WO1997030146A2 - Cardiotrophine et ses utilisations - Google Patents

Cardiotrophine et ses utilisations Download PDF

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WO1997030146A2
WO1997030146A2 PCT/US1997/002675 US9702675W WO9730146A2 WO 1997030146 A2 WO1997030146 A2 WO 1997030146A2 US 9702675 W US9702675 W US 9702675W WO 9730146 A2 WO9730146 A2 WO 9730146A2
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cells
ofthe
dna
cell
human
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PCT/US1997/002675
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WO1997030146A3 (fr
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Joffre Baker
Kenneth Chien
Kathleen King
Diane Pennica
William Wood
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Genentech, Inc.
The Regents Of The University Of California
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Priority to AU19654/97A priority Critical patent/AU733403B2/en
Priority to CA 2245635 priority patent/CA2245635A1/fr
Priority to EP97907730A priority patent/EP0885294A2/fr
Priority to IL12554897A priority patent/IL125548A/en
Priority to JP9529601A priority patent/JP2000504729A/ja
Publication of WO1997030146A2 publication Critical patent/WO1997030146A2/fr
Publication of WO1997030146A3 publication Critical patent/WO1997030146A3/fr

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/06—Antiabortive agents; Labour repressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/175—Cardiotrophin

Definitions

  • This application relates to a cardiac hypertrophy factor (also known as CT- 1 ) for modulating cardiac function in the treatment of heart failure, for modulating neural function in the treatment of neurological disorders, and for treatment of a variety of other disorders related to a CT-1 receptor, particularly the LIFR ⁇ BACKGROUND
  • Heart failure affects approximately three million Americans, developing in about 400,000 each year It is currently one ofthe leading admission diagnoses in the U S Recent advances in the management of acute cardiac diseases, including acute myocardial infarction, are resulting in an expanding patient population that will eventually develop chronic heart failure
  • ACE inhibitors While prolonging survival in the setting of heart failure, ACE inhibitors appear to slow the progression towards end-stage heart failure, and substantial numbers of patients on ACE inhibitors have functional class III heart failure Moreover, ACE inhibitors consistently appear unable to relieve symptoms in more than 60% of heart failure patients and reduce mortality of heart failure only by approximately 15-20% Heart transplantation is limited by the availability of donor hearts Further, with the exception of digoxin, the chronic administration of positive inotropic agents has not resulted in a useful drug without accompanying adverse side effects, such as increased arrhyth ogenesis, sudden death, or other deleterious side effects related to survival These deficiencies in current therapy suggest the need for additional therapeutic approaches A wide body of data suggests that pathological hypertrophy of cardiac muscle in the setting of heart failure can be deleterious, characterized by dilation ofthe ventricular chamber, an increase in wall tension/stress, an increase in the length vs width of cardiac muscle cells, and
  • Cardiac muscle hypertrophy is an important adaptive response ofthe heart to injury or to an increased demand for cardiac output
  • This hypertrophic response is characterized by the reactivation of genes normally expressed du ⁇ ng fetal heart development and by the accumulation of sarcome ⁇ c proteins in the absence of DNA replication or cell division (Kock an et al , Circulation, 87 VII14-VII21 (1993), Chien, FASEB J , 5 3037- 3046 ( 1991 ), Shubeita et al , J Biol Chem , 265 20555-20562 ( 1990))
  • non-myocytes are primarily fibroblast/mesenchymal cells, they aiso include endothelial and smooth muscle cells Indeed, although myocytes make up most ofthe adult myocardial mass they represent only about 30% ofthe total cell numbers present in heart Because of their close relationship with cardiac myocytes in vtvo non-myocytes are capable of influencing myocyte growth and/or development This interaction may be mediated directly through cell-cell contact or indirectly via production of a parac ⁇ ne factor Such association in vivo is important since both non-myocyte numbers and the extracellular matrix with which they interact are increased in myocardial hypertrophy and in response to injury and infarction. These changes are associated with abnormal myocardial function.
  • Cardiac myocytes are unable to divide shortly after birth. Further growth occurs through hypertrophy ofthe individual cells.
  • Cell culture models of myocyte hypertrophy have been developed to understand better the mechanisms for cardiac myocyte hypertrophy.
  • Most studies of heart myocytes in culture are designed to minimize contamination by non-myocytes. See, for example, Simpson etai., Cir. Cres., 50:101 -1 16 (1982); Libby, J. Mol. Cell. Cardiol, 16:803-81 1 (1984); Iwaki et ai, J. Biol. Chem., 265: 13809-13817 (1990).
  • This growth factor was compared to various growth factors known to be present in myocardium, including fibroblast growth factor (FGF), platelet derived growth factor (PDGF), tumor necrosis factor-alpha (TNF- ⁇ ), and transforming growth factor-beta 1 (TGF- ⁇ 1).
  • FGF fibroblast growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ tumor necrosis factor-alpha
  • TGF- ⁇ transforming growth factor-beta 1
  • the growth factor of Long et al. was found to be larger than these other known growth factors and to have a different heparin-Sepharose elution profile from that of all these growth factors except PDGF. Further, it was not neutralized by a PDGF-specific antibody. The authors proposed that it defines a paracrine relationship important for cardiac muscle cell growth and development.
  • Neurotrophic factors such as insulin-like growth factors, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, -4, and -5, and ciliary neurotrophic factor have been proposed as potential means for enhancing neuronal survival, for example, as atrearment forneurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, stroke, epilepsy, Huntington's disease, Parkinson's disease, and peripheral neuropathy. It would be desirable to provide an additional therapy for this purpose.
  • the 1L-6 family of cytokines (IL-6/LIF/CNTF/OSM7IL- 1 1 ) has a wide range of growth and differentiation activities on many cell types including those from the blood, liver, and nervous system (Akira et al. Adv. Immunol, 54: 1-78 (1993); Kishimoto et al, Science. 258:593-597 (1992)).
  • cytokine receptor family The biological effects induced by IL-6 and related proteins are mediated by a family of structurally similar cell surface receptors, the cytokine receptor family, that includes the receptors for growth hormone and prolattin as well as for many cytokines (Cosman et al. Trends Biochem. Sci., 15:265-270 (1990): Miyajima et al.,Ann. Rev. Immunol., 10:295-331 (1992); Taga et al., FASEB J, 6:3387-3396 (1992); Bazan, Immunol. Today, 1 1:350-354 (1990)).
  • the IL-6 receptor subfamily is composed of multi-subunit complexes that share a common signaling subunit, gpl30 (Davis et ai, Curr. Opin. Cell Biol., 5:281-285 (1993); Stahl et al, Cell. 74:587-590 (1993); Kishimoto et ai. Cell, 76:253-262 (1994)).
  • Some members ofthe IL-6 cytokine family induce the homodimerization of gp 130 (Murakami et al .
  • Jak/STAT signaling pathway includes the tyrosine phosphorylation and activation ofthe intracellular tyrosine kinases, Jakl, Jak2, and Tyk2 (L ⁇ tticken et ai , Science, 263-89-92 (1994), Stahl et al , Science, 263 92-95 (1994), Yin et al , Exp Hematol , 22 467-472 ( 1994), Narazaki et al , Proc Natl Acad USA.
  • the invention provides a method for assaying a test sample for hypertrophic activity comprising
  • test sample such as one suspected of containing a CT-1
  • test sample has hypertrophic activity
  • the invention provides isolated CT-1 polypeptide
  • This CT-1 polypeptide is preferably substantially homogeneous, may be glycosylated or unglycosylated, and may be selected from the group consisting of the native sequence polypeptide, a fragment polypeptide, a variant polypeptide, and a chimeric polypeptide
  • the CT- 1 polypeptide may be selected from the group consisting of the polypeptide that is isolated from a mammal, the polypeptide that is made by recombinant means, and the polypeptide that is made by synthetic means
  • this CT-1 polypeptide may be selected from the group consisting ofthe polypeptide that is human and the polypeptide that is non-immunogenic in a human
  • the isolated CT- 1 polypeptide shares at least 75% amino acid sequence identity with the translated CT-1 sequence shown in Fig 1
  • the polypeptide is the mature numan CT- 1 having the translated CT- 1 sequence shown in Fig 5
  • the invention provides an isolated polypeptide encoded by a nucleic acid having a sequence that hyb ⁇ dizes under moderately stringent conditions to the nucleic acid sequence provided in Fig 1
  • this polypeptide is biologically active
  • the invention provides a chimera comprising CT-1 fused to a heterologous polypeptide
  • the mvention provides a composition comprising biologically active CT- 1 and a pharmaceutically acceptable carrier or comprising biologically active CT-1 fused to an immunogenic polypeptide
  • the mvention provides an isolated antibody that is capable of binding CT-1 and a method for detecting CT-1 in vitro or in vivo comprising contactmg the antibody with a sample or cell suspected of containing CT-1 and detecting if binding has occurred, as with an ELISA
  • the invention provides a method for punfying CT-1 comp ⁇ sing passing a mixture of CT-1 over a column to which is bound the antibodies and recovering the fraction containing CT-1
  • the invention comp ⁇ ses an isolated nucleic acid molecule encoding CT-1 a vector comprising the nucleic acid molecule, preferably an expression vector comprising the nucleic acid molecule operably linked to control sequences recognized by a host cell transformed with the vector, a host ceil comprising the nucleic acid molecule, including mammalian and bacterial host cells, and a method of using a nucleic acid molecule encoding CT- 1 to effect the production of CT- 1 , comp ⁇ smg cultunng a host cell comp ⁇ sing the nucleic acid molecule
  • the host cell is transfected to express CT-1 nucleic acid and the CT-1 is recovered from the host cell culture, and if secreted, recovered from the culture medium
  • the invention provides an isolated nucleic acid molecule comprising the open reading frame nucleic acid sequence shown in Fig 1 or Fig 5
  • the mvention also provides an isolated nucleic acid molecule selected from the group consisting of
  • the invention also provides an isolated DNA molecule having a sequence capable of hybridizing to the DNA sequence provided in Fig 1 or Fig 5 under moderately stringent conditions, wherein the DNA molecule encodes a biologically active CT-1 polypeptide, excluding rat CT-1
  • a method is provided of determining the presence of a CT-1 nucleic acid molecule in a test sample comprising contacting the CT-1 nucleic acid molecule with the test sample and determining whether hybridization has occurred, or compnsing hybridizing the CT-1 nucleic acid molecule to a test sample nucleic acid and determining the presence of CT-1 nucleic acid
  • the invention provides a method of amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase chain reaction in the test sample with the CT-1 nucleic acid molecule
  • the invention provides a CT-1 antagonist and a method of identifying such antagonist comprising using cell supematants as the test sample in the hypertrophy assay as described above and screening for molecules that antagonize the hypertrophic activity of a CT-1 demonstrated in such assay
  • the mvention provides a method for treating a mammal having or at risk for heart failure, an motropic disorder, or an arrhythmic disorder comprising administering to a mammal in need of such treatment a therapeutically effective amount of a pharmaceutical composition comprising the CT-1 or a CT-1 antagonist in a pharmaceutically acceptable earner
  • the mvention also provides a method for treating a mammal having or at risk for a neurological disorder comprising administering to a mammal in need of such treatment a therapeutically effective amount of a pharmaceutical composition comprising the CT-1 in a pharmaceutically acceptable carrier
  • the mvention also provides a method for treating a mammal having or at risk for a disorder in which cytokines.
  • cytokines particularly the IL-6/LIF/CNTF/OSM/IL-l 1 cytokine family, more preferably LIF and OSM, more preferably LIF, their antagonists or their agonists, and most preferably a LIF-Receptor ⁇ subunit that interacts with gpl 0, play a role
  • the methods comprise administering to a mammal need of such treatment a therapeutically effective amount of a pharmaceutical composition comp ⁇ smg CT-1, its antagonist or its agonist, in a pharmaceutically acceptable carrier
  • the disorders involve a pathway regulated or induced by the activation of LIFR ⁇ by CT-1 binding and subsequent interaction with gpl30
  • the invention provides a CT-1 antagonist and a method of identifying such antagonist comprising using cell supematants or pu ⁇ fied or synthetic compounds as the test sample in an assay in which CT-1 has a demonstrated biological activity, receptor binding activity, or signaling pathway induction activity, preferably in a microassay, and screening for molecules that antagonize the activity of a CT-1 demonstrated in such an assay
  • the invention supplies a method of identifying a receptor for CT- 1 comprising using labeled CT-1, preferably radiolabeled CT-1, in a cellular receptor assay, allowing the CT-1 to bind to cells, or using the labeled CT-1 to pan for cells that contain the receptor
  • Figure 2 aligns the translated amino acid sequence ofthe mouse CT-1 clone (chf 781) (SEQ ID NO
  • FIG. 3 shows a graph of atrial natriuretic peptide (ANP) release for phenylephrine (standard curve) and transfections into 293 cells in a neonatal cardiac hypertrophy assay.
  • ADP atrial natriuretic peptide
  • Figure 4 shows a graph of survival of live ciliary ganglion neurons (measured by cell count) as a function of either the ciliary neutrotrophic factor (CNTF) standard (in ng/mL) or the transfected 293 conditioned medium (in fraction of assay volume), using a CNTF standard (circles), medium from a CT-1 DNA transfection of 293 cells (triangles), and medium from a control DNA transfection of 293 cells (squares).
  • CNTF ciliary neutrotrophic factor
  • Figures 5A and 5B depict the nucleotide sequence (sense and anti-sense strands) (SEQ ID NOS: 6 and 7) and deduced amino acid sequence (SEQ ID NO: 8) of a human CT-1 DNA clone.
  • Figure 6 aligns the translated amino acid sequence ofthe human CT-1 clone (humctl ) (SEQ ID NO: 8) with the translated amino acid sequence ofthe mouse CT-1 clone (chf.781 ) (SEQ ID NO: 3) to show the extent of sequence identity.
  • Figures 7A and 7B depict activity of CT-1 in hematopoietic cell assays. The induction by the human
  • EC 50 [LIF] 0.048 ( ⁇ 0.004) nM.
  • Figures 8A, 8B, and 8C depict activity of CT-1 in neuronal cell assays.
  • the induction by mouse (m) or rat (r) cytokines was performed as described in Example VI, Materials and Methods.
  • Figure 8 A shows the switch in transmitter phenotype of rat sympathetic neurons. Tyrosine hydroxylase (TH) and choiine acetyitransferase (ChAT) activities were determined in duplicate.
  • Figure 8B shows survival of rat dopaminergic neurons. Plotted are the average and standard deviation of triplicate determinations.
  • Figure 9 depicts activity of CT-1 in embryonic stem cells development.
  • Mouse embryonic stem cells were cultured in the presence ofthe mouse (m) cytokines as described in Example VI, Materials and Methods.
  • Figures 10A, 10B, 10C and 10D depict binding and cross-competition of CT-1 and LIF to mouse Ml cells. Assays contamed 0.047 nM I-mouse CT-1 ( 125 I-mCT-l) and unlabeled mouse (m) CT-l.
  • Figure 1 1 depicts cross-linking of CT-1 and LIF to Ml Cells.
  • 125 I-mouse CT-1 125 I-mCT-I
  • 125 I-mCT-I 125 I-mCT-I
  • J C mouse LIF ( l-mLIF) were bound and cross-linked to Ml cells in the absence (None) or presence of a 100 fold excess ofthe indicated mouse (m) cytokine, and the reaction products analyzed by SDS gel electrophoresis. The mobility of molecular weight standards is indicated.
  • Figure 12B depicts electrophoretic mobility shift of the DNA element S1E induced by CT-1 binding to Ml cells. Ml cells were incubated without (-) or with (+) 5 nM mouse (m) CT-1 or LIF, lysed, and the cell extract assayed for binding to the DNA element SIE as described in the Materials and Methods. Binding specificity was determined by the addition of unlabeled SIE DNA (Cold Oligo). The specific DNA complex is indicated (arrow).
  • Figure 13A and 13B depict binding and cross-competition of CT-1 and LIF to rat primary cardiac myocytes.
  • Duplicate assays contained either 0.047 nM 125 I-mouse CT-1 ( 125 I-mCT-l) or 0.042 nM 125 I- mouse LIF ( l2 - > I-mLIF) and unlabeled mouse (m) CT-1 or LIF as indicated.
  • Figures 14A, 14B, 14C and 14D depict binding of CT-1 to purified, soluble LIF receptor and gpl30.
  • Figures 14A-C show per cent binding of J I-mouse CT-1 (0.089 nM) to soluble mouse LIF receptor (s LIFR) and soluble mouse gpl30 (smgpHO) in the absence (-) or presence (+) of 164 nM unlabeled mouse CT-1 (mCT- /).
  • Figure 14A depicts binding to increasing concentrations soluble LIF receptor alone;
  • Figure 14B depicts binding to increasing concentrations of soluble gpl30 alone;
  • Figure 14C depicts binding at one soluble LIF receptor concentration with increasing concentrations of soluble gpl30. Plotted is the average and half the difference of duplicate determinations. The results for 0.84 nM soluble LIF receptor are shown twice for clarity.
  • Figure 14D depicts competition binding of I-mouse CT-1 (0.089 nM) to the soluble LIF receptor (2.8 nM) with increasing concentrations of unlabeled CT-1.
  • K d [CT-1] 1.9 ( ⁇ 0.2) nM.
  • Figures 15A and 15B depict similarity of IL-6 family ligands and subunit structure of their receptors.
  • Figure I5A shows per cent amino acid identity ofthe mature form ofthe IL-6 family ligands: (m) mouse, (h) human, (c) chicken. The bottom row gives the per cent identity ofthe cytokine to its human homologue. Shown in bold are the percentages greater than 40 %.
  • Figure 15B is a diagram ofthe IL-6 family receptors. The subunit stoichiometry ofthe various complexes is not known in most cases, although recent work has led to a conclusion that the IL-6 receptor complex is a hexamer containing two IL-6 molecules, two IL-6 receptors, and two gp 130 signaling subunits. Ward et ai, J. Biol. Chem., 269:23286-23289 (1994).
