WO1997048729A1 - Molecules bivalentes formant un complexe d'activation avec un recepteur d'erythropoietines - Google Patents

Molecules bivalentes formant un complexe d'activation avec un recepteur d'erythropoietines Download PDF

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WO1997048729A1
WO1997048729A1 PCT/US1997/010564 US9710564W WO9748729A1 WO 1997048729 A1 WO1997048729 A1 WO 1997048729A1 US 9710564 W US9710564 W US 9710564W WO 9748729 A1 WO9748729 A1 WO 9748729A1
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antibody
epo
monoclonal antibody
atcc
mab34
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PCT/US1997/010564
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English (en)
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Warak Lee Chaovapong
Lutz B. Giebel
Cyrus Karkaria
Michael J. Ross
Helmut H. Schneider
Kevin Shoemaker
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Arris Pharmaceutical Corporation
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Priority claimed from US08/876,813 external-priority patent/US6103879A/en
Application filed by Arris Pharmaceutical Corporation filed Critical Arris Pharmaceutical Corporation
Priority to AU34924/97A priority Critical patent/AU3492497A/en
Publication of WO1997048729A1 publication Critical patent/WO1997048729A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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 is directed to bivalent molecules that trigger cell proliferation and differentiation of erythroid precursors by activating signal transduction through the formation of a complex with erythropoietin receptors.
  • EPO Erythropoietin
  • erythropoiesis a 34-kDa glycoprotein hormone
  • EPO acts on erythroid progenitor cells by preventing apoptosis (Koury et al. (1990) Science 248, 378-381 ; Zhuang et al. (1995) J Biol Chem 270, 14500-14504), stimulating proliferation of erythroid precursor cells and by inducing differentiation into mature erythrocytes.
  • EPO-R erythropoietin receptor
  • EPO-R is a member of the cytokine receptor type I superfamily, which includes the receptors for interleukins (IL) 2-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-stimulating factor (G-CSF), growth hormone (GH), prolactin, thrombopoietin (TPO), leukemia inhibitory factor (LIF), and leptin (Bazan, J.F. (1990) Proc. Natl. Acad. Sci.
  • IL interleukins
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • G-CSF granulocyte-stimulating factor
  • GH growth hormone
  • prolactin thrombopoietin
  • LIF leukemia inhibitory factor
  • leptin Bazan, J.F. (1990) Proc. Natl. Acad. Sci.
  • EPO induced receptor dimerization is based primarily on constitutively active EPO-R mutants, which contain point mutations introducing cysteine substitutions into the extracellular domain at amino acid positions R129, E132, and E133 (Longmore et al. (1991) Cell 167, 1089-102; Yoshimura et al. (1990) Nature 348, 647-649; Watowich et al. (1992) Proc Natl Acad Sci USA 89, 2140-2144; Watowich et al.
  • EPO-R mutants form disulfide-linked homodimers in the endoplasmatic reticulum and on the cell surface (Watowich et al. (1992)). Based on sequence alignments with the related GH receptor, these mutations are expected to be in the receptor-dimer interface region.
  • Expression of the constitutively active EPO-R (R129C) mutant in BaF3 cells results iii factor-independent proliferation, and expression in primary cultures of mouse fetal liver cells induce EPO-independent erythroid differentiation (Pharr et al.
  • mice infected with a retrovirus carrying the EPO-R (R129C) mutant develop erythroleukemia (Longmore et ⁇ /.(1994)).
  • Truncated signal transduction inactive receptor mutants lacking part of the intracellular signaling domain are dominant-negative when coexpressed with wild-type EPO-R (Watowich et al. (1994); Barber et al. (1994) Mol Cell. Biol 14, 2257-2265).
  • Both wild-type and truncated receptors can be coimmunoprecipitated with an antibody directed against the C-terminus of the wild-type receptor, which is not present in the truncated form (Miura et al. (1993) Arch Biochem
  • EPO-R dimerization of EPO-R is required, it is not sufficient for complete activation of cells.
  • Other accessory cellular factors may be required to send a proliferation signal and, furthermore, these factors may be different from those required to send a differentiation signal. It is desirable to identify molecules other than EPO that activate the EPO-R and stimulate erythropoiesis and this invention meets that need.
  • An aspect of this invention is an antibody which activates erythropoietin receptors.
  • a second aspect of this invention is a bivalent molecule which activates erythropoietin receptors, which bivalent molecule contains at least one binding domain that selectively recognizes an epitope located on the extracellular domain of an erythropoietin receptor, which epitope is selectively recognized by the monoclonal antibody produced by Hybridoma
  • a third aspect of this invention is a hybridoma cell line which produces a monoclonal antibody which activates erythropoietin receptors, which monoclonal antibody contains two binding domains that selectively recognizes an epitope located on the extracellular domain of an erythropoietin receptor, which epitope is selectively recognized by the monoclonal antibody produced by Hybridoma #34.10.1 deposited at the ATCC as ATCC HB-12088.
  • a fourth aspect of this invention is a method of treating a condition in a mammal in which diminished erythropoietin receptor activity contributes to the pathology and/or symptomatology of the condition, which method comprises administering to such animal a tfierapeutically effective amount of an antibody which activates erythropoietin receptors.
  • a fifth aspect of this invention is method of treating a condition in a mammal in which diminished erythropoietin receptor activity contributes to the pathology and/or symptomatology of the condition, which method comprises administering to such animal a therapeutical ly effective amount of a bivalent molecule which activates erythropoietin receptors, which bivalent molecule contains at least one binding domain that selectively recognizes an epitope located on the extracellular domain of an erythropoietin receptor, which epitope is selectively recognized by the monoclonal antibody produced by Hybridoma
  • a sixth aspect of this invention is pharmaceutical composition comprising a therapeutical ly effective amount of an antibody which activates erythropoietin receptors in combination with a pharmaceutically acceptable excipient.
