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WO1994024160A2 - Erythropoïetines-muteines a activite renforcee - Google Patents

Erythropoïetines-muteines a activite renforcee Download PDF

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Publication number
WO1994024160A2
WO1994024160A2 PCT/US1994/004361 US9404361W WO9424160A2 WO 1994024160 A2 WO1994024160 A2 WO 1994024160A2 US 9404361 W US9404361 W US 9404361W WO 9424160 A2 WO9424160 A2 WO 9424160A2
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WIPO (PCT)
Prior art keywords
epo
amino acid
erythropoietin
leu
mutein
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PCT/US1994/004361
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WO1994024160A3 (fr
Inventor
Jean-Paul R. Boissel
H. Franklin Bunn
Danyi Wen
Mark O. Showers
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Brigham and Womens Hospital Inc
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Brigham and Womens Hospital Inc
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Priority to AU67097/94A priority Critical patent/AU6709794A/en
Publication of WO1994024160A2 publication Critical patent/WO1994024160A2/fr
Publication of WO1994024160A3 publication Critical patent/WO1994024160A3/fr
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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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to erythropoietin in general and more particularly to modified erythropoietin proteins (erythropoietin muteins) having improved biological activity.
  • Epo Erythropoietin
  • a glycoprotein hormone which, in its mature form in humans, is 166 amino acids long with a molecular weight of 34 to 38 kD (Jacobs et al., Nature 313: 806-809 (1985). Epo occurs in a, ⁇ , and asialo forms which differ slightly in their carbohydrate composition and biological activity (Dordal et al., Endocrinology 116: 2293-2299 (1985)).
  • Epo has been recognized as the hematopoietic cytokine that regulates the process of red blood cell production, known as erythropoiesis.
  • Erythropoiesis is a controlled physiological process which normally produces red blood cells in numbers which do not impede blood flow, but which are sufficient for oxygen transport.
  • Epo The binding of Epo to its cognate receptor (D'Andrea, A.D., et al., Cell 57:277-285 (1989)) on erythroid precursor cells in the bone marrow results in salvaging these cells from programmed cell death, known as apoptosis (Koury, M.J., et al., Science 248:378-381 (1990)), allowing them to proliferate and differentiate into circulating erythrocytes (red blood cells). Epo thus facilitates the formation of erythrocytes from erythroid precursor cells.
  • the level of Epo in circulation normally controls the rate of red blood cell production in the body.
  • Epo is present at a low plasma concentration sufficient to maintain a steady state concentration of red blood cells by stimulating formation of just enough red blood cells to replace those lost through normal processes, such as aging.
  • the amount of Epo in circulation is increased.
  • This red blood cell deficit may be caused, for example, by anemia, the general loss of blood through hemorrhage, over-exposure to radiation, prolonged unconsciousness, or exposure to high altitudes where oxygen intake is reduced.
  • Epo is produced in the fetal liver and adult kidney, circulates in the bloodstream, and binds to receptors on committed progenitor cells in the bone marrow and other hematopoietic tissues, resulting in proliferation and terminal maturation of erythroid cells (Jelkmann, W., Physiol. Rev. 72:449 (1992)).
  • the expression of Epo, both mRNA and protein, is markedly increased by hypoxia, owing to a 3' enhancer and to highly conserved elements in the promoter region (Semenza, G.L. et al. , Proc. Natl. Acad. Sci. USA SS:5680-1684 (1988); Beck, I. et al. , J. Biol. Chem.
  • Epo genes encoding Epo from a number of species have been studied.
  • the Epo genes of man Jacobs, K. et al. , Nature 373:806-10 (1985); Lin, F.-K. et al. , Proc. Natl. Acad. Sci. USA 82:7580-7584 (1985)), a monkey ⁇ Macacafascicularis) (Lin, F.-K. et al. , Gene 44:201-209 (1986)) and a rodent, the mouse (McDonald, J.D. et al. , Mol. & Cell Biol. 6:842-848 (1986); Shoemaker, C.B. et al. , Mol.
  • Recombinantly produced Epo has proven especially useful for the treatment of patients suffering from impaired red blood cell production (Physicians Desk Reference (PDR), 1993 edition, pp 602-605). Recombinant Epo has proven effective in treating anemia associated with chronic renal failure and HIV-Infected individuals suffering from lowered endogenous Epo levels related to therapy with Zidovudine (AZT) (See PDR, 1993 edition, at page 602).
  • Modifications of the Epo protein which would improve its utility as a tool for diagnosis or treatment of blood disorders are certainly desirable.
  • modified forms of Epo exhibiting enhanced biological activity would be more effective and efficient than native Epo in the therapy setting when it is necessary to administer Epo to the patient, enabling administration less frequently and/or at a lower dose.
  • Administration of reduced amounts of Epo would also presumably reduce the risk of adverse effects associated with Epo treatment, such as hypertension, seizures, headaches, etc. (See PDR, 1993 edition, at pp. 603-604).
  • Epo Epo-epoxysemiconductor
  • modifications of Epo including deletions, additions, and substitution of existing amino acids. These modifications have been proposed as a means of achieving a variety of effects, including separation of various Epo functions on individual protein fragments, improving the efficacy of recombinant Epo production, improving in vivo stability, etc.
  • modifications proposed to improve the biological activity (i.e. ability to stimulate red blood cell production) of Epo are those modifications proposed to improve the biological activity (i.e. ability to stimulate red blood cell production) of Epo.
  • Fibi also discusses a number of modified Epo proteins (Epo muteins). Fibi generally speculates about the alteration of amino acids 10-55, 70-85, and 130-166 of Epo. In particular, additions of positively charged basic amino acids in the carboxyl terminal region are purported to increase the biological activity of Epo.
  • Epo can be modified to increase its biological activity. This lack of guidance indicates that, based upon the present state of knowledge, other modifications of Epo which may increase biological activity cannot be predicted and may not even exist.
  • the present invention represents the inventors' achievement in overcoming the lack of guidance and unpredictability regarding modifications of the Epo protein which increase its biological activity.
  • Epo muteins with enhanced biological activity. It is a further object of the present invention to provide recombinant
  • Epo muteins DNA encoding Epo muteins and methods of producing biologically active Epo muteins in a host cell culture using such recombinant DNA.
  • Oligonucleotide Primer Sequences Oligonucleotide primers corresponding to Epo DNA sequences are shown. The localization of the primer coincides with previously published nucleotide sequences for the human (Lin, F.-K. et al , Proc. Natl. Acad. Sci. USA 82:7580-7584 (1985)) and murine (McDonald, J.D. et al. , ol. & Cell Biol. 6:842-848 (1986)) Epo genes. EX2R and EX5 are not completely conserved between human and mouse (respectively 93% and 95 % identity). An equal amount of each possible nucleotide was incorporated during the corresponding cycles of those primer syntheses. EX2R and NCO1 are reverse primers and their sequences represent the antisense DNA strand.
  • Figure 2 PCR Strategy used for the cloning of mammalian cDNAs containing the complete coding sequence of the mature Erythropoietin Protein.
  • SP species specific primers
  • Figure 1 Utilization of those SP primers and/or of, sequences that are 100% conserved between man and mouse 5' ATG and 3' NCO1 primers (Figure 1) on cDNA templates prepared from kidney of uninduced or hypoxia-induced animals, allows the amplification of a large variety of mammalian Epo clones.
  • Sense ( ⁇ ) and antisense (**-) primers are represented by the arrows. Dashed boxes correspond to the coding part (propeptide and mature protein) of the five Epo exons. Gray boxes represent the 5' and 3' untranslated regions.
  • FIG. 3 Comparison of IV1/EX2R sequences from various mammals. The numbering corresponds to the published human genomic sequence (Lin, F.-K. et al , Proc. Natl. Acad. Sci. USA 82:7580-7584 (1985)). The reported human sequence was obtained from the amplification of purified genomic DNA from Hep3B cells and agreed with the sequence previously reported. The mouse sequence is from McDonald, J.D. et al , Mol. & Cell Biol. 6:842-848 (1986). The horse sequence was obtained from kidney-extracted genomic DNA. All the other sequences were established from PCR amplification of genomic DNAs purified from several mammalian renal-derived cell lines.
  • the consensus sequence indicates the positions where a unique nucleotide was found in all the reported species.
  • the boundary between intron 1 and exon 2 is represented by the ascending arrows.
  • the locations of the two PCR primers, IVI and EX2R are shown by the dashed arrows.
  • FIG. 4 Alignment of the nucleotide sequences of mammalian Epo cDNAs.
  • the mouse sequence corresponds to the one previously reported (McDonald, J.D. et al., Mol. & Cell Biol 6:842-848 (1986)).
  • PCR-produced human sequence was obtained from hypoxia-induced Hep3B cell line and is in total agreement with the previously published sequence (Jacobs, K. et al , Nature 373:806-810 (1985); Lin, F.-K. et al , Proc. Natl. Acad. Sci. USA 82:7580-7584 (1985)).
  • Monkey (Macaca mulatto), rat, sheep, pig and cat were amplified from kidney-purified cDNAs. Amplifications of the human, monkey, rat and sheep were realized using the ATG (residues 1 to 20) and NCO1 (residues 723-742) primers. The line under residues 41 to 56 represents the specific SP1 primer used for the cloning of the pig and cat sequences. For these two mammals, 5' sequences for SP1 are derived from the data presented in Figure 3. Arrows indicate the start and the end of the coding sequence (propeptide and mature protein).