  • Figure 16 depicts alignment ofthe protein sequence of human CT-1, LIF and CNTF.
  • Encoded amino acid sequence of human CT-1 (hCT-1) aligned with that of human LIF (hLIF) and human CNTF (hCNTF).
  • Overlining indicates the location of four amphipathic helices based on their proposed locations in CNTF (Bazan, Neuron. 7: 197-208 (1991)).
  • Figures 17A and 17B depict the competition for the binding of human LIF to mouse M 1 or human Hela cell.
  • 1251-human LIF was bound in duplicate to M 1 (5 million cells per reaction) in the presence ofthe indicated competitors.
  • Figure 17B 1251-human LIF was bound in duplicate to Hela cells (2.5 million per reaction) in the presence ofthe indicated competitors.
  • CM is conditioned medium from 293 cells transfected with human CT- 1.
  • Figure 19 depicts the competition for the binding of human OSM to human WI-26 ceils. 1251-human
  • OSM was bound in duplicate to WI-26 VA4 cells (2.4 million cells per reaction) in the presence ofthe indicated competitors as described in the Examples.
  • Figure 20 depicts expression of CT-1 in human tissues, Northern blots containing poiyA+RNA from the indicated tissues were hybridized with a human CT- 1 cDN A probe as described in the Examples.
  • Figure 21 is a schematic depicting several biological activities of CT- 1
  • CHF cardiac hypertrophy factor
  • cardiotrophin cardiac hypertrophy factor
  • Card ⁇ otroph ⁇ n-1 or “CT-1 ”
  • CVF cardiac hypertrophy factor
  • Fig 1 or the human equivalent thereof shown in Fig 5 It does not include the rat homolog of CT-1, / e .
  • CT-1 from the rat species This definition encompasses not only the polypeptide isolated from a native CT-1 source such as murine embryoid bodies descnbed herein or from another source, such as another animal species except rat, including humans, but also the polypeptide prepared by recombinant or synthetic methods It also includes variant forms including functional derivatives, alleles, isoforms and analogues thereof
  • CT-1 fragment is a portion of a naturally occurring mature full-length CT-1 sequence having one or more amino acid residues or carbohydrate units deleted
  • the deleted amino acid res ⁇ due(s) may occur anywhere in the polypeptide, including at either the N-terminal or C-terminal end or internally
  • the fragment will share at least one biological property in common with CT- 1 CT-1 fragments typically will have a consecutive sequence of at least 10, 15, 20, 25, 30, or 40 ammo acid residues that are identical to the sequences ofthe CT- 1 isolated from a mammal including the CT- 1 isolated from murme embryoid bodies or the human CT- 1
  • CT-1 variants or "CT-1 sequence variants” as defined herein mean biologically active CT-ls as defined below having less than 100% sequence identity with the CT-1 isolated from recombinant cell culture or from mu ⁇ ne embryoid bodies having the deduced sequence described Fig 1 , or with the human equivalent desc ⁇ bed in Fig 5
  • a biologically active CT- 1 variant will have an amino acid sequence having at least about 70% ammo acid sequence identity with the CT- 1 isolated from murme embryoid bodies or the mature human CT-1 (see Figs 1 and 5), preferably at least about 75%, more preferably at least about 80% still more preferably at least about 85%, even more preferably at least about 90%, and most preferably at least about 95%
  • a "chimeric CT- 1" is a polypeptide comp ⁇ smg full-length CT- 1 or one or more fragments thereof fused or bonded to a second protein or one or more fragments thereof
  • the chimera will share at least one biological property m common with CT-1
  • the second protein will typically be a cytokine, growth factor, or hormone such as growth hormone, IGF-I, or a neurotrophic factor such as CNTF, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotroph ⁇ n-3 (NT-3), neurotroph ⁇ n-4 (NT-4), neurotroph ⁇ n-5 (NT-5), NT-6, or the like
  • Isolated CT-1 ", "highly punfied CT-1” and “substantially homogeneous CT-1” are used interchangeably and mean a CT-1 that has been purified from a CT-1 source or has been prepared b ⁇ recombinant or synthetic methods and is sufficiently free of other peptides or proteins ( 1 ) to obtain at least 15 and preferably 20 amino acid residues of the N-terminal or of an internal ammo acid sequence by using a spinning cup sequenator or the best commercially available ammo acid sequenator marketed or as modified bv published methods as of the filing date of this application, or (2) to homogeneity by SDS-PAGE under non- reducing or reducing conditions using Coomassie blue or preferably, silver stain Homogeneit y here means less than about 5% contamination with other source proteins
  • CT- 1 or "isolated CT- 1" means having myocardiotrophic motropic, anti-arrhythmic, or neurotrophic activity or having an in vivo effector or antigenic function or activity that is directly or indirectly caused or performed by a CT- 1 (whether its native or denatured conformation) or a fragment thereof
  • Effector functions mclude receptor binding and any carrier binding activity, agonism or antagonism of CT-1 , especially transduction of a proliferative signal including replication, DNA regulatory function, modulation of the biological activity of other growth factors, receptor activation, deactivation, up- or down-regulation, cell growth or differentiation, and the like
  • effector functions do not include possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against native CT- 1
  • an "antigenic function” means possession of an epitope or antigenic site that is capable of cross-reacting with annbodies raised against the native CT-1 whose sequence is shown in Fig 1 or another mammalian native CT-1, including the human homolog whose sequence is shown in Fig 5
  • the principal antigenic function of a CT- 1 polypeptide is that it binds with an affinity of at least about 10 L/mole to an antibody raised against CT- 1 isolated from mouse embryoid bodies or a human homolog thereof Ordinarily, the polypeptide binds with an affinity of at least about 10 L/mole
  • the antigenically active CT- 1 polypeptide is a polypeptide that binds to an antibody raised against CT-1 having one of the above-described effector functions
  • the antibodies used to define "biologically activity" are rabbit poiyclonal antibodies raised by formulating the CT- 1 isolated from recombinant cell culture or embryoid bodies in Freund's complete adjuvant, subcutaneously injecting the formulation, and boosting
  • Biologically active when used in conjunction with either "CT-1” or “isolated CT-1” mean a CT-1 polypeptide that exhibits hypertrophic, motropic, anti-arrhythmic, or neurotrophic activity or shares an effector function of CT- 1 isolated from murine embryoid bodies or produced in recombinant cell culture described herein, and that may (but need not) in addition possess an antigenic function
  • One principal effector function of CT-1 or CT-1 polypeptide herein is influencing cardiac growth or hypertrophy activity, as measured, e g , by atria!
  • CT-1 nat ⁇ uretic peptide release or by the myocyte hypertrophy assay desc ⁇ bed herein using a specific plating medium and plating density, and preferably using crystal violet stain for readout
  • the desired function of a CT-1 is to mcrease physiological (beneficial) forms of hypertrophy and decrease pathological hypertrophy
  • the CT-1 herein is expected to display anti-arrhythmic function by promoting a more normal electrophysiological phenotype
  • Another principal effector function of CT-1 or CT-1 polypeptide herein is stimulating the proliferation of chick ciliary ganglion neurons in an assay for CNTF activity
  • Antigenically active CT- 1 is defined as a polypeptide that possesses an antigenic function of CT- 1 and that mav (but need not) in addition possess an effector function
  • antigenically active CT- 1 is a polypeptide that binds with an affinity of at least about 10 L/mole to an antibody capable of binding CT-l
  • the polypeptide binds with an affinity of at least about 10 L/mole
  • Isolated antibody capable of binding CT-l is an antibody that is identified and separated from a component ofthe natural environment in which it may be present
  • the antigenically active CT-l is a polypeptide that binds to an antibodv capable of binding CT-l in its native conformation CT-l in its native conformation is CT-l as found in nature that has not been denatured by chaotropic agents, heat, or other treatment that substantially modifies the three-dimensional structure of CT-l as determined, for example, by migration on non-reducing, non-denaturing sizing gels
  • Antibody used in this determination is rabbit polyclonal antibody raised by formulating native CT-l from a non-rabbit species in Freund's complete adjuvant, subcutaneously injecting the formulation, and
  • Percent amino acid sequence identity with respect to the CT-l sequence is defined herein as the percentage of am o acid residues in the candidate sequence that are identical with the residues in the CT-l sequence isolated from murme embryoid bodies having the deduced ammo acid sequence desc ⁇ bed in Fig 1 or the deduced human CT-l ammo acid sequence desc ⁇ bed Fig 5, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part ofthe sequence identity None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the CT- 1 sequence shall be construed as affecting sequence identity or homology Thus, exemplary biologically active CT-l polypeptides considered to have identical sequences include prepro-CT-1 pro-CT- 1 , and mature CT- 1
  • CT-l microsequencing may be accomplished by any appropriate standard procedure provided the procedure is sensitive enough
  • highly purified polypeptide obtained from SDS gels or from a final HPLC step is sequenced directly by automated Edman (phenyl isothiocyanate) degradation using a model 470A Applied Biosystems gas-phase sequencer equipped with a 120A phenylthiohydantom (PTH) amino acid analyzer
  • CT-l fragments prepared by chemical (e g CNBr, hydroxylamine, or 2-n ⁇ tro-5- thiocyanobenzoate) or enzymatic (e g trypsin, clost ⁇ pain, or staphyiococcal protease) digestion followed by fragment purification (e g , HPLC) may be similarly sequenced PTH amino acids are analyzed using the ChromPerfectTM data system (Justice Innovations, Palo Alto, CA) Sequence mte ⁇ retation is performed on a VAX 1 1/7
  • Isolated CT-l nucleic acid is RNA or DNA containing greater than 16 and preferably 20 or more sequential nucleotide bases that encodes biologically active CT-l or a fragment thereof, is complementary to the RNA or DNA, or hybridizes to the RNA or DNA and remains stably bound under moderate to stringent conditions
  • This RNA or DNA is free from at least one contaminating source nucleic acid with which it is normally associated in the natural source and preferably substantially free of any other mammalian RNA or DNA
  • the phrase "free from at least one contaminating source nucleic acid with which it is normallv associated” includes the case where the nucleic acid is present in the source or natural cell but is in a different chromosomal location or is otherwise flanked by nucleic acid sequences not
  • Control sequences when referring to expression means DNA sequences necessary for the expression of an operably linked cod g sequence in a particular host organism
  • the control sequences that are suitable for prokarvotes include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood sequences
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
  • operably linked when referring to nucleic acids means that the nucleic acids are placed m a functional relationship with another nucleic acid sequence
  • DNA for a presequence or secretory leader is operablv linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide
  • a promoter or enhancer is operably linked to a codmg sequence if it affects the transcription of the sequence
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation
  • "operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase
  • enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice
  • Exogenous when referring to an element means a nucleic acid sequence that is foreign to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is ordina ⁇ ly not found
  • Cell Cell
  • cell lme cell culture
  • plasmids autonomously replicating circular DNA molecules possessing independent origins of replication and are designated herein by a lower case “p” preceded and/or followed by capital letters and/or numbers
  • the starting plasmids herein either are commercially available, are publicly available on an unrest ⁇ cied basis, or can be constructed from such available plasmids in accordance with published procedures
  • other equivalent plasmids are known in the art and
  • Southern analysis or “Southern blotting” is a method by which the presence of DNA sequences in a restriction endonuclease digest of DNA or a DNA-conta ing composition is confirmed by hybridization to a known.
  • labeled oligonucleotide or DNA fragment Southern analysis typically involves electrophoretic separation of DNA digests on agarose gels, denaturation ofthe DNA after electrophoretic separation, and transfer ofthe DNA to nitrocellulose, nylon, or another suitable membrane support for analysis with a radiolabeled, biot vlated, or enzyme-labeled probe as described in sections 9 37-9 52 of Sambrook et al , supra
  • RNA analysis or “Northern blotting” is a method used to identify RNA sequences that hybridize to a known probe such as an oligonucleotide, DNA fragment, cDN A or fragment thereof, or RNA fragment
  • a known probe such as an oligonucleotide, DNA fragment, cDN A or fragment thereof, or RNA fragment
  • the probe is labeled with a radioisotope such as P, or by biotinylation, or with an enzyme
  • the RNA to be analyzed is usually electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hyb ⁇ dized with the probe, using standard techniques well known in the art such as those desc ⁇ bed in sections 7 39-7 52 of Sambrook et al . supra
  • “Ligation” is the process of forming phosphodiester bonds between two nucleic acid fragments
  • the ends ofthe fragments must be compatible with each other In some cases, the ends will be directly compatible after endonuclease digestion However, it may be necessary first to convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation
  • the DNA is treated in a suitable buffer for at least 15 minutes at 15° C with about 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of the four deoxy ⁇ bonucleotide t ⁇ phosphates
  • the DNA is then purified by phenol-chloroform extraction and ethanol precipitation.
  • the DNA fragments that are to be ligated together are put in solution in about equimolar amounts.
  • the solution will also contain ATP.
  • ligase buffer an a ligase such as T4 DNA ligase at about 10 units per 0.5 ⁇ g of DNA.
  • the vector is first linearized by digestion with the appropriate restriction endonuclease(s).
  • the linearized fragment is then treated with bacterial alkaline phosphatase or calf intestinal phosphatase to prevent self-ligation during the ligation step.
  • Preparation of DNA from cells means isolating the plasmid DNA from a culture of the host cells. Commonly used methods for DNA preparation are the large- and small-scale plasmid preparations described in sections 1.25-1.33 of Sambrook et ai, supra. After preparation ofthe DNA, it can be purified by methods well known in the art such as that described in section 1.40 of Sambrook et ai, supra.
  • Oligonucleotides are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods such as phosphotriester, phosphite, or phosphoramidite chemistry, using solid-phase techniques such as described in EP 266,032 published 4 May 1988, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et ai, Nuci Acids Res., 14:5399-5407 (1986). Further methods include the polymerase chain reaction defined below and other autoprimer methods and oligonucleotide syntheses on solid supports. All of these methods are described in Engels et ai, Agnew. Chem. int. Ed.
  • oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands ofthe template to be amplified.
  • the 5' terminal nucleotides ofthe two primers may coincide with the ends ofthe amplified material.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA. bacteriophage or plasmid sequences, etc. See generally Mullis et ai. Cold Spring Harbor Symp. Quant.
  • PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sampie comprising the use of a known nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid.
  • “Stringent conditions” are those that (Chien et ai, Annu. Rev. Physioi, 55:77-95 (1993)) employ low ionic strength and high temperamre for washing, for example, 0.015 M NaCl 0.0015 M sodium citrate/0.1% NaDodSO ⁇ (SDS) at 50° C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol vol) formamide with 0.1% bovine serum albumin 0.1% Ficoll 0.1% polyvinylpyrrolidone/50 M sodium phosphate buffer at pH 6.5 with 750 M NaCl, 75 mM sodium citrate at 42° C.
  • formamide for example, 50% (vol vol) formamide with 0.1% bovine serum albumin 0.1% Ficoll 0.1% polyvinylpyrrolidone/50 M sodium phosphate buffer at pH 6.5 with 750 M NaCl, 75 mM sodium citrate at 42° C.
  • Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 M sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g mL), 0. ! % SDS, and 10% dextran sulfate at 42° C, with washes at 42° C in 0.2 x SSC and 0.1 % SDS.
  • 'Moderately stringent conditions are described in Sambrook et al .
  • moderately stringent conditions is a condition such as overnight incubation at 37° C in a solution comprising 20% formamide, 5 x SSC ( 150 mM NaCl, 15 mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH 7 6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50° C
  • 5 x SSC 150 mM NaCl, 15 mM t ⁇ sodium citrate
  • 50 mM sodium phosphate pH 7 6
  • 5 x Denhardt's solution 10% dextran sulfate
  • 20 mg/mL denatured sheared salmon sperm DNA followed by washing the filters in 1 x SSC at about 37-50° C
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc . as necessary to accommodate factors such as probe length and the like
  • Antibodies are glycoproteins having the same structural charactenstics While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity Polypeptides ofthe latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas
  • “Native antibodies and immunoglobulins” are usually heterotetrame ⁇ c glycoprotems of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chams Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies berween the heavy chains of different immunoglobulin isotypes
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains
  • Each light chain has a variable domain
  • va ⁇ able refers to the fact that certain portions ofthe va ⁇ able domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen
  • CDRs complementarity-determining regions
  • FR framework
  • the variable domains of native heavy and light cha s each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connectmg, and in some cases forming part of, the ⁇ -sheet structure
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation ofthe antigen-binding site of antibodies (see Kabat et al , Sequences of Proteins of Immunological Interest Fifth Edition, National Institute of Health, Bethes
  • Papain digestion of antibodies produces two identical antigen- binding fragments, called “Fab” fragments each with a single antigen-binding site, and a residual "Fc” fragment whose name reflects its ability to crystallize readily Pepsin treatment yields an F(ab' 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen "Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain va ⁇ able domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen- binding site on the surface ofthe j ⁇ -V ⁇ dimer.
  • the six CDRs confer antigen-binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three CDRs specific for an antigen
  • the Fab fragment also contains the constant domain ofthe light chain and the first constant domain (CH 1 ) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CHI domain including one or more cystemes from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) ofthe constant domains bear a free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cystemes between them. Other chemical couplings of antibody fragments are also known.
  • the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the ammo acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1 , IgG-2, IgG-3, IgG-4, IgA- 1, and IgA-2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , e, ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • antibody is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitopic specificity.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the monoclonal antibodies herein include hybrid and recombinant antibodies produced by splicing a variable (including hypervariable) domain of an anti-CT-1 antibody with a constant domain (e.g. "humanized” antibodies), or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous proteins, regardless of species of origin or immunoglobulin class or subclass designation, as well as antibody fragments (e.g., Fab, F(ab')->, and Fv), so long as they exhibit the desired biological activity.
  • Fab fragment antigen
  • F(ab')-> fusions with heterologous proteins
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et ai, Nature. 256:495 (1975). or may be made by recombinant DNA methods (Cabilly et ai.. supra).
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion ofthe heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (Cabilly et ai, supra; Morrison et ai, Proc. Natl. Acad. Sci USA, 81 :6851-6855 (1984)).