  • a seventh aspect of this invention is pharmaceutical composition comprising a therapeutically effective amount of a bivalent molecule which activates erythropoietin receptors, which bivalent molecule contains at least one binding domain that selectively recognizes an epitope located on the extracellular domain of an erythropoietin receptor, which epitope is selectively recognized by the monoclonal antibody produced by Hybridoma
  • FIG. 1 is a BIAcore sensogram recording and a graph of the data contained within the sensogram showing the binding kinetics of MAb34.
  • A BIAcore (Pharmacia Biosensor) sensograms of various concentrations of EPObp (ranging from 10 to 1500 nM) injected over immobilized MAb34, corrected by data from control surfaces. Data from 8 representative EPObp concentrations from a total of 16 are shown.
  • B The data were used for affinity determinations by plotting steady state values ( ⁇ R ⁇ ) versus EPObp concentrations. The solid line represents a fit of the data to the steady state model described by Karlsson et al. (Karlsson et al. (1991) J. Immunol Methods 145, 229-240).
  • FIG. 2 provides graphs showing MAb34-i ⁇ duced proliferation of BaF3/EPO-R cells and UT-7/EPO cells.
  • A Dose-dependent M Ab34-induced proliferation of BaF3/EPO-R cells. Cells were incubated in the presence of [ 3 H]thymidine and various concentrations of MAb34 (open squares) and Fab34 (open circles) as described in Example 4.
  • B Dose-dependent cell proliferation of UT-7/EPO in the presence of various concentrations of MAb34 (open squares), control anti-Axl antibody unrelated to EPO (solid circles), and EPO (open triangles), respectively as described in Example 4. Experiments were done in duplicate.
  • FIG. 3 provides graphs showing MAb34-mediated proliferation and differentiation.
  • EPO stimulates proliferation and differentiation of erythroid progenitor cells. Homodimerization of EPO-R by EPO on the cell surface is believed to be the key event in receptor activation and subsequent signal transduction (Youssoufian et al.).
  • Bivalent molecules that possess two identical binding sites for a given region on the extracellular domain of EPO-R, such as antibodies, for example, can bind two EPO P. molecules thereby bringing them into close proximity to permit dimerization. These bivalent molecules must bind so as to activate the signal transduction pathway and it is desirable that such activation result in both proliferation and differentiation of erythroid progenitors. It is also possible for the binding domains of the bivalent molecules to recognize different regions of EPO-R and have a similar or an enhanced effect. Such bivalent molecules are useful for researching the mechanism of EPO-R activation and signal transduction and have therapeutic and diagnostic applications as well.
  • Epitope means a region of an EPO-R recognized by a first antibody wherein the binding of the first antibody to the region prevents binding of a second antibody or other bivalent molecule to the region.
  • the region encompasses a pa ⁇ icular core sequence or sequences selectively recognized by a class of antibodies and can include additional EPO-R sequences.
  • several antibodies can selectively bind to the sequence XYZ and although they may bind with differing affinities to abcXYZ than to XYZdef, they are nevertheless binding to the same epitope.
  • the epitope can be contiguous or non-contiguous such that the first antibody selectively recognizes a peptide fragment containing the core sequence XYZ, but the second antibody or other bivalent molecule selectively recognizes a fragment containing the core sequence only in its native conformation on the EPO-R.
  • the ternary structure of the EPO-R can give rise to the epitope pqrXYZ from the primary sequence of pqr XYZ.
  • An antibody specific for pqrXYZ would not be expected to bind the peptide pqr XYZ as effectively as it would the EPO-R itself, yet it will selectively recognize either the peptide fragment or the EPO-R.
  • abcXYZ, XYZdef, and pqrXYZ are all the same epitope because they share the same core sequence.
  • the core sequence comprises at least 3 amino acid residues, preferably 4-20 amino acid residues, and can comprise as many as 35 amino acid residues.
  • the "MAb34 epitope” is an epitope that is selectively recognized by the MAb34 antibody described in more detail below.
  • “Selectively recognizes” or “selectively recognized” means that binding of the antibody or other bivalent molecule to an epitope is at least 2-fold greater, preferably 2-5 fold greater, and most preferably more than 5-fold greater than the binding of the bivalent molecule to an unrelated epitope or than the binding of an unrelated bivalent molecule to the epitope, as determined by techniques known in the an and described herein, such as, for example, ELISA and cold displacement assays.
  • Bivalent molecule or "BV” means a molecule capable of binding to two separate erythropoietin receptors at the same time, thereby forming a molecular complex.
  • the bivalent molecule is not limited to having two and only two binding domains and can be a polyvalent molecule or a molecule comprised of linked monovalent molecules so long as at least two EPO-R binding domains are provided.
  • the binding domains of the bivalent molecule can selectively recognize the same epitope or different epitopes located on the EPO-R extracellular domain, so long as at least one binding domain selectively recognizes an epitope recognized by MAb34 (i.e., the MAb34 epitope) and the resultant (EPO-R) 2 :BV complex is active in signal-transduction.
  • the bivalent molecule can be proteinaceous or non-proteinaceous provided that it can form a complex sufficiently stable to activate the EPO-R or to be detected.
  • the binding domains can be linked in any of a number of ways including, but not limited to, disulfide bonds, peptide bridging, amide bonds, and other natural or synthetic linkages known in the an (Spatola et al.
  • Proteinaceous means the molecule referred to comprises a polypeptide sequence.
  • a proteinaceous molecule can be comprised entirely of polypeptide sequences or it can comprise one or more non-peptidic substituents in addition to polypeptide sequences.
  • “Therapeutically effective amount” means an amount that provides a therapeutic effect for a given condition and administration regimen.
  • the therapeutic effect is an increase in erythrocyte levels, which can be evidenced by a rise in hematocrit in the patient being treated.
  • “Treating”, as in treating a condition means (1) preventing the condition from occurring in a mammal which may be predisposed to the condition but does not yet experience or display symptoms of the disorder, (2) inhibiting the condition (e.g., arresting development of the condition) or (3) ameliorating the condition (e.g., causing regression of the disorder).