  • FIG. 5 Predicted Amino Acid Sequences of Epo Propeptide.
  • the amino acid sequences are derived from data obtained from both genomic IV1/EX2R and cDNA amplifications. The numbering corresponds to the human sequence.
  • Ala 1 is the first amino acid of the human mature protein.
  • the ascending arrows show the site of the cleavage by the signal peptidase, as determined for the human and cynomolgus monkey proteins.
  • FIG. 6 Alignment of the Primary Structures of mature mammalian Epo Proteins.
  • the human, Macaca fascicularis and mouse amino acid sequences were previously reported (Lin, F.-K. et al. , Proc. Natl. Acad. Sci. USA 82:7580-7584 (1985); Lin, F.-K. et al , Gene 44:201-209 (1986); McDonald, J.D. et al , Mol. & Cell Biol. 6:842-848 (1986)).
  • the residue numbers correspond to the 166 aa of the mature human protein.
  • Plain boxes indicate the positions of the predicted four -helices in the human sequence (See Example II). Dashed boxes show the N- and O-glycosylation sites. Limits between exons are indicated by the vertical lines.
  • Figure 6A Schematic representation of the mammal erythropoietin.
  • the invariant amino acids among the eight sequences shown in Figure 6 are represented by the black boxes.
  • the localization of each of the four a-helices is underlined.
  • the three glycosylation sites are shown as diamonds.
  • the main disulfide bridge is indicated by the heavy line connecting noncontiguous sequences.
  • the small arrows under the sequence indicate the short SH-bridge, missing in the rodents.
  • Figures 7-7B Phylogenetic lineages derived from analyses of mammalian Epo cDNA sequences encoding the full length mature proteins from seven species.
  • This tree of lowest length required 374 base substitutions.
  • Each link between ancestral nodes has a circled number; this strength of grouping number is the minimum number of substitutions that must be added to the length of the maximum parsimony tree to find a tree that breaks down the barrier (moves one or more sequences) between the two groups separated by the interior link.
  • Figure 7A The phylogenetic tree derived from the maximum parsimony reconstruction. On the basis of other molecular evidence involving comparative amino acid sequence data from monotremes, marsupials, and many eutherian species (Czelusniak, J. et al., Meth. Enzymol.
  • Figure 7B Parsimony reconstruction on that portion of the cDN sequences that are codons for amino acids.
  • the numbers shown as a fraction on each link are, in the numerator, the number of amino acid changing base replacements and, in the denominator, the number of silent base replacements.
  • the computer algorithm that carried out this calculation is described in Czelusniak, J. et al , Nature 298:297-300 (1982) (see the legend for Figure 2).
  • Figures 8-8A Phylogenetic lineages derived from analyses of the mammalian Epo intron 1-exon 2 sequences.
  • Figure 8 Strength of groups in the maximum parsimony tree found on examining all 135,135 trees formed by nine terminal taxa. As the two feloid sequences (those of cat and lion) are nearly identical, they were treated as a single taxon in this strength of grouping analysis. The maximum parsimony tree for the segment (about 285 bp) of intron 1 and exon 2 from ten species required 361 base substitutions. The circled numbers are the strength of grouping numbers (as defined in the Figure 7 legend). Figure 8A. The near most parsimonious tree that groups dog with feloids. This tree required 365 base substitutions. The numbers on links represent numbers of base pairs by which the nodal ancestral and descendant sequences differ.
  • FIGS 9-9B Model of the Three-Dimensional Structure of Erythropoietin.
  • FIG. 9 Ribbon diagram of Epo tertiary structure.
  • the four a helices are labeled A to D; loops between helices are appropriately named.
  • Disulfide bridges are shown and N- and O-glycosylation sites are indicated respectively by dark and dotted segments.
  • Figure 9 A Schematic representation of Epo's primary structure depicting predicted up-up-down-down orientation of the four antiparallel helices (boxes with arrowhead). This folding pattern is strongly suggested by the large size of the two interconnecting loops AB and CD. The limits of each helix were drawn according to Table II of Example I. A predicted short region of 3-sheet is delineated by the dashed rectangle.
  • the N-glycosylation sites are represented by the dotted diamonds, the O-glycosylation site by the dashed oval.
  • the locations of the two disulfide bridges are shown as solid lines.
  • FIG. 9B Cross-section of the Epo molecule at the level of the four a helices.
  • the helical wheel projections are viewed from the NH 2 -end of each helix.
  • the hydrophobic residues, localized inside the globular structure, are indicated by filled circles.
  • Cos7 cells were transfected with pSG5, pSG5-Epo/wt or pSG5-Epo/ ⁇ 140-144. After three days, the cells were metabolically labeled with [ 35 S]-methionine and [ 35 S]-cysteine. Immunoprecipitations of cellular extracts and supernatants were performed with our polyclonal antibody, raised in rabbit against the native human Epo. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis.
  • Lanes 2 to 4 correspond to cellular extracts, and lanes 5 to 7 correspond to culture supernatants, from Cos7 transformed with the following: plasmid without insert (lanes 2 and 5), wild type Epo (lanes 3 and 6), and ⁇ 140-144 (lanes 4 and 7).
  • Figures 11-11B Interconnecting Loop AB.
  • FIG. 11 Schematic representation of the loop AB showing the localization of muteins with various deletions and amino acid replacements.
  • the dashed arrows point to the positions of the serine substitutions (in ⁇ 48-52).
  • the two N-glycosylation sites are represented by the gray diamonds.
  • Figure HA Amount and biological activities of secreted muteins.
  • the upper bar graphs show the relative secretion of wild type and loop AB muteins as determined by radioimmunoassay.
  • FIG 11B HCD57 cell proliferation as a function of increasing concentration of wild type and serine-substituted Epo muteins.
  • the line graphs show the cellular growth as measured by 3 H-thymidine uptake for cells cultured with wild type Epo (O), Epo mutein F48S ( O ), Epo mutein Y49S (X), Epo mutein A50S ( ⁇ ), Epo mutein W51S ( ⁇ ), and Epo mutein K52S ( ⁇ ).
  • Figures 12-12 A Interconnecting Loop CD.
  • Figure 12. Schematic representation of the loop CD showing the location of three deletion muteins: ⁇ 105-109, ⁇ lll-119, ⁇ 122-126, and the insertion of seven residues after Lysll ⁇ (myc epitope). The O-glycosylation site is indicated by the dashed oval.
  • FIG 12A Secretion and biological activities of the muteins located in loop CD.
  • the two bar graphs were created as described in Figure 11A.
  • the two mutants ⁇ l 11-119 and 116/myc were normally secreted and had full biological activities.
  • Figures 13-13A In Vitro Translation of the Epo Wild Type.
  • Figure 13 Analysis of the 35 S-labeled translation products by SDS-PAGE. One-step transcription/translation reactions were performed in the SP6-TnT rabbit reticulocyte lysate system. 1/30 of each reaction was resolved on a 15 % polyacrylamide gel. Lane 1-low Mr standard from Amersham; lane 2-in vitro reaction without added plasmid; lanes 3 and 4 are translation products obtained after incubation of 1 ⁇ g of circular p64T-Epo, in the presence or absence of canine pancreatic microsomal membranes, respectively.
  • FIG 13A Binding of the in vitro translated Epo wild type onto Epo Receptor-GTS-agarose beads. ⁇ xlO 3 cpm (counts per minute) of purified 35 S-labeled erythropoietin products, processed with microsomes (+) or not (-) were incubated in the presence of the extra-cytoplasmic domain of the Epo receptor (ERE X ), following the protocol described by Harris, K.W., et al, J. Biol Chem. 267: 15205-15209 (1992). Identical binding demonstrated that the conservation of the propeptide did not impair the hormone/receptor interaction.
  • Figures 14-14A COOH-end of Epo.
  • Figure 14 Schematic representation of the analyzed muteins, corresponding to the deletion of the four last amino acids ⁇ 163-166 and the replacements of the residues 162 to 166 by a KDEL or poly (His) sequences.
  • FIG 14A Relative secretion of these muteins.
  • the bioactivities in the supernatants (Krystal , G . , Exp. Hematol 11 : 649-60 ( 1983)) and the cell extracts ( Komatsu, N., et al, Cancer Res. 57:341-348 (1991)) of transformed Cos7 cells were measured by in vitro proliferation assay using HCD57. More KDEL mutant remained in the cytosol of the Cos7, when compared with the wild type Epo and ⁇ 163-166 or poly (His) muteins. However, all the analyzed muteins had the same specific activity as that of the wild type.
  • Figure 15. Bacterial Expression of Wild Type Epo.
  • FIG. 15 Panel A. Diagram of the fusion protein. An NH 2 - terminal 22 amino acid long peptide, containing a 10 histidine stretch, was fused to the mature erythropoietin sequence. Factor Xa cleavage allowed the recovery of the mature Epo with only two extra residues at its amino terminus.
  • FIG. 15 Panel B. IPTG induction of the fusion protein.
  • Lane 6 corresponds to an aliquot from transformed bacteria grown for 3 hours in a medium without IPTG.
  • Lane 1 is a low molecular weight standard.