  • chimeric antibodies immunoglobulins in which a portion ofthe heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in
  • Humanized forms of non-human (e.g., murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary-determining region
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Non-immunogenic in a human means that upon contacting the polypeptide in a pharmaceutically acceptable carrier and in a therapeutically effective amount with the appropriate tissue of a human, no state of sensitivity or resistance to the polypeptide is demonstratable upon the second administration ofthe polypeptide after an appropriate latent period (e.g., 8 to 14 days).
  • Neurodegenerative disorder refers to a disorder of neurons, including both peripheral neurons and neurons from the central nervous system.
  • disorders include all neurodegenerative diseases, such as peripheral neuropathies (motor and sensory), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, stroke, Huntington's disease, epilepsy, and ophthalmologic diseases such as those involving the retina, e.g , diabetic retinopathy, retinal dystrophy, and retinal degeneration caused by infantile malignant osteopetrosis, ceroid-lipofuscosis, or cholestasis, or caused by photodegeneration, trauma, axotomy, neurotoxic- excitatory degeneration, or ischemic neuronal degeneration.
  • peripheral neuropathies motor and sensory
  • ALS amyotrophic lateral sclerosis
  • Alzheimer's disease Parkinson's disease
  • stroke Huntington's disease
  • epilepsy epilepsy
  • ophthalmologic diseases such as those involving the retina, e.g
  • Pe ⁇ pheral neuropathy refers to a disorder affecting the peripheral nervous system most often manifested as one or a combination of motor, sensory, senso ⁇ motor, or autonomic neural dysfunction
  • peripheral neuropathies can be genetically acquired, can result from a systemic disease, or can be induced by a toxic agent
  • Examples include but are not limited to distal senso ⁇ motor neuropathy, or autonomic neuropathies such as reduced motility of the gastrointestinal tract or atony of the urinary bladder
  • Examples of neuropathies associated with systemic disease include post-polio syndrome, examples of hereditary neuropathies include Charcot-Ma ⁇ e-Tooth disease Refsum's disease Abeta poproteinemia, Tangier disease, Krabbe's disease.
  • Metachromatic leukodystrophy, Fabry's disease, and Deje ⁇ ne-Sottas syndrome, and examples of neuropathies caused by a toxic agent include those caused by treatment with a chemotherapeutic agent such as vinc ⁇ stine
  • Heart failure refers to an abnormality of cardiac function where the heart does not pump blood at the rate needed for the requirements of metabolizing tissues
  • Heart failure includes a wide range of disease states such as congestive heart failure, myocardial infarction, and tachyarrhythmia
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment mclude those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc
  • the mammal herein is human
  • ACE inhibitor refers to angiotensin-converting enzyme inhibiting drugs which prevent the conversion of angiotensin I to angiotensin II
  • the ACE inhibitors may be beneficial in congestive heart failure by reducing systemic vascular resistance and relieving circulatory congestion
  • the ACE inhibitors include but are not limited to those designated by the trademarks Accup ⁇ l (qumap ⁇ l), Altace® (ramip ⁇ l), Capoten® (captop ⁇ l), Lotensin® (benazep ⁇ l), Monop ⁇ l (fosinop ⁇ l), P ⁇ nivil® (Iisinop ⁇ i), Vasotec® (enalap ⁇ l), and Zest ⁇ l (lisinop ⁇ l)
  • Capoten Generically referred to as captop ⁇ l this ACE inhibitor is designated chemically as l-[(2S)-3-mercapto-2-methylprop ⁇ onyI]-L- proline
  • Preferred polypeptides of this invention are substantially homogeneous CT-l polypept ⁇ de(s), having the biological properties of being myocyte hypertrophic and of stimulating the development of chick ciliary neurons a CNTF assay More preferred CT-ls are isolated mammalian protem(s) having hypertrophic, a ⁇ ti- a ⁇ hythmic, motropic, and neurological activity Most preferred polypeptides of this invention are mouse and human CT-ls including fragments thereof having hypertrophic, anti-arrhythmic, motropic, and neurological activity Optionally these murine and human CT-ls lack glycosylation WO 9529237.
  • CT-l nucleic acid and protein sequences discloses CT-l nucleic acid and protein sequences and certain uses of CT-l
  • Optional preferred polypeptides of this mvention are biologically active CT-l variants) with an amino acid sequence having at least 70% amino acid sequence identity with the murine CT-l of Fig 1 , preferably at least 75%. more preferably at least 80% still more preferably at least 85%.
  • the preferred biologically active CT-l va ⁇ ant(s) have an am o acid sequence having at least 70%, preferably at least 75%, more preferably at least 80%, still more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% am o acid sequence identity with the human CT-l sequence of Fig 5 (l e , 70-100%, 75-100%, 80-100%, 85-100%, 90-100% and 95-100% sequence identity, respectively)
  • the CT- 1 cloned from murine embryoid bodies has the following characteristics
  • CT-l polypeptides are those encoded by genomic DNA or cDNA and having the ammo acid sequence of mu ⁇ ne CT-l desc ⁇ bed in Fig 1 or the amino acid sequence of human CT-l described in Fig 5
  • CT-l polypeptides of this invention include prepro-CT-1, pro-CT-1, pre-CT-1, mature CT-l, and glycosylation variants thereof
  • CT- 1 sequence variants and chimeric CT- 1 s
  • preferred CT-l sequence variants are biologically active CT- l variants that have an ammo acid sequence having at least 70% ammo acid sequence identity with the human or murine CT- 1 , preferably at least 75%, more preferably at least 80%, still more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%
  • An exemplary preferred CT- 1 variant is a C-terminal domain CT- 1 variant in which one or more of the basic or dibasic am o acid res ⁇ due(s) (e g , R or K) is substituted with a non-basic ammo acid res ⁇ due(s) (e g , hydrophobic, neutral, acidic, aromatic, gly, pro and the like)
  • CT-l sequence variant is a "domain chimera" that consists of the N- terminal residues substituted with one or more, but not all, ofthe human CNTF residues approximately aligned as shown in Fig 2
  • the CT-1 chimera would have individual or blocks of residues from the human CNTF sequence added to or substituted into the CT-l sequence at positions corresponding to the alignment shown in Fig 2
  • one or more of those segments of CNTF that are not homologous could be substituted into the corresponding segments of CT-I
  • this "CT-l -CNTF domain chimera" will have mixed hypertrophic/anti-arrhythmic/inotropic/neurotrophic biological activity
  • CT-l fragments having a consecutive sequence of at least 10, 15, 20, 25, 30, or 40 ammo acid residues, preferably about 10-150 residues, that is identical to the sequence of the CT- 1 isolated from murme embryoid bodies or to that of the correspondmg human CT- 1
  • CT-l fragments include those produced as a result of chemical or enzymatic hydrolysis or digestion of the purified CT- l
  • Another aspect ofthe invention is a method for pu ⁇ fying CT- 1 molecules comprising contacting a CT- 1 source contammg the CT-l molecules to be purified with an immobilized receptor or antibody polypeptide, under conditions whereby the CT-l molecules to be purified are selectively adsorbed onto the immobilized receptor or antibody polypeptide, washing the immobilized support to remove non-adsorbed material, and eluting the molecules to be purified from the immobilized receptor or antibody polypeptide to which they are adsorbed with an elution buffer
  • the source containing the CT-l may be a cell suspension of embryoid bodies Alternatively, the source containing the CT- 1 is recombinant cell culture where the concentration of CT-
  • pu ⁇ fying the CT-l from contaminant soluble proteins and polypeptides with the following procedures be g exemplary of suitable pu ⁇ fication procedures by fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation- exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation.
  • Toyopearl and MONO-Q or MONO-S chromatography gel filtration using, for example, Sephadex G-75, chromatography on columns that bind the CT-l, and protein A Sepharose columns to remove contaminants such as IgG
  • chromatography on columns that bind the CT-l, and protein A Sepharose columns to remove contaminants such as IgG
  • One preferred purification scheme for both native and recombinant CT-l uses a Butyl Toyopearl column followed by a MONO-Q column and a reverse-phase C4 column as described further below
  • this invention provides an isolated antibody capable of binding to the CT- l
  • a preferred isolated ant ⁇ -CT- 1 antibody is monoclonal (Kohler et al , Nature, 256 495-497 (1975), Campbell. Laboratory Techniques in Biochemistry and Molecular Biology, Burdon et al Eds, Volume 13, Elsevier Science Publishers, Amsterdam (1985); and Huse et al , Science, 246.1275-1281 (1989))
  • Preferred isolated ant ⁇ -CT-1 antibody is one that binds to CT-l with an affinity of at least about 10 L/mole More preferably, the antibody binds with an affinity of at least about 10 L/mole Most preferably, the antibody is raised agamst a CT-l having one ofthe above-desc ⁇ bed effector functions
  • the isolated antibody capable of binding to the CT-l may optionally be fused to a second polypeptide and the antibody or fusion thereof may be used to isolate and purify CT-l from a source as desc ⁇ bed above for immobil
  • the mvention also provides an isolated nucleic acid molecule encoding the CT-l or fragments thereof, which nucleic acid molecule may be labeled or unlabeled with a detectable moiety, and a nucleic acid molecule having a sequence that is complementary to, or hybridizes under stringent or moderately stringent conditions with, a nucleic acid molecule having a sequence encoding a CT-l
  • a preferred CT-l nucleic acid is RNA or DNA that encodes a biologically active CT-l sharing at least 75%, more preferably at least 80% still more preferably at least 85%, even more preferably 90%, and most preferably 95%, sequence identity with the murine or human CT- 1
  • More preferred isolated nucleic acid molecules are DNA sequences encodmg biological ly active CT-l, selected from, (a) DNA based on the coding region of a mammalian CT-l gene (e g , DNA comprising the nucleotide sequence provided in Fig 1 or Fig
  • the nucleic acid molecule is cDNA encoding the CT- 1 and further comprises a replicable vector in which the cDNA is operably linked to control sequences recognized by a host transformed with the vector
  • This aspect further includes host cells transformed with the vector and a method of using the cDNA to effect production of CT-l. comprising expressing the cDNA encoding the CT-l in a culture ofthe transformed host cells and recovering the CT-l from the host cell culture
  • the CT-l prepared in this manner is preferably substantially homogeneous murine or human CT- 1
  • the invention further includes a preferred method for treating a mammal having heart failure, or an arrhythmic, motropic, or neurological disorder, comp ⁇ smg administering a therapeutically effective amount of a CT-l to the mammal
  • a CT-l is administered in combination with an ACE inhibitor, such as captopnl, in the case of congestive heart failure, or with another myocardiotrophic.
  • CT-l Preparation of Natural-Sequence CT-l and Variants
  • a neurotrophic molecule such as, e g IGF-I, CNTF, NGF, NT-3, BDNF, NT-4, NT-5, etc in the case of a neurological disorder 2
  • CT-l of this invention may be produced by homologous recombination, as provided for in WO 91/06667 published 16 May 1991 Briefly, this method involves transforming primary mammalian cells containing endogenous CT-l gene (e g , human cells if the desired CT-l is human) with a construct (/ e , vector) comprising an amplifiable gene (such as dihydrofolate reductas
  • the DNA encoding CT-l may be obtained from any cDNA library prepared from tissue believed to possess the CT-l mRNA and to express it at a detectable level
  • the mRNA is suitably prepared, for example, from seven-day differentiated embryoid bodies
  • the CT-l gene may also be obtained from a genomic library or by in vitro oligonucleotide synthesis as defined above assuming the complete nucleotide or amino acid sequence is known
  • probes are screened with probes designed to identify the gene of interest or the protein encoded by it
  • suitable probes include, e g monoclonal or polyclonal antibodies that recognize and specifically bmd to the CT- 1 , oligonucleotides of about 20-80 bases in length that encode known or suspected portions of the CT-l cDNA from the same or different species, and/or complementary or homologous cDNAs or fragments thereof that encode the same or a similar gene
  • Appropriate probes for screening genomic DNA libraries include, but are not limited to, oligonucleotides, cDNAs, or fragments thereof that encode the same or a similar gene, and or homologous genomic DNAs or fragments thereof Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures as described in chapters 10- 12 of Sambrook et al supra
  • An alternative means to isolate the gene encoding CT-l is to use PCR methodology as described in section 14 of Sambrook et al , supra This method requires the use of oligonucleotide probes that will hybridize to the CT- 1 Strategies for selection of oligonucleotides are described below
  • a preferred method of practicing this mvention is to use carefully selected oligonucleotide sequences to screen cDNA libraries from various tissues, preferably mammalian differentiated embryoid bodies and placental, cardiac, and brain cell l es More preferably human embryoid, placental. cardiac, and brain cDNA libraries are screened with the oligonucleotide probes
  • the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are mmimized
  • the actual nucleotide sequence(s) is usually based on conserved or highly homologous nucleotide sequences
  • the oligonucleotides may be degenerate at one or more positions
  • degenerate oligonucleotides may be of particular importance where a library is screened from a species in which preferential codon usage is not known
  • the oligonucleotide must be labeled such that it can be detected upon hybridization to DNA in the library being screened
  • the preferred method of labeling is to use - > P-labeled ATP with polynucleotide kinase, as is well known in the art, to radiolabel the oligonucleotide
  • other methods may be used to label the oligonucleotide, including, but not limited to, biotinylation or enzyme labeling
  • the CT-l nucleic acid that encodes a full-length polypeptide
  • the nucleic acid sequence includes the native CT-l signal sequence
  • Nucleic acid having all the protem codmg sequence is obtained bv screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and. if necessary, using conventional primer extension procedures as described in section 7 79 of Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA B.
  • Ammo acid sequence variants of native CT-l are prepared by introducing appropriate nucleotide changes into the native CT-l DNA, or by in vitro synthesis of the desired CT-l polypeptide
  • Such variants clude. for example, deletions from, or insertions or substitutions of, residues withm the amino acid sequence shown for murme CT-l in Figure 1 and for human CT- 1 in Figure 5
  • Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characte ⁇ stics
  • CT-l variants or polypeptide sequences that are the rat homolog of CT- 1 The ammo acid changes also may alter post-translational processes ofthe native CT- 1 , such as changing the number or position of glycosylation sites
  • the location of the mutation site and the nature ofthe mutation will depend on the CT-l characte ⁇ st ⁇ c(s) to be modified
  • candidate CT- 1 antagonists or super agonists will be initially selected by locating sites that are identical or highly conserved among CT- 1 and other ligands binding to members of the growth hormone (GH)/cytok ⁇ ne receptor family, especially CNTF and leukemia inhibitory factor (LIF)
  • the sites for mutation can be modified individually or m se ⁇ es, e g , by ( 1) substituting first with conservative ammo acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting residues ofthe same or a different class adjacent to the located site, or combmations of options 1-3
  • a useful method for identification of certain residues or regions ofthe native CT- 1 polypeptide that are preferred locations for mutagenesis is called "alanine scanning mutagenesis.” as described by Cunningham et al , Science, 244 1081-1085 (1989).
  • a residue or group of target residues are identified (e g , charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction ofthe ammo acids with the surrounding aqueous environment in or outside the cell
  • a neutral or negatively charged amino acid most preferably alanine or polyalanine
  • Those domains demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at or for the sites of substitution
  • the site for introducing an ammo acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined
  • alanine scanning or random mutagenesis is conducted at the target codon or region and the CT-l vanants produced are screened for the optimal combination of desired activity.
  • ammo acid sequence variants there are two principal variables in the construction of ammo acid sequence variants the location of the mutation site and the nature ofthe mutation These are variants from the Fig 1 or Fig 5 sequence, and may represent naturally occurring alleles (which will not require manipulation of the native CT-l DNA) or predetermined mutant forms made by mutating the DNA, either to arrive at an allele or a variant not found in nature
  • the location and nature ofthe mutation chosen will depend upon the CT-l characteristic to be modified
  • Ammo acid sequence deletions generally range from about 1 to 30 residues, more preferablv about 1 to 10 residues, and typically are contiguous Contiguous deletions ordinarily are made in even numbers of residues, but smgle or odd numbers of deletions are withm the scope hereof Deletions may be introduced into regions of low homology among CT-l and other ligands binding to the GH cytokine receptor family which share the most sequence identity to the human CT- 1 amino acid sequence to modify the activity of CT- 1 Deletions from CT-
  • a third group of vanants are ammo acid substitution va ⁇ ants These va ⁇ ants have at least one amino acid residue in the native CT- 1 molecule removed and a different residue inserted in its place
  • the sites of greatest interest for substitutional mutagenesis mclude sites identified as the active s ⁇ te(s) of native CT- 1 and sites where the am o acids found in the known analogues are substantially different in terms of side-chain bulk, charge, or hydrophobicity, but where there is also a high degree of sequence identity at the selected site withm various animal CT-l species, or where the amino acids found in known ligands that bind to members ofthe GH/cytokine receptor family and novel CT- 1 are substantially different in terms of side-chain bulk, charge, or hydrophobicity, but where there also is a high degree of sequence identity
  • sites of mterest are those m which particular residues ofthe CT-l obtained from various species are identical among all animal species of CT-l and other ligands binding to GH/cytokine receptor family molecules, this degree of conformation suggesting importance in achievmg biological activity common to these enzymes
  • These sites are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and the products screened.