  • Phathology of a condition means the essential nature, causes and development of the condition as well as the structural and functional changes that result from the disease processes.
  • Symptomatology of a condition means any morbid phenomenon or departure from the normal in structure, function or sensation experienced by the patient and indicative of the condition, their production and the indications they furnish.
  • an antibody that selectively recognizes the extracellular domain of the erythropoietin receptor to form a molecular complex active in signal transduction.
  • the invention is not limited to use or identification of the specific monoclonal described in the Examples section below. Any antibody that selectively recognizes an epitope and that results in homodimerization of the erythropoietin receptor and subsequent cell proliferation and differentiation of erythroid precursors is encompassed by the present invention. Use of such antibodies is easily accomplished by one of ordinary skill in the an given the teachings of the present specification.
  • a bivalent molecule that selectively recognizes the extracellular domain of the erythropoietin receptor.
  • the bivalent molecule can bind the extracellular domains of two erythropoietin receptor to form a molecular complex active in signal transduction.
  • the invention is not limited to use or identification of the specific monoclonal described in the Examples section below. Any bivalent molecule as defined above that selectively recognizes an epitope selectively recognized by monoclonal antibody MAb34 (MAb34 epitope) and that results in homodimerization of the erythropoietin receptor and subsequent cell proliferation and differentiation of erythroid precursors is encompassed by the present invention.
  • the bivalent molecule can have a detectable label attached thereto, such as a fluorescent label (e.g., fluorescein, isothiocyanate (FITC)), an affinity label (e.g., biotin), an enzymatic label (e.g., horseradish peroxidase or alkaline phosphatase), or an isotopic label (e.g. , 125 I) or any other such detectable moiety.
  • FITC fluorescein, isothiocyanate
  • affinity label e.g., biotin
  • an enzymatic label e.g., horseradish peroxidase or alkaline phosphatase
  • an isotopic label e.g. , 125 I
  • a preferred bivalent molecule is an antibody that selectively recognizes the MAb34 epitope located on the extracellular domain of the erythropoietin receptor.
  • Antibodies can be produced by the immunization of various animals, including mice, rats, rabbits, goats, primates, humans and chickens with EPO-R or peptide fragments of EPO-R containing the MAb34 epitope.
  • the protein is purified prior to immunization of the animal.
  • the EPO-R can be purified by methods known in the art, for example, gel filtration, ion exchange, affinity chromatography, etc.
  • the EPO-R can be naturally occurring or genetically engineered as is apparent to one of ordinary skill in the art.
  • the antibody is purified. Affinity chromatography or any of a number of other techniques known in the art can be used to isolate polyclonal or monoclonal antibodies from serum, ascites fluid, or hybridoma supernatants.
  • the antibody can be of the isotypes IgA, IgE, IgM, preferably is an IgG antibody, and can be an IgD antibody, although this is less preferred.
  • IgG subclasses include IgGl, IgG2a, IgG2b and IgG3 in the mouse; IgGl, IgG2a, IgG2b, and IgG2c in the rat; and IgGl, IgG2, IgG3 and IgG4 in the human.
  • Useful antibodies of the invention are chosen upon consideration of such factors as complement fixation, autoaggregation, ability to mediate mast cell degranulation, ability to bind Fc receptors or macrophage receptors and resistance to proteolytic enzymes.
  • the bivalent molecules of the invention are resistent to proteolytic cleavage and are not excessively immunogenic.
  • fully human antibodies selectively recognizing the MAb34 epitope produced in genetically altered mice PCT Application No. 93/122227.
  • the antibody is a monoclonal antibody. Methods of generating monoclonal antibodies are known in the art and are described in detail, for example, by Oi and Herzenberg in "Selected Methods in Cellular Immunology” (1979) (B.B. Mishell and S.M. Shiigi, eds.), San Francisco: W.J. Freeman Publishers, pp. 351-352.
  • myeloma cells such as mouse myeloma cell lines P3X63Ag8.653 (ATCC CRL 1580); P3-NSl/lAg 4; and S194/5, XXO, BUI; human fusion partners such as UV 729-6 and SKO-007; and mouse-human hetero-myeloma lines, such as SHM-A6 and SHM-D33 (PCT Application No. 81/00957; Schlom et al., Proc. Natl. Acad. Sci. USA (1980) 77, 6841-6845; Croce et al.
  • myeloma cells such as mouse myeloma cell lines P3X63Ag8.653 (ATCC CRL 1580); P3-NSl/lAg 4; and S194/5, XXO, BUI; human fusion partners such as UV 729-6 and SKO-007; and mouse-human hetero-myeloma lines,
  • hybridoma cell lines are also encompassed by the invention and provide an inexhaustive supply of monoclonal antibody.
  • antibody titers are determined by methods such as enzyme linked immunosorbent assays (ELISA) (Engrail (1977) Med. Biol. 55, 193-200)to determine their affinity for their binding partners.
  • ELISA enzyme linked immunosorbent assays
  • the ability of the antibodies to activate EPO-R can be determined by cell proliferation or differentiation assays, some of which are described herein.
  • a preferred monoclonal antibody is MAb34, which is deposited at the ATCC as
  • the monoclonal antibody Mab34 directed against the extracellular domain of the EPO-R that activates EPO-R by dimerization and thus mimics EPO action as is demonstrated below in Example 4.
  • This bivalent IgG antibody triggers the proliferation of EPO-dependent cell lines and induces differentiation of erythroid precursors in vitro. Activation of cell proliferation and differentiation show a bell-shaped response curve over the range of antibody concentration tested with a maximum occuring at MAb34 concentrations in close vicinity to its K D . Indeed, a mathematical model (Perelson, A.S.