  • FIG. 15 Panel C. Purification of the fusion protein. After 3 hours of IPTG induction, the produced (His), 0 -Epo was solubilized in 6 M guanidine-HCl and purified on a nickel affinity resin by increasing imidazole concentrations following the pET-His system protocol (Novagen). Samples of the column eluants were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. Lane 1-elution by 20 mM imidazole; lane 2-elution by 100 mM imidazole, releasing the fusion protein; lane 3-chelation of the nickel by a 100 mM EDTA wash; lane 4-molecular weight standard.
  • FIG. 15 Panel D. Detection of the E. coli recombinant Epo on a Western blot. Solubilized proteins were separated on a 15 % SDS- polyacrylamide gel, transferred to a nitrocellulose membrane and probed with a 1/2000 dilution of our native wild type polyclonal antibody, as described under Materials and Methods. Lane 1 -analysis after oxidative reduction; lane 2-after dialysis against the factor Xa buffer; and lane 3-after factor Xa cleavage.
  • FIG. 16 Relationship Between Production of Muteins and Proposed Secondary Structure.
  • This bar graph shows the amount of secreted proteins in the supernatants of transiently expressed Epo mutants, as detected by radioimmunoassay.
  • the muteins were aligned over a schematic representation of the native Epo molecule. Each deletion is shown as a stippled bar, the width of which is proportional to the number of residues deleted.
  • the four helices are represented by the black rectangles. The two disulfide bridges are indicated.
  • FIG. 17 Biological Activity of Helix A Muteins.
  • the top panel shows a helical wheel projection of Epo helix A with arrows indicating the positions where modifications were made to create muteins.
  • FIG. 18 Biological Activity of Helix D Muteins.
  • the top panel shows a helical wheel projection of Epo helix D with arrows indicating the positions where modifications were made to create muteins.
  • the lower panel is a dose response curve representing the biological activity over a range of dosages (X-axis) of wild type Epo ( ⁇ ) and helix D muteins D136A ( ⁇ ), R139A (D), K140A ( O ) and 143A (x) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into Epo dependent HCD57 cells.
  • FIG. 19 Biological Activity of Muteins Adjacent to Helix D.
  • the top panel shows the region adjacent to the C-terminal boundary of Epo helix D with arrows indicating the positions where modifications were made to create muteins.
  • the lower panel is a dose response curve representing the biological activity over a range of dosages (X-axis) of wild type Epo (Q) and muteins adjacent to helix D K152A ( ⁇ ), K154A (Q), and Y156A ( O ) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into Epo dependent HCD57 cells.
  • Figure 20 Comparison of Muteins With Increased Biological Activity.
  • a dose response curve representing the biological activity of wild type Epo (-*-), and muteins R143A (D) and K154A ( ⁇ ) with increased biological activity relative to wild type Epo is shown over a range of dosages (X-axis) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into Epo dependent HCD57 cells, n.
  • FIG. 21 Comparison of Muteins With Increased Biological Activity in Non-malignant Cells.
  • FIG 22 Comparison of Muteins With Increased Biological Activity in a Human Cell Line.
  • a dose response curve representing the biological activity of wild type Epo (x), and muteins R143A (D), and K154A ( ⁇ ) with increased biological activity (relative to wild type Epo) is shown over a range of dosages (X-axis) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into human Epo-dependent UT-7/Epo cell line ( Komatsu, N. et al, Cancer Res. 51: 341-348 (1991).
  • Figure 23 Comparison of Muteins with Increased Biological
  • a dose response curve representing the biological activity of wild-type Epo (B) and mutein N147A ( ⁇ ) with increased biological activity (relative to wild-type Epo) is shown over a range of dosages (mU) (X-axis) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into human Epo-dependent UT-7/Epo cell line.
  • FIG. 24 Comparison of Muteins with Increased Biological Activity in a Human Cell Line.
  • a dose response curve representing the biological activity of wild-type Epo ( ⁇ ) and mutein N147A ( ⁇ ) with increased biological activity (relative to wild-type Epo) is shown over a range of dosages (mU) (X-axis) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into human Epo-dependent UT-7/Epo cell line.
  • FIG. 25 Comparison of Muteins with Increased Biological Activity in a Human Cell Line.
  • a dose response curve representing the biological activity of wild-type Epo ( ⁇ ) and mutein N147A ( ⁇ ,!!) with increased biological activity (relative to wild-type Epo) is shown over a range of dosages (mU) (X-axis) as determined by the relative incorporation of [ 3 H]- thymidine (Y-axis) into human Epo-dependent UT-7/Epo cell line.
  • mU dosages
  • Y-axis Biological Activities of Helix A Muteins.
  • Figure 26 Comparison of Muteins with Increased Biological
  • a dose response curve representing the biological activity of wild-type Epo (B) and mutein K20A with increased biological activity relative to the wild type Epo is shown over a range of dosages (X-axis) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into UT-7 Epo cells.
  • FIG. 27 Comparison of Muteins with Increased Biological Activity in a Human Cell Line.
  • a dose response curve representing the biological activity of wild-type Epo (B) and mutein Y49S with increased biological activity relative to the wild type Epo is shown over a range of dosages (X-axis) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into UT-7 Epo cells.
  • FIG. 28 Comparison of Muteins with Increased Biological Activity in a Human Cell Line.
  • a dose response curve representing the biological activity of wild-type Epo (B) and mutein A73G with increased biological activity relative to the wild type Epo is shown over a range of dosages (X-axis) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into UT-7 Epo cells.
  • Y-axis Biological Activities of Helix D Muteins.
  • Figure 29 Comparison of Muteins with Increased Biological
  • Figure 30a Comparison of Muteins with Increased Biological
  • a dose response curve representing the biological activity of wild-type Epo (B) and mutein R143A with increased biological activity relative to the wild type Epo is shown over a range of dosages (X-axis) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into UT-7 Epo cells.
  • FIG. 30b Comparison of Muteins with Increased Biological Activity in a Mouse Cell Line.
  • FIG. 31 Comparison of Muteins with Increased Biological Activity in a Human Cell Line.
  • FIG. 32 Comparison of Muteins with Increased Biological Activity in a Human Cell Line.
  • FIG. 33a Comparison of Muteins with Increased Biological Activity in a Human Cell Line.
  • a dose response curve representing the biological activity of wild-type Epo (B) and mutein K154A with increased biological activity relative to the wild type Epo is shown over a range of dosages (X-axis) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into UT-7 Epo cells.
  • FIG 33b Comparison of Muteins with Increased Biological Activity in a Mouse Cell Line.
  • Figure 33c Comparison of Muteins with Increased Biological
  • a dose response curve representing the biological activity of wild-type Epo (B) and mutein K154A with increased biological activity relative to the wild type Epo is shown over a range of dosages (X-axis) as determined by the relative incorporation of [ 3 H]-thymidine (Y-axis) into primary murine spleen cells.
  • FIG. 34 Bioactivity of Epo Replacement Muteins.
  • the letters in the column on the left designate predicted helices (A, B. C, D) or interhelical loops (A-B, C-D).
  • the 3'D designation (3' of D) in the table represents amino acids 153-166 which are at the carboxy-end of the D-helix.
  • the amino acid of wild type human Epo, its position from the N-terminus, and its replacement according to the single amino acid letter code, are listed under the mutein column, e.g. S9A, represents a mutation from the wild-type human Epo at the amino acid serine of position number 9 to alanine.
  • the three bioassay columns show specific bioactivity of each mutein, expressed as a percentage of wild type human Epo bioactivity, with the background COS supernatant alone subtracted from the value.
  • the mean and standard deviation of the bioassay values are listed for n > 3 determinations.
  • the numbers within the brackets indicate the number of separate COS cell transfections over (/) the number of separate bioassays that were performed.
  • NS not secreted from COS 7 cells. Definitions
  • Administration is meant to include introduction of the Epo muteins of the invention into an animal or human by any appropriate means known to the medical or veterinary art, including, but not limited to, injection, oral, enteral, and parenteral (e.g., intravenous) administration.
  • Amino Acid Codes The most common amino acids and their codes are described in the following table:
  • Animal is meant to include all animals whose production of red blood cells (erythrocytes) is dependent upon, or stimulated by, erythropoietin (Epo). Foremost among such animals are humans; however, the invention is not intended to be so limiting, it being within the contemplation of the present invention to treat any and all animals which may experience the beneficial effect of the Epo muteins of the invention.
  • Efficacious Amount An "efficacious amount" of an Epo mutein of the invention is an amount of such Epo mutein that is sufficient to bring about a desired result, particularly the stimulation of red blood cell production, especially upon administration to an animal or human.
  • Erythrocyte The term “erythrocyte” is intended to refer to a red blood cell.
  • Erythroid Precursor Cells By “erythroid precursor cells” is intended cells with a capability to proliferate and differentiate into erythrocytes that is dependent upon exposure or contact with Epo. Erythropoietin (Epo).
  • Epo Erythropoietin
  • the term "erythropoietin”, or “Epo” is primarily intended to refer to the mature form of this hormone.
  • wild type Epo and “native Epo” are used interchangeably to refer to Epo in its naturally occurring, unmodified form. Unless otherwise indicated, amino acid positions on the Epo protein referred to herein are made with reference to the mature, 166 amino acid human Epo sequence shown in Figure 6 and corresponding sequences from other species.
  • host or "host cell” is intended the cell in which a gene encoding an Epo mutein of the invention is incorporated and expressed.
  • An Epo mutein gene of the invention may be introduced into a host cell as part of a vector by transformation.