  • Substantial modifications in function or immunological identity ofthe native CT-l are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk ofthe side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another Such substituted residues also may be introduced mto the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites
  • it is desirable to activate one or more protease cleavage sites that are present in the molecule These sites are identified by spection ofthe encoded amino acid sequence, in the case of trypsin, e g , for an arginyl or lysmyl residue
  • protease cleavage sites are identified they are rendered inactive to proteolytic cleavage by substituting the targeted residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine, by deleting the residue, or by inserting a prolyl residue immediately after the residue
  • any methionyl residues other than the starting methionyl residue ofthe signal sequence, or any residue located with about three residues N- or C-terminal to each such methionyl residue is substituted by another residue (preferably in accord with Table 1 ) or deleted Alternatively, about 1 -3 residues are inserted adjacent to such sites Any cyste e residues not volved in maintaining the proper conformation of native CT- 1 also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking
  • Nucleic acid molecules encodmg ammo acid sequence variants of native CT-l are prepared bv a variety of methods known in the art These methods mclude, but are not limited to, isolation from a natural source (in the case of naturally occurrmg ammo acid sequence va ⁇ ants) or preparation by oligonucleotide-mediated (or site- directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of native CT- 1
  • Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution deletion, and insertion va ⁇ ants of native CT-l DNA
  • This technique is well known in the art as described by Adelman et al , DNA 2 183 ( 1983) Briefly, native CT- 1 DNA is altered by hyb ⁇ dizmg an oligonucleotide encodmg the desired mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of CT-l
  • a DNA polymerase is used to synthesize an entire second complementary strand ofthe template that will thus inco ⁇ orate the oligonucleotide primer, and will code for the selected alteration in the native CT-l DNA
  • oligonucleotides of at least 25 nucleotides m length are used
  • An optimal oligonucleotide will have 12 to 1 nucleotides that are completely complementary
  • the oligonucleotide is hybridized to the single-stranded template under suitable hybridization conditions
  • a DNA polymerizing enzyme usually the Klenow fragment of DNA polymerase I
  • Klenow fragment of DNA polymerase I is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis
  • a heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of native CT-l and the other strand (the o ⁇ gmal template) encodes the native, unaltered sequence of CT-l
  • This heteroduplex molecule is then transformed mto a suitable host cell, usually a prokaryote such as E coli JM 101 After the cells are grown, they are plated onto agarose plates and screened usmg the oligonucleotide primer radiolabeled with ⁇ * P to identify the bacterial colonies that contain the mutated DNA The mutated region is then removed and placed m an appropn
  • the method desc ⁇ bed immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutat ⁇ on(s)
  • the modifications are as follows
  • the single-stranded oligonucleotide is annealed to the single-stranded template as described above A mixture of three deoxy ⁇ bonucleotides.
  • deoxy ⁇ boadenosme dATP
  • deoxy ⁇ boguanosine dGTP
  • deoxy ⁇ bothymidine dTTP
  • dCTP-(aS) modified thio-deoxy ⁇ bocytosine
  • This mixture is added to the template-oligonucleotide complex
  • DNA polymerase Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated.
  • this new strand of DNA will contain dCTP-(aS) stead of dCTP, which serves to protect it from restriction endonuclease digestion
  • the template strand ofthe double-stranded heteroduplex is nicked with an appropriate restriction enzyme
  • the template strand can be digested with ExoIII nuclease or another appropriate nuclease past the region that contams the s ⁇ te(s) to be mutagenized.
  • the reaction is then stopped to leave a molecule that is only partially single-stranded
  • a complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxy ⁇ bonucleotide t ⁇ phosphates, ATP, and DNA ligase
  • This homoduplex molecule can then be transformed into a suitable host cell such as E coli JM101, as described above
  • DNA encoding mutants of native CT-l with more than one ammo acid to be substituted may be generated in one of several ways If the ammo acids are located close together in the polypeptide cham, they may be mutated simultaneously us g one oligonucleotide that codes for all ofthe desired ammo acid substitutions If, however, the ammo acids are located some distance from each other (separated by more than about ten amino acids), it is more difficult to generate a smgle oligonucleotide that encodes all ofthe desired changes Instead, one of two alternative methods may be employed In the first method, a separate oligonucleotide is generated for each am o acid to be substituted The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all ofthe desired ammo acid substitutions
  • the alternative method involves two or more rounds of mutagenesis to produce the desired mutant
  • the first round is as desc ⁇ bed for the single mutants wild-type DNA is used for the template, an oligonucleotide encodmg the first desired amino acid substitution s) is annealed to this template, and the heteroduplex DNA molecule is then generated
  • the second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template
  • this template already contains one or more mutations
  • the oligonucleotide encodmg the additional desired ammo acid subst ⁇ tut ⁇ on(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis
  • This resultant DNA can be used as a template in a third round of mutagenesis, and so on
  • PCR mutagenesis is also suitable for making ammo acid va ⁇ ants of native CT-l While the following discussion refers to DNA, it is understood that the technique also finds application with RNA
  • the PCR technique generally refers to the following procedure (see Erlich, supra, the chapter by R Higuchi, p 61-70)
  • primers that differ slightly in sequence from the correspondmg region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template
  • the sequence ofthe other primer must be identical to a stretch of sequence ofthe opposite strand ofthe plasmid, but this sequence can be located anywhere along the plasmid DNA It is preferred, however, that the sequence ofthe second primer is located within 200 nucleotides from that ofthe first, such that
  • DNA fragments that differ at the position ofthe mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone are DNA fragments that differ at the position ofthe mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone
  • template plasmid DNA (1 ⁇ g) is linearized by digestion with a rest ⁇ ction endonuclease that has a unique recognition site m the plasmid DNA outside of the region to be amplified Of this mate ⁇ al, 100 ng is added to a PCR mixture containing PCR buffer, which contains the four deoxynucleotide tnphosphates and is included in the GeneAmp® kits (obtained from Perkin-Elmer Cetus, Norwalk. CT and Emeryville, CA), and 25 pmole of each oligonucleotide primer, to a final volume of 50 ⁇ L The reaction mixture is overlayed with 35 ⁇ L mineral oil.
  • Thermus aquaticus (Taq) DNA polymerase (5 units ⁇ L, purchased from Perkin-Elmer Cetus) is added below the mineral oil layer.
  • the reaction mixture is then inserted into a DNA Thermal Cycler (purchased from Perkin-Elmer Cetus) programmed as follows
  • the reaction vial is removed from the thermal cycler and the aqueous phase transferred to a new vial, extracted with phenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA is recovered by standard procedures. This material is subsequently subjected to the appropriate treatments for insertion into a vector.
  • the starting material is the plasmid (or other vector) comprising the native
  • CT-l DNA to be mutated The codon(s) in the native CT-l DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side ofthe identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the native CT-l DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence ofthe DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have
  • This plasmid now contains the CT-l DNA sequence mutated from native CT-I .
  • the nucleic acid (e.g., cDNA or genomic DNA) encoding CT-l is inserted into a replicable vector for further cloning (amplification ofthe DNA) or for expression.
  • a replicable vector for further cloning (amplification ofthe DNA) or for expression.
  • Many vectors are available, and selection ofthe appropriate vector will depend on 1) whether it is to be used for DNA amplification or for DNA expression, 2) the size ofthe nucleic acid to be inserted into the vector, and 3) the host ceil to be transformed with the vector.
  • Each vector contains various components depending on its function (amplification of DNA or expression of
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • the CT-ls of this invention may be produced not only directly, but also as a fusion with a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus ofthe mature protein or polypeptide.
  • the signal sequence may be a component ofthe vector, or it may be a part ofthe CT-l DNA that is inserted into the vector.
  • the heterologous signal sequence selected should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase. penicillinase, Ipp, or heat-stable enterotoxin II leaders.
  • a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase. penicillinase, Ipp, or heat-stable enterotoxin II leaders.
  • yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, yeast alpha factor leader (including Saccharomyces and
  • Kluyveromyces ⁇ -factor leaders the latter described in U.S. Patent No. 5,010,182 issued 23 April 1991 ), yeast acid phosphatase leader, mouse salivary amylase leader, carboxypeptidase leader, yeast BAR1 leader. Humicola lanuginosa lipase leader, the C.
  • the native human signal sequence (; e , the CT-l presequence that normally directs secretion of native CT-l from human cells in vivo) is satisfactory, although other mammalian signal sequences may be suitable, such as signal sequences from other animal CT-ls, signal sequences from a ligand binding to another GH/cytokine receptor family member, and signal sequences from secreted polypeptides ofthe same or related species, as well as viral secretory leaders, for example, the he ⁇ es simplex gD signal
  • the DNA for such precursor region is ligated in reading frame to DNA encoding the mature CT-l
  • Both expression and clon g vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Generally, in cloning vectors this sequence is one that enables the vector to replicate independently ofthe host chromosomal DNA, and includes origins of replication or autonomously replicating sequences Such sequences are well known for a variety of bacteria, yeast, and viruses
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and va ⁇ ous viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 o ⁇ gm may typically be used only because it contains the early promoter)
  • Most expression vectors are "shuttle" vectors, i e , they are capable of replication in at least one class of organisms but can be transfected mto another organism for expression
  • a vector is cloned in E coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently ofthe host cell chromosome
  • DNA may also be amplified by insertion mto the host genome This is readily accomplished using Bacillus species as hosts, for example, by including in the vector a DNA sequence that is complementary to a sequence found in Bacillus genomic DNA Transfection of Bacillus with this vector results in homologous recombination with the genome and insertion of CT-l DNA
  • the recovery of genomic DNA encodmg CT-l is more complex than that of an exogenously replicated vector because restriction enzyme digestion is required to excise the CT- 1 DNA
  • Selection genes should contain a selection gene, also termed a selectable marker This gene encodes a protem necessary for the survival or growth of transformed host cells grown a selective culture medium Host cells not transformed with the vector contammg the selection gene will not survive in the culture medium
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicillin, neomycin. methotrexate, or tetracycline, (b) complement auxotrophic deficiencies or (c) supply critical nutrients not available from complex media, e g , the gene encoding D-alanine racemase for Bacilli
  • One example of a selection scheme utilizes a drug to arrest growth of a host cell Those cells that are successfully transformed with a heterologous gene produce a protem conferring drug resistance and thus survive the selection regimen Examples of such dominant selection use the drugs neomycin (Southern et al . J Molec
  • Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the CT-l nucleic acid, such as DHFR or thymidine kinase
  • the mammalian cell transformants are placed under selection pressure that only the transformants are uniquely adapted to survive by virtue of having taken up the marker
  • Selection pressure is imposed by cultu ⁇ ng the transformants under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes CT-l Amplification is the process by which genes m greater demand for the production of a protem c ⁇ tical for growth are reiterated in tandem with the chromosomes of successive generations of recombinant cells Increased quantities of CT-l are synthesized from the amplified DNA
  • amplifiable genes include metalloth ⁇ one ⁇ n-I and -II, preferably primate metallothionein genes, adenosine deaminase, orn
  • cells transformed with the DHFR selection gene are first identified by cultu ⁇ ng all ofthe transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR
  • Mtx methotrexate
  • An appropnate host cell when wild-type DHFR, is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al , Proc Natl Acad Sci USA, 77 4216 (1980)
  • the transformed cells are then exposed to increased levels of methotrexate This leads to the synthesis of multiple copies of the DHFR gene, and, concomitantly, multiple copies of other DNA comprising the expression vectors, such as the DNA encoding CT-l
  • This amplification technique can be used with any otherwise suitable host, eg , ATCC No CCL61 CHO-K1, notwithstanding the presence of endogenous DHFR if, for example, a mutant DHFR gene that is
  • host cells particularly wild-type hosts that contain endogenous DHFR transformed or co-transformed with DNA sequences encoding CT-l wild-type DHFR protein, and another selectable marker such as ammoglycoside 3-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminogiycosidic antibiotic, e g , kanamycin neomycin, or G418 See U S Patent No 4,965,199
  • a suitable selection gene for use in yeast is the trp ⁇ gene present in the yeast plasmid YRp7 (Stinchcomb et ai , Nature, 282 39 (1979), Kings an et al , Gene 7 141 (1979), or Tschemper et al , Gene, 10 157 (1980))
  • the trp ⁇ gene provides a selection marker for a mutant stram of yeast lackmg the ability to grow in tryptophan, for example, ATCC No 44076 or PEP4-1 (Jones, Genetics, 85 12 (1977))
  • the presence of the t ⁇ l lesion m the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan
  • Le ⁇ /2-defic ⁇ ent yeast strains (ATCC 20,622 or 38.626) are complemented by known plasmids bearing the Leul gene
  • vectors de ⁇ ved from the 1 6 ⁇ m circular plasmid pKDl can be used for
  • Kluyveromyces yeasts Bianchi et al Cure Genet..12 185 (1987) More recently, an expression system for large-scale production of recombinant calf chymosin was reported for K lactis Van den Berg, Bio Technologv. 8. 135 (1990) Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluvveromvces have also been disclosed Fleer et al , Bio/Techno log v. 9 968-975 (1991)
  • Promoter Component Expression and clonmg vectors usually contain a promoter that is recognized by the host organism and is operablv linked to the CT-l nucleic acid
  • Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence, such as the CT-l nucleic acid sequence, to which they are operably linked
  • Such promoters typically fall mto two classes, inducible and constitutive Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e g , the presence or absence of a nutnent or a change in temperature At this time a large number of promoters recognized by a variety of potential host cells are well known
  • These promoters are operably linked to CT-l -encoding DNA by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence mto the vector
  • Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems (Chang et al , Nature, 275 615 (1978), and Goeddel et al , Nature. 281 " 544 (1979)), alkaline phosphatase, a tryptophan (t ⁇ ) promoter system (Goeddel, Nucleic Acids Res.. 8. 4057 (1980) and EP 36.776), and hybrid promoters such as the tac promoter (deBoer et al . Proc Natl. Acad Sci. USA.
  • Promoters for use in bacterial systems also will contain a Shme-Dalgarno (S D ) sequence operably linked to the DNA encoding CT-l Promoter sequences are known for eukaryotes Virtually all eukaryotic genes have an AT- ⁇ ch region located approximately 25 to 30 bases upstream from the site where transc ⁇ ption is initiated Another sequence found 70 to 80 bases upstream from the start of transcnption of many genes is a CXCAAT region where X may be any nucleotide At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition ofthe poly A tail to the 3' end ofthe codmg sequence All of these sequences are suitably inserted into eukaryotic expression vectors
  • suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kmase (Hitzeman et al , J Biol Chem..2 ⁇ 5_ 2073 (1980)) or other glycolytic enzymes (Hess et al . J Adv. Enzvme Reg . 2 149 (1968), and Holland, Biochemistry. JJ 4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexok ase, pyruvate decarboxy lase, phosphofructokinase, glucose- 6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kmase. tnosephosphate isomerase, phosphoglucose isomerase, and glucokinase
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, lsocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phos- phate dehydrogenase. and enzymes responsible for maltose and galactose utilization.
  • Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et ai. EP 73.657.
  • Yeast enhancers also are advantageously used with yeast promoters.
  • CT- l transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, and from the promoter normally associated with the CT-l sequence, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus (UK 2,21 1,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcom
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. Fiers et al. , Nature. 273: 1 13 (1978); Mulligan and Berg, Science. 209: 1422-1427 (1980); Pavlakis et ai, Proc. Natl. Acad. Sci. USA. 7£: 7398-7402 (1981).
  • the immediate early promoter ofthe human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. Greenaway et ai , Gene. 18: 355-360 ( 1982).
  • a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S.
  • Patent No.4,419,446 A modification of this system is described in U.S. Patent No. 4,601 ,978. See also Gray et ai, Nature. 295: 503-508 (1982) on expressing cDNA encodmg immune interferon in monkey cells: Reyes et ai. Nature. 297: 598-601 (1982) on expression of human ⁇ -interferon cDNA in mouse cells under the control of a thymidine kinase promoter from he ⁇ es simplex virus: Canaani and Berg. Proc. Natl. Acad. Sci.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' (Laimins etai, Proc. Natl. Acad. Sci. USA.28: 993 (1981)) and 3' (Lusky et ai, Mol. Cell Bio.. 3_: 1108 (1983)) to the transcription unit, within an intron (Banerji et ai. Cell.
  • enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100-270), the cytomegalovirus early promoter enhancer. the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the CT-l -encoding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding CT-l (vnl Construction and Analysis of Vectors Construction of suitable vectors contammg one or more of the above listed components employs standard ligation techniques Isolated plasmids or DNA fragments are cleaved, tailored, and religated m the form desired to generate the plasmids required
  • the ligation mixtures are used to transform E colt K12 strain 294 (ATCC 31 ,446) and successful transformants selected by ampicillin or tetracycline resistance where appropnate Plasmids from the transformants are prepared, analyzed by restnction endonuclease digestion, and/or sequenced by the method of Messing et al , Nucleic Acids Res . 2 309 (1981 ) or by the method of Maxam et al , Methods in Enzvmologv. 6 > 499 ( 1980)
  • transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies ofthe expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector Sambrook et al , supra, pp 16 17 - 16 22
  • Transient expression systems comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired biological or physiological properties
  • transient expression systems are particularly useful in the mvention for pu ⁇ oses of identifying analogs and variants of native CT- 1 that are biologically active CT- 1
  • Suitable host cells for clonmg or expressing the vectors herein are the prokaryote yeast or higher eukaryote cells desc ⁇ bed above Suitable prokaryotes for this pu ⁇ ose mclude eubacte ⁇ a, such as Gram-negative or Gram-positive organisms, for example, Enterobacte ⁇ aceae such as Escherichia, e g , E coli, Enterobacter Erwmia, Klebsiella, Proteus, Salmonella, e g , Salmonella typhtmurium, Serratia, e g , Serrano marcescans, and Shigella, as well as Bacilli such as B subtilis and B licheniformis (e g , B licheniformis 41 P disclosed in DD 266 710 published 12 April 1989), Pseudomonas such as P aeruginosa.
  • Enterobacte ⁇ aceae such as Escherichia, e
  • E coli clonmg host is £ coli 294 (ATCC 31 ,446), although other strains such as E coli B, E coli XI 776 (ATCC 31,537), E. coli DH5 ⁇ . and £ coli W31 10 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
  • Stram W31 10 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations
  • the host cell secretes minimal amounts of proteolytic enzymes.
  • strain W31 10 may be modified to effect a genetic mutation in the genes encoding protems endogenous to the host, with examples of such hosts including £.