  • Example 4 demonstrates that EPO and MAb34 antagonize ligand-dependent cell proliferation with IC JO concentrations of approximately 20 ⁇ M and 2 ⁇ M, respectively.
  • MAb34 EPO-R interaction using a mathematical model describing antibody mediated receptor dimerization. The predicted receptor dimer formation on the cell surface was consistent with the observed proliferation and differentiation activity data.
  • Another preferred monoclonal antibody is a high-affinity variant of MAb34.
  • Such variants can be prepared by a number of methods known in the an or developed in the future, including the random or site-directed mutagenesis of the variable region of MAb34.
  • degenerate oligonucleotides can be substituted for the variable region in MAb34 and the resultant antibody population screened by competition with MAb34 /or EPO-R binding.
  • phage display can also be utilized for this purpose.
  • the antibody can be a chimeric antibody such as a humanized antibody or a
  • Another preferred chimeric antibody is a bispecific antibody in which one of the binding domains selectively recognizes an epitope located on the extracellular domain of EPO-R other than the M Ab34 epitope, provided that the chimeric antibody can form an (EPO-R) 2 :antibody complex active in signal transduction, a property possibly dependent upon the distance and geometry of the receptors.
  • Another preferred aspect is a bivalent proteinaceous molecule other than an antibody comprising two binding domains that selectively recognize the MAb34 epitope.
  • peptides can be designed based on an analysis of the amino acid sequence of the complementarity-determining region (CDR) of MAb34.
  • a bivalent molecule can be synthesized containing two or more of such peptides linked or fused to each other and possibly to an additional polypeptide structure provided as a scaffold for stability or some other purpose such as, for example, the recruitment of a tertiary molecule.
  • random peptide libraries prepared by techniques known in the art, such as, for example, peptide chemistry, phage display (Ladner et al; Huse; Devlin; Dower et al. ; Markland et al.) or the yeast two-hybrid system can be screened by competitive inhibition of MAb34 binding to EPO-R to identify a polypeptide sequence that selectively binds to the MAb34 epitope.
  • This peptide can then be synthesized in a bivalent form and tested for an ability to activate EPO-R by any of the methods herein described, known in the art, or developed in the future.
  • the bivalent proteinaceous molecule can be a bispecific molecule in which one of the binding domains selectively recognizes an epitope located on the extracellular domain of EPO-R other than the MAb34 epitope, such as, for example, an epitope corresponding to the
  • the bispecific molecule can be a bispecific antibody as mentioned above, a chimeric molecule comprising an immunoglobulin portion and a non-immunoglobulin portion, or an entirely non-immunoglobulin proteinaceous bispecific molecule, so long as the bispecific molecule is capable of forming an active molecular complex with EPO-R.
  • Another preferred aspect is a non-proteinaceous bivalent molecule.
  • Such a molecule can be identified and produced by techniques known in the art, such as, for example, peptidomimetics. Such mimetics can be produced by rational drug design based on molecular modeling and the polypeptide sequence of the CDR of MAb34.
  • combinatorial chemical libraries can be screened by competitive inhibition of MAb34 binding to EPO-R to identify a compound that selectively binds to the MAb34 epitope and then the compound can be synthesized in a bivalent form and tested for its ability to form an active (EPO-R) 2 :BV complex by any of the methods herein described.
  • Bivalent molecules selectively recognizing the MAb34 epitope can be produced in a number of methods using techniques known in the an. These methods are not pan of the invention but are provided herein for the convenience of the reader.
  • One method is initially to screen a population of candidate molecules for an ability to compete with MAb34 for binding of EPO-R, and then to perform a functional determination of an ability to activate EPO-R, such as, for example, thymidine uptake proliferation assays, erythroid cell differentiation assays, or any assays exploiting steps in the signal transduction cascade. Suitable assays are provided for illustrative purpose in the Examples section.
  • Another method is to synthesize peptides corresponding to the MAb34 epitope for use as a binding partner in binding assays.
  • the MAb34 epitope can be mapped by preparing overlapping synthetic peptides that span the extracellular domain EPO-R and screening them for binding to MAb34.
  • phage display Another procedure that can be used for mapping the MAb34 epitope is phage display (Ladner et al. , PCT Application No. PCT/US92/01456; Huse, PCT Application No. PCT/US91/07141; Devlin, PCT Application No. PCT/US91/03332; Dower et al. , PCT Application No. PCT/US91/02989 and PCT/US91/04384; Markland etal. , PCT Application No.
  • the invention also provides an (EPO-R) 2 :BV complex.
  • EPO-R EPO-R
  • Such a complex when present on the surface of a cell., is useful for modulating proliferation and differentiation of erythroid precursor cells.
  • Such a complex whether present on the surface of a cell or isolated from other cellular components, can also be used for EPO-R activator drug discovery where compounds that are potential candidate drugs for the treatment of disorders treatable by EPO-R activon are screened as described above for their ability to competitively inhibit complex formation between EPO-R and previously identified bivalent molecules of the invention.
  • the invention provides methods and kits for detecting the presence or absence of an erythropoietin receptor or an activatable EPO-R in a biological sample.
  • the detection is achieved by a binding assay in which the level of binding of a bivalent molecule to EPO-R is detected. Basically, the biological sample is contacted with the bivalent molecule and the level of binding is detected.
  • the biological samples include tissue specimens, intact cells, or extracts thereof.
  • Antibodies may be used as part of a diagnostic kit to detect the presence of EPO receptors in a biological sample.
  • Such kits employ bivalent molecules, preferably a monoclonal antibody, having an attached label to allow for detection. The detection can also be achieved by a functional assay in which it is the activation of the receptor that is detected.
  • any of a number of cellular products of the signal transduction cascade triggered by EPO-R activation can be monitored in a functional assay, such as nitric oxide, prostaglandins, cAMP, cGMP, Ca 2+ , inositol phosphates and the like.
  • a functional assay such as nitric oxide, prostaglandins, cAMP, cGMP, Ca 2+ , inositol phosphates and the like.