  • Mutein By “mutein” is meant a mutant or modified protein with one or more modifications to its native amino acid sequence in the form of amino acid additions, deletions, or substitutions.
  • An erythropoietin mutein refers to a modified erythropoietin protein.
  • Pharmaceutically Acceptable Vehicle The term “pharmaceutically acceptable vehicle” is intended to include solvents, carriers, diluents, and the like, which are utilized as additives to preparations of the Epo muteins of the invention so as to provide a carrier or adjuvant for the administration of such Epo muteins. Transformation.
  • transformation is intended the act of causing a host cell to contain a desired nucleic acid molecule, including either a native Epo gene or a gene encoding an Epo mutein of the invention, not originally part of that cell using methods known in the art.
  • Transfection is intended the introduction of a DNA or RNA vector carrying a desired nucleic acid molecule, including either a native Epo gene or a gene encoding an Epo mutein of the invention, into a host cell.
  • Vector is intended a DNA element used as a vehicle for cloning or expressing a desired gene, such as an Epo mutein gene of the invention, in a host.
  • Epo muteins of the invention provide a preferable alternative to native Epo where it is used to stimulate red blood cell production (e.g. treatment of blood disorders, cell culture, etc.).
  • Epo is predicted to have a four anti-parallel amphipathic ⁇ -helical bundle structure. Based upon this predicted structure, functionally important regions predicted to reside on the external surfaces of the helices (designated A-D, see Figures 9A-9B) are identified. In particular, the external surfaces of helices A and D, predicted to be involved in Epo receptor binding, are identified. According to the invention, these functionally important regions serve as targets for modifications which may enhance biological activity. Increased activity relative to the wild-type Epo can be found with amino acid substitutions in the A,B,C or D helix.
  • Epo muteins are provided in which one or more of the amino acids within the predicted external surfaces of helices A, B, C, D and 3' of D, or within the region immediately adjacent to the C-terminal end of helix D, have been replaced.
  • amino acid substitutions at positions 20, 49, 73, 140, 143, 146, 147 and 154 of wild type Epo have been replaced.
  • the inventors have discovered that substitution of the amino acids normally occurring at these positions results in Epo muteins with significantly higher biological activity than wild type Epo proteins.
  • the amino acids at position 20 are preferably alanine), 49 (typically serine), 73 (typically glycine), 140 (typically lysine), position 143 (typically arginine), position 146 (typically serine), position 147 (typically asparagine), and 154 (typically either lysine or threonine) of wild type Epo are preferably replaced with an alanine residue.
  • other amino acids with a chemical structure and properties similar to alanine, such as serine and threonine are contemplated by the invention as suitable substitutes for achieving Epo muteins of the invention with enhanced biological activity.
  • a portion of the predicted interloop region between helices A and B extending from amino acid 48-52 is identified as important to Epo function. According to the invention, this functionally important region serves as another target for modifications which may enhance biological activity. The inventors have discovered that substitution of the amino acid at position 49 results in Epo muteins with significantly higher biological activity than wild type Epo proteins.
  • the amino acid at position 49 (typically tyrosine) of wild type Epo is preferably replaced with a serine residue.
  • Other amino acids with a chemical structure and properties similar to serine, such as alanine and threonine, are contemplated as suitable substitutes for achieving Epo muteins of the invention with enhanced biological activity.
  • the modifications taught by the present invention occur in regions that are highly conserved among Epo proteins from evolutionarily divergent species (see Example I and Figure 6 in particular). These modifications are thus expected to be broadly applicable to Epo proteins which are substantially similar to the human Epo protein in the regions where the modifications occur, including, but not limited to, Epo proteins from monkey, mouse, rat, sheep, pig, cat, and dog.
  • Epo muteins can be determined by assaying the ability of these proteins to stimulate the proliferation of Epo responsive cells such as murine spleen cells (Krystal, G., Exp. Hematol. 77:649-60 (1983); Goldberg, M.A., et al, Proc. Natl Acad. Sci. USA 84:1912-1916 (1987)), murine Epo responsive murine erythroleukemia cells (Hankins, W.D., et al, Blood 70: 113a (1987)), and human Epo-dependent UT-7/Epo cells ( Komatsu, N., et ⁇ l, Cancer Res. 57:341-348 (1991)).
  • Epo responsive cells such as murine spleen cells (Krystal, G., Exp. Hematol. 77:649-60 (1983); Goldberg, M.A., et al, Proc. Natl Acad. Sci. USA 84:1912-1916 (1987)), mur
  • Epo mutein genes Genes Encoding Epo muteins (Epo mutein genes)
  • Epo muteins themselves, genes encoding these muteins are also contemplated by the present invention. Genes encoding the Epo muteins of the invention may be made by modification of the native Epo gene using standard methods well known to those of skill in the art.
  • the native Epo gene may be obtained using a variety of standard techniques. For example, a DNA molecule corresponding to the known sequence of the Epo gene from a number of species may be artificially synthesized (See Example I and Figure 4). The Epo gene may also be isolated from a cDNA or genomic library using nucleic acid probes based on available Epo DNA sequence information and standard hybridization techniques as taught in U.S. Pat. No. 4,703,008, issued Oct. 27, 1987, which is herein incorporated by reference (See also Jacobs et al, Nature 313: 806-810 (1985)). Alternatively, amplification of Epo gene sequences by polymerase chain reaction (PCR) may be accomplished using primers based on available sequence data as taught in Example I.
  • PCR polymerase chain reaction
  • the wild type Epo coding sequence can be altered to encode an Epo mutein of the invention using standard oligonucleotide-directed in vitro mutagenesis techniques (See Zoller, M. and Smith, M., Methods in Enzymology 100: 468-500 (1983); Dente, L. et al, Nucl. Acids Res. 11: 1645 (1983); Kunkel, T.A., et al, Meth. Enzymol. 154:361-382 (1987); Vandeyar, M. et al, Gene 65: 129 (1988)). These techniques generally involve hybridization of an oligonucleotide carrying the desired alteration to a single stranded target DNA.
  • Epo muteins of the invention can be introduced and expressed in a selected host cell system using conventional materials and techniques.
  • DNA elements such as promoters, enhancers, polyadenylation sites, transcription termination signals, and the like should be associated with the Epo mutein coding sequence so as to promote and control expression of the Epo mutein.
  • the specific regulatory elements used will depend upon the host cell system selected for expression, whether secretion of the protein is desired, and other considerations readily apparent to one of skill in the art.
  • Various vectors may be employed as vehicles for the introduction and expression of Epo mutein genes in a host cell.
  • Such vectors useful in the different host cell types include, for example, the mammalian expression vectors pSG5 (Stratagene), p-RKl (Genetics Institute), P-SVK3 (Pharmacia), p-EUK-Cl (Clontech), pCDM (Invitrogen), pc DNAI (Invitrogen), and the bacterial expression vectors pFLAG-1 (IBI), all pET system plasmids (Novagen), pTrcHis (Invitrogen), the pGEX series (Pharmacia), and pKK 233-2 (Clontech).
  • vectors may be maintained as episomes in the host cell or they may facilitate integration of the Epo mutein gene into the host cell genome, or both. Vectors may also include other useful features, such as genes which allow for the selection or detection of cells in which they have been successfully introduced.
  • Host cells suitable for expression of the Epo muteins of the invention include, but are not necessarily limited to, E. coli, yeast, insect cells, plant cells, and a variety of mammalian cell types, including in particular Chinese Hamster Ovary (CHO) cells, Cos7 cells, Cosl cells, baby hamster kidney cells, and CV1 cells. Epo mutein genes can be introduced into a host cell using standard transformation or transfection techniques.
  • Epo muteins are preferred for production of Epo muteins to be used in vivo since the carbohydrate structure of Epo is important for optimal biological activity in vivo (See Dordal, M.S. et al, Endocrinology 116(6): 2293-2299 (1985)).
  • Epo muteins of the invention may be recovered and purified from host cells in which they are expressed using known methods such as immunoaffinity chromatography with antibodies to human Epo. Therapeutic Use of Epo muteins of the Invention
  • Epo muteins of the invention are suitable for therapeutic use in animals, particularly humans, and may be used in the same manner as wild type Epo, except that lower dosages will be required to achieve the same level of biological activity.
  • Administration of the Epo muteins of the invention may be accomplished by any of the methods known to the skilled artisan, provided that the method effectively places the Epo mutein in the appropriate environment to exhibit its activity (i.e. in contact with erythroid precursor cells).
  • a preferred method is by parenteral routes, including intravenous and subcutaneous administration.
  • Administration will ordinarily include an efficacious amount of the Epo mutein supplied in a pharmaceutically acceptable vehicle.
  • the amount of Epo mutein which is efficacious will be determined by a trained professional depending upon a number of considerations, including the condition being treated and its severity, the sex and body weight of the subject being treated, the method of administration, and the presence of compositions in the pharmaceutically acceptable vehicle which affect Epo activity.
  • the efficacious amount of a Epo mutein as taught by the invention will be significantly less than the corresponding amount of unmodified Epo needed to achieve the same degree of efficacy.
  • an efficacious amount of an Epo mutein of the invention will be as low as 10-fold less than native Epo, or as low as about 5-10 units/kg body weight for treating chronic renal failure patients or about 10-30 units/kg body weight for treating HIV-infected patients with serum Epo levels less than or equal to 500 mU/ml (milliunits/milliliter) who are receiving a dose of AZT equal to, or less than, 4200 g/week.
