 to about 2,000 daltons. Typically, the average molecular weight is from about 300 to about 2,000 daltons. Preferably, the non-peptidic, polymeric linker is water soluble. Suitable non-peptidic, polymeric linkers include polyalkylene oxides (e.g., polyethylene glycol, polypropylene glycol, and the like), polyvinyl alcohol, polyvinylpyrrolidone, and the like, as well as derivatives and copolymers thereof.
[049J Polypeptide linkers that are employed as L2 and L3 linkers in the dual agonist compound typically function to increase the circulating serum half-life of the dual agonist compound in vivo and do not by themselves have detectable IL-3 or thrqmbopoeitin MpI receptor agonist activities as determined in the assays of Examples 3 and 4. Suitable polypeptide linkers include the Fc region of the IgG subclass of antibodies, a serum albumin, such as human serum albumin, and the like, as well as variants and fragments thereof. Fusions of proteins with the Fc region of the IgG subclass of antibodies and with serum albumin are described, for example, in U.S. Pat. No. 7,030,226 and U.S. Pat. No. 6,926,898, which are both incorporated herein by reference. Preferred polypeptide linkers employed in the practice of the present invention are resistant to proteolysis. Polypeptide linkers may have an attachment group in their sequences, such as a Cys or Lys, that can be utilized for subsequent attachment of a non-polypeptide conjugation moiety. Glycosylation sites, which are discussed hereinbelow, may also be incorporated into the polypeptide linker sequence.
[050J Dual agonist compounds of the present invention having peptide or polypeptide linkers can be readily expressed from a host cell without further processing using the methods described in Example 1. Dual agonist compounds having only peptide or polypeptide linkers are also referred to herein as "dual agonist polypeptides." When the linker is not a peptide or polypeptide, the dual agonist compound can be' prepared by separately expressing TM1 and TM' and IL-3 as described below, then chemically linking each linker substituent to TM1, TM2, and IL-3. Cross-linking sites may also be incorporated into components of the dual agonist compound to facilitate linking the substituents together. For example, a cassette containing TM1, L1, TM2, L2 (where L1 and L2 may be either peptide or non-peptide in nature) and a C-terminal thiol reactive or amine-reactive group may be synthesized. An IL-3 encoding polynucleotide can be . modified to add a cross-linkable N-terminal or C-terminal residue (e.g., cysteine). The recombinant IL-3 can then be expressed, refolded and cross-linked with the cassette to join all of the substituents together. - [051] Both linkers in the dual agonist compounds may be present, or one or both of the linkers in the dual agonist compounds may be absent. Therefore, dual agonist compounds may have the structure of formula (I) with any of the following combination _ of features; n=0 and m=l , n=l and m=0, n=l and m=l , and n=0 and m=0. Similarly, dual agonist compounds may have the structure of formula (II) with any of the following combination of features: m=0 and p=l, m=l and p=0, m=l and p=l , and m=0 and p=0. [052] The terms "IL-3", "IL-3 polypeptide", and "interleukin-3" are used interchangeably herein to refer to mature recombinant human IL-3 polypeptide (SEQ ID NO: 7) and muteins thereof that bind to and activate IL-3 receptor as measured by the proliferation of the TF-I cell line (German Resource Centre for Biological Material, DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) No. ACC 334; and described in Kitamura, et al., Blood (1989) 73(2):375-380, which is incorporated herein by reference) in the assay of Example 3. As used herein, the phrase "exhibits IL-3 receptor agonist activity" refers to detectable proliferation of the TF-I cell line in the assay of Example 3. Mature human IL-3 consists of 133 amino acid residues. It has one disulfide bridge and two potential N-glycosylation sites at the asparagines at positions 15 and 70. (Yang, et al. CELL, 47:3 (1986), which is incorporated herein by reference)
[053] Suitable IL-3 muteins may have from 1 to 31 amino acid substitutions relative to mature recombinant hu-IL-3 (SEQ ID NO: 7) and/or deletions of from 1 to 14 amino acid residues from the N-terminus and/or 1 to 15 amino acid residues from the C-terminus. In some embodiments, IL-3 muteins employed in the practice of the present invention may have from 1 to 25 amino acid substitutions, and some may have from 1 to 15 amino acid substitutions. Other suitable IL-3 muteins may have from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, or 14 amino acid substitutions.
[054] IL-3 polypeptides that are suitable for use in the dual agonist compounds of the present invention include IL-3 muteins that are well known in the art. A number of IL-3 muteins have been described in the literature. For example, although the human sequence for IL-3 was reported by Yang, et al. (Cell 47:3 (1986), which is incorporated herein by reference) as having a serine at position 8 of the mature protein sequence, others have reported the isolation of a proline at position 8 in the mature protein sequence for hJL-3. These apparent allelic forms of hIL-3 may be employed, along with other muteins described in the prior art. WO 88/04691 , which is incorporated herein by reference, reports that Alal can be deleted from hIL-3, while still retaining biological activity. This publication also describes other hIL-3 muteins, e.g., Alal Asp + Trpl 3Arg, Alal Asp + Met3Thr, and Alal Asp, Leu9Pro, and Trpl3Arg. The numbering of the above substitutions is with reference to mature recombinant hIL-3 (SEQ ID NO: 7). λ Additional IL-3 muteins suitable for use in the practice of the present invention are described in U.S. Pat. Nos. 5,677,149, 5,817,486, 5,604,116, 6,458,931, WO 91/00350, and WO 90/12874, all of which are incorporated herein by reference. [055] IL-3 muteins having deletions at either the N or C terminus are well known in the art. These truncated forms of hIL-3 are suitable for use in the dual agonist compounds of the present invention. For example, U.S. Pat. No. 5,677,149, which is incorporated herein by reference, describes hIL-3 muteins in which amino acid residues 1 to 14 have been deleted from the N terminus. U.S. Pat. No. 5,166,322, which is incorporated herein by reference, describes a mutein of the mature form of hIL-3 in which the alanine at position 2 is deleted (the "mp" mutein). It also describes a mutein in which the first two amino acids at the N-terminus of the mature hIL-3 are deleted, i.e., altering the N- terminus to begin with Met Thr GIn Thr (the "m3" mutein). U.S. Pat. No. 5,677,149, ■ which is incorporated herein by reference, reports that a mutein of the mature form of hIL-3 having a deletion of 8 amino acid residues from the C-terminus (i.e., amino acid residues 126 to 133) retains biological activity. An IL-3 mutein comprising residues 15- 1 18 of mature recombinant hIL-3 (SEQ ID NO: 7) was demonstrated by Olins, P.O. et al. ((1995) J. Biol. Chem. 270:23754-23760) which is incorporated herein by reference, to retain IL-3 receptor agonist activity. Accordingly, suitable IL-3 muteins employed in the dual agonist compounds of the present invention may have deletions of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid residues from the N-terminus and/or from 1 10 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 amino acids from the C-terminus. IL-3 muteins employed in the dual agonist compounds of the present invention may have any combination of deletions and substitutions described herein. [056] Those having ordinary skill in the art will appreciate that other suitable IL-3 muteins can be readily identified by employing methods for generating variant libraries that are well known in the art. For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides (such as, for example, the polynucleotide encoding mature rhIL-3 (SEQ ID NO: 6), or polynucleotides encoding any of the muteins specifically described herein or in the literature) to generate variant libraries that can be expressed, screened, and assayed using the methods described herein. Mutagenesis and directed evolution methods are well known in the art. See, e.g.. Ling, et al., "Approaches to DNA mutagenesis: an overview," Anal, Biochem., 254(2): 157-78 (1997); Dale, et al., "Oligonucleotide-directed random mutagenesis using the phosphorothioate method," Methods MoI. Biol., 57:369-74 (1996); Smith, "In vitro mutagenesis," Ann- Rev. Genet.. 19:423-462 (1985); Botstein, et al., "Strategies and applications of in vitro mutagenesis," Science, 229:1 193-1201 (1985); Carter, "Site-directed mutagenesis," Biochem. J.. 237:1-7 (1986); Kramer, et al., "Point Mismatch Repair," Cell, 38:879-887 (1984); Wells, et al., "Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites," Gene. 34:315-323 (1985); Minshull, et al., "Protein evolution by molecular breeding," Current Opinion in Chemical Biology, 3:284-290 (1999); Christians, et al., "Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling," Nature Biotechnology, 17:259-264 (1999); Crameri, et al., "DNA shuffling of a family of genes from diverse species accelerates directed evolution," Nature, 391 :288-291 ; Crameri, et al., "Molecular evolution of an arsenate detoxification pathway by DNA shuffling," Nature Biotechnology. 15:436-438 (1997); Zhang, et al., "Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening," Proceedings of the National Academy of Sciences. U.S.A.. 94:45-4-4509; Crameri, et al., "Improved green fluorescent protein by molecular evolution using DNA shuffling," Nature Biotechnology, 14:315-319 (1996); Stemmer, "Rapid evolution of a protein in vitro by DNA shuffling," Nature, 370:389-391 (1994); Stemmer, "DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution," Proceedings of the National Academy of Sciences, U.S.A., 91 : 10747-10751 (1994); WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651 ; and WO 01/75767 (all of which are incorporated herein by reference). IL-3 muteins suitable for use in the practice of the present invention can be readily identified by assaying candidate IL-3 muteins for IL-3 receptor agonist activity in accordance with the assay described in Example 3. [057] In some embodiments, IL-3 muteins employed in the practice of the present invention are those which have had one or more amino acid residues removed (e.g., by deletion or substitution) or introduced (e.g., by insertion or substitution) relative to mature recombinant hIL-3 (SEQ ID NO: 7), to facilitate conjugation of the dual agonist compound to a desired non-polypeptide conjugation moiety. Exemplary non-polypeptide conjugation moieties are described in further detail hereinbelow under the heading "Dual Agonist Conjugates."
[058] Modifications that remove or introduce attachment groups may also be used to optimize the distribution of the non-polypeptide conjugation moiety along the IL-3 backbone (these attachment sites may also be included in the TM1 and TM2 sequences and/or any peptide or polypeptide linkers that might be used in the invention dual agonist compounds). Attachment groups are typically removed or introduced by substitution of amino acid residues in the IL-3 sequence.
[059] Introduction or removal of attachment groups in any of the IL-3, TM1, TM", peptide or polypeptide linker sequences may facilitate a more efficient, specific and/or extensive conjugation of a desired non-polypeptide conjugation moiety during a subsequent conjugation reaction. The attachment group to be removed or introduced is selected based on the nature of the non-polypeptide conjugation moiety to be attached. For example, when the non-polypeptide conjugation moiety is a non-polypeptide polymer, such as, for example, a polyalkylene oxide derivative activated with a suitable reactive functional group, the attachment group may be cysteine, lysine, the N-terminal amino acid residue (i.e., via the terminal α-amino group), aspartic acid, glutamic acid, histidine, or arginine, or any combination of two or more thereof. Lysine and cysteine are the typical attachment groups which are introduced or removed in the context of adding or removing attachment sites for conjugation to a non-polypeptide polymer in the IL-3 component of the dual agonist compounds. [060] IL-3 muteins having one or more introduced cysteine residues have been reported in U.S. Pat. No. 5,166,322, which is incorporated herein by reference, as retaining the biological activity of hIL-3. The introduced cysteine residues provide attachment sites for sulfhydryl reactive compounds such as, for example, a polyalkylene glycol (including polyethylene glycol, polypropylene glycol, and the like) and derivatives thereof, dextran, colominic acids and other carbohydrate-based polymers, biotin, and the like. As used herein, the term "sulfhydryl reactive compound" refers to a compound that reacts and forms a covalent attachment to the sulfhydryl group (-SH) of a cysteine residue. Sulfhydryl reactive compounds include non-polypeptide polymers such as, for example, polyalkylene glycols that have been activated by the addition of a sulfhydryl group, thiol, triflate, tresylate, aziridine or oxirane, s-pyridyl, or malemeide.
[061] Specific IL-3 muteins having an introduced cysteine residue that are suitable for use in the practice of the present invention include those having cysteine substitutions or additions introduced within positions 1-14, inclusive, of the mature form of hIL-3, as described in U.S. Pat. No. 5,166,322, which is incorporated herein by reference. Other suitable IL-3 muteins include those having one or more of the following cysteine substitutions and substitution combinations relative to the mature form of recombinant hIL-3 (SEQ ID NO: 7): M3C, KlOC, T6C, S8C, S12C, M19C, KlOOC, T6C + KlOC, L9C + KlOC, T6C + S8C, T6C + S8C + KlOC, or S8C + L9C + KlOC. IL-3 muteins employed in the dual agonist compounds of the present invention may also have a cysteine residue inserted after the C-terminus (e.g., 134C). The above-described cysteine substitutions and insertions are described in U.S. Pat. No. 5,166,322, which is incorporated herein by reference. Cysteine residues may also be substituted in any one or more of the 8 C-terminal amino acid positions (i.e.,- 125C, 126C, 127C, 128C, 129C, 130C, 131C, 132C, and/or 133C). Cysteine attachment sites may also be removed from mature recombinant hIL-3 (SEQ ID NO: 7) or mutein thereof by substituting another amino acid for the cysteine residues at position 16, 84, or both (with amino acid position numbering corresponding to SEQ ID NO: 7), or by deleting one or both of these cysteine residues.
[062] Other suitable IL-3 muteins include those having one or more lysine residues introduced or having one or more lysine residues removed from mature recombinant hlL- 3 (SEQ ID NO: 7) or mutein thereof. The ε amino groups of lysine provide convenient attachment sites for non-polypeptide polymers and other desirable non-polypeptide conjugation moieties having amine reactivity. The lysine residues are thus attachment groups for amine reactive non-polypeptide conjugation moieties. As used herein, the term "amine-reactive" refers herein to a compound that reacts with and forms a covalent attachment to the amine group (-NH2) of a lysine residue.
(063) In some embodiments, IL-3 muteins employed in the practice of the present invention may have one or more of the following substitutions relative to mature recombinant hIL-3 (SEQ ID NO: 7): R54K, R55K, R63K, R94K, R108K, R109K. and any combination of two or more substitutions thereof, all of which introduce an attachment site for an amine reactive non-polypeptide conjugation moiety; and KlOR, K28R, K66R, K79R, KlOOR, Kl 1OR, Kl 16R, and any combination of two or more substitutions thereof which function to remove an attachment site for an amine reactive non-polypeptide conjugation moiety. Dual agonist compounds include those having an IL-3 mutein component (for example, an IL-3 polypeptide having an K-^R or an R->K substitution), where the IL-3 receptor activity (as measured by the assay of Example 3) is at least the same as or greater than the corresponding dual agonist compound having an IL-3 component that is mature recombinant hIL-3 (SEQ ID NO: 7) instead of an IL-3 mutein. [064] Suitable IL-3 muteins include those having an attachment site introduced or removed within the first 14 amino acid residues or last 8 amino acid residues of mature recombinant hIL-3 (SEQ ID NO: 7). In some embodiments, the lysine at position 10 in mature recombinant hIL-3 (SEQ ID NO: 7) may be substituted with another amino acid (i.e., Ala, Arg, Asn, Asp, Cys, GIn, GIu, GIy, His, He, Leu, Met, Pbe, Pro, Ser, Thr, Tip, Tyr, or VaI) or deleted to remove it as an attachment site, or may be substituted with a cysteine residue to substitute a lysine attachment group for introduce a cysteine attachment group, Lysine or cysteine may be substituted for any of the other amino acid residues in these regions, i.e., AlalLys/Cys, Pro2Lys/Cys, Met3Lys/Cys, Thr4Lys/Cys, Gln5Lys/Cys, Thr6Lys/Cys, Thr7Lys/Cys, Ser8Lys/Cys, Leu9Lys/Cys, LyslOCys, Thrl lLys/Cys, Serl2Lys/Cys, Trpl3Lys/Cys, VaI 14Lys/Cys, Thrl26Lys/Cys, Thrl27Lys/Cys, Leul28Lys/Cys, Serl29Lys/Cys, Leul30Lys/Cys, Alal31Lys/Cys, Ilel32Lys/Cys, Phel33Lys/Cys, and any combination of two or more thereof. Those having skill in the art will appreciate that one or more substitutions which introduce an attachment group for an amine reactive non-polypeptide conjugation moiety can be combined with one or more substitutions which remove such attachment sites to achieve a desired pattern of conjugation to a desired non-polypeptide conjugation moiety. [065] When the non-polypeptide conjugation moiety is a sugar moiety, the attachment group is typically an in vivo glycosylation site, such as, for example, an N-glycosylation or an O-glycosylation site. As used herein, the term "N-glycosylation site" refers to the sequence N-X-S/T/C, wherein X is any amino acid residue except proline, N is asparagine and S/T/C is either serine, threonine or cysteine, and typically, serine or threonine, and preferably threonine. Although the asparagine residue of the N- glycosylation site is the actual attachment site for the sugar moiety during glycosylation, such attachment cannot be achieved unless the other amino acid residues of the N- glycosylation site are present. Accordingly, when the non-polypeptide conjugation moiety is a sugar moiety and the conjugation is to be achieved by N-glycosylation, removal or introduction of an attachment group refers to an alteration in one, two, or all three of the amino acid residues that constitute an N-glycosylation site in order to introduce or remove a functional N-glycosylation site.