  • kinase and phosphatase activities resulting from EPO-R activation can also be monitored in a functional assay.
  • Such methods can be used in the diagnosis of anemia and other diseases characterized by a dysfunctional EPO-R or otherwise related to
  • the invention also provides methods of modulating the endogenous activity of an erythropoietin receptor in a mammal. Typically activation of the EPO-R in an erythroid precursor cell results in cell proliferation or differentiation. Such methods are useful for treating disorders characterized by low erythrocyte levels.
  • any condition treatable with EPO such as anemia, for example, is amenable to treatment with the bivalent molecules of the invention.
  • Any of the bivalent molecules of the invention can be used for this purpose, subject to such considerations as variations in bioavailability, antigenicity, and potency among the different bivalent molecules.
  • MAb34 may be more potent than another antibody, but for long-term use, a slightly less potent humanized antibody may be preferred.
  • non-immunoglobulin bivalent molecules may be preferred where stability or the need for an orally active drug is an issue.
  • the amount and method of administering the therapeutic molecules of the invention can be ascertained by one skilled in the art.
  • administration of large bivalent molecules is by injection, whether subcutaneous, intramuscular, or intravenous.
  • Smaller, less peptidic bivalent molecules can be administered orally as well as intravenously.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the bivalent molecule of the invention in a pharmaceutically acceptable adjuvant, which can be selected from one or more of a diluent, carrier, preservative, emulsifier, anti-oxidant and/or stabilizer.
  • a pharmaceutically acceptable adjuvant which can be selected from one or more of a diluent, carrier, preservative, emulsifier, anti-oxidant and/or stabilizer.
  • Pharmaceutically acceptable adjuvants are known to one skilled in the art (Remington's Pharmaceutical Sciences, 18th ed. A.R. Gennaro, ed. Mack, Easton, PA (1990)).
  • EXAMPLE 1 GENERATION OF EPO-R MONOCLONAL ANTIBOD ⁇ ES In order to obtain an antibody capable of dimerizing an EPO-R, monoclonal antibodies were raised against the extracellular, ligand binding domain of the human EPO-R (EPObp). Expression and affinity purification of soluble human EPO-R:
  • DNA encoding a soluble truncated EPObp was generated by the polymerase chain reaction (PCR) using the full-length cDNA as template.
  • the amplification product introduces a TAG termination codon 5' of the transmembrane region and encodes the extracellular domain comprising amino acids 1 through 249 of the published sequence (Jones et al. (1990) Blood 76, 31-35).
  • the PCR product was subcloned into expression vector pRc/CMV (Invitrogen) and stably transfected into CHO cells. Individual clones secreting EPObp were selected by limiting dilution cloning.
  • Roller bottles (surface area 1 ,700 cm 2 , Corning, Corning, NY) were seeded with the stable cell line and grown to confluence in RPMI plus 10% FBS. Cells were washed twice in serum free RPMI medium and cultured in 200 ml of serum-free RPMI. Cell supernatant was collected after two days and fresh medium was added for another two days. The EPObp was secreted at an approximate concentration of 0.2 ⁇ g/ml and was purified by EPO affinity chromatography.
  • EPO was oxidized with 10 mM NaIOont and biotinylated using 10 mM biotin hydrazide (Pierce) following the manufacturer's instructions.
  • a ligand affinity column was prepared by immobilizing biotinylated EPO (10 mg) on Streptavidin 3M Emphaze beads (3 ml; Pierce), overnight in Dulbecco's phosphate buffered saline (PBS, Irvine Scientific) at 4°C. The beads were separated from the supernatant by centrifugation, and incubated with 10 mM biotin in PBS for 2 h at 4°C to saturate all biotin binding sites.
  • EPO coated beads were packed in a glass column (Omnifit).
  • Cell supernatant (10 L) was concentrated and diafiltered to 1 L in 20 mM Tris HCI, pH 7.6 and loaded on the column at a flow rate of 0.7 ml/min.
  • the column was washed with 50 ml of 20 mM Tris/HCl, pH 7.6.
  • Bound EPObp was eluted with 750 mM NaCl in 20 mM Tris/HCl, pH 7.6.
  • SDS-PAGE analysis showed a single 30 kD EPObp band.
  • the EPObp fractions were pooled, concentrated and buffer exchanged with PBS to a final concentration of 0.8 mg/ml.
  • Monoclonal antibodies were generated essentially as described ir Galfre et al. (1981) Methods. Enzymol., 73, 3-46).
  • Five Balb/c mice were immunized by seven subcutaneous injections at two sites over a period of 14 weeks. Each 50 ⁇ l injection contained 25 ⁇ g EPObp in Freud's adjuvant.
  • Antibody titers were measured by ELISA after 12 weeks. Polysorb microtiter plates
  • EPObp was covalently immobilized.
  • EPObp was oxidized in 1 mM NaIOont, 50 mM sodium acetate, pH 5.5 at 4 °C for 30 min in the dark.
  • the protein was separated from periodate on a NAP-5 column (Pharmacia Biotech Inc., Piscataway, NJ) and incubated on hydrazide-activated microtiter plates (Unisyn, San Diego, CA) at 2 ⁇ g/ml (100 ⁇ l per well) for 1 h at room temperature. Plates were blocked with PBS, 20 mg/ml BSA for 1 h.
  • EPObp was immobilized via MAb 2E12, a specific, non-neutralizing rat monoclonal antibody directed against EPObp: Polysorb microtiter plates were incubated with 10 ⁇ g/ml MAb 2E12 for 1 h at 37°C, washed and blocked with PBS 16 containing 20 mg/ml BSA for 1 h at 37°C. EPObp (1 ⁇ g/ml) was added in PBS, 1 mg/ml BSA, 0.02 % Tween-20 for 1 h at 37°C. After the immobilization of EPObp, both ELISA protocols were identical. Hybridoma supernatants were added and incubated for 1 h at 37°C.