  • Epo muteins of the invention can be used effectively at lower dosages relative to wild type Epo, their use may reduce potential adverse effects associated with administration of Epo such as exacerbation of hypertension, seizures, headaches, tachycardia, nausea, clotted vascular access, shortness of breath, hyperkalemia, and diarrhea (see PDR, 1993 edition, at pp. 603-604).
  • human and monkey Epos are 94% and 91 % identical in nucleotide and amino acid sequence respectively.
  • human and mouse Epos are 76% identical in nucleotide sequence and 80% identical in amino acid sequence.
  • Human hepatoma Hep3B cell line and multiple kidney-derived cell lines from hamster (BHK), sheep (MDOK), pig (LLC- PK1), dog (MDCK), cat (CRF-K), lion (PAL1-K) and spotted dolphin (SP1-K) were obtained through the American Type Culture Collection (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852). Monolayer cells were grown in 100 x 20 mm tissue culture dishes using recommended media and maintained in a humidified 5 % C0 2 /95 % air incubator at 37°C. In some experiments, cells were made hypoxic by overnight incubation in 1 % O 2 , 5 % CO 2 , 94% N, at 37°C.
  • RNA prepared from Rhesus monkey kidney was purchased from Clontech (Palo Alto, CA). Kidneys from anemic cat and horse were obtained from veterinarians at Tufts School of Veterinary Medicine and the University of Kansas, respectively. Anemia was induced by three consecutive daily intraperitoneal injections of phenylhydrazine (60 mg/kg body weight) into male Sprague-Dawley rats (SD-strain) and by repeated bleeding of the sheep and the pig. Kidneys were aseptically removed after induction and stored immediately in liquid nitrogen.
  • Epo4 A 4 kb genomic human Epo clone (g Epo4) was provided by Genetics Institute (Boston, MA).
  • D ⁇ As from cell lines or homogenized kidneys were extracted using pancreatic R ⁇ Aase/SDS/proteinase K following a procedure modified from Blin and Stafford (Blin, ⁇ . et al , Nucleic Acids Res. 3:2303-2308 (1976)). Kidneys were homogenized in 4 M guanidine isothiocyanate (10 ml/g of frozen organ), containing 0.1 M beta-mercaptoethanol and 10% ⁇ -lauryl- sarcosine. Total R ⁇ As were isolated by centrifugation over 5.7 M CsCl (Chirgwin, J.M. et al , Biochemistry 78:5294 (1979)).
  • RNAs were isolated as described above for the kidneys.
  • RNA samples were first converted into single strand cDNA. 2 to 4 ⁇ g of total RNA from kidney or 500 ng to 1 ⁇ g of total RNA from cultured cells were denatured at 68°C in the presence of 2 ⁇ g of oligo dT (15) . Reverse transcription was carried out in a 20 ⁇ l final volume, containing 50 mM Tris- HC1 pH 8.3, 6 mM MgCl 2 , 40 mM KC1, 10 mM DTT, 500 ⁇ M dNTPs, 20 u RNAsin (Promega. Madison, WI) and 20 u of avian myeloblastosis virus reverse transcriptase (AMV-SuperRT, Molecular Genetic Resources. Tampa, FL). The reaction was allowed to proceed at 37°C for 20 minutes, then at 42°C for one hour. Inactivation of the enzyme was performed at 100°C for 10 minutes. cDNA samples were stored at -70°C.
  • Standard PCR reactions were performed in a 100 ⁇ l volume containing 50 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 0.01 % w/v gelatin, 200 ⁇ M dNTPs, 30 pM of each sense (5') and antisense (3') primers and 0.5 units of Taq DNA polymerase (Perkin Elmer Cetus, Emeryville, CA). After an initial denaturation step for 5 minutes at 95°C, routinely 35 cycles of PCR were performed on the DNA Thermal Cycler (Perkin Elmer Cetus). The denaturation step of each cycle was carried out at 94 °C for 1 min. For each sample and/or the primer pair used, annealing temperatures were optimized (from 45°C to 60°C) for a 2-minute reaction. A 3-minute elongation step at 72 °C ended each cycle.
  • Amplification products were analyzed on 1 to 3% agarose gel-TAE. Nu Sieve or Seaplaque agaroses (FMC, Rockland, ME) were used for preparative purposes. Specific PCR products were recovered from the gel, by the use of the Gene Clean II kit (Bio 101, La Jolla, CA), by Spin X centrifugation (Costar, Cambridge, MA) or by standard phenol/chloroform extractions of melted gel slices. Subcloning and Sequencing. Small (250-330 bp) amplified genomic fragments were directly subjected to automated sequencing, using the PCR primers as DNA sequencing primers.
  • PCR-generated cDNA products were blunt-ended using Klenow fragment and 5' phosphorylated by T4 DNA kinase (Boehringer Mannheim,
  • Sequencing reactions were carried out on an Applied Biosystems 373 A Automated DNA Sequencer utilizing ABI's DyeDeoxy Terminator Sequencer kit (Foster City, CA) and thermal cycling with Taq DNA polymerase (Promega, Madison, WI) as previously described (Tracy, T.E. et al , Biotechniques 77:68-74 (1991)). To avoid errors, for each species, samples derived from different amplification/cloning experiments were prepared and both strands were sequenced.
  • Epo mRNA is expressed at barely detectable levels in all tissues except in hypoxic kidney and liver (Goldberg, M.A. et al , Proc. Natl. Acad. Sci. USA 84: 7972-7976 (1987); Fandrey, J. et al, Blood 81: 617-623 (1993)). Therefore it would be necessary to generate libraries for each of the species of interest.
  • Epo Primer Design for PCR Amplification of Epo.
  • the sequences of Epo genes from three species have been already published: two primates, human (Jacobs, K. et al, Nature 373:806-810 (1985); Lin, F.-K. et al, Proc. Natl. Acad. Sci. USA 82:7580-7584 (1985)) and Cynomolgus monkey (Macaca fascicularis) (Lin, F.-K. et al, Gene 44:20 ⁇ -209 (1986)) and a rodent, the mouse (Mus muscuius) (McDonald, J.D. et al , Mol. & Cell Biol.
  • IV1 is a 25-mer oligonucleotide localized in intron 1, 216/217 bases upstream from the start of exon 2.
  • Human and mouse are identical except that the human sequence contains an extra G nucleotide at position 996.
  • IV 1 primer corresponding to the mouse sequence was synthesized.
  • the 23-mer EX2R ends 28 nucleotides upstream from the 3' end of exon 2.
  • EX5 is a 22-mer, beginning 34 bases downstream from the 5' end of exon 5.
  • NCO1 represents a 20 bp, 100% conserved DNA fragment, starting 112/113 bases downstream from the TGA stop codon and contains the unique NCO1 site present in the human and mouse gene.
  • An ATG primer was also synthesized, corresponding to a 20 oligonucleotide stretch, extending both 5' and 3' from the initiator methionine codon. This sequence is also totally conserved between primates and mouse.
  • Epo protein was detectable by radioimmunoassay in the supernatants of cells maintained overnight in 1 % 0 2 , 5% CO 2 , 94% N 2 at 37°C (Goldberg, M.A. et al , Proc. Natl. Acad. Sci. USA 84:1912-1916 (1987)).
  • the amplification of species specific IV1/EX2R and EX5/NCO1 genomic fragments allowed us to design a strategy shown in Figure 2 for obtaining cDNA clones containing the complete coding sequence of mature Epo protein from various mammals.
  • the IV1/EX2R primer pair resulted in a genomic fragment ( ⁇ 330 bp) corresponding to the 3' third of the first intron and 80% of exon 2, encoding the COOH-end of the propeptide and the NH 2 - terminal amino acids of the mature protein.
  • EX5/NCO1 primers amplified a segment of exon 5, containing the COOH-end of the Epo protein and a 3' untranslated sequence of about 140 bp downstream of the stop codon: DNA sequencing of these 5' and 3' fragments permitted, if necessary, the design of a second generation of species-specific primers (SP), localized outside the coding part of the mature protein (i.e., in the sequences encoding the propeptide and the 3' untranslated region).
  • SP species-specific primers
  • the nucleotide alignment includes 6 different orders of mammals: one primate, human (Homo sapiens); two artiodactyls, sheep (Ovis aries) and pig (Sins scrofa); one perissodactyl, horse (Equus caballus); one cetacean, spotted dolphin (Stenella plagiodon); three carnivores, dog (Canis familiaris), cat (Felis catus) and lion (Panthera led); two rodents, mouse (Mus musculus) and hamster (Cricetus cricetus). Cat and lion exhibited an almost complete sequence identity, with only one T to G nucleotide substitution near the 5' end of the amplified portion of the first intron.