  • E coli W31 10 stram 1 A2 which has the complete genotype tonA ⁇ : E coli W3110 strain 9E4, which has the complete genotype tonA ⁇ ptr3; E coli W31 10 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA ⁇ EJS ⁇ (argF-lac) 169 ⁇ degP ⁇ ompT karf; E colt W31 10 strain 37D6, which has the complete genotype tonA ptr3 phoA ⁇ EI ⁇ ⁇ (argF-lac)169 ⁇ degP ⁇ ompT ⁇ rbs7 ilvG karf; E coli W31 10 stram 40B4.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for CT- 1 -encoding vectors.
  • Saccharomyces cerevisiae. or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms.
  • Schtzosaccharomyces pombe Beach and Nurse, Nature. 290: 140 (1981); EP 139,383 published 2 May 1985
  • Kluyveromyces hosts U.S. Patent No 4,943,529; Fleer et ai, supra
  • K e.g., K.
  • lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al , J Bacteriol.. 737 (1983)), K fragilis (ATCC 12,424), K bulgartcus (ATCC 16.045), K wickeramii (ATCC 24,178), K. waltu (ATCC 56,500), A:, drosophilarum (ATCC 36,906; Van den Berg et ai, supra), K thermotolerans, and K marxianus, yarrowia (EP 402,226); Pichia pastor is (EP 183,070; Sreekrishna et al , J. Basic Microbiol..
  • Suitable host cells for the production of CT- 1 are derived from multicellular organisms. Such host cells are capable of complex processmg and glycosylation activities. In principle, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and va ⁇ ants and correspondmg permissive insect host cells from hosts such as Spodopterafrugiperda (cate ⁇ illar).
  • Aedes aegypti (mosquito), Aedes alboptctus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mort have been identified See, e g , Luckow el al , Bio/Technologv. 6 47-55 (1988); Miller et ai, in Genetic Engineering. Setlow, J.K. et al , eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al , Nature. 315: 592-594 (1985).
  • a variety of viral strains for transfection are publicly available, e g , the L-l variant of Autographa califormca NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodopterafrugiperda cells.
  • Plant cell cultures of cotton, com, potato, soybean petunia, tomato, and tobacco can be utilized as hosts Typically, plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens.
  • the DNA encoding the CT-l is transferred to the plant cell host such that it is transfected, and will, under appropriate conditions, express the CT-l DNA
  • regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences Depicker et al . J. Mol. Appl. Gen..
  • DNA segments isolated from the upstream region ofthe T-DNA 780 gene are capable of activatmg or increasing transcription levels of plant- expressible genes in recombinant DNA-containing plant tissue EP 321,196 published 21 June 1989
  • interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture
  • tissue culture has become a routine procedure in recent years (Tissue Culture. Academic Press, Kruse and Patterson, editors (1973))
  • useful mammalian host cell lines are a monkey kidney CV1 cell line transformed by S V40 (COS-7, ATCC CRL 1651 ), a human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al , J. Gen Virol . 36 . 59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chmese hamster ovary cellsADHFR (CHO, Uriaub and Chasm, Proc. Natl Acad Sci. USA. 22 4216 (1980)), mouse sertoli cells (TM4, Mather, Biol.
  • monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL- 1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A. ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2 HB 8065), mouse mammary tumor cells (MMT 060562, ATCC CCL51 ), TRI cells (Mather et al , Annals N Y. Acad Sci . 383 44-68 (1982)), MRC 5 cells, FS4 cells, and a human hepatoma line (Hep G2)
  • Host cells are transfected and preferably transformed with the above-desc ⁇ bed expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired se ⁇ uences Transfection refers to the taking up of an expression vector by a host cell whether or not any codmg sequences are fact expressed Numerous methods of transfection are known to the ordinarily skilled artisan for example, CaPO ⁇ and electroporation Successful transfection is generally recognized when anv indication ofthe operation of this vector occurs within the host cell
  • Transformation means introducing DNA into an organism so that the DNA is replicable either as an extrachromosomal element or by chromosomal integrant Depending on the host cell used, transformation is done usmg standard techniques appropriate to such cells
  • the calcium treatment employing calcium chlo ⁇ de as described in section 1 82 of Sambrook et al , supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers
  • Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al , Gene.
  • plants may be transfected using ultrasound treatment as desc ⁇ bed in WO 91/00358 published 10 January 1991
  • the calcium phosphate precipitation method of Graham and van der Eb, Virology. 5.2 456-457 ( 1978) is preferred
  • Transformations into yeast are typically carried out according to the method of Van Solingen et al , J. Bact.. J Q 946 ( 1977) and Hsiao et al , Proc. Natl Acad.
  • Prokaryotic cells used to produce the CT-l polypeptide of this invention are cultured in suitable media as described generally in Sambrook et al , supra
  • the mammalian host cells used to produce the CT-l of this invention may be cultured m a variety of media
  • Commercially available media such as Ham's F-10 (Sigma), F-12 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI- 1640 (Sigma), Dulbecco's Modified Eagle's Medium ([D-MEM], Sigma), and D- MEM F-12 (Gibco BRL) are suitable for cultunng the host cells
  • any ofthe media described for example, m Ham and Wallace, Methods in Enzvmologv. 5.8. 44 (1979), Barnes and Sato, Anal Biochem . 102 255 (1980), U S Patent Nos 4.767,704, 4,657.866.
  • the host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal
  • Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl Acad. Sci. USA.
  • Gene expression may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.
  • immunohistochemical staining techniques a cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product coupled, where the labels are usually visually detectable, such as enzymatic labels, fluorescent labels, luminescent labels, and the like.
  • a particularly sensitive staining technique suitable for use in the present invention is described by Hsu et ai, Am. J. Clin. Path... 7_5_: 734-738 (1980).
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polycional, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native CT- 1 polypeptide or against a synthetic peptide based on the DNA sequences provided herein as described further in Section 4 below.
  • CT-l preferably is recovered from the culture medium as a secreted polypeptide. although it also may be recovered from host cell lysates when directly produced without a secretory signal.
  • CT-l is produced in a recombinant cell other than one of human origin, the CT- 1 is completely free of proteins or polypeptides of human origin.
  • the particulate debris is removed, for example, by centrifugation or ultrafiltration; optionally, the protein may be concentrated with a commercially available protein concentration filter, followed by separating the CT-l from other impurities by one or more steps selected from immunoaffinity chromatography, ion-exchange column fractionation (e.g., on DEAE or matrices containing carboxymethyl or sulfopropyl groups), chromatography on Blue-Sepharose, CM Blue-Sepharose, MONO-Q.
  • immunoaffinity chromatography e.g., ion-exchange column fractionation (e.g., on DEAE or matrices containing carboxymethyl or sulfopropyl groups), chromatography on Blue-Sepharose, CM Blue-Sepharose, MONO-Q.
  • MONO-S lentil lectin-Sepharose, WGA-Sepharose, Con A- Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl, or protein A Sepharose, SDS-PAGE chromatography, silica chromatography, chromatofocusing, reverse phase HPLC (e g., silica gel with appended aliphatic groups), gel filtration using, e.g., Sephadex molecular sieve or size-exclusion chromatography, chromatography on columns that selectively bind the CT-l, and ethanol or ammonium sulfate precipitation.
  • a protease inhibitor may be included in any ofthe foregoing steps to inhibit proteolysis.
  • protease inhibitors examples include phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin. aprotinin, 4-(2- aminoethy -benzenesulfonyl fluoride hydrochloride-bestatin, chymostatin, and benzamidine.
  • PMSF phenylmethylsulfonyl fluoride
  • leupeptin leupeptin
  • pepstatin aprotinin
  • 4-(2- aminoethy -benzenesulfonyl fluoride hydrochloride-bestatin chymostatin
  • benzamidine examples include benzamidine.
  • a preferred purification scheme involves adjusting the culture medium conditioned by cells transfected with the relevant clone to 1.5 M NaCl and applying to a Butyl ToyopearlTM column.
  • the column is washed with Tris[hydroxymethyl]aminomethane hydrochlo ⁇ de (TRJS-HCl), pH 7.5, containing NaCl, and the activity eluted with TRIS-HCl, pH 7.5, containing 10 mM ZwittergentTM 3- 10 surfactant.
  • TRJS-HCl Tris[hydroxymethyl]aminomethane hydrochlo ⁇ de
  • TRIS-HCl pH 7.5
  • the peak of activity is adjusted to 150 mM NaCl, pH 8.0, and applied to a MONO-Q Fast Flow column.
  • This column is washed with TRJS-HCl, pH 8.0. containing NaCl and octyl glucoside.
  • Activity is found in the flow-through fraction.
  • the active material is then applied to a reverse phase C4 column in 0.1% TFA, 10% acetonitrile, and eluted with a gradient of 0.1% TFA up to 80%.
  • the activity fraction ates at about 15-30 kDa on gel filtration columns. It is expected that a chaotrope such as guanidine-HCl is required for resolution and recovery.
  • CT-l variants in which residues have been deleted, inserted, or substituted are recovered in the same fashion as native CT-l, taking account of any substantial changes in properties occasioned by the variation.
  • preparation of a CT-l fusion with another protein or polypeptide e.g.. a bacterial or viral antigen
  • an immunoaffinity column containing antibody to the antigen can be used to adsorb the fusion polypeptide.
  • Immunoaffinity columns such as a rabbit polyclonal anti-CT-1 column can be employed to absorb the CT-l variant by binding it to at least one remaining immune epitope.
  • a protease inhibitor such as those defined above also may be useful to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants.
  • purification methods suitable for native CT-l may require modification to account for changes in the character of CT-l or its variants upon production in recombinant cell culture.
  • H. Covalent Modifications of CT-l Polypeptides Covalent modifications of CT-l polypeptides are included within the scope of this invention. Both native CT- 1 and amino acid sequence variants of native CT- 1 may be covalently modified.
  • One type of covalent modification included within the scope of this invention is the preparation of a variant CT- 1 fragment.
  • Variant CT-l fragments having up to about 40 amino acid residues may be conveniently prepared by chemical synthesis or by enzymatic or chemical cleavage ofthe full-length or variant CT-l polypeptide.
  • Other types of covalent modifications ofthe CT-l or fragments thereof are introduced into the molecule by reacting targeted amino acid residues of the CT-l or fragments thereof with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
  • Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ -(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino-terminal residues are reacted with succinic or other carboxyiic acid anhydrides.
  • Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • suitable reagents for derivatizing -amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4- pentanedione, and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1 ,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because ofthe high pK a ofthe guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • aromatic diazonium compounds or tetranitromethane Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Tyrosyl residues are iodinated using I or 13 to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable.
  • R and R' are different alkyl groups, such as l-cyclohexyl-3-(2-mo ⁇ holinyl-4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Derivatization with bifunctional agents is useful for crosslinking CT-l to a water-insoluble support matrix or surface for use in the method for purifying anti-CT-1 antibodies, and vice-versa.
  • Commonly used crosslinking agents include, e.g., l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido- 1 ,8- octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming, crosslinks in the presence of light.
  • reactive water- insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated form of these residues falls within the scope of this invention.
  • altering is meant deleting one or more carbohydrate moieties found in native CT-l, and/or adding one or more glycosylation sites that are not present in the native CT- 1.
  • N-linked refers to the attachment ofthe carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X- serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment ofthe carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation refers to the attachment of one ofthe sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • Addition of glycosylation sites to the CT-l polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more ofthe above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the native CT- 1 sequence (for O-linked glycosylation sites).
  • the native CT- 1 amino acid sequence is preferably altered through changes at the DNA level, particularly by mutating the DNA encoding the native CT-l polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • the DNA mutation(s) may be made using methods described above under Section 2B.
  • Another means of increasing the number of carbohydrate moieties on the CT-l polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. These procedures are advantageous in that they do not require production ofthe polypeptide in a host cell that has glycosylation capabilities for N- or O-linked glycosylation.
  • the sugar(s) may be attached to (a) arginine and histidine.
  • (c) free sulfhydryl groups such as those of cysteine,
  • free hydroxyl groups such as those of serine, threonine, or hydroxyproline,
  • aromatic residues such as those of phenylalanine. tyrosine.
  • Removal of any carbohydrate moieties present on the CT-l polypeptide may be accomplished chemically or enzymatically.
  • Chemical deglycosylation requires exposure of the polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact.
  • Chemical deglycosylation is described by Hakimuddin, et ai. Arch. Biochem. Biophvs.. 259: 52 (1987) and by Edge et ai. Anal. Biochem.. 1 18: 131 (1981).
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et ai, Meth. EnzvmoL J3J: 350 (1987).
  • CT-l Another type of covalent modification of CT-l comprises linking the CT- 1 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791.192 or 4,179,337.
  • CT-l also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly- [methylmethacylate] microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • CT-l preparations are also useful in generating antibodies, as standards in assays for CT-l (e.g., by labeling CT- 1 for use as a standard in a radioimmunoassay, enzyme-linked immunoassay, or radioreceptor assay), in affinity purification techniques, and in competitive-type receptor binding assays when labeled with radioiodine, enzymes, fluorophores, spin labels, and the like.
  • the receptor can be expression cloned, then a soluble form of the receptor is made by identifying the extracellular domam and excis g the transmembrane domam therefrom The soluble form ofthe receptor can then be used as an antagonist, or the receptor can be used to screen for small molecules that would antagonize CT-l activity Transfected cells expressing recombinant receptor find use in screening molecules both for receptor binding and receptor activation agonis or antagonism
  • va ⁇ ants of native CT- 1 are made that act as antagonists Smce the GH/cytokine receptor family is known to have two b ding sites on the ligand, the receptor binding sites of CT-l can be determined by binding studies and one of them eliminated by standard techniques (deletion or radical substitution) so that the molecule acts as an antagonist
  • Figure 16 indicate regions that can act as antagonists
  • Antagonist activity can be determined by several means, including the hypertrophy assay, the neurotrophic assay, and the other CT-l assays presented herein J Hypertrophy Assay
  • a miniatured assay is preferably used to assay for hypertrophic activity
  • the medium used allows the cells to survive at a low platmg density without serum By plat g directly into this medium, washmg steps are eliminated so that fewer cells are removed
  • the plating density is important many fewer cells and the survival is reduced, many more cells and the myocytes begin to self-induce hypertrophy
  • the steps involved are (a) platmg 96-well plates with a suspension of myocytes at a cell density of about 7 5 x 10 cells per mL in D-MEM F-12 medium supplemented with at least insulin, transfemn, and aprotmin,
  • the medium can be supplemented with additional elements such as EGF that ensure a longer viability of the cells, but such supplements are not essential D-MEM/F-12 medium is available from Gibco BRL, Gaithersburg, MD, and consists of one ofthe following media (Table 2)
  • the preferred hypertrophy assay comprises: (a) precoating the wells of 96-well tissue culture plates with a medium containing calf serum, preferably
  • the medium used in step (c) is a serum-free medium also containing penicillin streptomycin (pen strep) and glutamine.
  • the medium contains 100 mL D-MEM/F- 12.
  • 100 ⁇ L transferrin 10 mg/mL.
  • 20 ⁇ L insulin 5 mg/mL
  • 50 ⁇ L aprotinin 2 mg/mL
  • 1 mL pen/strep JRH Biosciences No. 59602- 77P
  • 1 mL L-glutamine 200 mM.
  • the assay capacity of 1000 single samples a week coupled with the small sample size requirement of 100 ⁇ L or less has enabled an expression cloning and protein purification that would have been impossible to accomplish using the current methods available.
  • Another method for assaying hypertrophy involves measuring for atrial natriuretic peptide (ANP) release by means of an assay that determines the competition for binding of I-rat ANP for a rat ANP receptor A-IgG fusion protein.
  • the method suitable for use is similar to that used for determining gp 120 using a CD4-IgG fusion protein described by Chamow et ai, Biochemistry. 22: 9885-9891 (1990). The basis for the isolation and characterization of the novel hypertrophy factor.
  • CT- 1 is the miniaturized high through-put hypertrophy assay system, which was developed in a 96 well format, in which hypertrophy is scored on individual myocardial cells following crystal-violet staining of neonatal rat cardiac myocytes. This assay was used in combinatio with an in vitro model of embryonic stem cell cardiogenesis (Miller-Hance et ai, Journal of Biological Chemistry, 268:25244-25252 (1993)). These totipotent stem cells can differentiate into multi-cellular cystic embryoid bodies (EBs) when cultured in the absence of a fibroblast feeder layer, or without LIF.
  • EBs multi-cellular cystic embryoid bodies
  • the hypertrophy assay system was then used to expression clone this factor, which proved to be the novel cytokine, CT- 1.
  • CT- 1 novel cytokine
  • These studies document the utility of using expression cloning approaches to identify novel growth factors and cytokines from this in vitro model of embryonic stem cell differentiation.
  • This assay system will be of interest in the isolation of other novel cytokines derived from precursors of other differentiated cell types found in EBs. i.e., neurogenic, skeletal myogenic, and hematopoietic precursors.
  • K. Neurotrophic Assay The assay used for ciliary ganglion neurotrophic activity described in Leung, Neuron. £: 1045-1053 (1992) is suitable herein.
  • ciliary ganglia are dissected from E7-E8 chick embryos and dissociated in trypsin-EDTA (Gibco 15400-013) diluted ten fold in phosphate-buffered saline for 15 minutes at 37°C.
  • the ganglia are washed free of trypsin with three washes of growth medium (high glucose D-MEM supplemented with 10% fetal bovine serum, 1.5 M glutamine, 100 ⁇ g/mL penicillin, and 100 ⁇ g/mL strepomycin). and then gently triturated in 1 L of growth medium into a single-cell suspension.
  • Neurons are enriched by plating this cell mixture in 5 mL of growth media onto a 100-mm tissue culture dish for 4 hours at 37° C in a tissue culture incubator. During this time the non-neurona) cells preferentially stick to the dish and neurons can be gently washed free at the end ofthe incubation. The enriched neurons are then plated into a 96-well plate previously coated with collagen. In each well, 1000 to 2000 cells are plated, in a final volume of 100 to 250 ⁇ L, with dilutions ofthe CT-l to be tested. Following a 2-4-day incubation at 37° C, the number of live cells is assessed by staining live cells using the vital dye metal lothionine (MTT).