  • EXAMPLE 2 SCREENING OF EPO-R MONOCLONAL ANTIBODIES FOR RECEPTOR AGONIST ACTIVITY
  • EPO-dependent BaF3/EPO-R cell line was generated by transfecting the full-length human EPO receptor into BaF3 cells, a murine IL-3 dependent cell-line (Palacios et al. (1985) Cell 41, 727-734).
  • a cDNA encoding the full-length human EPO receptor was generated by transfecting the full-length human EPO receptor into BaF3 cells, a murine IL-3 dependent cell-line (Palacios et al. (1985) Cell 41, 727-734).
  • EPO-dependent growth Individual clones were selected by limited dilution cloning. The EPO-dependent cell line chosen for this study proliferates in the presence of EPO with an
  • BaF3/EPO-R and IL-3-dependent BaF3 parental cells were incubated with individual hybridoma supernatants in the absence of EPO and IL-3, respectively, and proliferation was measured by [ 3 H]thymidine incorporation.
  • BaF3 and BaF3/EPO-R cells were grown to late logarithmic phase, collected by centrifugation, washed three times with RPMI 1640 media (containing 10% FBS and 10 mM HEPES pH 7 in the absence of EPO and IL-3), then starved in the same media for 2 hours.
  • Antibody test samples (hybridoma supernatants or purified proteins) were diluted at least 4-fold into 100 ⁇ l media and 100 ⁇ l cells were added (25,000 cells per well).
  • EPO was dialyzed against 10 mM HEPES pH 7.0, and 100 ⁇ l test samples were combined with 100 ⁇ l ceils (25,000 cells per well) in 2-fold concentrated medium. Plates were incubated for 4 hours at 37°C and 5% CO 2 in a humidified tissue culture incubator. Then 0.5 ⁇ Ci [ 3 H]thymidine (Amersham), diluted into 20 ⁇ l medium, was added and incubation continued for another 15 hours. Cells were harvested onto glass fiber filtermats using a Tomtec cell harvester (Wallac Oy), and incorporated radiolabel was determined using a Microbeta 1450 scintillation counter (Wallac, Turk ⁇ , Finland). The hybridoma supernatant of MAb clone 34 stimulated thymidine uptake in
  • Hybridoma clone MAb34 was subcloned twice by limiting dilution. Ig-isotyping was performed using an IsoStrip Mouse Monoclonal Antibody Isotyping Kit from Boehringer Mannheim (Indianapolis, IN). MAb34 is an IgG monoclonal which was subtyped as IgG ⁇ l .
  • EXAMPLE 3 PURIFICATION AND DETERMINATION OF BINDING CHARACTERISTICS OF AN EPO-R AGONIST
  • MAb34 was purified and used in cold displacement binding assays. Binding kinetics were examined using the BIAcore system.
  • MAb34 was purified by protein G affinity chromatography, and Fab fragments (Fab34) were prepared by papain cleavage. 0.75 ug and 1.5 ug of EPObp was heat-denatured and analyzed by reducing SDS-PAGE on a 12% acrylamide gel and subsequent transfer to nitrocellulose.
  • the blot was incubated with MAb34 (10 ⁇ g/ml) and subsequently with anti-mouse IgG coupled to horseradish peroxidase. A robust signal was obtained for both 0.75 ug and 1.5 ug samples of EPObp. This immunoblot analysis of heat-denatured and reduced EPObp suggested that MAb34 recognizes a linear continuous epitope.
  • Hybridomas were grown in 47.5% RPMI, 47.5% DMEM, 5% FBS. Culture supernatant was filtered through a 0.2 ⁇ m membrane. A 6 ml protein G Sepharose 4 fast flow column (Pharmacia) was packed at 80 psi pressure. A 1 1 sample was loaded at 4 ml/min at 4 C C, followed by washing with >5 column volumes of PBS. MAb34 was then eluted from the column with ImmunoPure IgG elution buffer (Pierce) at 2 ml/min. The eluate was immediately neutralized to pH 7.5 by adding 3 M Tris. The purity was evaluated by non-reducing SDS-PAGE.
  • F, b fragments were generated by papain cleavage using ImmunoPure IgGl F lb Preparation Kit (Pierce) following the manufacturer's instructions.
  • the F ⁇ was further purified by gel filtration, using a Superdex 75 column (1.6 cm x 60 cm, Pharmacia), eluted with PBS, and analyzed for purity by SDS-PAGE.
  • EPO cold displacement assay :
  • OCIM1 cells a human erythroleukemia cell line which expresses EPO-R on the cell surface (Broudy et al. (1988) Proc. Nat. Acad. Sci. USA 85, 6513-6517), were grown in IMDM, 10% FBS, 1 % penicillin-streptromycin-fungisone to approximately 2-5 X 10 5 cells/ml. Cells were collected by centrifugation, washed two times in binding buffer (RPMI 1640, 1 % BSA, 25 mM HEPES pH 7.3), then resuspended in binding buffer containing 0.1 % sodium azide and 10 ⁇ g/ml cytochalisin B at 1-2 X 10 7 cells/ml.
  • binding buffer RPMI 1640, 1 % BSA, 25 mM HEPES pH 7.3
  • MAb34 did not compete with [ 12i I]-EPO for binding to EPO-R in a displacement assay using human OCIM 1 cells (Broudy et al. (1988) Proc. Nat. Acad. Sci. USA 85, 6513-6517), indicating that the binding sites of EPObp for EPO and for MAb34 are different. Analysis of MAb34 and Fab34 binding kinetics:
  • EXAMPLE 4 EPO-R AGONIST STIMULATES PROLIFERATION IN BOTH RECOMBINANT AND NON-RECOMBINANT EPO-DEPENDENT CELL LINES
  • Purified MAb34 was tested for its ability to stimulate proliferation of both recombinant and non-recombinant cell lines. Purified MAb34 was able to stimulate proliferation in the EPO-dependent BaF3/EPO-R cell line using the procedure described in Example 2. A dose dependent response evaluation in a [ 3 H]thymidine uptake cell proliferation assay revealed ECj o values of approximately 10 nM (FIG. 2A). The effect of MAb34 was specific to EPO-R, because it did not stimulate growth of the parental BaF3 cell line. Based on the maximal amount of f 3 H]thymidine incorporation, the potency of MAb34 was 8 to 10 -fold lower than the potency of EPO (FIG. 2A).