  • Propeptides The predicted amino acid propeptide sequences of several mammals are presented in Figure 5. In the primates, there was strong conservation of the sequences of deduced propeptides, with only two amino acid (aa) substitutions at positions -11 (Leu in humans vs. Val in both monkeys) and -2 (Leu in humans vs. Pro in both monkeys). Expression of Cynomolgus monkey Epo gene in cultured mammalian cells resulted in the production of mature monkey Epo that was elongated at the N-terminus by three additional residues: Val-Pro-Gly (Lin, F.-K. et al , Gene 44:201-209 (1986)). As the Rhesus monkey has the same substitutions, an identical site
  • GH growth hormone
  • GM-CSF granulocyte- macrophage colony-stimulating factor
  • IL-3 IL-4
  • IL-5 IL-5
  • IL-4 IL-5
  • IL-5 granulocyte- macrophage colony-stimulating factor
  • Mammalian Epo cDNAs were subcloned into the pSG5 plasmid and transiently expressed in the monkey Cos7 cell line. Supernatants of the transfected cells were able to sustain the cellular proliferation of the murine Epo-dependent HCD57 cell line, demonstrating the biological cross-reactivity between species. As the human Epo antisera used in our radioimmunoassay bind to Epos from other species with variable affinity, the amount of produced protein was difficult to determine accurately. Experiments are in progress to further characterize the relative affinities of the various mammalian erythropoietins toward murine and human Epo receptors.
  • An "all trees" set of computer programs determined the parsimony length of each of the 945 unrooted trees formed by the seven cDNA sequences (the terminal taxa) ( Figure 4) and, on ordering those 945 trees according to increasing length, identified the minimum number of extra nucleotide substitutions needed to break up each group in the maximum parsimony (lowest length) tree.
  • Figure 7 and 7A the mouse and rat are strongly grouped, as are the human and monkey.
  • the pig and sheep are more weakly grouped, in accord with accepted phylogenetic views, which place the divergence of these animals about 55-60 million years ago.
  • silent base substitutions occurred much more readily than amino acid replacements (Figure 7B).
  • Figure 8 shows strength of grouping results based on all 135,135 unrooted trees formed by nine terminal taxa (in this case, cat and lion sequences, which are nearly identical, were treated as a single taxon), and Figure 8 A shows a phylogenetic tree that combines maximum parsimony with established phylogenetic evidence.
  • Figure 7A is a near most parsimonious tree in which dog joins the feloids.
  • the rodents are strongly grouped, as are the feloids (cat and lion), and at more moderate strength the cetacean (dolphin) is grouped with the artiodactyls (sheep and pig).
  • deletions at the N-terminus ( ⁇ l-6), the C-terminus ( ⁇ 163- 166), or in predicted interhelical loops (AB: ⁇ 32-36, ⁇ 53-57; BC: ⁇ 78-82; CD: ⁇ l 11-119) resulted in the export of immunologically detectable Epo muteins that were biologically active.
  • the mutein ⁇ 48-52 could be readily detected by RIA but had markedly decreased biological activity.
  • replacement of each of these deleted residues by serine resulted in Epo muteins with full biological activity.
  • Replacement of Cys29 and Cys33 by tyrosine residues also resulted in the export of fully active Epo. Therefore this small disulfide loop is not critical to Epo's stability or function.
  • the properties of the muteins that we tested are consistent with our proposed model of tertiary structure. Introduction
  • Epo The binding of Epo to its cognate receptor (D' Andrea, A.D., et al, Cell 57:277-285 (1989)) on erythroid progenitors in the bone marrow results in salvaging these cells from apoptosis (Koury, M.J., et al, Science 248:378-381 (1990)), allowing them to proliferate and differentiate into circulating erythrocytes.
  • the Epo receptor is a member of an ever enlarging family of cytokine receptors (Bazan, F., Biochem. Biophys. Res. Commun. 764:788-796 (1989)).
  • Epo shares weak sequence homology with other members of a family of cytokines which also include growth hormone, prolactin, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, GCSF, GM- CSF, M-CSF, oncostatin M, leukemia inhibitory factor and ciliary neurotrophic factor (Parry, D.A.D., et al, J. Mol. Recognition 7: 107-110 (1988); Bazan, F. , Immunology Today 77:350-354 (1990); Manavalan, P., et al, J. Protein Chem. 77:321-331 (1992)).
  • HGH X-ray diffraction
  • the algorithm for tertiary structure generation is divided into four computations.
  • the program aapatch identifies clusters of hydrophobic residues within the putative helices that could mediate helix-helix interactions (Richmond, T.J., et al, J. Mol. Biol. 779:537-55 (1978)).
  • Aafold generates all possible helix pairings according to the location and geometric preferences of the interaction sites.
  • Aabuild generates the three-dimensional models of all possible structures from the list of helix pairings (from aafold) and subject to stearic restrictions and geometric constraints on chain folding.
  • aavector applies the user defined distance constraints (e.g., disulfide bridges) to the structures generated.
  • coordinates have been specified only for residues in the core ⁇ -helices. For residues in sequentially distinct loops, lower bounds on the inter-residue distances can be inferred from the relevant helix terminus.
  • M13 plasmid, containing a 1.4 Kb EcoRI-EcoRI human Epo cDNA insert was a gift from Genetics Institute (Cambridge, MA) (Jacobs, K., et al, Nature 373:806-810 (1985)).
  • Annealing of the phosphorylated primers (10: 1 oligonucleotide/DNA template molecular ratio) was performed in 10 ⁇ l of a 20 mM Tris-HCl pH 7.4, 2 mM MgCl 2 , 50 mM NaCl solution. The reactions were incubated at 80°C for 5 minutes, then allowed to cool slowly to room temperature over a one hour period.
  • the DNA polymerization was initiated by the addition as a mix of 1 ⁇ l of 10X synthesis buffer (100 mM Tris-HCL pH 7.4, 50 mM MgCl 2 , 10 mM ATP, 5 mM each dNTPs, 20 mM DTT), 0.5 ⁇ l (8 units) of T4 DNA ligase and 1 ⁇ l (1 unit) of T4 DNA polymerase (Boehringer Mannheim). After 2 hours at 37°C, 80 ⁇ l of IX TE was added. 5 ⁇ l of the diluted reaction mix was used to transform competent E. coli NM522 (ung + , dut + ).
  • RNAs were prepared from cultured Cos7 cells (Chirgwin, J.M., et al, Biochemistry 78:5294-5299 (1979)) and 2 ⁇ g samples electrophoresed on a 1.1 % agarose gel containing 2.2 M formaldehyde. Transfer to GeneScreen Plus filters (New England Nuclear) and hybridization with a 32 P-labeled wt Epo probe were carried out as previously described (Faquin, W.C., et al, Blood 79: 1987-1994 (1992)). Quantitation of Transiently-Expressed Recombinant Epos. The amount of secreted protein in the supernatants of transfected Cos7 was determined by a radioimmune assay (RIA).
  • RIA radioimmune assay
  • the RIA was performed using a high titer rabbit polyclonal antiserum raised against the human wild type Epo and produced in our laboratory.
  • 125 I-labeled recombinant Epo was obtained from Amersham. Details of the protocol have been published elsewhere (Faquin, W.C., et al, Exp. Hematol. 21: 420-426 (1993)).
  • radioimmune precipitation buffer 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% (w/v) sodium azide, 0.1 % (w/v) SDS, 0.5 % (w/v) sodium deoxycholate, 1 % (w/v) Triton X-100, 1 mM phenylmethanesulfonyl fluoride and 1 ⁇ g/ml aprotinin).
  • Samples were precleared with rabbit preimmune serum/protein A-Sepharose CL-4B (Pharmacia) for two hours.
  • Immunoprecipitations were performed overnight with our polyclonal antibody specific for human recombinant wild type Epo and immunoadsorbed with protein A-Sepharose CL-4B. Immunoprecipitates were run on 15 % SDS-polyacrylamide gels (Laemmli, U.K. , Nature 227:680-685 (1970)) and analyzed by autoradiography after treatment with Enhance (New England Nuclear). Bioassays. The dose-dependent proliferation activities of wild type Epo and Epo muteins were assayed in vitro using three different target cells: murine spleen cells, following a modification of the method of Krystal (Krystal, G., Exp. Hematol.
  • the wild type Epo target corresponding to the nucleotide sequence coding for the mature protein, was PCR-amplified using appropriate primers.
  • an Ndel site (CATATG) was placed immediately 5' to the Alal codon of the mature protein.
  • a Bglll site was placed 3' to the TGA stop codon.
  • the 516 bp PCR fragment was inserted in an Ndel/BamHI-cut pETl ⁇ b plasmid (Novagen), which has a T7 promoter followed immediately by the lac operator. IPTG induction of transformed E.
  • coli BL21 (DE3) (T7 RNA polymerase "1" , Ion " , ompT) resulted in high levels of expression of a fusion protein with a ten histidine stretch at the amino terminus.
  • the oligo- His tag allowed the binding of the produced (His 10 )-Epo on a nickel affinity resin and its elution by increasing imidazole concentrations in presence of phenylmethylsulfonyl fluoride (Sigma). Most of the produced protein formed insoluble aggregates and was solubilized and affinity-purified under denaturing conditions in 6 M guanidine-HCl.
  • Oxidative refolding was performed by overnight dialysis at 4°C against 50 mM Tris-HCl pH 8.0, 40 ⁇ M CuSO 4 and 2% (wt/vol) Sarkosyl. Soluble protein was further dialyzed against 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM CaCl 2 and subjected to factor Xa (New England Biolabs) cleavage to remove the NH 2 -polyHis sequence. Monitoring of the fusion protein following induction and during the various steps of purification was done by electrophoresis on a 15 % polyacrylamide- SDS gel, stained with Coomassie Brilliant Blue.