[066] The mature form of hIL-3 has two natural N-glycosylation asparagine attachment sites, N15 and N70. IL-3 muteins suitable for use in the practice of the present invention include those having these attachment sites removed by substituting the asparagines at these positions for another amino acid, or alternatively, introducing a substitution at S 17 and/or S72 with an amino acid that is not threonine or cysteine.
[067] It is often advantageous to introduce one or more additional N-glycosylation sites into the sequence of the IL-3 polypeptide. Locations in the IL-3 polypeptide in which an N or an S/T in "position 1 " or "position 3" of the N ("position 1 ")-X ("position 2")-S/T ("position 3")-Z N-glycosylation site pattern are preferable sites for introducing a new N- glycosylation site. Even more preferable are positions already holding an S/T in position 3. Thus, suitable IL-3 muteins having introduced N-glycosylation sites that may be employed in dual agonist compounds of the present invention include those having a substitution selected from the following: P2N, T4N, Q5N, L9N, KlON, I20S/T, I23N, L40S/T, N41 S/T, E43S/T, L53S/T, R54S/T, A64S/T, V65N, I74N, L87N, F107S/T, Kl ION, Ll 15N, Q122S/T, Q124N, Q125N, T127N, or any combination of two or more thereof, where position numbering is based on correspondence with mature recombinant hIL-3 (SEQ ID NO: 7). [068] In introducing the glycosylation site, it may be desirable to utilize an already existing asparagine residue in the IL-3 sequence. Therefore, in some embodiments, the ■ one or more introduced glycosylation sites are made by making one or more of the following substitutions: I20S/T, L40S/T, N41S/T, E43S/T, L53S/T, R54S/T, A64S/T, F107S/T, Q122S/T, or any combination of two or more thereof. Typically, the substitution is I20T, L40T, N42T, E43T, L53T, R54T, A64T, F107T, Q122T, or any combination of two or more thereof. In some embodiments, one or more of the following substitutions is made: I20S/T, N41 S/T, E43S/T, L53S/T, Q122S/T, or any combination of two or more thereof. Of these embodiments, the substitution is usually I20T, N41T, E43T, L53T, and/or Q122T. Of the substitutions listed in the previous paragraph, one or more of the following substitutions two residues "downstream" from an existing S/T can be made to introduce a glycosylation site: P2N, T4N, Q5N. L9N. KlON, I23N, V65N, I74N, L87N, Kl 10N, Ll 15N, Q124N, Q125N, T127N, or any combination or two or more thereof. Typically, the substitution is P2N, T4N, Q5N, L9N, KlON, L87N, Q124N, Q125N, and/or T127N. More typically, the substitution is P2N, T4N, Q5N, L9N, KlON, L87N, Q124N, Q125N, and/or Tl 27N. [069] Glycosylated dual agonist compounds comprising the IL-3 muteins described above typically have sugar moieties attached to the asparagine residues at any one or more of positions 2, 4, 5, 9, 10, 18, 23, 38, 39, 41 , 51, 52, 62, 65, 74, 87, 105, 110, 115, 120, 124, 125, and/or 127, respectively (with amino acid position numbering corresponding to that of SEQ ID NO: 7). [070] In some embodiments, the introduced N-glycosylation site is not within about 20 amino acid residues from the C-teπninal end of the IL-3 (e.g., an IL-3 with a substitution selected from I20S/T, L40S/T, N41S/T, E43S/T, L53S/T, R54S/T, or A64S/T). Usually, it is not within about 30, 40, and in some embodiments, 50 amino acid residues from the C-terminal end. Desirable substitutions that create the third position of an N- glycosylation site in the IL-3 component include I20S/T, L40S/T, N41 S/T, E43S/T, L53S/T, R54S/T, and A64S/T, usually I20S/T, N41 S/T, E43S/T, and L53S/T, and more typically, I20S/T, N41 S/T, E43S/T, and L53S/T. Desirable substitutions that introduce an asparagine to create an N-glycosylation site in the IL-3 component include P2N, T4N, Q5N, L9N, Kl ON, I23N, V65N, I74N, T4N, Q5N, L9N, or KlON. Preferable substitutions of either type include I20S/T, N41S/T, E43S/T, L53S/T, P2N, T4N, Q5N, L9N, KlON, and particularly I20T, N41T, E43T, and L53T.
[071] In constructing the dual agonist compounds of the present invention, those having ordinary skill in the art will appreciate that they can readily optimize combinations of TM1 and TM2 and the linkers, by assessing TPO receptor c-Mpl receptor agonist activity of TM1 and TM2 in the assay of Example 4 in one or more of the following contexts: TM'-(L')n-TM2,
Figure imgf000027_0001
TM'-(L2)m-IL-3, IL-3-(L3)p-TM2, or TM1- (L2)m-IL-3 -(L3VTM2. Illustrative TM'-L'-TM2 combinations are provided as SEQ ID NOS: 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, and 308 (encoded by polynucleotides corresponding to SEQ ID NOS: 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281 , 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, and 307, respectively).
[072] Similarly, the combination of substituents that make up the invention dual agonist compounds can be optimized by screening different combinations for IL-3 receptor agonist activity using the assay of Example 3. [073] Exemplary dual agonist compounds include those in which TM1 and TM2 each have a molecular weight of at least about 900 daltons and less than about 1 ,800 daltons, the linkers (either L1 and L2 for compound (T) or L2 and L3 for compound (H)) are both present and are peptide linkers, and the IL-3 is mature recombinant hIL-3 (SEQ ID NO: 7) or an IL-3 mutein having from 1 to 15 amino acid substitutions, and/or a deletion of 1 to 14 amino acid residues from the N-terminus, and/or a deletion of 1 to 8 amino acid residues from the C-terminus. Typically, the substitutions in the IL-3 are those which either introduce or remove an attachment site for a non-polypeptide conjugation moiety. Specific dual agonist compounds are illustrated in SEQ ID NOS: 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, and 362. [074] In another embodiment, the present invention provides a method for identifying a dual agonist compound that exhibits a desired dual agonist activity, said method comprising: providing a library of peptide dimers having the structure
P' - L' - P2 wherein P1 and P2 are each independently non-naturally occurring peptides having a molecular weight of less than about 2,500 daltons, wherein L1 is a linker that covalently links the C terminus of P1 to the N terminus of P2, screening the library of peptide dimers to identify one or more peptide dimers having a desired thrombopoeitin c-Mpl receptor agonist activity, providing a library of IL-3 fusion compounds comprising one or more of the identified dimers of peptides fused to an IL-3 polypeptide, wherein each IL-3 fusion compound has the structure pi _ L' -P2 - (L2)m — IL-3 wherein m is 0 or 1 , wherein P1 -L1 -P2 is the one or more identified dimers or peptides, wherein L2 is a linker that covalently links the C terminus of TM2 to IL-3, and screening the library of IL-3 fusion compounds to identify one or more IL-3 fusion compounds having a desired combination of thrombopoeitin c-Mpl receptor agonist activity and IL-3 receptor activity (i.e., a desired dual agonist activity). [075] Typically at least one, and often both of P1 and P2 are random peptides, which are described in more detail hereinabove. P1 and P: may also be mutated variants of one or more random peptides previously selected by panning against immobilized human thromobopoeitin MpI receptor in a prior step. P1 and P2 typically each exhibit thrombopoeitin c-Mpl receptor agonist activity in accordance with the assay of Example 4. The molecular weights and typical lengths of P1 and P2 are the same as described above for TM1 and TM2. P1 and P2 may be the same or different in both sequence and length. Suitable L1 and L2 linkers are the same as described for the dual agonist compounds described hereinabove. In the libraries of dimers and IL-3 fusion compounds, linkers L1 and L2 may be the same or different from library member to library member. Suitable IL-3 polypeptides are the same as described above for the dual agonist compounds.
[076] The library of peptide dimers is screened using the proliferation assay of Example 4 to identify one or more peptide dimers having a desired level of thrombopoeitin c-Mpl receptor agonist activity. Those dimers having a desired level of thrombopoetin c-Mpl receptor agonist activity are subsequently employed in the construction of IL-3 fusion compounds using, for example, the methods described in Example 1 or other suitable methods. [077] With respect to the IL-3 fusion compound library, the sequence of the IL-3 polypeptide may differ among the IL-3 fusion compound library members. Likewise, linker L2 may differ among the IL-3 fusion compounds in the library, and may not be present at all (i.e., m = 0) in some or all of the library members. The library of IL-3 fusion compounds can be screened using the proliferation assays of both Example 3 and Example 4 to identify a dual agonist compound that has the desired combination of thrombopoeitin c-Mpl receptor agonist activity and IL-3 receptor activity.
[078] Dual agonist compounds of the present invention typically exhibit agonist activities with respect to either thrombopoeitin c-Mpl receptor or hJL-3 receptor that are from about 0.1 to about 5 or more fold more potent than that observed for native ligands recombinant human thrombopoeitin (SEQ ID NO: 373, encoded by the polynucleotide of SEQ ID NO: 372) and mature recombinant IL-3 (SEQ ID NO: 7) in the assays of
Examples 4 and 3, respectively (i.e., an EC50 in either assay that is from about 10 times to about 0.2 or less times the ECJO with respect to either recombinant human thrombopoietin (SEQ ID NO: 373) or mature rhIL-3 (SEQ ID NO: 7) in their respective assays). It may be desirable in certain circumstances to utilize a dual agonist compound that is less potent than either or both rh-TPO (SEQ ID NO: 373) and/or mature rhIL-3 (SEQ ID NO: 7) with respect to agonizing the TPO c-Mpl receptor or hIL-3 receptor, respectively. For example, lower potency dual agonist molecules may be more suitable for use in an extended release composition or in the form of a dual agonist conjugate, which is discussed in more detail hereinbelow.
[079J Dual agonist compounds of the present invention stimulate the proliferation of hematopoietic bone marrow stem cells in the assay of Example 8. These cells have both the human TPO c-Mpl and human IL-3 receptors. Typically, dual agonist compounds of the present invention stimulate the proliferation of more hematopoeitic stem cells in this assay than are stimulated by each individual growth factor, rhTPO (SEQ ID NO: 373) and mature rhIL-3 (SEQ ID NO: 7), as measured individually on an equimolar basis. Usually, dual agonist compounds of the present invention stimulate the proliferation of more hematopoeitic stem cells in the assay of Example 8 than the sum of stem cells stimulated by each individual growth factor, rhTPO (SEQ ID NO: 373) and mature rhlL- 3 (SEQ ID NO: 7), as measured individually on an equimolar basis in the assay of Example 8. [080] Thus, dual agonist compounds of the present invention typically exhibit synergistic dual agonist activity. As used herein, the phrase, "synergistic dual agonist activity" refers to a compound that stimulates the proliferation of more stem cells in the assay of Example 8 than the sum of combined individual effects of rhTPO (SEQ ID NO: 373) and mature rhIL-3 (SEQ ID NO: 7), i.e., as measured individually, in the assay of Example 8, where all are compared on an equimolar basis.
Dual Agonist Polynucleotides
[081] The present invention provides polynucleotides encoding invention dual agonist polypeptides. Exemplary dual agonist polynucleotides include those corresponding to SEQ ID NOS: 309, 311 , 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, and 361. These polynucleotides encode the dual agonist polypeptides corresponding to SEQ ID NOS: 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, and 362 respectively. [082] Those having ordinary skill in the art will readily appreciate that due to the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding dual agonist polypeptides of the present invention exist. Table I is a Codon Table that provides the synonymous codons for each amino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the polynucleotides of the invention where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence. Table I: Codon Table
Amino acids- Codon
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
Glutamic acid GIu E GAA GAG
Phenylalanine Phe F UUC UUU
Glycine GIy G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine He I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA ULIG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine GIn Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Seπne Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG' ACU
Valine VaI V GUA GUC GUG GUU
Tryptophan Trp W UGG
Tyrosine Tvr Y UAC UAU
[083) Such "silent variations" are one species of "conservative" variation. One of ordinary skill in the art will recognize that each codon in a polynucleotide sequence (except AUG, which is ordinarily the only codon for methionine) can be modified by Standard techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a polynucleotide which encodes a polypeptide is implicit in any described sequence. The invention contemplates and provides each and every possible variation of polynucleotide sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code (set forth in Table I), as applied to the polynucleotide sequences of the present invention. 1084) A group of two or more different codons that, when translated in the same context, all encode the same amino acid, are referred to herein as "synonymous codons." Dual agonist polypeptides of the present invention may be codon optimized for expression in a particular host organism by modifying the polynucleotides to conform with the optimum codon usage of the desired host organism. Those having ordinary skill in the art will recognize that tables and other references providing preference information for a wide range of organisms are readily available. See e.g., Henaut and Danchin in "Escherichia coli and Salmonella," Neidhardt, et al. Eds., ASM Press, Washington D.C. (1996), pp. 2047-2066, which is incorporated herein by reference. [085] The terms "conservatively modified variations" and "conservative variations" are used interchangeably herein to refer to those polynucleotides that encode identical or essentially identical amino acid sequences, or in the situation where the polynucleotides are not coding sequences, the term refers to polynucleotides that are identical. One of ordinary skill in the art will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are considered conservatively modified variations where the alteration results in one or more of the following: the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. When more than one amino acid is affected, the percentage is typically less than 5% of amino acid residues over the length of the encoded sequence, and more typically less than 2%. References providing amino acids that are considered conservative substitutions for one another are well known in the art.
[086] Polynucleotides of the present invention can be prepared using methods that are well known in the art. Typically, oligonucleotides of up to about 50 to 120 bases are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form essentially any desired continuous sequence. Dual agonist polynucleotides of the present invention can be prepared by chemical synthesis using, for example, the classical phosphoramidite method described by Beaucage, et al. (1981) Tetrahedron Letters. 22:1859-69, or the method described by Matthes, et al. (1984) EMBO J,, 3:801-05, both of which are incorporated herein by reference. According to the phosphoramidite method, oligonucleotides are synthesized, purified, annealed, ligated and cloned in appropriate vectors. In addition, essentially any oligonucleotide can be custom ordered from any of a variety of commercial sources, such as, for example. The Midland Certified Reagent Company (Midland, TX), The Great American Gene Company (Ramona, CA), ExpressGen Inc. (Chicago, IL), and others. [087] Polynucleotides may also be synthesized by well-known techniques as described in, for example, Carruthers, et al., Cold Spring Harbor Syrnp. Quant. Biol., 47:411-418 (1982) and Adams, et al., J. Am. Chem. Soc. 105:661 (1983), both of which are incorporated herein by reference. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by generating the complementary strand in a polymerase chain reaction using DNA polymerase with an appropriate primer sequence. [088] General texts which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other relevant topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, vol. 152, Academic Press, Inc., San Diego, CA ("Berger"); Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook"), and Current Protocols in Molecular Biology, F.M. Ausubel, et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 1999) ("Ausubel"), all of which are incorporated herein by reference. Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Qβ-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as well as Mullis, et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis, et al., eds.) Academic Press Inc., San Diego, CA (1990) (Innis); Arnheim & Levinson ((October 1, 1990) C&EN 36-47; The Journal of NIH Research (1991) 3:81-94; Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA. 86:1173; Guatelli, et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874; Lomell, et al. (1989) J. Clin. Chem. 35:1826: Landegren, et al. (1988) Science 241:1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560; Barringer, et al. (1990) Gene 89:1 17, and Sooknanan and Malek (1995) Biotechnology 13:563-564, all of which are incorporated herein by reference. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace, et al., U.S. Pat. No. 5,426,039. which is incorporated herein by reference. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng, et al. (1994) Nature 369:684-685, which is incorporated herein by reference. One of ordinary skill in the art will readily appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase as described in Ausebel, Sambrook, and Berger, supra.