  • Test samples 100 ⁇ l diluted in assay medium at least 5-fold, were added to wells. Then 50 ⁇ l cells were added (5,000 cells per well) and plates were incubated at 37°C and 5 % CO 2 . After 72 hours, 50 ⁇ l methyl-[ 3 H]thymidine (1 mCi/ml; 20 Ci/mmol) diluted 1: 100 in assay medium was added. Cells were incubated for another 4 hours at 37°C and 5 % CO 2 . Labeled cells were harvested onto glass fiber filtermats using a PHD cell harvester (Cambridge Technology, Inc.). Filters were rinsed with 2-propanol, dried and counted in a Beckman Model LS6000IC scintillation counter.
  • MAb34 was more active in the UT-7/EPO cell line: In UT-7/EPO cells MAb34 stimulated cell proliferation with an EC 50 of approximately 300 pM (FIG. 2B) as compared to 10 nM for BaF3/EPO-R. The maximum of incorporation was close to the value obtained with EPO. This may be due to the higher concentration of EPO-R molecules on the surface of UT-7/EPO cells, which contain 2400 receptors per cell ( Komatsu et al. (1993) Blood 82, 456-464; Nicolis et al. (1993) Exp Hematol 21, 665-670) compared to 800 for BaF3/EPO-R.
  • BaF3/EPO-R cells proliferate with an EC50 of 15 pM, which is six orders of magnitude lower.
  • parental BaF3 cells were incubated with EPO at identical concentrations and in the presence of IL-3 containing WEHI media. No decrease in the IL-3 dependent proliferation was observed.
  • EXAMPLE 5 EPO-R AGONIST INDUCES DIFFERENTIATION OF CD34 + ERYTHROID PROGENITOR CELLS IN THE PRESENCE OF SCF. MAb34 was tested for its ability to induce differentiation of erythroid progenitor cells.
  • CD34 + cells The differentiation of CD34 + cells to BFUe is dependent on EPO and SCF (Iscove et al. (1974) J. Cell. Physiol 83, 309-320).
  • MAb34 was able to induce in vitro differentiation of human CD34 + erythroid cell precursors.
  • CD34 + erythroid cell precursors normal human donors were lymphopheresed according to standard protocols.
  • the lymphopheresed cells were washed, resuspended in Hank's Balanced Salt Solution (HBSS) and separated by density centrifugation over a gradient (ficoll-paque).
  • HBSS Hank's Balanced Salt Solution
  • LD low-density cells
  • the LD cells were then further purified using a CD34 progenitor Cell Isolation Kit (QBend/10) made by Miltenyl Biotech GmbH.
  • the in vitro BFUe assay was done on purified CD34 + cells in methyl cellulose.
  • the medium contains 20% FBS, 0.33X IMDM (Gibco), salts, 2-mercaptoethanol, nucleosides, cholesterol, sodium pyruvate, Hu-transferrin, lipids, Hu-insulin, deionized BSA and 100 ng/ml Stem Cell Factor (SCF) (Ponting et al. (1994) Exp. Hematol 22, 810. To plate out duplicate 1 mi samples, an excess of 3 ml was prepared in sterile polystyrene tubes.
  • SCF Stem Cell Factor
  • Each tube received CD34 + cells (10,000 cells/ml), 0.015 ml SCF (20 ⁇ g/ml), and a combination of sample and medium totaling 3 ml.
  • the tubes were mixed and 1 ml was aliquoted onto 35x100 mm tissue culture plates. The plates were incubated at 37°C and 10% CO 2 in a humidified tissue culture incubator. Erythroid colonies (orange to red in color) were scored after 20 days.
  • EXAMPLE 6 AGONIST ACTIVITY OF MAB34 CORRELATES WELL W ⁇ A MODEL FOR ANTIBODY-MEDIATED RECEPTOR DIMERIZATION
  • Perelson postulates a two step mechanism, where the formation of 1 : 1 complexes is driven by the affinity constant K A ( 1/K D ). Subsequent dimer formation is dependent on a "cross-linking" constant K x , which includes K A but also depends on the effective concentration of receptors on the cell surface and other factors.
  • the concentration of dimer is given by:
  • FIG. 3 fits the data of the MAb34 cell proliferation and differentiation assays to the equation.
  • the resulting bell-shaped curves for the proliferation assays correlate well the assay data.
  • the obtained 2: 1 complex maxima were 114 nM for BaF3/EPO-R cells and 26 nM for UT-7/EPO cells. According to the model, this translates to apparent K D values of 228 nM and 52 nM respectively, in good agreement with the K D value of 54 nM determined by BIAcore (Pharmacia) analysis (Table 1). A larger number of parallel experiments would minimize data scattering as is apparent to one of ordinary skill in the art.

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Abstract

Molécules bivalentes qui activent des érythropoïétines et induisent la prolifération ou la différentiation de cellules progéniteurs d'érythroïdes. L'invention décrit également des procédés d'utilisation de ces molécules bivalentes pour la recherche de médicaments, pour le diagnostic et le traitement de troubles relatifs à l'activation d'un récepteur d'érythropoïétines.