  • the His-Epo fusion protein was detected on Western Blot (Gershoni, J.M., et al, Anal. Biochem. 737: 1-15 (1983)), using a 1/2000 dilution of our wt native Epo polyclonal antibody and a second biotinylated rabbit-specific antibody which is detected with a streptavidin-alkaline phosphatase conjugate (Amersham).
  • Sense and antisense primers creating new Bglll sites respectively 5' and 3' of the initiator and stop codons, were used in a polymerase chain reaction on pSG5-Epo/WT template.
  • aapatch identified eight possible helix-helix interaction sites. In principle, these sites could be used to generate 1.6 x 10 4 structures. Of these, only 706 maintained the connectivity of the chain and were sterically sensible. These structures resembled four helix bundles, an increasingly common motif in protein structure (Presnell, S.R., et al, Proc. Natl Acad. Sci. 86:6592-6596 (1989)). The structures that were not compatible with the native disulfide bridge between Cys 7 and Cys 16I were eliminated. This reduced the total number of structures from 706 to 184 (total computer time approximately 1 hr. on a Silicon Graphics IRIS 4D/35G).
  • the remaining structures were then rank ordered by solvent-accessible surface contact area, a measure of the validity of model structures.
  • the most compact structures were right handed, all anti- parallel four-helix bundles with no overhand connections, but this may be an artifact of a failure to add the polypeptide chain that forms the loops to the helical core constructed by aabuild.
  • the other less compact structures were left-handed four-helix bundles with two overhand loops, a topology previously seen in the structures of IL-4 and growth hormone. We suspect that this is the likely structure for Epo.
  • the consensus for assignments of putative ⁇ -helices in human Epo are summarized in Table III.
  • Epo seems to be more closely related to growth hormone, prolactin, IL-6 and GM-CSF rather than the other members of the helical cytokine superfamily. Nevertheless, recent improvements in algorithms for the identification of distant evolutionary relationships between proteins from structural fingerprints suggested that it might be possible to align the IL-4 structure to the Epo sequences.
  • the Eisenberg (Bowie, J.U., et al, Science 253: 164-170 (1991)) structural environment and 3D-1D profile methods are a powerful tool for recognizing that a sequence is compatible with a known structure, e.g., a four-helix bundle.
  • the NMR structure of IL-4 from Smith, L.J., et al, J. Mol. Biol. 224:899-904 (1992) was used to construct a 3D- ID profile.
  • a mixture of sequences including four helix bundles, globins, and non-helical structures were aligned against the IL-4 profile.
  • the other known four-helix bundle cytokines known to share a similar fold with IL-4 e.g., human growth hormone (Abdel-Meguid, S.S., et al, Proc. Natl. Acad. Sci.
  • Z-scores are used to describe the normalized weight associated with a profile score.
  • a distribution is built from a collection of sequences with a mean 2 score of 0.0 and a standard deviation of 1.0.
  • Z-scores greater than 6.0 are associated with significant alignments.
  • Z-scores between 3.0 and 6.0 may or may not be structurally relevant.
  • transiently expressed in Cos7 cells Northern blot analyses demonstrated that all the mutant plasmids produced about the same amount of mRNA as that of the wild type (data not shown). Yet, no detectable amount of Epo protein could be found in the Cos7 supernatants, either by radioimmunoassay or by bioassay using various Epo-dependent cell lines. Table IV summarizes these findings.
  • FIG. 10 An example of SDS-PAGE of immunoprecipitants from in vivo 35 S- labeling is presented in Figure 10.
  • Figure 10 shows the cytoplasmic retention of the mutein ⁇ 140-144, lacking four residues in the middle of the predicted D helix. The apparent molecular weight (around 28 kD) is less than expected for a five-amino acid deletion. Therefore not only the secretion, but also the glycosylation, seem to be impaired.
  • AB loop consists of 36 amino acids. Two N-glycosylation sites and a small disulfide bridge are located in the first half and their biological implications will be discussed later.
  • the COOH end of the AB loop contains a stretch of amino acids that is strongly conserved among mammals (See Example I). Alignments of human, monkeys, cat, mouse, rat, pig, and sheep Epos showed a consensus sequence: DTKVNFYAWKR(M/I)(E/D)VG (residues 43 to 57) [SEQ. ID NO: 4].
  • Helix B is linked to helix C by a much shorter segment (residues 77 to 89) and contains in its center the third N-glycosylation site (Asn83).
  • Asn83 the third N-glycosylation site
  • the residues 111-119 are predicted to be at the surface of the molecule.
  • Primary amino acid alignments of mammalian Epo showed a large variation in the sequence of residues 116 to 130, including amino acid deletion, insertion, and substitution (See Example I).
  • Ser 126 which removed the O-glycosylation site
  • Both rodents, rat and mouse lack the O-glycosylation site because of a Serl26 to Pro replacement.
  • the proline residue at position 122 is invariant among mammals.
  • the C-terminal part of the protein (residues 162 to 166) is clearly not involved in any structural or functional feature.
  • the deletion of the four last amino acids or the replacement of residues 162-166 by either a KDEL sequence or a poly-histidine sequence 4 did not modify the specific activity of the erythropoietin ( Figure 14).
  • Radioimmunoassay revealed that the secretion of the KDEL-tail mutein in the media of transfected cells was 45 % less than normally obtained with the wild type Epo. However, when compared to the wild type, this mutein had more biological activity in the hypotonic Cos7 cell extracts.
  • the KDEL COOH-terminal sequence has been shown to be essential for the retention of several proteins in the lumen of the endoplasmic reticulum (Andres, D.A., et al, J. Biol. Chem. 266: 14288-14282 (1991)). Nevertheless, because of overproduction in transiently expressed cells, a large percentage of recombinant protein escaped into the media. Disulfide Bridges. Wang et al. demonstrated that the biological activity of Epo was lost irreversibly if the sulfhydryl groups were alkylated (Wang, F.F., et al, Endocrinology 776:2286-2292 (1985)).
  • Cys7 Cysl61
  • Cys33 was previously changed to Pro by site-directed mutagenesis, and the resulting protein was reported to have greatly reduced in vitro biological activity (Lin, F.-K., Molecular and Cellular Aspects of
  • the poly-His tail wild type mutant was purified by means of nickel affinity chromatography which will enable quantitation of cytosolic-retained mutants.
  • the (His) 6 COOH-terminal sequence has been appended to all the muteins described in this example. Erythropoietin and Erythropoiesis H8:23-36, NATO AS1 Series, Springer- Verlag, Berlin Heidelberg (1987)).
  • rat and mouse Epos have the same substitution and yet exhibit full cross-species bioactivity.
  • To resolve the role of the small disulfide bridge in human Epo function we created a C29Y/C33Y double mutation. The resulting mutein was normally processed and showed the same in vitro bioactivity as the wild type ( Figure 11A).
  • Natural or recombinant human Epo is a heavily glycosylated protein; 40% of its molecular weight is sugars (Davis, J.M., et al, Biochemistry 26:2633-2638 (1987)).
  • the protein has three N-linked oligosaccharide chains, located at amino acid positions 24 and 38 (in predicted loop AB) and position 83 (in loop BC). It has one O- linked carbohydrate chain at position 126 (in loop CD), which is missing in rodents.
  • the role of these sugar chains in the biological activity of the human hormone has been extensively studied.
  • Epo is among a large number of biologically important proteins which have not yet been analyzed by X-ray diffraction or NMR. The problem is simplified by cumulative evidence that the structures of most proteins are likely to be variations on existing themes (Levitt, M. , et al, Nature 267:552-558 (1976)). Indeed, as mentioned above, Epo appears to share common structural features with a large group of cytokines (Parry, D.A.D., et al, J. Mol Recognition 7: 107 -110 (1988); Bazan, F., Immunology Today 77:350-354 (1990); Manavalan, P., et al, J. Protein Chem. 77:321-331 (1992)).
  • Secondary structure is predicted from the primary amino acid sequence and, when available, optical measurements. Analysis of Epo by circular dichroism reveals about 50% ⁇ helix and no detectable ⁇ sheet (Lai, P.-H. , et al, J. Biol. Chem. 267:3116-3121 (1986); Davis, J.M., et al, Biochemistry 26:2633-2638 (1987)). With the knowledge of disulfide bonds, secondary structural elements are then packed into a set of alternative tertiary structures. The number of plausible arrangements can be reduced by empirical knowledge of preferred helix-helix packing geometries and the need for globular structure to form a hydrophobic core.
  • the putative tertiary structure is then refined by standard force field calculations. Since there are a large number of alternate tertiary structures, the availability of experimentally determined structure of a homologous protein is critically important. Thus the predicted model of Epo structure gains considerable validity by knowledge of the structures of GH (Abdel-Meguid, S.S. , et al, Proc. Natl. Acad. Sci. USA 84:6434-6437 (1987); deVos, A.M., et al, Science 255:306-312 (1992)) and IL4 (Redfield, C , et al, Biochemistry 30: 1 1029- 1 1045 ( 1991 ) ; Powers, R. , et al.
  • Epo receptor is a member of an ever enlarging family of cytokine receptors (Bazan, F., Biochem. Biophys. Res. Commun. 764:788-796 (1989)). In like manner, Epo shares weak sequence homology with other members of a family of cytokines which also include growth hormone, prolactin, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, G-CSF, GM-CSF, M-CSF, oncostatin M, leukemia inhibitory factor and ciliary neurotrophic factor (Parry, D.A.D. et al , J. Mol.