Vectors. Promoters, and Expression Systems
[089] The present invention also includes recombinant constructs comprising one or more of the dual agonist polynucleotides, which are described above. The term
"construct" or "nucleic acid construct" refers herein to a nucleic acid, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature. The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a dual agonist encoding polynucleotide sequence of the present invention. [090] In a specific embodiment, the present invention also provides an expression vector comprising a dual agonist polynucleotide of the present invention operably linked to a promoter. The term "expression vector" refers herein to a DNA molecule, linear or circular that comprises a segment encoding a dual agonist polypeptide of the invention, which is operably linked to additional segments that provide for its transcription. As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Example 1 provides a description of how to make expression constructs for expression of dual agonist polypeptides of the present invention.
ii [091] The skilled artisan will readily appreciate that the peptide and polypeptide components of the invention dual agonist compounds can be separately expressed using the methods of Example 1 and chemically linking the separate components together using linkers that are non-peptide or non-polypeptide in nature in one or more reactions using known methods. (See paragraph 050)
[092] Nucleic acid constructs of the present invention typically include a control sequence, such as a promoter. The term "control sequences" refers herein to all of the components that are necessary or advantageous for the expression of a dual agonist polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter and a transcriptional and a translational stop signals. The control sequences may be provided with additional sequences that introduce specific restriction sites, which facilitate ligation of the control sequences with the coding region of the nucleotide sequence encoding the polypeptide.
[093] The term "operably linked" refers herein to a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polypeptide. [094] When used herein, the term "coding sequence" refers to a polynucleotide sequence that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon. The coding sequence typically includes a DNA, cDNA, and/or recombinant polynucleotide sequence. [095] Dual agonist polynucleotides of the present invention can be incorporated into any one of a variety of expression vectors that are well known in the art. Suitable vectors include chromosomal, nonchromosomal, and synthetic DNA sequences. Exemplary vectors include a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a plasmid, such as, for example, a bacterial plasmid or a yeast plasmid, a cosmid, a phage, vectors derived from viral DNA, such as, for example, vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses, and the like, as well as vectors derived from combinations of plasmids and phage DNA. Any vector that transduces genetic material into a cell, and if replication is desired, which is replicable and viable in the relevant host can be used.
[096] When incorporated into an expression vector, a polynucleotide of the invention is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis, such as, for example, T5 promoter. Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses and which can be used in some embodiments of the invention include SV40 promoter, E. coli lac or trp promoter, phage lambda PL promoter, tac promoter, T7 promoter, and the like. An expression vector optionally contains a ribosome binding site for translation initiation, and a transcription terminator, such as Pin II. The vector also optionally includes appropriate sequences for amplifying expression, such as, for example, an enhancer. [097] In addition, the expression vectors of the present invention optionally contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Suitable marker genes include those coding for resistance to the antibiotic spectinomycin or streptomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycinn resistance. Additional selectable marker genes include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance in E. coli.
[098] Vectors of the present invention can be employed to transform an appropriate host to permit the host to express a dual agonist polypeptide, or the separate components of specific dual agonist compounds (e.g., TM1, TM2, TM'-L'-TM2 (where L1 is a peptide linker), IL-3, TM2-L2-IL-3 (where L2 is a peptide or polypeptide linker), and the like). Examples of appropriate expression hosts include bacterial cells, such as E. coli, B. subtilis, and Streptomyces. In bacterial systems, a number of expression vectors may be selected, such as, for example, multifunctional E. coli cloning and expression vectors. [099] Dual agonist polypeptides of the invention can also be fused, for example, in- frame to nucleic acids encoding a secretion/localization sequence, to target polypeptide expression to a desired cellular compartment, membrane, or organelle of a cell, or to direct polypeptide secretion to the periplasmic space or into the cell culture media. Such sequences are known to those of skill in the art, and include secretion leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, endoplasmic reticulum (ER) retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization/anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.
Expression Hosts
[100] The present invention also relates to engineered host cells that are transduced (transformed or transfected) with a vector or construct of the invention, as well as the production of polypeptides of the invention by recombinant techniques. As used herein, the term "host cell" refers to any cell type which is susceptible to transformation with a nucleic acid construct of the present invention. Therefore, the present invention includes host cells composing any polynucleotide of the present invention that is described hereinabove. Typically, the polynucleotide is operably connected to one or more promoters and/or enhancers that provide for expression of the polynucleotide of the host cell.
[101] The host cell can be a eukaryotic cell, such as a mammalian cell, a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell (e.g., E. coli, Bacillus sp., and the like). Introduction of the nucleic acid construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation, gene ir vaccine gun, injection, or other common techniques (see, e.g., Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in Molecular Biology), which is incorporated herein by reference, for in vivo, ex vivo or in vitro methods. [102] A host cell strain is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "pre" or a "prepro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as E. coli, Bacillus sp., yeast or mammalian cells such as CHO, HeLa, BHK, MDCK, HEK 293, WI38, and the like, have specific cellular machinery and characteristic mechanisms for such post- translational activities and may be chosen to ensure the correct modification and processing of the introduced foreign protein. -
[103] Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express a polypeptide of the present invention are transduced using expression vectors which contain viral on gins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. For example, resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. [104] Host cells transformed with a polynucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The polypeptide produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides encoding polypeptides of the invention can be designed to include signal sequences which direct secretion of mature polypeptides through a prokaryotic or eukaryotic cell membrane.
Production and Recovery of Dual Agonist Polypeptides
[105] In another embodiment, the present invention provides a method of making a dual agonist polypeptide having both IL-3 receptor agonist activity and thrombopoeitin MpI receptor agonist activity, said method comprising: culturing a host cell transformed with a dual agonist polynucleotide of the present invention under conditions suitable for expression of the encoded dual agonist polypeptide; and recovering the dual agonist polypeptide from the culture medium or from the transformed and cultured host cells. [106] Following transduction of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means, such as, for example, by temperature shift or chemical induction, and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well known to those skilled in the art. [107] As noted, many references are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archebacterial origin. These references include Sambrook, Ausubel, and Berger (supra), as well as Freshney (1994) Culture of Animal Cells, a Manual of Basic Techique, third edition, Wiley-Liss, New York (and the references cited therein); Doyle and Griffiths
(1997) Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, fourth edition, W. H. Freeman and Company; and Rjcciardelli, et al. (1989) In vitro Cell Dev. Biol. 25: 1016-1024, all of which are incorporated herein by reference. References that describe plant cell culture and regeneration include, Payne, et al. (1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (Eds) (1995) Plant Cell. Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg, New York); Jones, Ed. (1984) Plant Gene Transfer and Expression Protocols, Humana Press, Totowa, New Jersey and Plant Molecular Biology (1993) R.R.D. Croy, Ed. Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6, all of which are incorporated herein by reference. [108] Cell culture media in general is described in Atlas and Parks (Eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL, which is incorporated herein by reference. Additional information for cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue
(1998) from Sigma-Adrich, Inc. (St. Louis, MO) and The Plant Culture Catalogue and supplement (1997) from Sigma- Aldrich, Inc. (St. Louis, MO), both of which are incorporated herein by reference. [109] Dual agonist polypeptides of the present invention can be recovered/isolated and optionally purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or solvent precipitation (such as, for example, by using a solvent like ethanol, acetone, and the like), acid extraction, ion (anion or cation) exchange chromatography, high performance liquid chromatography (HPLC), phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Suitable protein purification methods are described in Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition. Wiley- Liss, NY; Walker (1996) The Protein Protocols Handbook, Humana Press, NJ; Harris and Angal (1990) Protein Purification Applications: A Practical Approach, IRL Press at Oxford, Oxford, England; Harris and Angal , Protein Purification Methods: A Practical Approach, IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3rd Edition. Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition. wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM. Humana Press, NJ, all of which are incorporated herein by reference.
[11 OJ Bacterial production of dual agonist polypeptides may require further processing steps to generate active dual agonist polypeptides, or IL-3, if generated as a separate component that is subsequently fabricated into a dual agonist compound. This further processing entails the separation and solubilization of inclusion bodies, unfolding the protein, then refolding the protein into the correct biologically active tertiary structure. The present invention provides a method of producing a dual agonist polypeptide having both IL-3 receptor agonist activity and thrombopoeitin MpI receptor agonist activity, said method comprising: culturing a host cell transformed with a dual agonist polynucleotide of the present invention under conditions suitable for expression of a dual agonist polypeptide; recovering inclusion bodies comprising the encoded dual agonist polypeptide from the transformed and cultured host cells; solubilizing the recovered inclusion bodies comprising the encoded dual agonist polypeptide with a solubilizing agent; purifying the solubilized encoded dual agonist polypeptide; allowing the encoded dual agonist polypeptide to refold; and purifying the refolded dual agonist polypeptide. [111] Inclusion bodies are typically solubilized in solvent, such as, for example urea. Refolding can be accomplished by incubating solubilized dual agonist polypeptide in a solution of urea This process is illustrated in Example 2D-E The refolded dual agonist polypeptides of the present invention may be purified as described above and in Example 2E.
Dual Agonist Conjugates
[112] The present invention provides dual agonist conjugates comprising a dual agonist compound of the present invention covalently bound to at least one non-polypeptide conjugation moiety, either directly or indirectly via a linker moiety. Thus, dual agonist conjugates have a dual agonist compound covalently bound to one or more of a non- polypeptide polymer, a sugar moiety, a non-polypeptide, non-polymeric lipophilic moiety, or any combination of two or more thereof.- The term "non-polypeptide polymer" refers herein to a water soluble polymer that may be a natural or synthetic polymer (homopolymer, copolymer, terpolymer, and the like), that is not a peptide, polypeptide, or protein. As used herein, the term "sugar moiety" refers to a carbohydrate molecule attached by an in vivo or in vitro glycosylation process, such as an N- or O-glycosylation process. Non-polypeptide conjugation moieties are typically selected to alter specific attributes of the dual agonist compounds, such as, for example, in vivo serum half life or functional in vivo half life, stability, immunogenicity, and the like. [113] As used herein, the term in vivo serum half-life refers to the time at which 50% of the compound of interest circulates in the bloodstream of a non-human mammal such as a rat, mouse, rabbit, or monkey. The term "serum" is used herein to refer to its normal meaning, i.e., as blood plasma without fibrinogen and other clotting factors. The term "functional in vivo half-life" refers herein to the time at which 50% of the biological activity of the compound of interest is still present in the body or target organ, or the time at which the activity of the compound of interest is 50% of the initial value. The functional in vivo half-life may be determined in a non-human mammal, such as a rat, mouse, rabbit, dog, or monkey. Methods for determining both in vivo serum half-life and functional in vivo half-life are well known in the art. For example, dual agonist compounds and conjugates thereof may be administered to a non-human mammal, and blood samples collected at fixed time intervals. The blood samples may be analyzed for levels of both platelets and dual agonist compound by a standard blood count and quantitative IL-3 ELISA, respectively. The half life can be determined from a plot of dual agonist concentration versus time.
[114] Dual agonist conjugates of the present invention typically exhibit greater functional in vivo half-life and/or greater serum half-life as compared to the corresponding non-conjugated dual agonist compound. These dual agonist conjugates usually have a non-polypeptide polymer or a sugar moiety conjugated to the dual agonist compound. For example, each non-polypeptide polymer and/or sugar moiety may be conjugated either directly or indirectly via a linker, to the dual agonist compound. The non-polypeptide conjugation moiety is typically bound, either directly or indirectly via a linker, to an attachment group in the dual agonist compound. IfL1 and L2 are not a peptide or polypeptide, the non-polypeptide conjugation moiety may be bound to them, either directly or indirectly via a linker, via functional groups that are the same as the attachment sites described herein. [115] The term "greater" as it is used in connection with the functional in vivo half-life or serum half-life is used herein to indicate that the relevant half-life of the dual agonist conjugate is statistically significantly greater than a reference molecule, such as the corresponding non-conjugated dual agonist compound or a component thereof, when determined under comparable conditions. Thus, dual agonist conjugates include those which have a functional in vivo half-life or a serum half-life that is greater than that of the corresponding non-conjugated dual agonist compound.
[116] Dual agonist conjugates include those where the ratio between the funcational in vivo half-life (or serum half-life) of the conjugate and that of the corresponding non- conjugated dual agonist compound is at least 1.25, at least 1.5, at least 1.75, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8. [117] In a further aspect, the dual agonist conjugate may exhibit greater bioavailability than the corresponding non-conjugated dual agonist compound. An indication of bioavailability is provided by the "Area Under the Curve when administered subcutaneously" parameter or "AUCsc". The term "AUCsc"- or "Area Under the Curve when administered subcutaneously" is used in its normal meaning to refer to the area under the activity-in-serum vs. time curve, where the dual agonist conjugate has been administered subcutaneously to an experimental animal. Once the experimental activity time points have been determined, the AUCsc may conveniently be calculated by a computer program, such as GraphPad Prism 3.01, GraphPad Software Inc., San Diego, CA. The term "greater" as it is used in connection with AUCsc is used to indicate that the Area Under the Curve for a dual agonist conjugate, when administered subcutaneously, is statistically significantly greater than the corresponding non- conjugated dual agonist or component thereof, when determined under comparable conditions. In order to make direct comparisons between different molecules, the AUCsc values should typically be normalized, e.g., expressed as AUCsc/dose administered. [118] Exemplary dual agonist conjugates include those in which the ratio between the AUCsc of the conjugate and the AUCsc of the corresponding non-conjugated dual agonist compound is at least 1.25, at least, 1.5, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 when administered under comparable conditions to the same species of experimental animal (e.g., rat, monkey, and the like). [119] Certain dual agonist conjugates of the present invention exhibit greater activity after a longer duration post-administration than the corresponding non-conjugated dual agonist compound. These dual agonist conjugates exhibit a Tmax that is greater than the Tmax for the corresponding non-conjugated dual agonist compound. As used herein, the term "Tmax" refers to the time point in the activity-in-serum vs. time curve where the highest activity in serum is observed. The ratio of Tmax for this dual agonist conjugates to the Tmax of the corresponding non-conjugated dual agonist compound is at least 1.2, at least 1.4, at least 1.6, at least 1.8, at least 2, at least 2.5, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, when administered subcutaneously, in particular when administered subcutaneously in an experimental animal such as a rat or a monkey.
[120] Exemplary attachment groups for attaching a non-polypeptide conjugation moiety are described below in Table 2: Table 2
Useful attachment groups and examples of corresponding non-polypeptide conjugation moieties
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
*Each publication is incorporated herein by reference.
[121] Polymers suitable for use in the practice of the present invention may be branched (i.e., having two or more linear polymer chains linked together by a linker group) or linear and typically have an average molecular weight in the range of from about 300 to about 100,000 daltons and typically from about 1,000 daltons to about 50,000 daltons, or from about 2,000 to about 30,000 or 20,000, or 10,000 daltons, and in some embodiments from about 1,000 daltons to about 5,000 daltons. More particularly, the polymer molecule will typically have an average molecular weight of about 2,000 daltons, 5,000 daltons, 10,000 daltons, 10,000 daltons, 12,000 daltons, 15,000 daltons, 20,000 daltons, 30,000 daltons, 40,000 daltons, or 50,000 daltons.
[122] When used in the context of describing the molecular weight of a polymer, the term "about" indicates an approximate average molecular weight and reflects the fact that there will normally be a certain molecular weight distribution in a given polymer preparation.
[123] Exemplary polymers that are suitable for use in the conjugates of the present invention include polyalkylene oxides (PAO), such as a polyalkylene glycol (PAG) that may, for example, be a polyethylene glycol (PEG), a monomethoxypolyethylene glycol (mPEG), a polypropylene glycol (PPG), a branched polyethylene glycol having two or more polyethylene glycol chains linked together by a linker group (a linker, such as, for example, lysine, glycerol, and the like), polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone), polyethylene-co-maleic acid anhydride, a dextran (such as, for example, carboxymethyl dextran), and other like polymers. [124] Polyalkylene glycol-derived polymers are typically employed in the conjugates of the present invention because they are generally biocompatible, non-toxic, non-antigenic, non-irnmunogenic, water soluble, and are easily excreted from living organisms. Polyethylene glycol (PEG) in particular is favored because it has only a few reactive groups that are capable of cross-linking to other compounds, such as polysacchararides (e.g., dextran). Monofunctional PEG, such as, for example, a monomethoxypolyethylene glycol (mPEG), is particularly suitable for use in the conjugates of the present invention because its coupling chemistry is relatively simple. There is only one reactive group available for conjugating with the dual agonist compound. Consequently, the risk of crosslinking is eliminated. Therefore, the resulting population of dual agonist conjugates is more homogeneous with respect to having one PEG conjugated to only one attachment site. The reaction of the polymer molecules with the dual agonist compound is also easier to control.