  • MTT vital dye metal lothionine
  • CT- 1 activates downstream cellular responses via the heterodimerization of gp 130 and LIFR ⁇ .
  • the expression pattern of CT-l and pleiotropic activities suggest that it may have important functions, not only in the cardiac context, but in extra-cardiac tissues as well.
  • CT-l acts to maintain normal embryonic growth and mo ⁇ hogenesis, as well as physiological homeostasis in the adult.
  • CT- 1 is believed to find use as a drug for treatment of mammals (e.g. , animals or humans) in vivo having heart failure, arrhythmic or inotropic disorders, and/or peripheral neuropathies and other neurological disorders involving motor neurons or other neurons in which CNTF is active.
  • CT-l has additional uses as shown herein.
  • CT-l may be useful in treating congestive heart failure in cases where ACE inhibitors cannot be employed or are not as effective.
  • CT-l optionally is combined with or administered in concert with other agents for treating congestive heart failure, including ACE inhibitors.
  • the effective amount of ACE inhibitor to be administered, if employed, will be at the physician's or veterinarian's discretion. Dosage administration and adjustment is done to achieve optimal management of congestive heart failure and ideally takes into account use of diuretics or digitalis, and conditions such as hypotension and renal impairment. The dose will additionally depend on such factors as the type of inhibitor used and the specific patient being treated. Typically the amount employed will be the same dose as that used if the ACE inhibitor were to be administered without CT- 1.
  • a test dose of enalapril is 5 mg, which is then ramped up to 10-20 mg per day, once a day, as the patient tolerates it.
  • captopril is initially administered orally to human patients in a test dose of 6.25 mg and the dose is then escalated, as the patient tolerates it, to 25 mg twice per day (BID) or three times per day (TID) and may be titrated to 50 mg BID or TID. Tolerance level is estimated by determining whether decrease in blood pressure is accompanied by signs of hypotension. If indicated, the dose may be increased up to 100 mg BID or TID.
  • Captopril is produced for administration as the active ingredient, in combination with hydrochlorothiazide, and as a pH stabilized core having an enteric or delayed release coating which protects captopril until it reaches the colon. Captopril is available for administration in tablet or capsule form. A discussion ofthe dosage, administration, indications and contraindications associated with captopril and other ACE inhibitors can be found in the Physicians Desk Reference, Medical Economics Data Production Co., Montvale, NJ. 2314-2320 (1994).
  • CT-l is also potentially useful in the generation, maturation, and survival of oligodendrocytes in vitro for protection of oligodendrocytes against natural and tumor necrosis factor-induced death, in the survival and differentiation of astrocytes and the induction of type-2 astrocyte development, and in the stimulation of the recombinant production of low-affinitv nerve growth factor receptor and CD-4 bv rat central nervous system (CNS) microglia
  • CNS central nervous system
  • CT-l is also potentially useful in having a trophic effect on denervated skeletal muscle
  • it is expected to have the proliferative responses and binding properties of hematopoietic cells transfected with low-affinity receptors for leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor to regulate fibrinogen gene expression in hepatocytes by binding to the ⁇ nterleukm-6 receptor to have trophic actions on murme embryonic carcinoma cells, to be an endogenous pyrogen, and to have a mitogenic effect on human IMR 32 neuroblastoma cells
  • CT- 1 is expected to enhance the response to nerve growth factor of cultured rat sympathetic neurons, to mamtam motoneurons and their target muscles m developmg rats, to mduce motor neuron sprouting in vivo to promote the survival of neonatal rat corticospinal neurons in vitro, to prevent degeneration of adult rat substantia nigra dopaminergic neurons in vivo, to alter the threshold of hippocampal pyramidal neuron sensitivity to excitotoxm damage, to prevent neuronal degeneration and promote low-affinity NGF receptor production in the adult rat CNS, and to enhance neuronal survival in embryonic rat hippocampal cultures
  • CT- 1 shall benzo the response to nerve growth factor of cultured rat sympathetic neurons, to mamtam motoneurons and their target muscles m developmg rats, to mduce motor neuron sprouting in vivo to promote the survival of neonatal rat corticospinal neurons in vitro, to prevent degeneration of adult rat substanti
  • CT-l shares at least some of the growth inhibitory activities of the IL-6 family cytokmes CT-l has the potential for use as a therapeutic non-proliferative agent for suppressing some forms of myeioid leukaemia as well as a reagent for modifying macrophage function and other responses to infections CT-l was 6 fold more potent than LIF in inhibiting the uptake of 3H-thym ⁇ dme by Ml cells and thus the growth ofthe myeioid leukemia cell l e CT- 1 inhibits the growth of the mouse myeioid leukemia cell line, M 1 , and induces its differentiation into a macrophage-like phenotype CT-l does not mimic the activity of
  • CT-l can be used in vitro to eliminate malignant cells from marrow for autologous marrow transplants or to inhibit proliferation or eliminate malignant cells in other tissue, e.g. blood, prior to reinfusion.
  • CT-l can also be used as a treatment in disorders ofthe hematopoietic system, especially as a means of stimulating hematopoiesis in patients with suppressed bone marrow function, for example, patients suffering from aplastic anemia, inherited or acquired immune deficiency, or patients undergoing radiotherapy or chemotherapy.
  • Antagonists to CT-l can also be used for treating a wide variety of wounds including substantially all cutaneous wounds, corneal wounds, and injuries to the epithelial-lined hollow organs ofthe body and those involving myocytes and neurons.
  • Wounds suitable for treatment include those resulting from trauma such as bums, abrasions, cuts, and the like as well as from surgical procedures such as surgical incisions and skin grafting.
  • Other conditions suitable for treatment with the CT-l antagonists include chronic conditions, such as chronic ulcers, diabetic ulcers, and other non-healing (trophic) conditions.
  • a CT-l antagonist is inco ⁇ orated in physiologically-acceptable carriers for local or site-specific application to the affected area.
  • CT-l antagonist compositions in bandages and other wound dressings to provide for continuous exposure ofthe wound to the peptide. Aerosol applications also find use.
  • the antagonist will be present in an amount effective to suppress CT-l inhibition of epithelial cell proliferation.
  • the compositions will be applied topically to the affected area, typically as eye drops to the eye or as creams, ointments or lotions to the skin. In the case of eyes, frequent treatment is desirable, usually being applied at intervals of 4 hours or less. On the skin, it is desirable to continually maintain the treatment composition on the affected area during healing, with applications of the treatment composition from two to four times a day or more frequently.
  • CT- 1 maintains the undifferentiated phenotype of embryonic stem cells.
  • CT- 1 can promote cell survival and acts as an anti-apoptotic factor during mouse embryogenesis.
  • CT- 1 will find use in techniques in which undifferentiated ES cells are useful as well as techniques in which control of their differentiation is useful
  • CT-l will find use to maintain the undifferentiated state of embryonic stem cells during recombinant DNA transformation and their synchronized differentiation in methods such as gene cloning and creating transgenic animals.
  • CT-l also find use in artificial insemination techniques.
  • CT- 1 is used in the enhancement of development and maintenance of animal or mammalian embryos and to enhance impregnation.
  • IVF in vitro fertilization
  • ET embryo transfer
  • the practice of transferring more than one embryo to the recipient animal to ensure pregnancy can result in unwanted multiple births.
  • One major constraint with embryo transfer is the need to hold embryos in culture media for either relatively short periods of time, perhaps only a few hours p ⁇ or to transfer or for longer periods of some days, after micromanipulation
  • the fertilized egg passes through a number of stages including the morula and the blastocyst stages
  • the cells form an outer cell layer known as the trophectoderm (which is the precursor of the placenta) as well as an inner cell mass (from which the whole of the embryo proper is derived)
  • the blastocyst is surrounded by the zona pellucida.
  • the present mvention also extends to a method for in vitro fertilization and subsequent implantation of a mammalian embryo which is characterized in that the embryo is cultured in vitro in a culture medium contammg an effective amount of mammalian CT-l prior to transfer into animal or mammalian host, where "host" is defined as a suitably receptive female animal or mammal.
  • a further aspect ofthe present invention relates to a non-human animal and in particular a chimeric non-human animal or transgenic progeny of said animai generated by known techniques using ES cells which have been maintained in vitro in CT-I -containing culture medium.
  • ES cells are derived from animal embryos passaged in a culture medium containing CT-l wherein said ES cells have additional genetic material inserted therein.
  • the transgenic animals contemplated include nonhuman mammals such as livestock and ruminant animals and domestic animals.
  • the present invention is also directed to composition comprising an effective amount of CT-l in combination with an animal (e.g. mammalian) embryo maintaining medium.
  • the present invention also provides a composition having embryotrophic and/or embryo protective properties comprising CT-l .
  • the amount of CT-l used in accordance with the present invention is that required to maintain and/or develop embryos and/or enhance impregnation. Generally it is in the range of 0.1 ng ml to 10,000 ng/ml, preferably 1 ng/ml to 1000 ng/ml.
  • CT-l also finds use to produce a mammalian pluripotential embryonic stem cell composition which can be maintained on feeder layers and give rise to embryoid bodies and multiple differentiated cell phenotypes in monolayer culture.
  • a method of making a pluripotential embryonic stem cell by administering a growth enhancing amount of basic fibroblast growth factor, CT-l, membrane associated steel factor, and soluble steel factor to primordial germ cells under cell growth conditions, thereby making a pluripotential embryonic stem cell.
  • a "pluripotential embryonic stem cell” as used herein means a cell which can give rise to many differentiated cell types in an embryo or adult, including the germ cells (sperm and eggs).
  • ES cell This cell type is also referred to as an "ES cell.” Only those mammals which can be induced to form ES cells by the described methods are within the scope ofthe invention. Although not a requirement for application of this embodiment ofthe invention, the ES cells may be capable of indefinite maintenance, typically at least 15 days. Once the ES cells are established, they can be genetically manipulated to produce a desired characteristic. For example, the ES cells can be mutated to render a gene non-functional, e.g. the gene associated with cystic fibrosis or an oncogene. Alternatively, functional genes can be inserted to allow for the production of that gene product in an animal. e.g. growth hormones or valuable proteins.
  • a gene non-functional e.g. the gene associated with cystic fibrosis or an oncogene.
  • functional genes can be inserted to allow for the production of that gene product in an animal. e.g. growth hormones or valuable proteins.
  • the invention also provides a composition comprising pluripotential ES cells and/or primordial germ cells and/or embryonic ectoderm cells and CT-l.
  • an FGF. membrane associated SF, and soluble SF wherein the factors are present in amounts to enhance the growth of and allow the continued proliferation of the cell.
  • Growth and proliferation enhancing amounts can vary. Generally, 0.5 to 500 ng factor/ml of culture solution is adequate. Preferably, the amount is between 10 to 20 ng/ml.
  • CT-l can be used to maintain ES cells. In this case, the amounts of CT-l, FGF, and SF necessary to maintain ES cells can be much less than that required to enhance growth or proliferation to become ES cells.
  • CT-l, FGF, or SF may not be required for maintenance of ES cells.
  • the invention also provides a method of making a pluripotential ES cell comprising administering a growth enhancing amount of CT-l.
  • basic FGF, membrane associated SF, and soluble SF to primordial germ cells and/or embryonic ectoderm cells under cell growth conditions, thereby making a pluripotential ES cell.
  • This method can be practiced utilizing any animal cell, especially mammal cells including mice, rats, rabbits, guinea pigs, goats, cows, pigs, humans, etc.
  • the ES cell produced by this method is also contemplated.
  • Cell growth conditions are set forth in the Examples.
  • the invention provides a method of using the ES cells to contribute to chimeras in vivo comprising injecting the cell into a blastocyst and growing the blastocyst m a foster mother Alternatively, aggregating the cell with a morula stage embryo and growing the embryo m a foster mother can be used to produce a chimera
  • the ES cells can be manipulated to produce a desired effect in the chimeric animal Alternatively, the ES cells can be used to derive cells for therapy to treat an abnormal condition For example, derivatives of human ES cells could be placed in the brain to treat a neurodegenerative disease
  • CT-l will stimulate the proliferation of satellite cells and the subsequent development of myoblasts
  • methods of stimulating the proliferation and/or differentiation of mammalian satellite cells mto myoblasts which includes the steps of contactmg said cells with a stimulation-effective amount of CT- 1 for a time and under conditions sufficient for said satellite cells to proliferate and/or differentiate into myoblasts
  • the stimulation-effective amount of CT-l can be administered simultaneously or in sequential combination with one or more other cytokmes, for a time and under conditions sufficient for said satellite cells to proliferate and/or differentiate into myoblasts
  • methods of myoblast transfer therapy which include the steps of contactmg mammalian satellite cells with a proliferation- and/or differentiation-effective amount of CT-l for a time and under conditions sufficient for said satellite cells to proliferate and or differentiate into myoblasts and then admmiste ⁇ ng said myoblasts at multiple sites mto muscles
  • CT-l is used simultaneous or sequential combination with one or more other
  • CT-l is a multi-functional cytokine which shares several biological activities with other members ofthe IL-6 cytokine family CT-l and LIF have similar activities in the in vitro assay systems examined thus far Accordingly, CT-l is expected to find use in the medical treatment uses known for LIF
  • Figure 21 is a schematic that summa ⁇ zes the diverse bioactivities of CT-l in a wide variety
  • fibrinogen is responsible for the modifications m the plasma viscosity and for the mcrease the speed of sedimentation which are observed in the inflammation
  • fibrinogen is responsible for the modifications m the plasma viscosity and for the mcrease the speed of sedimentation which are observed in the inflammation
  • a method for treating a mammal afflicted with arthritis or an inflammatory disease including those related to autoimmune diseases
  • the method includes the step of administering to the mammal an amount of compound which is effective for alleviation of the condition
  • Inflammatory states in mammals include, but are not limited to allergic and asthmatic manifestations, dermatological diseases, inflammatory diseases, collagen diseases, reperfusion injury and stroke, infections, and lupus erythematosus. Treatment of both acute and chronic diseases are possible.
  • Preferred diseases for treatment are arthritis, asthma, allergic rhinitis, inflammatory bowel disease (IBD), psoriasis, reperfusion injury and stroke.
  • Other disorders involving acute phase proteins are acute lymphoblastic leukemia (ALL), acute graft versus host disease (aGvHD), chronic lymphocytic leukemia (CLL).
  • cutaneous T-cell lymphoma (CTCL) type 1 diabetes, aplastic anemia (AA), Crohn's Disease, and scleroderma. Additional inflammatory conditions include patients with severe burns, kidney transplants, acute infections ofthe central nervous system and septic shock.
  • CT- 1 like LIF, inhibits the proliferation and induces the differentiation of a mouse myeioid leukemia cell line. Similar to the activity seen for LIF and CNTF, CT-l has neuronal function, in that it promotes the survival of cultured dopaminergic neurons and ciliary ganglion neurons and induces a switch in the transmitter phenotype of sympathetic neurons. Thus, while CT-l was initially isolated on die basis of its actions on cardiac muscle cells, it may also have pleiotropic functions in other organ systems that overlap to a significant extent with the activities other IL-6 family cytokines, preferably those of LIF and OSM, and more preferably those of LIF.
  • IL-6 family cytokines preferably those of LIF and OSM, and more preferably those of LIF.
  • CT-l signals through and induces tyrosine phosphorylation ofthe gpl30/LlFR ⁇ - heterodimer in cardiac myocytes and other cell types. This does not exclude the possibility that CT-l may use an alternative signaling pathway via an additional private receptor in some cell types.
  • IL-6 cytokine family including, IL- 11, LIF, CNTF, and OSM trigger downstream signaling pathways in multiple cell types through the homodimerization of gpl30 or through the heterodimerization of gpl30 and a related transmembrane signal transducer, the LIF receptor subunit LIFR ⁇ ( Figure 15B: Gearing et ai, Science, 255: 1434-1437 (1992); Vp et al, Cell, 69:1 121-1132 (1992); Murakami et ai, Science, 260: 1808-1810 (1993); Davis et ai, Science, 260:1805-1808 (1993).
  • An anti-gpl30 monoclonal antibody was used to determine its effect on CT-l binding to Ml cells. This neutralizing antibody inhibited CT-l binding to Ml cells indicating that gp 130 is a component ofthe CT-l receptor complex.
  • CT-l and LIF also cross-compete for binding to rat cardiac myocytes and mouse Ml cells indicating that these two ligands act on these cells via the LIF receptor.
  • c-fos induction by CT-l and LIF in cardiac myocytes was antagonized by the anti-gpl30 monoclonal antibody as well as by a mutated human LIF protein, acting as a LIFR ⁇ -antagonist.
  • CT-l will find use in disorders, diseases or condition relating to cells expressing the LIFR ⁇ and to its signaling pathways.
  • LIF induced the tyrosine phosphorylation of an additional -200 kDa protein, which was not phosphorvlated upon stimulation with the IL-6/sIL-6R complex Based on previous results this protein most likely corresponds to the LIF receptor subunit LIFR ⁇ (Ip et al Cell, 69 1121-1 132 (1992), Davis et al , Science 260 1805- 1808 ( 1993), Boulton et al Journal of Biological Chemistry.
  • CT-l like LIF, induces the tyrosine phosphorylation of LIFR ⁇
  • CT-l and LIF appear to have functional redundancy m these assay systems, the possibility exists that CT-l compensated for the complete loss of LIF durmg embryonic development and adulthood in these LIF deficient embryos Alternatively, smce LIF is not expressed at very high levels in the embryo, CT- 1 may be the endogenous ligand which normally performs this function during mouse embryonic development If the latter is the case, one might expect severe embryonic defects CT- 1 deficient embryos, analogous to either the LIFR ⁇ deficient or gpl 30 deficient phenotypes CT- 1 will be involved in the maintenance of normal cardiac growth, mo ⁇ hogenesis, and hypertrophy, which can be analyzed in the basal state and in response to the imposition of a mechanical stimulus for hypertrophy via miniaturized physiological technology (Rockman et al Proc Natl Acad Sci USA, 88 8277-8281 (1991)) This system will allow screening and identification of CT-l agonists and antagonists Interestingly, a large disparity
  • CT-l may also be useful as an adjunct treatment of neurological disorders together with such neurotrophic factors as. e g , CNTF, NGF, BDNF, NT-3, NT-4, and NT-5
  • the nucleic acid encodmg the CT- 1 may be used as a diagnostic for tissue-specific typing For example, such procedures as in situ hybridization, northern and Southern blotting, and PCR analysis may be used to determine whether DNA and/or RNA encodmg CT-l is present in the cell type(s) bemg evaluated
  • Isolated CT-l polypeptide may also be used in quantitative diagnostic assays as a standard or control against which samples containing unknown quantities of CT-l may be prepared
  • Therapeutic formulations of CT-l for treating heart failure, neurological disorders, and other disorders are prepared for storage by mixing CT-l having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, supra), in the form of lyophilized cake or aqueous solutions
  • Acceptable earners, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinyipyrrolidone, ammo acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccha ⁇ des, and other carbohydrates including glucose, mannose, or dextrins.