  • Epo has not yet been analyzed by either X-ray diffraction or by NMR.
  • M13 plasmid, containing a 1.4 Kb Ec ⁇ RI-Ec ⁇ RI human ⁇ po cDNA insert was a gift from Genetics Institute (Cambridge, MA) (Jacobs, K. et al , Nature 373:806-810 (1985)).
  • Oligonucleotides (24 to 46 mer) were synthesized with their 5' and 3' ends complementary to the target wild-type ⁇ po sequence. A large variety of mutations (base substitutions, deletions and insertions) were created at the centers of the mutagenic primer sequences. Annealing of the phosphorylated primers (10:1 oligonucleotide/DNA template molecular ratio) was performed in 10 ⁇ l of a 20 mM Tris-HCL pH 7.4, 2 mM MgCl 2 , 50 mM NaCl solution. The reactions were incubated at 80°C for 5 minutes, then allowed to cool slowly to room temperature over a one hour period.
  • the DNA polymerization was initiated by the addition as a mix of 1 ⁇ l of 10X synthesis buffer (100 mM Tris-HCl pH 7.4 50, mM MgCl 2 , 10 mM ATP, 5 mM each dNTPs, 20 mM DTT), 0.5 ⁇ l (8 units) of T4 DNA ligase and 1 ⁇ l (1 unit) of T4 DNA polymerase (Boehringer Mannheim). After 2 hours at 37°C, 80 ⁇ l of IX TE was added. 5 ⁇ l of the diluted reaction mix was used to transform competent E. coli NM522 (ung + , dut + ).
  • RNA Blot-Hybridization Analysis Total R ⁇ As were prepared from cultured Cos7 cells (Chirgwin, J.M. et al., Biochemistry 78:5294-5299 (1979)) and 2 ⁇ g samples electrophoresed on a 1.1 % agarose gel containing 2.2 M formaldehyde. Transfer to GeneScreen Plus filters (New England Nuclear) and hybridization with a 32 P-labeled wt Epo probe were carried out as previously described (Faquin, W.C. et al, Blood 79: 1987-1994 (1992)). Quantitation of Transiently-Expressed Recombinant Epos.
  • the amount of secreted protein in the supernatants of transfected Cos7 was determined by a radioimmune assay (RIA).
  • the RIA was performed using a high titer rabbit polyclonal antiserum raised against the human wild-type Epo and produced in our laboratory.
  • 125 I-labeled recombinant Epo was obtained from Amersham. Details of the protocol have been published elsewhere (Faquin, W.C. et al, Exp. Hematol. 21: 420-426 (1993)).
  • Bioassays The dose-dependent proliferation activities of wt and Epo muteins were assayed in vitro using three different target cells: murine spleen cells, following a modification of the method of Krystal (Krystal, G., Exp. Hematol. 77:649-660 (1983); Goldberg, M.A. et al, Proc. Natl. Acad. Sci. USA 84:7972-7976 (1987)); murine Epo responsive murine cell line, developed by Hankins (Hankins, W.D.
  • our objective is to identify the functionally important domains on the Epo molecule by preparing muteins that have altered specific activity, i.e. significantly high or low biological activity per unit mass of protein.
  • Such muteins must satisfy the following criteria: efficient secretion by Cos7 cells; full recognition (near normal binding affinity) by the polyclonal anti-Epo antiserum that we use in our radio-immune assay; preservation of the overall folding and three dimensional structure.
  • the muteins that we employ in this study have only single amino acid replacements.
  • a comprehensive amino acid replacement scan of Epo would of necessity require the preparation and testing of a minimum of 166 muteins. Such an undertaking would be prohibitively labor intensive and costly.
  • mouse erythroid progenitors prepared from the spleens of animals challenged with phenylhydrazine. This bioassay obviates the possible confounding effects of a malignant clonal cell line such as HCD57 which was derived from mouse erythroleukemia virus. As shown in Figure 21, results with erythroid cells from mouse spleen were very similar to those obtained with HCD57 cells.
  • NAME Cimbala, Michele A.
  • the amino acid at position 12 may also be isoleucine.
  • the amino acid at position 13 may also be aspartic acid.”
  • CTCTCTTTCC TAGACTGTAC TCCGCTGCTG CTGCTGCTGC TGTCTCTTCT GCTGTTTCCT 240 CTGGGCCTCC CAGTCTTGGG CGCCCCCA CGCCTCATCT GTGACAGC 288
  • GGGGAGGCAG AACCTGAACG CTGGGAAGGT GGGGGTCGGG CGCGACTAGT TGGGGGCAGA 120
  • CCGAACGTCC CACCCTGCTG CTTTTACTAT CCTTGCTACT GATTCCTCTG GGCCTCCCAG 60
  • ATCACTGTCC CAGACACCAA GGTTAACTTC TATGCCTGGA AGAGGATGGA GGTCCAGCAG 240
  • ACTTTGTGCA AACTTTTCCG CAACTACTCC AATTTCCTGC GGGGAAAGCT GACGCTGTAC 540
  • ATCACTGTCC CAGACACCAA GGTCAACTTC TATACCTGGA AGAGGATGCA CGTCGGGCAG 240
  • Gly Ala Gin Lys Glu Ala lie Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125

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Abstract

Nouvelles modifications de l'érythropoïétine (Epo) améliorant l'activité biologique de la protéine. Les protéines de type Epo modifiées (Epo mutéines) peuvent être utilisées, comme il a été démontré que doivent l'être les Epos de type sauvage, mais à des doses plus faibles, en raison de leur activité biologique renforcée. Des méthodes d'utilisation des Epo mutéines pour le traitement des affections du sang sont présentées ainsi qu'une méthode de production d'Epo mutéines additionnelles, à activité biologique renforcée.
PCT/US1994/004361 1993-04-21 1994-04-21 Erythropoïetines-muteines a activite renforcee Ceased WO1994024160A2 (fr)

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US5614184A (en) * 1992-07-28 1997-03-25 New England Deaconess Hospital Recombinant human erythropoietin mutants and therapeutic methods employing them
WO1997049729A1 (fr) * 1996-06-25 1997-12-31 Diapharm Limited Polypeptides imitant l'activite de l'erythropoietine de l'homme
US5952226A (en) * 1996-11-05 1999-09-14 Modex Therapeutiques Hypoxia responsive EPO producing cells
WO2000068376A1 (fr) * 1999-05-07 2000-11-16 Genentech, Inc. Nouveaux polypeptides d'erythropoietine du chimpanze (chepo) et acides nucleiques codant pour ces memes polypeptides
US6153407A (en) * 1992-07-28 2000-11-28 Beth Israel Deaconess Medical Center Erythropoietin DNA having modified 5' and 3' sequences and its use to prepare EPO therapeutics
WO2001036489A3 (fr) * 1999-11-12 2001-11-08 Merck Patent Gmbh Formes d'erythropoietine dotees de proprietes ameliorees
US6504007B1 (en) 1996-03-14 2003-01-07 Genentech, Inc. GDNF receptor
US6555343B1 (en) 1999-05-07 2003-04-29 Genentech Inc. Chimpanzee erythropoietin (CHEPO) polypeptides and nucleic acids encoding the same
WO2004009627A1 (fr) * 2002-07-19 2004-01-29 Cangene Corporation Composes erythropoietiques pegyles
US6831060B2 (en) 1999-05-07 2004-12-14 Genentech, Inc. Chimpanzee erythropoietin (CHEPO) polypeptides and nucleic acids encoding the same
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US6967092B1 (en) 1996-10-25 2005-11-22 Mc Kearn John P Multi-functional chimeric hematopoietic receptor agonists
US6987006B2 (en) 1992-01-31 2006-01-17 Aventis Behring L.L.C. Erythropoietin and albumin fusion protein, nucleic acids, and methods thereof
EP1552298A4 (fr) * 2002-07-01 2006-11-08 Kenneth S Warren Inst Inc Cytokines protectrices des tissus recombinees et acides nucleiques codants associes pour la protection, la restauration et l'amelioration de cellules, de tissus et d'organes sensibles
US7141547B2 (en) 2001-12-21 2006-11-28 Human Genome Sciences, Inc. Albumin fusion proteins comprising GLP-1 polypeptides
WO2008065372A3 (fr) * 2006-11-28 2008-10-23 Nautilus Biotech S A Polypeptides d'érythropoïétine modifiés et utilisation de ceux-ci dans des traitements
US7459538B2 (en) 2001-05-03 2008-12-02 Merck Patent Gmbh Recombinant tumor specific antibody and use thereof
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US7507414B2 (en) 2000-04-12 2009-03-24 Human Genome Sciences, Inc. Albumin fusion proteins
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US10010583B2 (en) 2005-04-29 2018-07-03 Peter C. Dowling Methods for treating inflammatory disorders and traumatic brain injury using stabilized non-hematopoietic EPO short peptides
US10047132B2 (en) * 2005-04-29 2018-08-14 Peter C. Dowling Erythropoietin-derived short peptide and its mimics as immuno/inflammatory modulators
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US8278418B2 (en) * 2008-09-26 2012-10-02 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
CN112770766A (zh) * 2018-09-27 2021-05-07 利托瑞尔国立大学 经修饰的人促红细胞生成素
WO2022159414A1 (fr) 2021-01-22 2022-07-28 University Of Rochester Érythropoïétine pour un dysfonctionnement gastrointestinal

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