[125] To effect covalent attachment of a hydroxylated polymer like polyethylene glycol to the dual agonist compound, at least one terminal hydroxyl group of the polymer molecule is typically provided in activated form, i.e., derivatized with functional groups that are reactive with the target attachment group in the dual agonist compound. Exemplary reactive functional groups include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl propnonate (SPA), succinimidyl carboxymethylate (SCM), benzotπazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)). [126] Suitably activated polymer molecules are commercially available, e.g., from Nektar Therapeutics, Inc., San Carlos, CA, NOF Corporation, Japan, and DowPharma, Midland, MI. Alternatively, the polymer molecules can be activated by conventional methods known in the art, such as those disclosed in WO 90/13540, which is incorporated herein by reference. Specific examples of activated linear and branched polymer molecules that are suitable for use in the dual agonist conjugates of the present invention are described in the Nektar 2005-2006 Advanced Pegylation Catalogue, which is incorporated herein by reference. Specific examples of these activated polyethylene glycol polymers include the following linear PEGs: NHS-PEG (e.g., SPA-PEG, SSPA- PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PET, SG-PEG, and SCM-PET), and NOR- PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES- PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as mPEG2-NHS, disclosed in the Nektar 2005-2006 "Advanced Pegylation" Catalogue (which is incorporated herein by reference) and those disclosed in U.S. Pat. Nos. 5,932,462 and 5,643,575, both of which are incorporated herein by reference. [127] Other examples of activated linear and branched polymer molecules that are suitable for use in the dual agonist conjugates of the present invention are described in NOF Corp. 2005 Catalogue': "PEG Derivatives, Phospholipids and Drug Delivery Materials for Pharmaceutical Products and Formulations", which is incorporated herein by reference. Additional examples of activated polyethylene glycol polymers include the following PEGs: NHS-PEG (e.g., SUNBRIGHT ME-020(-050, -100, -20O)-CS (CH3O(CH2CHO)n-CO-CH2CH2-COO-NHS); SUNBRIGHT MEGC-20 (-5O)-HS and SUNBRIGHT MEGC-10(-20, -3O)-TS (CH3O(CH2CH2O)n-CO-CH2CH2CH2-COO- NHS); SUNBRIGHT ME-020 (-05O)-AS (CH3O(CH2CH2O)n-CH2-COO-NHS); SUNBRIGHT ME-050HS (CH3O(CH2CH2O)n-(CH2)S-COO-NHS)); Aldehyde PEG (e.g., SUNBRIGHT ME-050 (-100, -200, -30O)-AL (CH3O(CH2CH2O)n-CH2CH2CHO)); Maleimido-PEGs (e.g., SUNBRIGHT ME-020 (-050, -120, -200, -20O)-MA
(CH3O(CH2CH2O)n-(CH2)3NHCO(CH2)2-(C4H2NO2)); Branched PEG Maleimides (e.g., SUNBRIGHT GL2-200 (-40O)-MA); Branched PEG NHS-glutaryl (e.g., SUNBRIGHT GL2-200 (-400)-GS2); Branched PEG NHS-carboxymethyl (e.g., SUNBRIGHT GL2- 200 (-40O)-HS); Branched PEG aldehyde (e.g., SUNBRIGHT GL3-200 (-400)AL2); all disclosed in NOF Corp. 2005 Catalogue: "PEG Derivatives, Phospholipids and Drug Delivery Materials for Pharmaceutical Products and Formulations" (which is incorporated herein by reference) and those disclosed in U.S. Pat. No. 6,875,841 and U.S. Patent Application Publications US 2005/0058620 and US 2005/0288490, all of which are incorporated herein by reference. [128] The following publications disclose further useful polymers and/or PEGylation chemistries that are suitable for use in connection with the dual agonist conjugates of the present invention: U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,476,653, U.S. Pat. No. 6,875,841, U.S. Pat. No. 5,872,191, U.S. Pat. No. 5,767,284, EP 0 839 850, WO 97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502, U.S. Pat. No. 5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No. 5,219,564, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, U.S. Pat. No. 5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No. 5,629,384, WO 96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat. No. 5,516,673, EP 605 963, US 5,382,657, EP 510 356, EP 400472, EP 183 503 and EP 154 316, all of which are incorporated herein by reference.
[129] Specific examples of activated PEG polymers particularly preferred for coupling to cysteine residues include the following linear PEGs: Vinyl sulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG 9VS-mPEG); maleimide-PEG (MAL-PEG, as well as other maleimide PEGs described herein) preferably maleimide-mPEG (MAL-mPEG, as well as other maleimide mPEGs described herein) and orthopyridyl disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG). Typically such PEG or mPEG polymers will have an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 12,000 daltons, about 20,000 daltons, about 30,000 daltons, or about 40,000 daltons.
[130] The conjugation of the dual agonist compound and the activated polymer molecule(s) is conducted in accordance with any conventional method, e.g. as described in the following references (which also describe suitable methods for activation of polymer molecules): Harris and Zalipsky, eds., Poly(ethylene glycol) Chemistry and Biological Applications, AZC, Washington; R.F. Taylor, (1991), "Protein Immobilisation: Fundamentals and Applications", Marcel Dekker, N. Y.; S.S. Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; G.T. Hermanson et al., (1993), "Immobilized Affinity Ligand Techniques", Academic Press, N. Y., all of which are incorporated herein by reference. The process of conjugating an activated polyethylene glycol to the dual agonist compound is referred to herein as "PEGylation". Covalent coupling of a polyethylene glycol moiety to the dual agonist compound can be targeted to a specific attachment site by selection of the appropriate activated polyethylene glycol and reaction conditions. These are well known in the art and are described in more detail hereinbelow. Furthermore, the conjugation may be achieved in one step or in a stepwise manner using known methods, such as those described in WO 99/55377, which is incorporated herein by reference. [131] For PEGylation of cysteine residues the polypeptide is usually treated with a reducing agent, such as dithiothreitol (DDT) prior to PEGylation. The reducing agent is subsequently removed by any conventional method, such as by desalting. Conjugation of PEG to a cysteine residue typically takes place in a suitable buffer at pH 6-9 at temperatures varying from 4°C to 25°C for periods up to about 16 hours. [132] PEGylation of lysines often employs PEG moieties activated with active esters, such as, for example, N-hydroxysuccinimidyl (NHS) ester (e.g., mPEG-N- hydroxylsuccinimide (e.g., mPEG-NHS or mPEG2-NHS), esters such as PEG succinimidyl propionate (e.g., mPEG-SPA) or PEG succinimidyl butanoate (e.g., mPEG- SBA), and the like, including other activated PEG NHS esters described herein). One or more PEG moieties can be attached to a protein within 30 minutes at pH 8-9.5 at room temperature if about equimolar amounts of PEG and protein are mixed. A molar ratio of PEG to protein amino groups of 1-5 to 1 will usually suffice. Increasing pH increases the rate of reaction, while lowering pH reduces the rate of reaction. These highly reactive active esters can couple at physiological pH, but less reactive derivatives typically require higher pH. Low temperatures may also be employed if a labile protein is being used. Under low temperature conditions, a longer reaction time may be used. [133] Covalent attachment of a PEG moiety to the N-terminal amino group of a polypeptide is referred to herein as "N-terminal PEGylation". N-terminal PEGylation is facilitated by the difference between the pKa values of the α-amino group of the N- terminal amino acid (-7.6 to 8.0) and the ε-amino group of lysine (~10). PEGylation of the N-terminal amino group often employs PEG-aldehydes, which are more selective for amines and thus are less likely to react with the imidazole group of histidine; in addition, PEG reagents used for lysine conjugation may also be used for conjugation of the N- terminal amine. Conjugation of a PEG-aldehyde to the N-terminal amino group typically takes place in a suitable buffer (such as, 100 mM sodium acetate or 100 ήiM sodium bisphosphate buffer with 20 mM sodium cyanόborohydride) at pH ~ 5.0 overnight at temperatures varying from about 4°C to 25°C. Useful N-terminal PEGylation methods and chemistries are also described in US Pat. 5,985,265 and US Pat. 6,077,939, both of which are incorporated herein by reference.
[134] A PEG moiety may be conjugated to any one or more components of the dual agonist compound, i.e., TM1, L3, TM2, L2, and/or the IL-3. The TM1, L1, TM2, L2, and IL-3 components may be designed to have one or more attachment groups (or functionality) that are suitable for reaction with the desired PEG moiety. Suitable attachment groups are described hereinabove.
[135] Often, at least one or more PEG moieties is conjugated to the IL-3 component of the dual agonist conjugate at one or more of the following attachment groups in the IL-3 (with reference to the amino acid position number corresponding to that of mature recombinant huIL-3 (SEQ ID NO: 7): KlO, C 16, K28, K66, K79, C84, KlOO, Kl 10, Kl 16, the α amino group on the N-terminal amino acid residue of IL-3 if that group is not bound to a linker moiety, and any combination of two or more thereof. Typically, dual agonist conjugates of the present invention comprise a PEG moiety bound to a lysine attachment group in the IL-3 component selected from KlO, K28, K66, K79, KlOO, Kl 10, Kl 16, or any combination of two or more thereof. Usually, dual agonist conjugates of the present invention comprise a PEG moiety bound to K28. Often, dual agonist conjugates of the present invention comprise a PEG moiety bound to KlO, K28, and K66 or KlO and K28. In some embodiments, dual agonist conjugates of the present invention comprise a PEG moiety bound to Kl O5 K28, K66, K79, Kl 00, Kl 10, and Kl 16.
[136] As described above, for example in paragraphs 057-059 and 062-064, any one of these amine reactive PEG attachment sites may be removed to eliminate attachment at that particular site. For example, the dual agonist conjugate may have an IL-3 mutein component that has a substitution selected from Kl OR, K28R, K66R, K79R, Kl 0OR, Kl 1OR, Kl 16R or any combination of two or more substitutions thereof (where numbering is with reference to mature recombinant hIL-3 (SEQ ID NO: 7). Dual agonist conjugates typically contain an IL-3 mutein with a substitution that is a substitution selected from KlOR or K28R. [137] Similarly, reactive PEG attachment sites may be introduced into the IL-3 component of the dual agonist component as described above, for example in paragraphs 057-064.
[138] Dual agonist conjugates typically have one or more polyethylene glycol moieties attached to a lysine residue in the IL-3 moiety of the dual agonist conjugate. The polyethylene glycol moiety may be linear or branched, and typically has an average molecular weight of about 20,000 to about 40,000 daltons, usually about 30,000 to about 40,000 daltons, and often about 40,000 daltons. [139] If cysteine pegylation methods are utilized, dual agonist conjugates of the present invention typically comprise a PEG moiety bound to the IL-3 component at the following attachment groups: C 16, C84, or both. PEG moieties may be bound to any combination of the aforedescribed attachment groups, as well as any introduced attachment groups described herein. When cysteine pegylation methods are utilized, the polyethylene glycol moiety may be linear or branched, and typically has an average molecular weight of about 20,000 to about 40,000 daltons, usually about 30,000 to about 40,000 daltons, and often about 40,000 daltons.
[140] In some instances, it may be desirable to conjugate as many of the available polymer attachment groups as possible with polymer. This may be achieved by utilizing a molar excess of polymer relative to the dual agonist compound. Such molar excess may be achieved by utilizing a molar ratio of activated polymer to dual agonist compound of up to about 100: 1 or 200: 1 to about 1000: 1. In some cases, the molar ratio may be somewhat lower, such as up to about 50:1, 10:1 or 5:1. An equimolar ratio of activated polymer to dual agonist compound may also be used. [141] As described above, dual agonist conjugates of the present invention also include conjugates having one or more sugar moieties (i.e., carbohydrate molecules) bound to the dual agonist conjugate. These conjugates are also referred to herein as "glycosylated" dual agonist compounds. The glycosylation sites on the dual agonist compound are typically either an N- or O-glycosylation site. As used herein, the term "N-glycosylation site" refers to the sequence N-X-S/T/C", wherein X is any amino acid residue except proline, N is asparagines and S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine. An "O-glycosylation site" refers herein to the —OH group of a serine or threonine residue.
[142] Dual agonist compounds may be glycosylated by either an in vitro or in vivo process. Depending on the coupling method employed, the carbohydrate(s) may be attached to the following attachment groups: a) arginine and histidine, as described in Lundblad and Noyes, "Chemical Reagents for Protein Modification", CRC Press me. Boca Raton, FI, which is incorporated herein by reference b) free carboxyl groups (e.g. of the C-terminal amino acid residue, asparagine or glutamine), c) free sulfhydryl groups such as that of cysteine, d) free hydroxyl groups such as those of serine, threonine, tyrosine or hydroxyproline, e) aromatic residues such as those of phenylalanine or tryptophan or f) the amide group of glutamine. which may be introduced and/or removed in the polypeptide of the invention. Suitable methods of in vitro coupling are described in WO 87/05330 and in Aplin et al./'CRC Crit Rev. Biochem." pp. 259-306, 1981, both of which are incorporated herein by reference. The in vitro coupling of sugar moieties to protein- and peptide-bound Gin-residues can also be carried out by transglutaminases (TGases), e.g. as described by Sato et al., 1996 Biochemistry 35:13072-13080 and EP 725145, both of which are incorporated herein by reference.
[143] Dual agonist polypeptides of the present invention may be glycosylated in vivo by introducing a polynucleotide encoding a dual agonist polypeptide having one or more N- or O-glycosylation sites into a glycosylating eukaryotic expression host cell. The glycosylation site may be one of the natural glycosylation sites in mature recombinant ML-3, SEQ ID NO: 7 (i.e., Nl 5 and N70, with amino acid position numbering corresponding to SEQ ID NO: 7), or a glycosylation site that is introduced into any of the components of the dual agonist polypeptide (i.e., TM1, TM2, L1, L2, L3, or the IL-3). Substitutions in mature recombinant hIL-3 (SEQ ID NO: 7) that are useful for introducing one or more glycosylation sites into the IL-3 component of the dual agonist compound are described in more detail in the discussion of IL-3 muteins hereinabove. [144] The glycosylating eukaryotic expression host cell may be selected from a fungal cell (e.g., a filamentous fungal cell or a yeast cell), an insect cell , a mammalian cells, a plant cell, or any other glycosylating eukaryotic expression host cell known in the art. [145] Typically, dual agonist conjugates of the present invention comprise a sugar moiety bound to one or both of the natural glycosylation sites in mature recombinant hlL- 3 (SEQ ID NO: 7) or a mutein thereof which retains the two natural glycosylation sites, i.e., Nl 5 and N70 (with amino acid position numbering corresponding to SEQ ID NO: 7). Often, dual agonist conjugates of the present invention comprise one or more sugar moieties bound to one or more introduced glycosylation sites in the IL-3, TM1, L1, TM2, or combination of any two or more thereof. These conjugates may further comprise a sugar moiety bound to one or both of the natural glycosylation (Nl 5 and N70) sites in mature recombinant hIL-3 (SEQ ID NO: 7). When one or more sugar moieties is bound to an introduced glycosylation site in the IL-3, it is typically bound to an asparagine residue at one or more of the following amino acid positions corresponding to SEQ ID NO: 7: P2N, T4N, Q5N, L9N, KlON, Nl 8, 123N> N38, N39, N41, N51, N52, N62, N63, N72, L87N, N105, Kl ION, Ll 15N5 N120, Q124N, Q125N, T127N, and any combination or two or more thereof (with substitutions shown relative to SEQ ID NO: 7). IL-3 muteins having introduced glycosylation sites are described in more detail hereinabove. Dual agonist conjugates of the present invention may also comprise a combination of one or more non-polypeptide polymers (such as, for example, PEG, mPEG, and the like) and one or more sugar moieties bound to the dual agonist compound. [146] Non-polypeptide lipophilic moieties that are suitable for conjugation to the dual agonist compounds of the present invention include a natural compound such as a saturated or an unsaturated fatty acid, a fatty acid diketone, a te'rpene, a prostaglandin, a vitamin, a carotenoid or a steroid, a phospholipid, or alternatively, a synthetic compound, such as a linear or branched aliphatic, aryl, alkaryl acid (e.g., carboxylic, sulphonic, and the like), alcohol, amine, and the like. Conjugation to non-polypeptide lipophilic moieties may take place at any one of the following exemplary attachment sites: the N- terminus or the C-terminus of the dual agonist compound, the hydroxyl groups of the amino acid residues Ser, Thr or Tyr, the ε-amino group of Lys, the SH group of Cys or the carboxyl group of Asp and GIu. The dual agonist compound and the non-polypeptide lipophilic moiety may be conjugated to each other either directly or indirectly via a linker in accordance with methods known in the art, such as those described in Bodanszky, "Peptide Synthesis", John Wiley, New York (1976) and WO 96/12505, both of which are incorporated herein by reference.