  • buffers such as phosphate, citrate,
  • chelating agents such as EDTA.
  • sugar alcohols such as mannitol or sorbitol, salt-forming counte ⁇ ons such as sodium, and/or nonionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG) CT-l to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes, p ⁇ or to or following lyophilization and reconstitution CT- 1 ordinarily will be stored in lyophilized form or in solution.
  • PEG polyethylene glycol
  • Therapeutic CT-l compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle
  • a sterile access port for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle
  • the route of CT-l or CT-l antibody administration is in accord with known methods, e g , injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, lntraarte ⁇ al.
  • CT- 1 is administered continuously by infusion or by bolus injection
  • CT- 1 antibody is administered the same fashion, or by administration into the blood stream or lymph
  • CT-l or its antagonist is administered locally or site-specifically to better obtain a local or site-specific effect
  • suitable delivery methods are known in the art including implants, pumps, patches, direct injection, and transmucosal delivery
  • Site-specific delivery can be obtained by gene delivery vectors and viruses and by transplantation of cells expressing CT-l or an antagonist
  • sustamed-release preparations include semipermeable mat ⁇ ces of solid hydrophobic polymers contammg the protein, which matrices are in the form of shaped articles, e g , films, or microcapsules
  • sustamed-release mat ⁇ ces mclude polyesters, hydrogels (e g , poly(2-hydroxyethyl- methacry late) as desc ⁇ bed by Langer et al , J. Biomed Mater. Res . H 167-277 ( 1981 ) and Langer, Chem Tech . 12 98-105 (1982) or poly(v ⁇ nylalcohol)), polylactides (U.S Patent No 3,773,919.
  • EP 58,481 copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al , Biopolvmers.22. 547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer et al , supra), degradable lactic acid-glycolic acid copolymers such as the Lupron DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybuty ⁇ c acid (EP 133,988)
  • CT-l are prepared by methods known per se DE 3,218.121. Epstein et al . Proc Natl. Acad Sci USA. 82 3688-3692 (1985). Hwang et al . Proc. Natl. Acad. Sci USA. 77 4030-4034 (1980) EP 52,322. EP 36.676, EP 88,046, EP 143,949, EP 142,641, Japanese patent application 83-118008, U S Patent Nos 4 485,045 and 4,544.545, and EP 102,324 Ordinarily the liposomes are ofthe small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal CT- 1 therapy.
  • CT-l an effective amount of CT-l to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.
  • a typical daily dosage might range from about 1 ⁇ g/kg to up to 100 mg/kg of patient body weight or more per day, depending on the factors mentioned above, preferably about 10 ⁇ g/kg/day to 10 mg/kg/day.
  • the clinician will administer CT- 1 until a dosage is reached that achieves the desired effect for treatment ofthe heart, neural, or other dysfunction.
  • the amount would be one which increases ventricular contractility and decreases peripheral vascular resistance or ameliorates or treats conditions of similar importance in congestive heart failure patients. The progress of these therapies is easily monitored by conventional assays. 4.
  • Immunoglobulins (Ig) and certain variants thereof are known and many have been prepared in recombinant cell culture. For example, see U.S. Patent No. 4,745,055; EP 256,654; EP 120.694; EP 125,023; EP 255,694; EP 266,663; WO 88/03559; Faulkner et al.. Nature. 298: 286 (1982); Morrison, J. Immun.. Ul: 793 (1979); Koehler et at, Proc. Natl. Acad. Sci. USA. 22: 2197 (1980); Raso et al., Cancer Res.. 4J_: 2073 (1981); Morrison etai.
  • immunoglobulin chains are also known. See. for example, U.S. Patent No. 4,444,878; WO 88/03565; and EP 68,763 and references cited therein.
  • the immunoglobulin moiety in the chimeras ofthe present invention may be obtained from IgG- 1. IgG-2, IgG-3, or IgG-4 subtypes, IgA, IgE, IgD, or IgM, but preferably from IgG-1 or IgG-3.
  • Polvclonal antibodies Polyclonal antibodies to CT-l polypeptides or CT-l fragments are generally raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of CT- 1 or CT- 1 fragment and an adjuvant.
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor
  • Animals are immunized against the CT-l polypeptide or CT-l fragment, immunogenic conjugates, or derivatives by combining 1 mg or 1 ⁇ g of the peptide or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • 1 mg or 1 ⁇ g of the peptide or conjugate for rabbits or mice, respectively
  • 3 volumes of Freund's complete adjuvant injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • Seven to 14 days later the animals are bled and the serum is assayed for CT-l or CT-l fragment antibody titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate ofthe same CT- 1 or CT- 1 fragment, but conjugated to a different protein and/or through a different cross-linkmg reagent Conjugates also can be made in recombinant cell culture as protein fusions Also, aggregating agents such as alum are suitably used to enhance the immune response
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, e , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present mmor amounts Thus, the modifier "monoclonal" indicates the character ofthe antibody as not bemg a mixture of discrete antibodies
  • CT-l monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler and Milstein, Nature. 256 495 (1975), or may be made by recombinant DNA methods (Cabilly et al , supra)
  • lymphocytes may be immunized in vitro Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hyb ⁇ doma cell (Godi ⁇ g, Monoclonal Antibodies Principles and Practice, pp 59- 103 [Academic Press, 1986))
  • a suitable fusing agent such as polyethylene glycol
  • the hyb ⁇ doma cells thus prepared are seeded and grown in a suitable culture medium that preferably contams one or more substances that inhibit the growth or survival ofthe unfused, parental myeloma cells
  • a suitable culture medium that preferably contams one or more substances that inhibit the growth or survival ofthe unfused, parental myeloma cells
  • the culture medium for the hybridomas typically will include hypoxanthme, aminopterm, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells
  • Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-1 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland USA
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed agamst CT-l
  • the binding specificity of monoclonal antibodies produced by hyb ⁇ doma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA)
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem . 107 220 (1980)
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra) Suitable culture media for this pu ⁇ ose include, for example, D-MEM or RPMI- 1640 medium
  • the hybridoma cells may be grown tn vivo as ascites tumors in an animal
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis. dialysis, or affinity chromatography
  • DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures (e g , by usmg oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies)
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E colt cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protem, to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • host cells such as E colt cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protem
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (Morrison, et al , Proc. Nat Acad. Sci..).
  • chimeric or “hybrid” antibodies are prepared that have the binding specificity of an ant ⁇ -CT-1 monoclonal antibody herein
  • non-immunoglobulin polypeptides are substituted for the constant domains of an antibody ofthe mvention, or they are substituted for the variable domains of one antigen-combining site of an antibody ofthe mvention to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for a CT-l and another antigen-combining site having specificity for a different antigen
  • Chimeric or hybrid antibodies also may be prepared in vitro using known methods synthetic protein chemistry, mcludmg those mvolvmg crosslmkmg agents.
  • immunotoxms may be constructed using a disulfide-exchange reaction or by forming a thioether bond.
  • suitable reagents for this pu ⁇ ose m clude lminothiolate and methyl-4-mercaptobutyr ⁇ m ⁇ date.
  • the antibodies ofthe invention typically will be labeled with a detectable moiety
  • the detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, j2 P, j5 S, or 125 I, a fluorescent or chemiluminescent compound, such as fluorescein lsothiocyanate, rhoda ⁇ une. or lucife ⁇ n, radioactive isotopic labels, such as, e g , 2 I, 3 P, C, or H; or an enzyme, such as alkaline phosphatase. beta-galactosidase, or horseradish peroxidase.
  • any method known m the art for separately conjugating the antibody to the detectable moiety may be employed, includmg those methods described by Hunter et al , Nature. 144 945 (1962); David et al , Biochemistry. 13 1014 (1974V Pain et ai A. Immunol. Meth.. 40 219 (19811: and Nvpren. J Histochem. and CytQchem come 20.: 407 (1982).
  • the antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)
  • ком ⁇ онентs rely on the ability of a labeled standard (which may be a CT-l or an immunologically reactive portion thereof) to compete with the test sample analyte (CT-l) for binding with a limited amount of antibody.
  • CT-l analyte
  • the amount of CT-l m the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
  • the antibodies generally are lnsolubihzed before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound
  • Sandwich assays mvolve the use of two antibodies, each capable of binding to a different immunogenic portion or epitope, ofthe protem (CT- 1 ) to be detected
  • the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex David and Greene, U S Patent No 4,376,1 10
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay)
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme (e g , horseradish peroxidase)
  • a humanized antibody has one or more ammo acid residues mtroduced into it from a source which is non-human
  • These non- human ammo acid residues are often referred to as "import" residues, which are typically taken from an "import" va ⁇ able domam Hu anization can be essentially performed following the method of Winter and co-workers (Jones et al , Nature 321. 522-525 ( 1986), Riechmann et al , Nature 332. 323-327 (1988), Verhoeyen et al , Science 239.
  • humanized antibodies are chimeric antibodies (Cabilly et al , supra), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
  • variable domains both light and heavy
  • FR human framework
  • humanized antibodies are prepared by a process of analysis ofthe parental sequences and va ⁇ ous conceptual humanized products usmg three-dimensional models of the parental and humanized sequences Three-dimensionai immunoglobulin models are commonly available and are familiar to those skilled in the art Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences Inspection of these displays permits analysis of the likely role of the residues in the functioning ofthe candidate immunoglobulin sequence, i.e.. the analysis of residues that influence the ability ofthe candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • Human monoclonal antibodies can be made by the hybridoma method. Human myeloma and mouse- human heteromyelo a cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor, J. Immunol. 133. 3001 (1984); Brodeur, et ai, Monoclonal Antibodv Production Techniques and Applications, pp.51-63 (Marcel Dekker. Inc.. New York. 1987): and Boerner et ai. J. Immunol.. 142: 86-95 (1991).
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • the homozygous deletion ofthe antibody heavy-chain joining region (J ⁇ ) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.
  • Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge.
  • Jakobovits et ai Proc. Natl. Acad. Sci. USA. 9_Q: 2551 (1993); Jakobovits et al, Nature.
  • phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
  • V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M 13 or fd, and displayed as functional antibody fragments on the surface ofthe phage particle.
  • the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties ofthe antibody also result in selection ofthe gene encoding the antibody exhibiting those properties.
  • the phage mimicks some ofthe properties ofthe B-cell.
  • Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology. 3: 564-571 (1993).
  • V-gene segments can be used for phage display. Clackson et ai. Nature.
  • the affinity of "primary" human antibodies obtained phage display can be improved by sequentially replacing the heavy and light chain V region genes with repertoires of naturally occurrmg va ⁇ ants (repertoires) of V domam genes obtained from unimmunized donors
  • This technique allows the production of antibodies and antibody fragments with affinities in the nM range
  • a strategy for making very large phage antibody repertoires has been desc ⁇ bed by Waterhouse et al , Nucl. Acids ⁇ £S appeal 21 2265-2266 (1993)
  • Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibodv has similar affinities and specificities to the starting rodent antibody Accordmg to this method, which is also referred to as "epitope imprinting", the heavy or light chain V domam gene of rodent antibodies obtained by phage display technique is replaced with a repertoire of human V domain genes, creating rodent-human chimeras Selection on antigen results m isolation of human vanable capable of restoring a functional antigen- binding site, i e the epitope governs (imprints) the choice of partner When the process is repeated in order to replace the remaining rodent V domam, a human antibody is obtained (see PCT WO 93/06213, published 1 Ap ⁇ l 1993) Unlike traditional humanization of rodent antibodies by CDR grafting, this technique provides completely human antibodies, which have no framework or CDR residues of rodent origin (vi) Bispecific antibodies
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens
  • one ofthe binding specificities is for a CT-l
  • the other one is for any other antigen, and preferably for another ligand that binds to a GH/cytokme receptor family member
  • bispecific antibodies specifically binding a CT-l and neurotrophic factor, or two different types of CT-l polypeptides are within the scope ofthe present invention
  • Methods for making bispecific antibodies are known m the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light cham pairs, where the two heavy chams have different specificities (Milstein and Cuello, Nature.
  • antibody-variable domains with the desired binding specificities are fused to immunoglobulm constant-domain sequences
  • the fusion preferably is with an immunoglobulm heavy-cham constant domain, comprising at least part ofthe hmge, CH2. and CH3 regions It is preferred to have the first heavy-chain constant region (CH 1 ).
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm.
  • Heteroconiugate antibodies are also within the scope of the present invention Heteroconjugate antibodies are composed of two covalently joined antibodies Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U S Patent No 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/00373, and EP 03089) Heteroconjugate antibodies may be made usmg any convenient cross-linking methods Suitable cross-linking methods Suitable cross-link
  • CT-l antibodies are useful in diagnostic assays for CT-l , e g , its production in specific cells, tissues, or serum
  • the antibodies are labeled in the same fashion as CT- 1 described above and/or are immobilized on an insoluble matrix
  • an antibody composition that binds to all or a selected plurality of CT-ls is immobilized on an insoluble matrix
  • the test sample is contacted with the immobilized antibody composition to adsorb all CT-ls
  • the immobilized CT-ls are contacted with a plurality of antibodies specific for each CT-l , each ofthe antibodies being ⁇ nd ⁇ v> ⁇ ally identifiable as specific for a predetermined CT-l, as by unique labels such as discrete fluorophores ur the like
  • unique labels such as discrete fluorophores ur the like
  • CT-l antibodies also are useful for the affinity purification of CT-l from recombinant cell culture or natural sources CT- 1 antibodies that do not detectably cross-react with the rat CT- 1 can purify CT- 1 free from such protein
  • Suitable diagnostic assays for CT-l and its antibodies are well known per se
  • the competitive and sandwich methods employ a phase-separation step as an integral part ofthe method, while stenc inhibition assays are conducted in a smgle reaction mixture Fundamentally, the same procedures are used for the assay of CT- 1 and for substances that bind CT- 1 , although certain methods will be favored depending upon the molecular weight ofthe substance being assayed Therefore, the substance to be tested
  • the label used (and this is also useful to label CT-l nucleic acid for use as a probe) is any detectable functionality that does not interfere with the binding of analyte and its binding partner
  • Numerous labels are known for use m immunoassay, examples including moieties that may be detected directly, such as fluorochrome, chemiluminscent and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected
  • examples of such labels include the radioisotopes P, C, I, H, and I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbeliiferone, luciferases, e g , firefly luciferase and bacterial luciferase (U S Patent No 4,737 456), lucife ⁇ n, 2,3-d ⁇ hydrophthalazmed ⁇ ones, malate dehydrogen
  • peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases, e g , glucose oxidase galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uncase and xanthine oxidase coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase. or microperoxidase, biotm/avidin, spin labels, bacteriophage labels, stable free radicals, and the like
  • HRP horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase glucoamylase
  • lysozyme saccharide oxidases
  • e g glucose
  • enzyme labels are a preferred embodiment No single enzyme is ideal for use as a label m every conceivable assay Instead, one must determine which enzyme is suitable for a particular assay system C ⁇ te ⁇ a important for the choice of enzymes are turnover number of the pure enzyme (the number of substrate molecules converted to product per enzyme site per unit of time), purity ofthe enzyme preparation, sensitivity of detection of its product, ease and speed of detection ofthe enzyme reaction absence of interfering factors or of enzyme-like activity in the test fluid, stability of the enzyme and its conjugate availability and cost of the enzyme and its conjugate, and the like Included among the enzymes used as preferred labels the assays ofthe present invention are alkaline phosphatase, HRP, beta-galactosidase
  • Immobilization of reagents is required for certain assay methods. Immobilization entails separating the binding partner from any anaiyte that remains free in solution. This conventionally is accomplished by either insolubilizing the binding partner or analyte analogue before the assay procedure, as by adso ⁇ tion to a water- insoluble matrix or surface (Bennich et ai. U.S. Patent No. 3,720,760), by covalent coupling (for example, using glutaraldehyde cross-linking), or by insolubilizing the partner or analogue afterward, e.g., by immunoprecipitation.
  • ком ⁇ онент rely on the ability of a tracer analogue to compete with the test sample analyte for a limited number of binding sites on a common binding partner.
  • the binding partner generally is insolubilized before or after the competition and then the tracer and analyte bound to the binding partner are separated from the unbound tracer and analyte. This separation is accomplished by decanting (where the binding partner was preinsolubilized) or by centrifuging (where the binding partner was precipitated after the competitive reaction).
  • the amount of test sample analyte is inversely proportional to the amount of bound tracer as measured by the amount of marker substance.
  • Dose-response curves with known amounts of analyte are prepared and compared with the test results to quantitatively determine the amount of analyte present in the test sample. These assays are called ELISA systems when enzymes are used as the detectable markers. Another species of competitive assay, called a "homogeneous" assay, does not require a phase separation.
  • a conjugate of an enzyme with the analyte is prepared and used such that when anti-analyte binds to the analyte, the presence of the anti-analyte modifies the enzyme activity.
  • CT-l or its immunologically active fragments are conjugated with a bifunctional organic bridge to an enzyme such as peroxidase. Conjugates are selected for use with anti-CT-1 so that binding of the anti-CT-1 inhibits or potentiates the enzyme activity ofthe label. This method per se is widely practiced under the name of EMIT.