PCT/US1997/010564 1996-06-21 1997-06-19 Molecules bivalentes formant un complexe d'activation avec un recepteur d'erythropoietines WO1997048729A1 (fr)

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Cited By (11)

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EP1169352A1 (fr) * 1999-04-14 2002-01-09 SmithKline Beecham Corporation Anticorps du recepteur d'erythropoietine
EP1578779A2 (fr) * 2002-10-14 2005-09-28 Abbott Laboratories Anticorps se liant au recepteur de l'erythropoietine
US7048934B2 (en) 2001-08-30 2006-05-23 Stem Cell Therapeutics Inc. Combined regulation of neural cell production
WO2005100403A3 (fr) * 2004-04-09 2006-07-13 Abbott Lab Anticorps diriges contre le recepteur de l'erythropoietine et utilisations associees
WO2007120767A2 (fr) * 2006-04-14 2007-10-25 Amgen Inc. Agonistes limités à durée prolongée du récepteur d'érythropoïétine
US7368115B2 (en) 2002-07-31 2008-05-06 Stem Cell Therapeutics Inc. Method of enhancing neural stem cell proliferation, differentiation, and survival using pituitary adenylate cyclase activating polypeptide (PACAP)
US7393830B2 (en) 2001-09-14 2008-07-01 Stem Cell Therapeutics Inc. Prolactin induced increase in neural stem cell numbers
US7534765B2 (en) 2005-09-27 2009-05-19 Stem Cell Therapeutics Corp. Pregnancy-induced oligodendrocyte precursor cell proliferation regulated by prolactin
US7846898B2 (en) 2004-02-13 2010-12-07 Stem Cell Therapeutics Corp. Pheromones and the luteinizing hormone for inducing proliferation of neural stem cells and neurogenesis
EP2319531A2 (fr) 2004-10-07 2011-05-11 Stem Cell Therapeutics Corp. Stimulation de la proliferation de cellules souches pluripotentes par administration de composés associés à la gestation
US8333974B2 (en) 2006-03-17 2012-12-18 Stem Cell Therapeutics Corp. Continuous dosing regimens for neural stem cell proliferating agents and neural stem cell differentiating agents

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WO1996003438A1 (fr) * 1994-07-26 1996-02-08 Amgen Inc. Anticorps activant un recepteur de l'erythropoietine
WO1996040231A1 (fr) * 1995-06-07 1996-12-19 Smithkline Beecham Corporation Procede d'obtention d'anticorps antagonistes de recepteurs

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1169352A4 (fr) * 1999-04-14 2005-05-04 Smithkline Beecham Corp Anticorps du recepteur d'erythropoietine
EP1169352A1 (fr) * 1999-04-14 2002-01-09 SmithKline Beecham Corporation Anticorps du recepteur d'erythropoietine
US7048934B2 (en) 2001-08-30 2006-05-23 Stem Cell Therapeutics Inc. Combined regulation of neural cell production
US7604993B2 (en) 2001-08-30 2009-10-20 Stem Cell Therapeutics Inc. Combined regulation of neural cell production
US8222212B2 (en) 2001-09-14 2012-07-17 Stem Cell Therapeutics Inc. Prolactin induced increase in neural stem cell numbers
US7884072B2 (en) 2001-09-14 2011-02-08 Stem Cell Therapeutics Inc. Prolactin induced increase in neural stem cell numbers
US7393830B2 (en) 2001-09-14 2008-07-01 Stem Cell Therapeutics Inc. Prolactin induced increase in neural stem cell numbers
US7368115B2 (en) 2002-07-31 2008-05-06 Stem Cell Therapeutics Inc. Method of enhancing neural stem cell proliferation, differentiation, and survival using pituitary adenylate cyclase activating polypeptide (PACAP)
EP1578779A2 (fr) * 2002-10-14 2005-09-28 Abbott Laboratories Anticorps se liant au recepteur de l'erythropoietine
EP1578779A4 (fr) * 2002-10-14 2011-11-16 Abbott Lab Anticorps se liant au recepteur de l'erythropoietine
US8217002B2 (en) 2004-02-13 2012-07-10 Stem Cell Therapeutics Corp. Pheromones and the luteinizing hormone for inducing proliferation of neural stem cells and neurogenesis
US7846898B2 (en) 2004-02-13 2010-12-07 Stem Cell Therapeutics Corp. Pheromones and the luteinizing hormone for inducing proliferation of neural stem cells and neurogenesis
JP2008505612A (ja) * 2004-04-09 2008-02-28 アボット・ラボラトリーズ エリスロポエチン受容体に対する抗体およびこの使用
EP2062917A3 (fr) * 2004-04-09 2009-09-30 Abbott Laboratories Anticorps du récepteurs de l'érythropoïétine et leurs utilisations
WO2005100403A3 (fr) * 2004-04-09 2006-07-13 Abbott Lab Anticorps diriges contre le recepteur de l'erythropoietine et utilisations associees
US8343920B2 (en) 2004-10-07 2013-01-01 Stem Cell Therapeutics Corp. Stimulation of proliferation of pluripotential stem cells through administration of pregnancy associated compounds
EP2319531A2 (fr) 2004-10-07 2011-05-11 Stem Cell Therapeutics Corp. Stimulation de la proliferation de cellules souches pluripotentes par administration de composés associés à la gestation
US7994131B2 (en) 2004-10-07 2011-08-09 Stem Cell Therapeutics Corp. Stimulation of proliferation of pluripotential stem cells through administration of pregnancy associated compounds
US7534765B2 (en) 2005-09-27 2009-05-19 Stem Cell Therapeutics Corp. Pregnancy-induced oligodendrocyte precursor cell proliferation regulated by prolactin
US8333974B2 (en) 2006-03-17 2012-12-18 Stem Cell Therapeutics Corp. Continuous dosing regimens for neural stem cell proliferating agents and neural stem cell differentiating agents
WO2007120767A3 (fr) * 2006-04-14 2008-04-10 Amgen Inc Agonistes limités à durée prolongée du récepteur d'érythropoïétine
WO2007120767A2 (fr) * 2006-04-14 2007-10-25 Amgen Inc. Agonistes limités à durée prolongée du récepteur d'érythropoïétine

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