[147] Non-polypeptide conjugation moieties may be bound, either directly or indirectly via a linker moiety, to any one or more components of the dual agonist compound, i.e., TM1, L1, TM2, L2, or IL-3. Linker moieties that are suitable for conjugating the non- polypeptide conjugation moieties indirectly to the dual agonist compound include any of the linker types described hereinabove for L1 and L2. Other suitable linkers will be readily apparent to the skilled person. For example, the non-polypeptide polymer may be conjugated to the dual agonist compound via a cyanuric chloride linker as described in Abuchowski et al., (1977), J. Biol. Chem..252, 3578-3581; US 4,179,337; and Shafer et al., (1986), , 24, 375-378, all of which are incorporated herein by reference. [148] It has been discovered that dual agonist conjugates (for example, PEGylated with a branched or a linear PEG moiety of from 20 kDa to about 40 KDa) may induce the production of at least about 2 times more platelets in a mammal (non-human or human) than that observed for the corresponding non-conjugated form of the dual agonist measured 6 days after intravenous administration of a single equivalent dose of the dual agonist conjugate or the non-conjugated form of the dual agonist, respectively. Suitable dual agonist conjugates include those described hereinabove, and in particular the dual agonist conjugates having a dual agonist of formula I, and TMP1 and TMP2 moieties of sequence formula (1) or sequence formula (13), and PEGylated with a branched or a linear PEG moiety, for example a PEG having a molecular weight of from 20 kDa to about 40 kDa. The foregoing dual agonist conjugates of the present invention may induce the production of at least about 2.5 and 3 times more platelets in a mammal, than that observed for the corresponding non-conjugated form of the dual agonist measured 6 days after intravenous administration of a single equivalent dose of the dual agonist conjugate or the non-conjugated form of the dual agonist, respectively. Exemplary mammals include a rat (using the assay of Example 9 and a single dose of 100 μg/kg), a mouse, a monkey, a human, and the like. [149] The inventors have also discovered that dual agonist conjugates having one or more PEG moieties are more stable to proteolysis. Biotinylated PEGylated dual agonist compounds (i.e., dual agonist conjugates) were incubated with rat plasma at 370C overnight and the samples were analyzed by western blot probed with avidin-horse radish peroxidase to detect peptides cleaved from the molecules by proteases in the rat plasma. The results suggested that the bands detected as likely degradation products were less than 1% compared to the full length protein. Accordingly, the present invention provides dual agonist conjugates that are more resistant to proteolytic degradation in vivo as compared to the corresponding non-conjugated dual agonist compound. [150] Dual agonist conjugates may exhibit reduced immunogenicity as compared to hTPO. The reduction may be on the order of at least 10%, 25%, 50%, or at least 75% as compared to hTPO. Irnmunogenicity may be determined by use of any suitable method known in the art. The term "reduced immunogenicity" is intended to indicate that the dual agonist compound or conjugate of the present invention gives rise to a measurably lower immune response than that of a reference molecule (such as, for example, hTPO, hIL3, or a fragment or variant thereof), as determined under comparable conditions. Reduced antibody reactivity (e.g., reduced reactivity towards antibodies present in serum from patients treated with, for example, hTPO or a fragment thereof, or hIL3 or a fragment thereof) is an example of an indication of reduced immunogenicity. [151] Dual agonist conjugates of the present invention may exhibit reduced or no neutralization when contacted with antibodies or antisera isolated from patients treated with hTPO or a fragment thereof. Neutralization may be evidenced by a decrease in biological activity (such as, receptor binding activity or agonist activity) of a test molecule when an antibody or antisera raised against a control molecule is incubated with the test molecule. In this example, the test molecule is the conjugate of the invention and the control molecule is the hTPO or fragment thereof. Reduced neutralization may be expressed as the percent difference between the decrease in biological activity of the test molecule and the decrease in biological activity of the control molecule and assayed under comparable conditions. For example, a conjugate of the invention may exhibit at least 25% reduced neutralization, such as at least 50% reduced neutralization, e.g., at least 75% reduced neutralization, for example at least 90% reduced neutralization, e.g., at least 95% reduced neutralization, relative to the neutralization observed when the hTPO or fragment thereof is contacted with antibodies or antisera isolated from patients treated with hTPO or a fragment thereof. [152] In another aspect, a polypeptide comprising the sequence of hTPO or a fragment thereof may exhibit reduced or no neutralization when contacted with antibodies or antisera isolated from patients treated with a conjugate of the invention, relative to the neutralization observed when contacted with antibodies or antisera isolated from patients treated with hTPO or a fragment thereof. For example, a polypeptide comprising the sequence of hTPO or a fragment thereof may exhibit at least 25% reduced neutralization, such as at least 50% reduced neutralization, e.g., at least 75% reduced neutralization, when contacted with antibodies or antisera isolated from patients treated with a conjugate of the invention, relative to the neutralization observed when the TPO or fragment thereof is contacted with antibodies or antisera isolated from patients treated with a conjugate of the invention, relative to the neutralization observed when the hTPO or fragment thereof is contacted with antibodies or antisera isolated from patients treated with hTPO or a fragment thereof.
Dual Agonist Derivatives
[153] The present invention also provides dual agonist derivatives comprising a dual agonist compound modified by an organic derivatizing agent. Typically, the organic derivatizing agent is employed to alter an amino acid residue in the dual agonist compound. Such derivatizing agents and methods are well known in the art. [154] For example, cysteinyl residues most commonly are reacted with oc-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(4-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2- pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7- nitrobenzo-2-oxa-l,3-diazole. Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing α-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase- catalyzed reaction with glyoxylate. Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as wen as the arginine guanidino group. Carboxyl side groups (aspartyl or glutamyl or C- terminal amino acid residue) are selectively modified by reaction with carbodiimides (R- N=C=N-R'), where R and R' are different alkyl groups, such as l-cyclohexyl-3-(2- morpholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
Dual Agonist Compositions
[155] The present invention provides a composition comprising a dual agonist compound of the present invention or conjugate thereof as described hereinabove, together with a pharmaceutically acceptable carrier or excipient.
[156] Suitable pharmaceutically acceptable excipients include processing agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β- cyclodextrin, polyvinylpyrrolidone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof. Other suitable pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences", 18th edition, A.R. Gennaro, Ed., Mack Pub. Co. New Jersey (1991), "Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis (2000), and "Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), all of which are incorporated herein by reference.
[157] Pharmaceutical compositions containing an invention dual agonist compound or conjugate thereof may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion. Liquid carriers contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils and fats, and the like, as well as mixtures of any two or more thereof. The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, and the like. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like. For parenteral administration, the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like. Compositions of the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof. [158] The dual agonist compounds and conjugates of the present invention may be administered orally, parenterally, sublingually, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoretic devices. The term "parenteral" as used herein includes subcutaneous injections, intravenous administration, intramuscular administration, intrasternal injections, transdermal or transmucosal administration, or infusion techniques. [159] Injectable preparations (such as, for example, sterile injectable aqueous or oleaginous suspensions) may be formulated using standard methods and materials known in the art, such as, for example, suitable dispersing, wetting, and suspension agents. The sterile injectable preparation may also be a solvent, for example, as a solution in 1,3- propanediol. Among the acceptable vehicles and solvents that may be employed are water. Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. [160] Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise additional substances other than inert diluents, such as, for example, lubricating agents (e.g., magnesium stearate), and the like. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
[161] Compounds of the present invention may also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in lipid form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. Typical lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods of forming liposomes are known in the art and are described in Prescott, Ed., "Methods in Cell Biology", Volume XIV, Academic Press, New York, N. W., p. 33 et seq. (1976), which is incorporated herein by reference.
Methods of Using Dual Agonist Compounds. Dual Agonist Conjugates, and Compositions Thereof [162] In accordance with a further embodiment, the present invention provides a method for stimulating platelet production in a human or non-human animal subject (e.g., a patient in need thereof), said method comprising administering to a subject an amount of dual agonist compound or conjugate thereof (or composition comprising such compound or conjugate) effective to stimulate platelet production in the subject. The present invention also provides a method for stimulating cell proliferation comprising administering to the cell an amount of invention dual agonist compound, conjugate or composition thereof effective to induce proliferation of the cell. Typical cells include platelets and/or megakaryocytes. The term "cell proliferation stimulation" refers herein to detectable stimulation of cell proliferation as compared to a control. [163] A composition of the invention may stimulate megakaryocyte production by inducing proliferation of early and late platelet progenitor cells and maturation of megakaryocytes to produce platelets. When given prior to chemotherapy, a composition of the invention may increase the reservoir of platelet precursors and protect megakaryocytes from chemotherapy induced apoptosis. Such compositions are useful in a number of therapeutic and/or prophylactic applications, primary of which is the ability to enhance platelet production in a subject, for example to prevent or reduce the duration of thrombocytopenia in a subject undergoing chemotherapy, without the risk of the subject generating neutralizing antibodies, for example, to endogenous hTPO. Use of a composition of the invention may reduce or eliminate the need for platelet transfusions, in instances such as prior to, concurrent with or following myelosuppressive chemotherapy.
[164] Thrombocytopenia is a blood disorder characterized by an abnormally low number of platelets in the bloodstream. A normal platelet count in human blood is usually at least about 150,000 cells per microliter (μl) of blood, with a range generally between about 150,000 and 350,000 platelets/μl of blood. When the platelet number drops below 150,000/μl, such as below 100,000/μl, the patient is considered to be ' thrombocytopenic. Patients with thrombocytopenia are predisposed to a risk of hemorrhage which increases with decreasing platelet levels. Treatment for thrombocytopenia may be initiated when platelet counts are, for example, below 50,000/μl, below 40,000/μl, or below 30,000/μl, such as in the range of 50,000/μl to 20,000/μl, e.g., in the range of 30,000/μl - 20,000/μl. At platelet levels below 10,000/μl, spontaneous, and sometimes fatal, hemorrhage can occur.
[165] Thrombocytopenia may occur as a result of decreased platelet production in the subject, increased platelet destruction in the subject, and/or other factors associated with platelet loss, and may be associated with a variety of diseases, disorders, treatments or other factors. [166] For example, thrombocytopenia may arise from decreased platelet production, which, for example, may be associated with chemotherapy or radiation therapy, may be associated with intake of drugs or other substances (e.g., alcohol, thiazide diuretics), may be associated with a congenital or inherited disorder ( such as May Hegglin Anomaly, Bernard Soulier syndrome, Alport syndrome), or may result from an acquired condition, such as a viral infection (for example, chicken pox, mumps, rubella) or bone marrow infiltration (e.g., associated with cancer).
[167] Thrombocytopenia may occur as a result of increased platelet destruction. For example, platelet destruction may be associated with immune-related disorders (such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus), may be drug induced (e.g., heparin, depakote, quinine), may be associated with HIV, thrombotic thrombocytopenic purpura, hemolytic uraemic syndrome, disseminated intravascular coagulation, may be associated with physical trauma (e.g., cardiopulmonary bypass, artificial heart valve), or certain infections (such as mononucleosis, cytomegalovirus). [168] Thrombocytopenia may occur as a result of platelet loss associated with, for example, large volume transfusion or splenomegaly (e.g., cirrhosis of the liver, Gaucher's disease).
[169] Accordingly, the invention includes a method of increasing the number of platelets in a subject suffering from thrombocytopenia or at risk for thrombocytopenia. In one aspect, the method comprises administering an effective amount of a composition comprising a dual agonist polypeptide or conjugate thereof to the subject (e.g., a mammalian host) such that an increased number of platelets are produced in the subject compared to the number of platelets present in the subject prior to such administration. The subject in need of such treatment may be suffering from thrombocytopenia associated with, for example, chemotherapy for treatment of cancer, AIDS, and the like, or radiation therapy, may be undergoing a bone marrow transplant procedure, or may be suffering from a disorder characterized by low platelet numbers, such as idiopathic thrombocytopenic purpura or thrombotic thrombocytopenic purpura. [170] For example, a patient with a myeloid or nonmyeloid malignancy who is at risk for thrombocytopenia (based on, e.g., the type of chemotherapy regimen, or prior history of thrombocytopenia) may be administered a composition of the invention prior to, concurrent with, or following the chemotherapy regimen.
[171] In another example, a patient undergoing a bone marrow transplant procedure may be administered a composition of the invention prior to, concurrent with, or following the procedure.
[172] The composition of the invention may be administered prophylactically, for example, to a patient prior to or concurrent with the start of a course of chemotherapy or radiation therapy or prior to a procedure (such as, bone marrow transplant), to prevent or reduce the duration of thrombocytopenia known or suspected to be associated with such' therapy or procedure.
[173] The composition of the invention may be administered therapeutically to a patient exhibiting a low platelet count (such as, a thrombocytopenic patient) to increase the number of platelets in the patient. For example, the composition may be administered to the patient exhibiting a low platelet count (e.g., less than 100,000 platelets/μl of blood, such as less than 50,000 platelets/μl of blood, e.g., less than 30,000 platelets/μl of blood, for example, between 20,000-30,000 platelets/μl/blood) in an amount sufficient to raise the platelet count to greater than, e.g., 100,000 platelets/ μl of blood, such as to at least about 150,000 platelets/μl of blood, e.g., to between about 150,000 and 350,000 platelets/μl of blood. [174] Effective amounts of compounds, conjugates, and compositions of the invention generally include any amount sufficient to detectably stimulate platelet stimulation which can be assessed by a complete blood count (CBC) from whole blood drawn from the subject in question. Successful treatment of a subject in accordance with the invention may result in the inducement of a reduction or alleviation of symptoms in a subject afflicted with thrombocytopenia, chemotherapy-induced, idiopathic pupura (ITP), liver disease, bone marrow failure, myelodysplastic syndrome (MDS), and other similar disorders which are characterized by platelet deficiency.
[175] The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the disorder. The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.
[176] For purposes of the present invention, a therapeutically effective dose will generally be from about 0.1 μg/kg/day to about 10 μg/kg/day of a dual agonist compound or conjugate of the present invention, which may be administered in one or multiple doses. [177] While the compounds and conjugates of the invention can be administered as the sole active pharmaceutical agent, they may also be used in combination with one or more other agents used in the treatment of other disorders, as well as thrombocytopenia. For example, the present invention provides combining a dual agonist compound or conjugate thereof, with a cytokine (such as, for example, stem cell factor (SCF), leukemia inhibitory factor (LIF), interferon-α, consensus interferon, IFN-β, IFN-γ, an interleukin (e.g.. IL-7, IL-8, IL-9, IL-IO, and the like), and the like) and/or a hematopoietic factor (such as, for example, erythropoietin (EPO), granulocyte colony stimulating factor (G- CSF), and the like) and/or an angiopoeitin (such as, for example, angiopoeitin-1 (Ang-1), angiopoeitin-2 (Ang-2), and the like, the human angiopoetin-like polypeptide, and the like), and/or other therapeutically active agent in a pharmaceutical composition that is administered in accordance with the methods provided herein. [178] The compounds and conjugates of the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower dosages. Dosage levels of the active compounds and conjugates in the compositions of the invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease, and the response of the patient. The combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition. [179] The foregoing and other aspects of the invention may be better understood in connection with the following non-limiting examples.
EXAMPLES
EXAMPLE 1.
Construction of DNA Constructs for Expression of a Dual Agonist Polypeptide A. Preparation of Nucleic Acid Encoding a TM -L -TM2 peptide sequence
[180] Variants were designed with different amino acid substitutions within TM ' and TM2. Once the amino acid sequences were selected, DNA sequences encoding each TM1 -L1 -TM2 component were designed using codons optimal for E. coli and mammalian expression. In addition, codons were selected at each position in order to avoid repeat sequences in TM1 versus TM2. For example:
ATC GAA GGT CCA ACG CTT ACT CAG TGG TTG GCA GCC CGT GCA GGC GGTGGC TCCATT GAG GGC CCT ACC CTT AGT CAA TGG TTG GCC GCA CGT GCA (SEQ ID NO: 263); and
ATC AGC GGT CCA ACG CTT CGT CAG TGG TTG GAA GCC CGT GCA GGC GGTGGC TCCATT GCG GGC CCTACC CTT CGT CAATGG TTG GAG GCA CGT GCA (SEQ IDNO: 275).
[181] The encoded TM'-L'-TM2 peptide sequences are provided as SEQ ID NOS: 264 and 276, respectively. The first underlined section is TM1, the sequence in italics is the linker, L1, and the second underlined sequence is TM2.
[182] Further exemplary nucleic acids encoding TM'-L^TM2 are illustrated in SEQ ID NOS: 255, 257, 259, 261, 265, 267, 269, 271, 273, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, and 307. The corresponding TM'-L^TM2 encoded sequences are provided as SEQ ID NOS: 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, and 308, respectively. B. Assembly of IL-3 gene
[183] Mature recombinant human IL-3 (SEQ ID NO: 7) was back translated using codon biases for E. coli and mammalian cells. The gene was designed and optimized based on E. coli codon biases and was predicted to be acceptable for mammalian expression as well. Overlapping 50-mers were assembled using PCR, and the full-length gene was rescued using terminal primers. IL-3 gene: get cct atg ace caa ace ace tec ttg aaa acg tec tgg gtc aat tgt tec aat atg ate gac gag ate ate act cac ttg aaa caa cct cct ttg cca ctt ctt gac ttc aat aac ttg aac ggt gag gac caa gat att ttg atg gag aat aat ttg cgt cgt cca aat ttg gaa get ttc aat cgt get gtc aaa agt ctt cag aac gcc tea gcc att gaa tct ate ctt aag aac ctt ctt cct tgt ttg cct ttg gca ace get get cct ace cgt cac ccc ate cac ate aag gat ggt gac tgg aac gag ttt cgt cgt aaa ttg ace ttt tac ttg aaa ace ttg gag aac gca cag gcc caa caa acg acg ttg tec ctt gca ate ttt (SEQ ID NO: 6)
C. Assembly of Expression Constructs
[184] In order to generate a fusion of the TM^L1 -TM2 peptide sequence and mature recombinant human IL-3, bridging oligos were designed to overlap with one end of the TM1 -L1 -TM2 peptide sequence and one end of the IL-3 gene (for example, linking the 3' end of the TM '-L1 -TM2 peptide sequence to the 5' end of the IL-3 sequence). The bridging oligos can also encode for an "in-frame" second linker "L2" comprising neutral amino acids such as Glycine, Serine, and Threonine to create a linker between the TM1- L'-TM2 peptide and IL-3 of lengths such as 6 or 12 amino acids residues (for example: GGGSGG (SEQ ID NO: 368) or GGGSGGGSGGGS (SEQ ID NO: 364). [185] Illustrative dual agonist polynucleotides are provided as SEQ ID NOS: 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, and 361. These dual agonist polynucleotides encode the dual agonist polypeptides corresponding to SEQ ID NOS-/310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348-, 350, 352, 354, 356, 358, 360, and 362 respectively. [186] The assembled nucleic acids encoding the dual agonist polypeptides were cloned into the pCR4 vector (Invitrogen, Carlsbad, CA) using a TOPO Cloning® kit and sequenced. The nucleic acids were subsequently subcloned into vector pCDNA3.1 (+) (Invitrogen, Carlsbad, CA) for protein expression in mammalian cells or subcloned into vector pCKQ3 (Figure 2) for E. coli expression. Mammalian expression constructs were made carrying these assembled genes under the transcriptional control of the eukaryotic CMV promoter and carrying an amino terminal TPA signal peptide for secretion.
Expression in the bacterial expression vector was under the transcriptional control of a T5 lac promoter and was restricted to the cytoplasm to favor production of inclusion bodies. Both mammalian and bacterial expression constructs were made with and without amino- terminal 6HIS tags for purification.
EXAMPLE 2
Production of Dual Agonist Polypeptides A. Transient Mammalian Expression [1871 The green monkey kidney cell line COS-7 (ATCC Acession No.CRL- 1651) was maintained in a medium of DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin/glutamine (PSG) (GIBCO brand, Invitrogen, Carlsbad, CA). One day prior to transfection, Tl 75 flasks were seeded with 2 x 106 cells in 50 mis of the media. After overnight incubation, the media was replaced with Opti-MEM (GIBCO brand, Invitrogen, Carlsbad, CA) supplemented with PSG or fresh media and the cells were transfected with 20 μg of the pCDNA3.1 (+) DNA construct carrying the dual agonist polynucleotides from Example 1C:6O μl FuGENE 6 Transfection Reagent (Roche Applied Science, Mannheim, Germany). Cell supernatant containing the expression product was harvested after 3 days of cultivation. Since, the human IL-3 portion of the dual agonist polypeptide contains natural occurring, mammalian glycosylation recognition sites, expression in COS-7 results in glycosylated protein. Glycosylated dual agpnist polypeptide was isolated from the supernatant using the method described in part C below.
B. Stable Mammalian Expression [188] The Chinese hamster ovary cell line CHO-Kl (ATCC Accession No. CCL-61) was maintained in DMEM/F12 (GIBCO brand, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (GIBCO brand, Invitrogen, Carlsbad, CA)3 1% penicillin/streptomycin/glutamine (PSG) (GIBCO brand, Invitrogen, Carlsbad, CA). Cells were resuspended to IxIO7 / ml in Opti-MEM (GIBCO brand, Invitrogen, Carlsbad, CA) and transfected with 20 μg of the pCDNA3.1(+) DNA construct carrying the dual agonist polynucleotides from Example 1 C by electoporation using a Gene Pulser II electroporator (Bio-Rad, Hercules, CA). The pCDNA3.1(+) DNA construct carries a G418 resistance gene for selection of stably transfected cells. After 5 days of culture in selection media A [DMEM/F12 (GIBCO brand, Invitrogen, Carlsbad, CA) supplemented with 10% FBS and G418 antibiotic at 1 mg/ml (GIBCO brand, Invitrogen, Carlsbad, CA)] to eliminate untransfected cells, the cells were sorted in a DakoCytomation MoFIo cell sorter (Dako Colorado, Inc. Fort Collins, CO) into 96 well plates containing 200 μl of selection medium. The plates were incubated at 37°C for 10-14 days. Subclones exhibiting high expression, as determined by western blot analysis using anti-IL-3 antibodies (PeproTech #500-P23Bt, PeproTech, Inc. Rocky Hill, NJ) for the dual agonist polypeptides of the present invention were expanded. The best expressing clones were selected for large scale cultivation in roller bottles containing expression media DMEM/ Fl 2 supplemented with insulin-transferrin-selenium A (GIBCO brand, Invitrogen, Carlsbad, CA)]. Since, the human IL-3 portion of the dual agonist polypeptide contains naturally occurring, mammalian glycosylation recognition sites, expression in CHO cells results in glycosylated recombinant protein. Glycosylated, dual agonist polypeptide was isolated from the supernatant using the method described in part C below.
C. Protein Recovery and Purification from Mammalian Culture Supernatants [189] Glycosylated, recombinant dual agonist proteins carrying 6His tags were isolated and purified from mammalian culture supernatants (COS transient from Example 2A or CHO stable supernatant from Example 2B) by affinity to immobilized metal affinity chromatography (IMAC) columns. The supernatants were adjusted to 25nM HEPES pH 7.5, 200 mM NaCl5 10 mM Imidazole, then loaded on a POROS MC metal affinity column (Applied Biosystem, Foster City, CA) on a BioCAD 700E Workstation for Perfusion Chromatography (Applied Biosystems, Foster City, CA). Affinity purified proteins were further purified by size on a Superdex 75 size exclusion chromatography column (GE Healthcare, Piscataway, NJ).
D. E. coli expression [190] E. coli strain W3110 (E. coli Genetic Stock Center, New Haven, CT) was transformed with plasmids encoding dual agonist polypetides as described in Example 1C. Liquid cultures were inoculated from glycerol stocks into shake flasks containing 2XYT medium (EMD, Gibbstown, NJ) with 50 μg/ml kanamycin (Sigma, St. Louis, MO) and 0.5 % glucose (Sigma, St. Louis, MO). The culture was grown overnight at 370C, shaking at 250 RPM. The culture was then diluted 1 :3.5 into 2XYT medium with 50 μg/ml kanamycin and 2 mM PTG (Calbiochem, La Jolla, CA) for a four hour induction at 370C and 250 JAPM. The cells were harvested by centrifugation and resuspended in 7 ml PBS (GIBCO brand, Invitrogen, Carlsbad, CA) per pellet. The cells were lysed using a French Press (Thermo Electron Corporation, Waltham, MA). Three passes were done at 1500 psi. The inclusion bodies were harvested by centrifugation and washed twice with 1 % Triton X-IOO and once with water for irrigation (WFI).
E. Protein purification for E. coli expression
[191] The inclusion bodies were solubilized at 40 mg inclusion body/ml 8M urea (JT Baker, Phillipsburg, NJ) in 1/2X PBS and incubated for one hour at room temperature. Insoluble material was removed by centrifugation. The solubilized inclusion bodies were prepared for cation exchange purification by 1 :4 dilution with equilibration buffer, pH adjustment to 6.5 and filtration through a 0.2 μ filter. The material was loaded at <7 AU/ml gel onto a SP Sepharose HP column (GE Healthcare, Piscataway, NJ) equilibrated with 25 mM sodium phosphate (Sigma, St. Louis, MO), 8 M urea, pH 6.5. The protein was eluted using a 0 - 80 mM NaCl (Sigma, St. Louis, MO)5 20 column volume gradient. [192] The eluate pool was refolded overnight at 0.1 mg/ml in 3 M urea with 1:10 oxidizingrreducing glutathione (Sigma, St. Louis, MO) (0.1:1 mM) atpH 8. The refold mixture was then adjusted to pH 6.5 and filtered using a 0.2 μ filter. This material was loaded onto a SP Sepharose HP column equilibrated with 25 mM sodium phosphate, pH 6.5. The protein was eluted with a 0 - 150 mM NaCl, 30 column volume gradient. The eluate pool was either concentrated to between 0.4 and 0.7 mg/ml and dialyzed against PBS, pH 6.5 or dialyzed against 50 mM sodium borate (Sigma, St. Louis, MO), pH 9 for a subsequent PEGylation reaction.
EXAMPLE 3
Assay for IL-3 receptor agonist activity - TF-I Proliferation Assay [193] Human erythroleukemia cell line, TF-I , DSMZ Accession No. ACC 334, was . obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). This cell line is described in Kitamura, et al., Blood, 73:375 (1989), Kitamura; et al., J. CellPhysio., 140:323 (1989), and Drexler, et al., Leukiemia, 11:701 (1997), all of which are incorporated herein by reference. The TF-I cells were maintained in growth media A [RPMI 1640 (GIBCO brand, Invitrogen, Carlsbad, CA), 10% fetal bovine serum (FBS) (GIBCO brand, Invitrogen, Carlsbad, CA), 1 ng/ml GM-CSF (R&D Systems, Minneapolis, MN), 1 ng/ml penicillin/streptomycin (PS) (GIBCO brand, Invitrogen, Carlsbad, CA)] in a humidified incubator at 370C and 5% CO2. For the proliferation assay, cells were washed three times in phosphate buffer solution (PBS) and grown in starvation media A [RPMI 1640 (GIBCO brand, Invitrogen, Carlsbad, CA), 1% fetal bovine serum (FBS) (GIBCO brand, Invitrogen, Carlsbad, CA), 1 ng/ml penicillin/streptomycin (PS) (GIBCO brand, Invitrogen, Carlsbad, CA)] overnight. On the day of the assay, 5 x 103 cells in 50 μl starvation media were seeded into each well of a 96-well white opaque plate. Test proteins were serially diluted in starvation media. A to various concentrations and 50 μl was added to the cells. Proliferation was measured after 3 days using the CellTiter-Glo reagent (Promega, Madison, WI) which detects metabolic ATP by luminescence as a measure of viable cell numbers. Purified, recombinant human IL-3 (SEQ ID NO: 7) (R&D Systems, Minneapolis, MN) was tested on each plate to generate a standard curve of 10 points on a 7x dilution series for comparison. ' EXAMPLE 4 Assay for Human Thrombopoeitin MpI Receptor Agonist Activity- Ba/F3-Mpl Cell
Proliferation Assay A. Generation of Ba/F3-Mpl Cells [194] Ba/F3, a murine IL-3 dependent pro-B cell line, was engineered to express human c-Mpl (SEQ ID NO: 2) in accordance with the protocol described in Abe, M., et al. (2002) Leukemia 16:1500-1506, which is incorporated herein by reference. Ba/F3 was obtained from DSMZ5 accession no. ACC 300, and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 10 ng/ml penicillin/streptomycin (P/S) and 10 ng/ml recombinant mouse IL-3 (rmIL-3, R&D Systems, Minneapolis, MN)- To clone c-Mpl, mRNA was isolated from TF-I cells (DMZ No. ACC 334) and cDNA encoding a c-Mpl was amplified by RT-PCR using MpI primers (5' TCAAG GCTGC TGCCA ATAGC TTAGT GG; SEQ ID NO: 368) and (g( ATGCC CTCCT GGGCC CTCTT CATGG; SEQ ID NO: 369). The PCR fragment was cloned into the pCR4 TOPO vector (Invitrogen, Carlsbad, CA) and sequenced. The cDNA encoding c-Mpl was subsequently subcloned into pCDNA3.1(+) (Invitrogen, Carlsbad, CA) along with an upstream Kozak sequence for optimal transcription. Ba/F3 cells were transfected by electroporation withl5 μg of the pcDNA3.1+c-Mpl construct that had been linearized with Mfe I. Transfected cells were selected in selection media B [RPMI 1640 (GIBCO brand, Invitrogen, Carlsbad, CA), 10% fetal bovine serum (FBS) (GIBCO brand, Invitrogen, Carlsbad, CA), 1 ng/ml purified, recombinant human TPO (rhTPO; PeproTech, Inc. Rocky Hill, NJ), 1 ng/ml penicillin/streptomycin (PS) (GIBCO brand, Invitrogen, Carlsbad, CA)and 1 mg/ml G418]. A pool of cells resistant to G418 was sorted for single cells using flow-cytometric cell sorting. Individual clones were selected for robust growth in 1 ng/ml rhTPO (SEQ ID NO: 373) (PeproTech, Inc. Rocky Hill, NJ) and 1 mg/ml G418 (GIBCO brand, Invitrogen, Carlsbad, CA)]. Stable Ba/F3-Mρl cell clones were evaluated based on their responsiveness to rhTPO (SEQ ID NO: 373) in the cell proliferation assay described below in part B. B. Proliferation Assay
[195] Ba/F3-Mpl cells were maintained in growth media B [RPMI 1640 (GIBCO brand, Invitrogen, Carlsbad, CA)5 10% fetal bovine serum (FBS) (GIBCO brand, Invitrogen, Carlsbad, CA), 1 πg/ml rhTPO (SEQ ID NO: 373) (PeproTech, Inc. Rocky Hill, NJ), 1 mg/ml G418 (GIBCO brand, Invitrogen, Carlsbad, CA), and 1 ng/ml penicillin/streptomycin (PS) (GIBCO brand, Invitrogen, Carlsbad, CA)]. For the proliferation assay, Ba/F3-Mpl cells were washed three times in PBS, resuspended in starvation media B (growth media B without rhTPO or G418), and maintained overnight in a humidified incubator at 37°C and 5% CO2. On the day of the assay, 1 xlO4 cells in 50 μl were seeded into each well of a 96-well white opaque plate. Test proteins were serially diluted in starvation media B to various concentrations and 50 μl was added to the cells. Proliferation Was measured after 3 days using the CellTiter-Glo reagent (Promega, Madison, WI) which detects metabolic ATP by luminescence as a measure of viable cell numbers. Purified, recombinant human TPO (SEQ ID NO: 373) (rhTPO; PeproTech, Inc. Rocky Hill, NJ) was tested on each plate to generate a standard curve of 10 points on a 4x dilution series for comparison.
EXAMPLE 5 PEGylation of Dual Agonist Polypeptides [196] For the PEGylation reaction, the dual agonist protein was concentrated to 4 mg/ml in 50 mM sodium borate, pH 9. The protein was mixed with a 3X molar excess of 4OK branched PEG-NHS reagent (GL2-400GS2, NOF Corporation, Tokyo, Japan). The reaction was incubated at room temperature for 1.5 hours. The PEGylation mixture was buffer-exchanged into 50 mM sodium acetate (Sigma, St. Louis, MO), pH 5.5, using a PD-IO desalting column (GE Healthcare, Piscataway, NJ). This mixture was loaded at 1 mg/ml gel onto an SP Sepharose FF column (GE Healthcare, Piscataway, NJ). The monoPEG pool was eluted using a 0 - 125 mM NaCl, 15 column gradient. The eluate pool was concentrated to between 0.4 and 0.7 mg/ml and dialyzed against PBS, pH 6.5. EXAMPLE 6
Characterization of Refolded and Pegylated Dual Agonist Polypeptides A. SDS-PAGE Analysis of Purity
[197] The purity of the dual agonist protein was assessed by SDS-PAGE before and after PEGylation. Five micrograms of protein was loaded onto a 4 - 12 % bis-tris gel (Invitrogen, Carlsbad, CA), which was stained with SimplyBlue SafeStain (Invitrogen, Carlsbad, CA). Purity was assessed by presence or absence of impurity bands in the unPEGylated sample and unPEGylated or multiPEGylated bands in the PEGylated sample. B. Size Exclusion Chromatography:
[198] Size exclusion analysis was used to detect aggregates in the unPEGylated sample and unPEGylated or multiPEGylated species in the PEGylated sample. Five micrograms of protein was injected onto a TSK-GeI G3000SWXL size exclusion column (Tosoh Bioscience LLC5 Montgomeryville, PA), which is run in PBS5 pH 6.5 at 1 ml/minute.
C. Peptide Mapping Analysis of unPEGylated and PEGylated Dual Agonist Polypeptides [199] A 25mg protein sample was adjusted to pH 8.0 by addition of 1/10 volume of IM Tris-HCl buffer (pH 8), then digested with 0.83 ug of Lys-C endopeptidase at a protein to enzyme ratio of 30: 1 for 20 hours at 370C. The sample was then reduced with 5mM DTT for 30 min at 37°C. An appropriate volume equivalent to 5 ug of starting sample was injected onto a Jupiter C18, 2.1xl50mm column. The mobile phases used were, buffer A: 5% acetonitrile in 0.05% TFA/H2O and buffer B: 80% acetonitrile in 0.04% TFATH2O. The peptides were resolved using a gradient fromlO-60% B over 45 min at the flow rate of 0.15 mL/min on an Agilent HP 1100. The peptide identities were confirmed by on-line measurement of their mass using an ABI Q-TOF mass spectrometer. The degree of PEGylation at each PEGylation site was calculated based on the ratio of chromatographic peak areas of peptides from the PEGylated vs unPEGylated sample. EXAMPLE 7
' Exemplary Invention Dual Agonist Polypeptides
[200] Dual agonist polypeptides having the sequences of SEQ ID NOS: 318 and 33O5 were prepared in accordance with the methods of Examples -1 and 2 using encoding • polynucleotide sequences corresponding to SEQ ID NOS: 317 and 329.
[201] IL-3 receptor agonist activity and human thrombopoeitin MpI receptor agonist activity were assessed in accordance with the assays provided in Examples 3 and 4 and the data is provided in Table 3 below.
Table 3
TF-I (IL-3) assay BaF3-mpl (TPO) assay EC50 r+/- STDEVl EC50 \+l- STDEVI
RhIL-3 (SEQ ID NO: 7) from CHO 115 pM [+/- 30 pM] rhTPO (Peprotech, SEQ ID NO: 373) — . 5O pM [+/- 3.7 pM]
Dual- Agonist Polypeptide 66 pM [+/- 23 pM] 10 pM [+/- 4.7 pM]
(mean and standard deviation of 10 experiments)
[202] The TF-I human erythroleukemia cell line proliferates in response to several growth factors, including human IL-3 and human TPO (Drexler H.G. and Quentmeier H. (1996) Leukemia 10(9): 1405- 1421, both incorporated herein by reference) and can be used to measure the bioactivity of both of these cytokines. Dual agonist polypeptides isolated and purified from mammalian expression or E. coli expression were tested in the TF-I proliferation assay described in Example 3, above. TF-I cells proliferated in the presence of dual agonist polypeptides and the hIL-3 control (SEQ ID NO: 7), but not in the presence of conditioned media controls. Because TF-I cells proliferate in response to both hIL-3 and hTPO, the TF- 1 proliferative activity of the polypeptides of the invention could arise from signaling either through the hIL-3 receptor or through the hTPO c-Mpl receptor, or, through both of these receptors. However, differential cytokine sensitivity and neutralization data suggest that the TF-I proliferative activity of the polypeptides of the invention is largely due to IL-3 receptor activation. TF-I cells are approximately 3 log units more sensitive to IL-3 than to TPO. Neutralizing antibodies directed against the alpha chain of the IL-3 receptor (mAb rhIL-3 sRa, R&D Cat. 203-IL) reduced the TF- 1 cell proliferative activity of dual agonist polypeptides by 2 log units, suggesting that the TF-I cell proliferative activity of the polypeptides of the invention is primarily due to signaling through the IL-3 receptor.
[203] Similarly, BaF3-mpl cell line is a specific biological indicator for TPO agonism because the original naive BaF3 cell line from mouse (Abe, M., et al. 2002 Leukemia 16: 1500-1506, which is incorporated herein by reference ) does not proliferate in response to TPO or TPO mimetic peptides. Although the BaF3 cell line responds to species-specific IL-3 from mouse, the response to human IL-3 is at least 3 log units lower. Furthermore, once BaF3 cells had been stably transfected with the human (or rhesus monkey, SEQ ID NO: 370) mpl receptor gene (the rhesus monkey mpl receptor sequence is provided in SEQ ID NO: 371), the cell line proliferates in response to human TPO or TPO mimetic peptides at sensitivities 3-4 log units more potent than the proliferative responses induced from human IL-3. Therefore, the BaF3-mpl proliferative response induced by dual agonist polypeptides is due to the TPO mimetic portion of the molecule and not due to human IL-3 portion of the molecule.
EXAMPLE 8 Determination of dual agonism of TPO receptor and IL-3 receptor on human bone marrow stem cells A. Summary
[204] Stem cell experiments were used to determine whether dual agonist polypeptides with IL-3 and TPO activities had the capacity to induce human CD34+ hematopoietic stem cells from the bone marrow of healthy human or rhesus donors to form megakaryocytes in culture. Stem cell experiments were set up similarly to proliferation assays in 96 well plates and then analyzed for general cell proliferation (Cell-Titer GIo reagent) or maturation by FACS with cell surface markers. CD41 is a cell surface marker specific for CFU-Mk cells (immature megakaryocytes), mature megakaryocytes, and platelets. FACS analysis with anti-CD41is definitive of the megakaryocyte lineage. Cells can also stained for other cell surface markers (e.g. CD66 for granulocytes and their progenitors). B. Proliferation Assay
[205] Human or rhesus CD34+ hematopoietic bone marrow stem cells (Cambrex Bio Science, Walkersville, Maryland) were suspended in assay media [STEMSPAN SFEM (Stemcell Technologies, Vancouver, BC ), 1% bovine serum albumin, 10 ug/ml rh insulin, 200 ug/ml human transferrin (iron saturated), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine5 Iscove's MDM and 1 ng/ml penicillin/streptomycin (PS) (GIBCO brand, Invitrogen, Carlsbad, CA)] without cytokines and adjusted to 20,000 cells/ml cell concentration. 50 μl of cell suspension was seeded into each well (1000 cells per well) of a 96-well white opaque plate. Test proteins were serially diluted in assay media to various concentrations and 50 μl was added to the cells. Plates were covered and incubated at 37 0C with 5 % CO2 for 10-14 days. Proliferation was measured using the CellTiter-Glo reagent (Promega, Madison, WI) which detects metabolic ATP by luminescence as a measure of viable cell numbers.
C. Megakaryocyte maturation
[206J Human or rhesus CD34+ hematopoietic bone marrow stem cells were treated as in section B for 10-14 days. Maturation to megakaryocytes was measured by staining for CD41 surface expression (a megakaryocyte-specific marker) and analyzed using FACS. Cells were pre-treated with purified human IgG (10 μL of 1 mg/mL/106 cells for 15 minutes at room temperature in order to block Fc-mediated interactions). Then cells were stained with Mouse IgGl, anti-CD4 Ia-FITC (BD Pharmingen, Franklin Lakes, NJ) or isotype control antibody IgGl-FITC, loaded on a FACS Caliber (Becton Dickinson, Franklin Lakes, NJ), and gated for live and CD41 positive cells.
D. Results
[207] Dual agonist polypeptides exhibited agonist activities on human hematopoietic stem cells with respect to both human TPO c-Mpl and H-3 receptors that was greater than that observed for each single growth factor (human thrombopoietin or IL-3) or the theoretical additive effects of both human IL-3 and thrombopoietin in the bone marrow stem cell assay. Dual agonist polypeptides exhibited activities on human hematopoietic stem cells that were at least as potent as that observed with the equal molar mixture of human IL-3 and thrombopoietin. Dual agonist polypeptides stimulated more stem cells to proliferate than equal concentrations of the single growth factors human thrombopoietin or IL-3. Dual agonist polypeptides were more potent than equal concentrations of human thrombopoietin or IL-3 at stimulating stem cells to mature into the megakaryocyte lineage as evidenced by more cells detected by FACS as expressing the cell surface marker CD41.
Example 9 Pharmacokinetic and Pharmacodynamic Study in Male Rats
[208] The pharmacokinetic and pharmacodynamic properties of dual agonist compounds and dual agonist conjugates are assessed in male Sprague Dawley rats. [209] To assess the serum half-life and ability of the dual agonist compounds and conjugates to stimulate platelet production in healthy animals, male Sprague Dawley rats (10 animals per compound) are injected intravenously with a single dose of each compound at 100 μg/kg or a similar volume of formulation buffer. The animals are bled from a canula at various timepoints for complete blood counts (including platelets) and collection of plasma. The quantity of each compound in each plasma sample is measured by quantitative ELISA specific for the IL-3 portion of the dual agonist compound or conjugate thereof. The IL-3 ELISA is a standard sandwich ELISA containing mouse monoclonal anti-human-IL-3 coating and detection antibodies. The coating antibody is unconjugated and the detection antibody is conjugated with biotin. High sensitivity of detection of the complex is achieved by exploiting the affinity of biotin to a streptavidin- horseradish peroxidase conjugate. The quantity of active compound in each plasma sample may be measured by a semi-quantitative method using the TF-I cell and BaF3- mpl cell assays of Example 3 and 4.
[210] Pharmacokinetic parameters, such are the area under the curve (AUC) and half life (t ■/,) can be calculated from concentration-time profiles (concentration of dual agonist or dual agonist conjugate vs. time) observed by IL-3 ELISA. The platelet counts are plotted as a function of time. . . [211] While preferred embodiments of the invention have been illustrated and described, it will be readily appreciated that various changes can be made therein without departing from the spirit and scope of the invention. S

Claims

We claim:
1. A dual agonist compound that exhibits both IL-3 receptor agonist activity and thrombopoeitin MpI receptor agonist activity, wherein said compound comprises a structure of the following formula:
Figure imgf000080_0001
wherein TM1 and TM2 are non-naturally occurring peptides that each independently have a molecular weight of less than about 2,500 daltons and wherein each exhibits thrombopoeitin c-Mpl receptor agonist activity, wherein n and m are each independently 0 or 1 , wherein L1 is a linker that covalently links the C terminus of TM1 to the N terminus of TM2, wherein L2 is a linker that covalently links the C terminus of TM2 to IL-3, wherein the IL-3 is an IL-3 polypeptide that exhibits IL-3 receptor agonist activity, and wherein the dual agonist compound exhibits both IL-3 receptor agonist activity and thrombopoeitin c-Mpl receptor agonist activity.
2. A dual agonist compound that exhibits both IL-3 receptor agonist activity and thrombopoeitin c-Mpl receptor agonist activity, wherein said compound comprises a structure of the following formula:
TM1 - (L2)ra - IL-3 - (L3)p - TM2 (II) wherein TM1 and TM2 are non-naturally occurring peptides that each independently have a molecular weight of less than about 2,500 daltons and wherein each exhibits thrombopoeitin c-Mpl receptor agonist activity, wherein m and p are each independently 0 or 1, wherein L2 is a linker that covalently links the C-terminus of TM1 to IL-3, wherein the IL-3 is an IL-3 polypeptide that exhibits IL-3 receptor agonist activity wherein L is a linker that covalently links IL-3 to the N-terminus of TM , and wherein the dual agonist compound exhibits both IL-3 receptor agonist activity and human thrombopoeitin c-Mpl receptor agonist activity.
19
3. The dual agonist compound of claim 1 or 2, wherein TM1 and TM2 are each independently selected from the group consisting of:
I-X2-G-P-T-L-X7 rX8-X9 ' (1) wherein X2 is E, R5 H, Q5 T, A, G, or S; X7 is R, S, or T; X8 is A, D5Q, S, or T; and X9 is A, C, or W;
X2-X3-X4-X5-X6-X7-X8-X9-X10 (2) wherein X2 is D, G, K5 R, S5 or T; X3 is L or V; X4 is K5 R5 or W; X5 is D5 E5 G5 or S; X6 is C5 Q5 or T; X7 is G5 I5 or V; X8 is A, K5 L5 M5 S5 W, or Y; X9 is A, G515 K5 L5 M, Q5 R5 W5 or Y; and X10 is A5 C5 G5 H5 L5 S5 W, or Y; X1 -V-R-X4-X5-X6-X7-X8-X9-X10-Xn (3) wherein X1 is G5 R5 S5 or T; X4 is D5 E5 or Q; Xs is Q or V; X6 is I or V; X7 is C, D5 E5 G, K5NP5R1 or S; X8 is A515 K5 L5 M5 S, W or Y; X9 is F, H5 R5 W5 or Y; X10 is F5 L5 M, V, W5 or Y; and X1 ' is A5 C5 F5 H515 L5 M5 S5 or V;
X4-X5-X6-X7-X8-X9-X10-X"-X12-X13-X14-X15 (4) wherein X4 is W5 T, Q5 or C; X5 is N5 G5 S5 T5 or A; X6 is L, V5 or R; X7 is T5 R5 K5 A5 H5 S5 or N ; X8 is E5 S5 D5 Q5 G5 or K; X9 is F5 V5 Q5 W5 Y5 L5 or M; X10 is V5 R5 K5 L5 or I; X11 is L5 D, Q5 A5 V5 K515 G5 S5 or R; X12 is D5 Q5 G5 R5 L5 Q5 or K; X13 is T5 V5 C5 D5 N5 E5 G5 M5 H5 or A; X14 is H5 C5 T5 R5 A5 or N; and X15 is P5 A5 W5 G5 T5 V5 C, L5 F5 D5 Y5OrR5 X3-X4-X5-X6-X7-X8-W-X10 (5) wherein X3 is G5 R5 or S; X4 is E, M5 P5 Q, or R; X5 is H5 Q5 R5 S5 or T; X6 is C, L5 P5 or V; X7 is A5 F5 K5 M5 R5 S5 or V; X8 is E5 G5 M5 P5 Q5 S5 or T; and X10 is L or M;
X1-X2-G-C-X5-L-X7-X8-W-X10-Xn-G-X13-C (6) wherein X1 is F515 L5 P5 or Y; X2 is E5 H5 K5 L5 Q5 R5 S5 W5 or Y; X5 is R or T; X7 is K or R; X8 is A5 S5 or V; X10 is L5 R5 or S; X1 ' is A or G; and X13 is I5 L5 M5 or V;
X'-X2-T-X4-X5-X6-X7-X8 • (7) wherein X1 is G5 S5 or Y; X2 is C5 L, or P; X4 is F5 L5 or V; X5 is K5 P5 Q5 R5 or S; X6 is D5 E5 H5 Q5 or Y; X7 is C, F5 L5 or W; and X8 is I5 K5 L5 M5 R5 or V;
C-X2-X3-X4-X5-X6-X7-X8-X9-C (8) wherein X2 is N5 Q, R, S, or T; X3 is C, F, I, L, or R; X4 is A, E, G5 H5 K, N, Q, R5 or S; X5 is D5 E, or Q; X6 is F, L, V5 or Y; X7 is I5 K, L, M, N5 R5 or V; X8 ϊs C, F515 M5 P, T5 V5 W, or Y; and X9 is A5 C, F5 G, I5 L5 Q5 R5 or S;
X1-X2-X3-X4-X5-X6-X7-X8-X9 (9) wherein X1 is C or E; X2 is R or T; X3 is F5 G3 L5 R5 or S; X4 is A5 G, P, Q, R5 S5 or T; Xs is E5 F5 Q5 or S; X6 is F or W; X7 is A5 K5 L5 or R; X8 is D, E, G5 H5 K5 L5 Q, R5 or S; and X9 is A, C5 D5 G5 or R;
C-X2-X3-G-X5-X6- X'-X^-W-X1 !-X12-X13-C (10) wherein X2 is A5 G5 L, M5 R5 or S; X3 is A5 D5 E5 Q5 T5 or absent; X5 is L or P ; X6 is T5 S, or F; X7 is L or V; X8 is R5 T5 or L; X9 is E, P5 Q5 or A; X11 is L5 M5 or I; X12 is Y5 L5 T5 E, S, or absent; and X13 is V5 L5 E5 H, F5 or absent;
C-X2-X3-X4-X5-X6-X7-X8-X9-X10-Xn-X12-X13-C (11) wherein X2 is^D, K5 S5 T5 or V; X3 is F5 L5 or M; X4 is A5 K5 L5 Q5 R5 or S; X5 is D5 E5 or Q; X6 is F5 L5 W5 or Y; X7 is K or L; X8 is A5 D5 E5 H5 L5 M5 Q5 S5 T5 or V; X9 is S5 N5 F5 Y5 L5 H5 A5 or P; X10 is G5 H5 S5 or T; X1 ' is E, G5 L5 M5 T5 V5 or Y; X12 is A5 E, G5 M5 N5 S, V5 or Y; and X13 is A5 E5 L5 Q5 S5 V5 W5 or Y; and
C-X2-X3-X4-Xs-X6-X7-X8-X9-X10-Xπ-C (12) wherein X2 is L5 R5 S5 or T; X3 is F or L; X4 is G, K5 L5 M5 Q5 R5 or S; X5 is A5 D, E5 Q5 S5 or V; X6 is F or W; X7 is I5 K5 L5 or V; X8 is C515 K5-N5 P5 T, W, or Y; X9 is A5 D5 E, G, L, M5 N5 S5 W5 or Y; X10 is A5 E5 G, H5 L P5 Q5 R, S5 or W; and X11 is E5 H515 K5 R5 or Y.
4. The compound of claim 1 or claim 25 wherein TM1 and TM2 are each independently selected from SEQ ID NOS: 8-254 and 374-378.
5. The compound of any of claims 1 , wherein n is 1.
6. The compound of any of claims I5 wherein n is 0.
7. The compound of claim I5 wherein L1 is selected from the group consisting of a peptide, a non-peptidic non-polymeric aliphatic moiety, and an oligonucleotide.
8. The compound of claim 7, wherein L1 is a peptide.
9. The compound of claim 8, wherein L1 is resistant to proteolytic cleavage.
10. The compound of claim 8 or 9, wherein L1 consists essentially of from 1 to 20 amino acid residues.
11. The compound of claim 10, wherein L1 consists essentially of from 1 to 12 amino acid residues.
12. The compound of claim 103 wherein L1 comprises amino acid residues selected from the group consisting of glycine, serine, alanine, and threonine.
13. The compound of claim 12, wherein L1 comprises an amino acid residue selected from the group consisting of glycine and serine.
14. The compound of claim 13, wherein L1 consists essentially of glycine and serine.
15. The compound of claim 13, wherein L1 consists essentially of glycine.
16. The compound of any of claims 1 -4 , wherein m is 0.
17. The compound of any of claims 1 -4 , wherein m is 1.
18. The compound of claim 17, wherein L2 is selected from the group consisting of of a peptide, a non-peptidic non-polymeric aliphatic moiety, an oligonucleotide, a non-peptidic polymer, a polypeptide lacking IL-3 and thrombopoeitin MpI receptor agonist activities, and a polynucleotide.
19. The compound of claim 18, wherein L is a peptide.
20. The compound of claim 19, wherein L2 is resistant to proteolytic cleavage.
2i . The compound of claim 19 or 20, wherein L2 consists essentially of from 1 to 20 amino acid residues.
22. The compound of claim 21, wherein L2 consists essentially of from 1 to 12 amino acid residues.
23. The compound of claim 21, wherein L2 comprises amino acid residues selected from the group consisting of glycine, serine, alanine, and threonine.
.
24. The compound of claim 23, wherein L2 comprises amino acid residues selected from the group consisting of glycine and serine.
25. The compound of claim 23, wherein L2 consists essentially of glycine and serine residues.
26. The compound of claim 23, wherein 1? consists essentially of glycine.
27. The compound of claim 2, wherein p is 0.
28. The compound of claim 2, wherein p is 1.
29. ■ The compound of claim 28, wherein L3 is selected from the group consisting of of a peptide, a non-peptidic non-polymeric aliphatic moiety, an oligonucleotide, a non-peptidic polymer, a polypeptide lacking IL-3 and thrombopoeitin MpI receptor agonist activities, and a polynucleotide.
30. The compound of claim 29, wherein L3 is a peptide.
31. The compound of claim 30, wherein L3 is resistant to proteolytic cleavage.
32. The compound of claim 30 or 31 , wherein L3 consists essentially of from 1 to 20 amino acid residues.
33. The compound of claim 32, wherein L3 consists essentially of from 1 to 12 amino acid residues.
34. The compound of claim 32, wherein L3 comprises amino acid residues selected from the group consisting of glycine, serine, alanine, and threonine.
35. The compound of claim 30, wherein L3 comprises amino acid residues selected from the group consisting of glycine and serine.
36. The compound of claim 35, wherein L3 consists essentially of glycine and serine residues.
37. The compound of claim 35, wherein L3 consists essentially of glycine.
38. The compound of claim 1 , wherein TM1 and TM2 are identical and n and m are 1.
39. The compound of claim 1, wherein TM1 and TM2 are different and n and m are 1.
40. The compound of claim 2, wherein TM1 and TM2 are identical and m and p are 1.
41. The compound of claim 2, wherein TM1 and TM2 are different and m and p are 1.
42. The compound of claim 1, wherein TM1 and TM2 are identical and at least one of n and m is 0.
43. The compound of claim 1 , wherein TM1 and TM2 are different and at least one of n and m is 0.
44. The compound of claim 2, wherein TM1 and TM2 are identical and at least one of m and p is 0.
45. The compound of claim 2, wherein TM1 and TM2 are different and at least one ofm andp is O.
46. The compound of any of claims 1-45, wherein the IL-3 is mature rhuIL3 having the sequence corresponding to SEQ ID NO: 7.
47. The compound of any of claims 1-45, wherein the IL-3 has from 1 to 15 substitutions with respect to mature rhuIL3 (SEQ ID NO: 7).
48. The compound of claim 46 or 47, wherein the IL-3 has a truncation of 1 to
14 amino acid residues from the N-terminus and/or 1 to 15 amino acid residues from the C-terminus.
49. The compound oi claim 4 /, wherein the 1 to 15 substitutions comprises an introduced pegylation attachment group.
50. The compound of claim 49, wherein the introduced pegylation group is an introduced lysine or cysteine residue.
51. The compound of claim 47, wherein the 1 to 15 substitutions comprises a substitution that removes a pegylation attachment group.
52. The compound of claim 51 , wherein the substitution that removes a pegylation attachment group is a Lysine to Arginine substitution.
53. The compound of claim 47, wherein the 1 to 15 substitutions comprises an introduced N-glycosylation site.
54. The compound of claim 47, wherein the 1 to 15 substitutions comprises a substitution that removes an N-glycosylation site.
55. The compound of any of claims 1 -54, wherein the compound has greater agonist activity with respect to human thrombopoeitin c-Mpl receptor as compared to human thrombopoeitin in the assay of Example 4, such that the ratio of EC50 (human thromobopoeitin (SEQ ID NO: 373)) : EC50 (dual agonist compound) is greater than 1.
56. The compound of any of claims 1 -54, wherein the compound has greater agonist activity with respect to the IL-3 receptor as compared to mature rhIL-3 (SEQ ID NO: 7) in the assay of Example 3, such that the ratio of EC50 (mature rhIL-3 (SEQ ID NO: 7)) : EC50 (dual agonist compound) is greater than 1.
57. The compound of any of claims 1 -54, wherein the dual agonist compound stimulates the proliferation of more hematopoietic stem cells in the assay of Example 8 than are stimulated by each individual growth factor, rhTPO (SEQ ID NO: 373) and rhIL-3 (SEQ ID NO: 7), as measured individually in the assay of Example 8.
58. The compound of any of claims 1 -54, wherein the dual agonist compound stimulates the proliferation of more hematopoietic stem cells in the assay of Example 8 than the sum of hematopoietic stem cells stimulated by each individual growth factor, rhTPO (SEQ ID NO: 373) and rhIL-3 (SEQ ID NO: 7), as measured individually in the assay of Example 8.
59. The dual agonist compound of claim 1, wherein the dual agonist compound is a polypeptide having a TM1 -L1 -TM with an amino acid sequence selected from the group consisting of SEQ ID NO: 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, and 308
60. ■ The dual agonist compound of claim 1 , wherein the dual agonist compound is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, and 362.
61. A dual agonist conjugate comprising a dual agonist compound of any of claims 1-60 covalently bound to a non-polypeptide conjugation moiety selected from the group consisting of a non-polypeptide polymer, a sugar moiety, and a non-polypeptide non-polymeric lipophilic moiety.
62. The dual agonist conjugate of claim 61 , wherein the dual agonist conjugate induces the production of at least about 2 times more platelets as measured using the assay of Example 9 than the corresponding non-conjugated form of the dual agonist as measured 6 days after intravenous administration of a single dose of the dual agonist conjugate or the corresponding non-conjugated form of the dual agonist.
63. The dual agonist conjugate of claim 61, wherein the dual agonist conjugate induces the production of at least about 2.5 times more platelets in a rat as measured using the assay of Example 9 than the corresponding non-conjugated form of the dual agonist as measured 6 days after intravenous administration of a single dose of the dual agonist conjugate or the corresponding non-conjugated form of the dual agonist.
64. The dual agonist conjugate of claim 61 , wherein the dual agonist conjugate induces the production of at least about 3 times more platelets as measured in a rat using the assay of Example 9 than the corresponding non-conjugated form of the dual agonist as measured 6 days after intravenous administration of a single dose of the dual agonist conjugate or the corresponding non-conjugated form of the dual agonist.
65. The dual agonist conjugate of any of claims 61 -64, wherein the non- polypeptide conjugation moiety is covalently bound to an amino acid residue of the IL-3.
66. The dual agonist conjugate of claim 65, wherein the non-polypeptide conjugation moiety is a non-polypeptide polymer.
67. The dual agonist conjugate of claim 66, wherein the synthetic non- polypeptide polymer is selected from the group consisting of a polyalkylene oxide, a polyvinyl alcohol, and a polyvinylpyrrolidone.
68. The dual agonist conjugate of claim 67, wherein the synthetic non- polypeptide polymer is a polyalkylene oxide.
69. The dual agonist conjugate of claim 68, wherein the polyalkylene oxide is a polyethylene glycol.
70. The dual agonist conjugate of claim 69, wherein the polyethylene glycol is a branched polyethylene glycol.
71. The dual agonist conjugate of claim 69, wherein the polyethylene glycol is a linear polyethylene glycol.
72. The dual agonist conjugate of claim 70 or 71 , wherein the polyethylene glycol is covalently bound to a lysine residue in the IL-3 component of the dual agonist compound, selected from the group consisting of KlO, K28, K66, K79, KlOO, Kl 10, and Kl 16, wherein amino acid position is with reference to mature human IL-3 (SEQ ID NO: 7).
73. The dual agonist conjugate of claim 70 or 71, wherein the IL-3 has from 1 to 15 substitutions with respect to mature rhuIL3 (SEQ ID NO: 7), wherein at least one or more of the substitutions are selected from the group consisting of KlOR, K38R, K66R, K79R, Kl 00R5 Kl 1 OR, Kl 16, wherein amino acid position is with reference to mature human IL-3 (SEQ ID NO: 7).
74. The dual agonist conjugate of claim 70 or 71, wherein the IL-3 has from 1 to 15 substitutions with respect to mature rhuIL3 (SEQ ID NO: 7), wherein at least one or more of the substitutions introduce a lysine residue selected from the group consisting of R54K, R55K, R63K, R94K, Rl 08K, Rl 09K, wherein the polyethylene glycol is covalently bound to the introduced lysine, and wherein amino acid position is with reference to mature human IL-3 (SEQ ID NO: 7).
75. The dual agonist conjugate of claim 61 -64, wherein the non-polypeptide conjugation moiety is a sugar moiety.
76. The dual agonist conjugate of claim 75, wherein the IL-3 is mature rhuIL3 (SEQ ID NO: 7) and wherein the sugar moiety is covalently bound to an amino acid residue selected from the group consisting of Nl 5 and N70.
77. The dual agonist conjugate of claim 75, wherein the sugar moiety is conjugated to an introduced glycosylation site.
78. The dual agonist conjugate of any of claims 61-64, wherein the non- polypeptide conjugation moiety is a non-polypeptide non-polymeric lipophilic moiety.
79. A pharmaceutical composition comprising a dual agonist compound or conjugate thereof of any of claims 1-78 and a pharmaceutically acceptable carrier.
80. A polynucleotide encoding the dual agonist compound of any of claims 1 , 59, and 60 wherein L1 is a peptide linker, and L2 is selected from the group consisting of a peptide linker and a polypeptide linker.
81. A polynucleotide encoding the dual agonist compound of claim 2, wherein
L and L are selected from the group consisting of a peptide linker and a polypeptide linker.
82. A vector comprising the nucleic acid of claim 80 or 81 , operably linked to a promoter.
83. A host cell transformed or transfected with the vector of claim 82.
84. The host cell of claim 83, wherein the host cell is a bacterial cell.
85. The host cell of claim 84, wherein the bacterial cell is E. coli.
86. The host cell of claim 83, wherein the host cell is a mammalian cell.
87. The host cell of claim 86, wherein the mammalian cell is a CHO cell.
88. A method of producing a dual agonist polypeptide having both IL-3 receptor agonist activity and thrombopoeitin MpI receptor agonist activity, said method comprising: (a) culturing a host cell transformed with the polynucleotide of claim 80 or
81 under conditions suitable for expression of the encoded dual agonist polypeptide; and
(b) recovering the dual agonist polypeptide from the culture medium or from the transformed and cultured host cells.
89. A method of producing a dual agonist polypeptide having both IL-3 receptor agonist activity and thrombopoeitin MpI receptor agonist activity, said method comprising:
(a) culturing a host cell transformed with the dual agonist polynucleotide of claim 80 or 81 under conditions suitable for expression of the encoded dual agonist polypeptide;
(b) recovering inclusion bodies comprising the encoded dual agonist polypeptide from the transformed and cultured host cells;
(c) solubilizing the recovered inclusion bodies comprising the encoded dual agonist polypeptide with a solubilizing agent,
(d) purifying the solubilized encoded dual agonist polypeptide,
(e) incubating a solution of DTT, cysteine, or reduced/oxidized glutathione and the encoded dual agonist polypeptide to allow the purified, encoded dual agonist polypeptide to refold; and (f) purifying the refolded dual agonist polypeptide by ion exchange chromatography.
90. The method of claim 89, further comprising attaching at least one non-polypeptide conjugation moiety to an attachment group of the dual agonist polypeptide, wherein the resulting conjugate has both IL-3 receptor agonist activity and thrombopoeitin c-Mpl receptor agonist activity.
91. A method for stimulating platelet production in a human or non-human animal subject, said method comprising administering to the subject an amount of the dual agonist compound of any of claims 1-60 effective to stimulate platelet production in the subject.
92. A method for stimulating platelet production in a human or non-human animal subject, said method comprising administering to the subject an amount of the dual agonist conjugate of any of claims 61-78 effective to stimulate platelet production in the subject.
93. A method for stimulating platelet production in a human or non-human animal subject, said method comprising administering to the subject an amount of the the pharmaceutical composition of claim 79 effective to stimulate platelet production in the subject.
94. A method of preventing or reducing the duration of thrombocytopenia in a patient, comprising administering to the patient an effective amount of a composition comprising the dual agonist compound or dual agonist conjugate of any of claims 1-78. - '
95. A method of preventing or reducing the duration of thrombocytopenia in a patient, comprising administering to the patient an effective amount of the pharmaceutical composition of claim 79.
96. The method of claim 95 , wherein the pharmaceutical composition is administered prior to or concurrent with a course of chemotherapy or radiation therapy.
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