  • Steric conjugates are used in steric hindrance methods for homogeneous assay. These conjugates are synthesized by covalently linking a low-molecular-weight hapten to a small analyte so that antibody to hapten substantially is unable to bind the conjugate at the same time as anti-analyte. Under this assay procedure the analyte present in the test sample will bind anti-analyte, thereby allowing anti-hapten to bind the conjugate, resulting in a change in the character ofthe conjugate hapten, e.g., a change in fluorescence when the hapten is a fluorophore.
  • Sandwich assays particularly are useful for the determination of CT- 1 or CT- 1 antibodies.
  • an immobilized binding partner is used to adsorb test sample analyte, the test sample is removed as by washing, the bound analyte is used to adsorb labeled binding partner, and bound material is then separated from residual tracer. The amount of bound tracer is directly proportional to test sample analyte.
  • sandwich assays the test sample is not separated before adding the labeled binding partner.
  • a sequential sandwich assay using an anti-CT-1 monoclonal antibody as one antibody and a polyclonal anti-CT- 1 antibody as the other is useful in testing samples for CT-l activity.
  • the foregoing are merely exemplary diagnostic assays for CT-l and antibodies. Other methods now or hereafter developed for the determination of these analytes are included within the scope hereof, including the bioassays described above.
  • the assay used for hypertrophy is an in vitro neonatal rat heart hypertrophy assay described in general as follows:
  • the preparation ofthe myocyte cell suspension is based on methods outlined in Chien et ai, J. Clin.
  • a different protocol has been developed in accordance with this invention for plating and culturing these cells to increase testing capacity with a miniaturized assay.
  • the wells of 96-well tissue-culture plates are precoated with D-MEM/F12/4% fetal calf serum for 8 hr at 37°C.
  • This medium is removed and the cell suspension is plated in the inner 60 wells at 7.5 x 10 cells per mL in D-MEM/F-12 supplemented with insulin, transferrin. and aprotinin.
  • the medium typically also contains an antibiotic such as penicillin/streptomycin and glutamine. This medium allows these cells to survive at this low plating density without serum. Test substances are added directly into the wells after the cells have been in culture for 24 hours.
  • neonatal rat myocardial cells in culture display several features ofthe in vivo cardiac muscle cell hypertrophy seen in congestive heart failure, including an increase in cell size and an increase in the assembly of an individual contractile protein into organized contractile units.
  • Chien et ai FASEB J.. supra. These changes can be viewed with an inverted phase microscope and the degree of hypertrophy scored with an arbitrary scale of 7 to 0, with 7 being fully hypertrophied cells and 3 being non-stimulated cells.
  • the 3 and 7 states may be seen in Simpson et ai, Circulation Research. J.: 787-801 (1982), Figure 2, A and B, respectively.
  • the myocytes are fixed and stained after the appropnate testmg penod with crystal violet stain in methanol Crystal violet is a commonly used protem stain for cultured ceils
  • an aliquot can be taken from the 96-well plates and monitored for the expression of protein markers ofthe response such as release of ANF or ANP B.
  • Embryoid bodies were generated by the differentiation of plu ⁇ potent embryonic stem (ES) cells (Doetschman et al , J. Embrvol Exp Mo ⁇ hol .
  • the embryonic stem cell line ES-D3 (ATCC No CRL 1934) was maintained in an undifferentiated state in a medium contammg LIF (Williams et al . Nature. 336 684-687 (1988)) This medium contamed D-MEM (high glucose), 1% glutamine, 0 1 mM 2-mercaptoethanol, penicillin-streptomycin, 15%heat- mactivated fetal bovme serum, and 15 ng/mL mouse LIF When these cells were put into suspension culture in the same medium without LIF and contammg 20% heat-inactivated fetal bov e serum (day 0), they aggregated and differentiated mto multicellular structures called embryoid bodies By day 8 of culture, beating primordial heart- like structures formed on a fraction ofthe bodies The embryoid bodies were evaluated for the production of CT-l activity by changing the differentiating ES cells to serum-free medium (D-MEM/F-12, 1% glutamine, pen
  • a cDNA library in the plasmid expression vector, pRK5B was prepared following a vector priming strategy (Strathdee et al , Nature.
  • the vector, pRK5B was linearized at the Noil site, treated with alkaline phosphatase, and ligated to the single- stranded oligonucleotide, ocdl 1.3, having the sequence 5'-GCGGCCGCGAGCTCGAATTCTTTTTTTTl 1 H I ITI 1 1 ITlTI ' l 1 1 H I (SEQ ID NO 5)
  • the ligated product was then cut with Z?5/XI, and the 4700-bp vector fragment was isolated by agarose gel electrophoresis
  • the vector was further purified by oligo dA chromatography
  • the expression library was constructed using 1 ⁇ g ofthe poly (A) RNA, 5 ⁇ g of vector primer, and reagents from Amersham Following first- and second-strand synthesis and T4 DNA polymerase fill-in reactions, the material was sized for inserts of greater than 500 bp by gel electrophoresis and circularized by blunt-end ligation without the addition of linkers The ligations were used to transform E colt strain DH5 ⁇ by electroporation From 1 ⁇ g of poly(A) + RNA, 499 ng of double-stranded cDN A were generated Seventeen nanograms of cDNA were ligated, and 3 3 ng were transformed to yield 780,000 clones.
  • the 3' untranslated region of clone pchf 781 contams a common mouse repeat known as bl (bp -895-1015) Hyb ⁇ dization of 7-day embryoid body poly(A) ⁇ RNA with a probe from clone pchf 781 shows a smgle band of - 1 4 kb, which is about the same size as the insert from the cDNA clones
  • the encoded sequence is not highly similar (> 35% ammo acid identity) to any known protem sequences in the Dayhoff database It does, however, show a low degree of similarity to a family of distantly related protems including CNTF, mter
  • Mouse CT- 1 has 24% ammo acid identity with mouse LIF (Rose and Todaro, WO 93/05169) and 21% amino acid identity with human CNTF (McDonald et ai , Biochim. Biophvs Acta. 1090 70-80 (1991)) See Figure 2 for an alignment of mouse CT-l and human CNTF sequences CNTF, IL-6, IL-1 1, LIF. and OSM use related receptor signaling proteins includmg gpl 30 that are members ofthe GH/cytokine receptor family (Kishimoto et al , £eJl, 76.
  • CNTF like CT- 1 , lacks an N-terminal secretion signal sequence C.
  • Identity and Activity of Clone To demonstrate that clone pchf 781 encodes a CT-l, expression studies were performed both by transfection of 293 cells and by utilizing a coupled tn vitro SP6 transcription translation system S-methionme and cysteme labeling of the proteins produced by pchf 781 -transfected 293 cells (in comparison with vector- transfected cells) showed that the conditioned medium contained a labeled protein of about 21 8 kDa, and that the cell extract showed a protem of 22 5 kDa Conditioned media from these transfections gave a mo ⁇ hology score of 6 when assayed for cardiac hypertrophy at a dilution of 1 4 using the assay described above Conditioned media from unlabeled transfections gave a mo ⁇ hology score of 5 5-6 5 at a dilution of 1 1
  • microtiter wells were coated with 100 ⁇ L of rat anti-human IgG antibody (2 ⁇ g/mL) overnight at 4° C After washmg with phosphate-buffered saline containing 0 5% bov e serum albumin, the wells were incubated with 100 ⁇ L of 3 ng/mL rat ANP receptor A-IgG (produced and purified in a manner analogous to the human ANP receptor A-lgG (Bennett et al , J. Biol Chem .
  • rat cardiac fibroblasts were cultured Conditioned medium from these primary cultures does have cardiac hypertrophy in the tn vitro neonatal rat heart hypertrophy assay herem Blot hybridization of rat fibroblast mRNA isolated from these cultures shows a clear band of 1 4 kb when probed with a coding region fragment of clone pchf 781 (Hyb ⁇ dization was performed 5 x SSC, 20% formamide at 42° C with a final wash in 0 2 x SSC at 50° C ) P. Purification of Factor
  • the culture medium conditioned by cells transfected with clone pchf 781 or a human clone is adjusted to 1 5 M NaCl and applied to a ToyopearlTM Butyl-650M column The column is washed with 10 mM TRIS- HCl, pH 7 5, 1 M NaCl.
  • the purified CT-l from Example I is tested m normal rats to observe its effect on cardiovascular parameters such as blood pressure, heart rate, systemic vascular resistance, contractility, force of heart beat concent ⁇ c or dilated hypertrophy, left ventricular systolic pressure, left ventricular mean pressure, left ventricular end-diasto c pressure, cardiac output, stroke index, histological parameters, ventricular size, wall thickness, etc
  • the purified CT-l is also tested in the pressure-overload mouse model wherein the pulmonary artery is constricted, resulting in right ventricular failure.
  • RV Murine Dysfunctional Model A retroviral murine model of ventricular dysfunction can be used to test the purified CT-l, and the dP/dt, ejection fraction, and volumes can be assayed with the hypertrophy assay described above. In this model, the pulmonary artery ofthe mouse is constricted so as to generate pulmonary hypertrophy and failure.
  • Transgenic mice that harbor a muscle actin promoter-IGF-I fusion gene display cardiac and skeletal muscle hypertrophy, without evidence of myopathy or heart failure. Further, IGF-I- gene-targeted mice display defects in cardiac myogenesis (as well as skeletal) including markedly decreased expression of ventricular muscle contractile protein genes. The purified CT-l is tested in these two models.
  • the purified CT-l is also tested in a post-myocardiai infarction rat model, which is predictive of human congestive heart failure in producing natriuretic peptide.
  • a post-myocardiai infarction rat model which is predictive of human congestive heart failure in producing natriuretic peptide.
  • male Sprague-Dawley rats (Charles River
  • Rats are fed a pelleted rat chow and water ad libitum and housed in a light- and temperature-controlled room.
  • Myocardial infarction is produced by left coronary arterial ligation as described by Greenen et ai, L Appl. Phvsiol.. 9 S: 92-96 (1987) and Buttrick et ai, Am. J. PhvsioL 260: 1 1473-1 1479 (1991 ).
  • the rats are anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally), intubated via tracheotomy, and ventilated by a respirator (Harvard Apparatus Model 683). After a left-sided thoracotomy, the left coronary artery is ligated approximately 2 mm from its origin with a 7-0 silk suture.
  • Electrocardiograms One week after surgery, electrocardiograms are obtained under light metofane anesthesia to document the development of infarcts.
  • the ligated rats of this study are subgrouped according to the depth and persistence of pathological Q waves across the precordial leads. Buttrick et ai, supra; Kloner et ai, Am. Heart J.. 5 .: 1009- 1013 (1983). This provides a gross estimate of infarct size and assures that large and small infarcts are not differently distributed in the ligated rats treated with CT- 1 or CT- 1 antagonist and vehicle. Confirmation is made by precise infarct size measurement.
  • CT-l or CT-l antagonist (10 ⁇ g/kg to 10 mg/kg twice a day for 15 days) or saline vehicle is injected subcutaneously in both ligated rats and sham controls. Body weight is measured twice a week during the treatment.
  • CT-l or CT-l antagonist is administered in saline or water as a vehicle.
  • rats are anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally).
  • a catheter (PE 10 fused with PE 50) filled with heparin-saline solution (50/U/mL) is implanted into the abdominal aorta through the right femoral artery for measurement of arterial pressure and heart rate.
  • a second catheter (PE 50) is implanted into the right atrium through the right jugular vein for measurement of right atrial pressure and for saline injection.
  • dP/dt left ventricular pressures and contractility
  • PE 50 is implanted into the left ventricle through the right carotid artery.
  • a thermistor catheter (Lyons Medical Instrument Co., Sylmar, CA) is inserted into the aortic arch.
  • the catheters are exteriorized at the back ofthe neck with the aid of a stainless-steel wire tunneled subcutaneously and then fixed. Following catheter implantation, all rats are housed individually.
  • MAP Mean arterial pressure
  • SAP systolic arterial pressure
  • HR heart rate
  • RAP right atrial pressure
  • LVSP left ventricular systolic pressure
  • LVMP left ventricular mean pressure
  • LVEDP left ventricular end-diastolic pressure
  • dP/dt left ventricular maximum
  • 0.1 mL of isotonic saline at room temperature is injected as a bolus via the jugular vein catheter.
  • the thermodilution curve is monitored by VR- 16 simultrace recorders (Honeywell Co., NY) and cardiac output (CO) is digitally obtained by the microcomputer.
  • 1 mL of blood is collected through the arterial catheter. Serum is separated and stored at -70° C for measurement of CT- 1 levels or various biochemical parameters if desired.
  • the rats are anesthetized with pentobarbital sodium (60 mg/kg) and the heart is arrested in diastole with intra-atrial injection of KCI ( 1 M).
  • the heart is removed, and the atria and great vessels are trimmed from the ventricle.
  • the ventricle is weighed and fixed in 10% buffered formalin.
  • the right ventricular free wall is dissected from the left ventricle
  • the left ventricle is cut in four transverse slices from apex to base
  • Five micrometer sections are cut and stained with Massons' t ⁇ chrome stain and mounted
  • the endocardial and epicardial circumferences ofthe mfarcted and non-mfarcted left ventricle are determmed with a planimeter Digital Image Analyzer
  • the mfarcted circumference and the left ventricular circumference of all four slices are summed separately for each of the epicardial and endocardial surfaces and the sums are expressed as a ratio of mfarcted circumference to left ventricular circumference for each surface These two ratios are then averaged and expressed as a percentage for infarct size 7.
  • Results are expressed as mean + SEM Two-way and one-way analysis of variance (ANOVA) is performed to assess differences in parameters among groups Significant differences are then subjected to post hoc analysis usmg the Newman-Keuls method p ⁇ 0 05 is considered significant 8 Results
  • the mean body weight before and after treatment with CT-l or CT-l antagonist or vehicle is not expected to be different among the experimental groups
  • Infarct size in ligated rats is not expected to differ between the vehicle-treated group and the CT-l- or CT-l- antagonist-treated group
  • CT-l or CT-l antagonist to the ligated rats in the doses set forth above would result in improved cardiac hypertrophy by increasing ventricular contractility and decreasing peripheral vascular resistance over that observed with the vehicle-treated sham and ligated rat controls
  • This expected result would demonstrate that administration of CT-l or CT-l antagonist improves cardiac function m congestive heart failure In sham rats, however, CT-l or CT-l antagonist administration at this dose is not expected to alter significantly cardiac function except possibly slightly lowe ⁇ ng arterial pressure and peripheral vascular resistance
  • the rat data herein may be extrapolated to horses, cows, humans, and other mammals, correcting for the body weight ofthe mammal in accordance with recognized veterinary and clinical procedures Usmg standard protocols and procedures, the veterinarian or clinician will be able to adjust the doses, scheduling, and mode of administration of CT-l or a CT-l antagonist to achieve maximal effects in the desired mammal being treated Humans are believed to respond in this manner as well EXAMPLE III
  • CT-l or CT-l antagonist at an initial dose of 10-150 ⁇ g/kg/day is proposed The dose would be adjusted downward for adverse effects If no beneficial effects and no limiting adverse effects are determined at the time of re-evaluation, the dose would be adjusted upward Concurrent medication doses (e g , captopril as an ACE inhibitor and diuretics) would be adjusted at the discretion of the study physician After the maximum dose is administered for 8 weeks, the CT-l or CT-l antagonist administration is stopped, and re-evaluation is performed after a similar time period off treatment (or a placebo) B Inclusion Criteria
  • DCM -Dilated cardiomyopathy
  • LV left ventncle
  • EDV LV end-diastolic dimension
  • valvular heart disease operable or not
  • specific treatable etiologies including alcohol, if abstinence has not been attempted
  • operable coronary artery disease -Exercise limited by chest pam or obstructive peripheral vascular disease
  • EXAMPLE IV Testing for in vitro Neurotrophic Activity An assay used for ciliary ganglion neurotrophic activity was performed as described m Leung, Neuron.
  • ciliary ganglia were dissected from E7-E8 chick embryos and dissociated in trypsin-EDTA (Gibco 15400-013) diluted ten fold in phosphate-buffered saline for 15 minutes at 37° C
  • the ganglia were washed free of trypsin with three washes of growth medium (high glucose D-MEM supplemented with 10% fetal bovme serum, 1 5 mM glutamine, 100 ⁇ g/mL penicillin, and 100 ⁇ g/mL strepomyci ⁇ ), and then gently tnturated in 1 L of growth medium into a smgle-cell suspension
  • Neurons were enriched by plating this cell mixture m 5 mL of growth media onto a 100-mm tissue culture dish for 4 hours at 37° C in a tissue culture incubator During this time the non-neuronal cells preferentially stuck to the dish and neurons were gently washed free at the end ofthe in
  • a source of mRNA encoding human CT- 1 (also known as human cardiotrophin- 1 (CT- 1 ]) was identified by screening poly(A)+RNA from several adult tissues with a probe from the mouse CT-l cDNA clones.
  • Heart, skeletal muscle, colon, ovary, and prostate showed a 1.8 kb band upon blot hybridization with a 180-bp mouse CT-l probe (extending from 19 bp 5' ofthe initiating ATG through amino acid 50) in 20% formamide, 5 X SSC at 42° C with a final wash at 0.25 X SSC at 52° C.
  • Clones encoding human CT- 1 were isolated by screening a human heart cDNA library (Clontech) with the same probe and conditions (final wash at 55° C).
  • the DNA sequence from clone h5 (SEQ ID NOS: 6 and 7 for the sense and anti-sense strands, respectively) is shown in Figure 5 and includes the whole coding region.
  • Clone h5 (pBSSK+.hu.CTl .h5) was deposited on July 26, 1994 in the American Type Culture Collection as ATCC No. 75,841.
  • the DNA sequence of another clone, designated h6, matches that of clone h5 in the region of overlap.
  • Clone h6 begins at base 47 of clone h5 and extends 3' of clone h5 for an additinal 521 bases.
  • the encoded protein sequence of human CT-l (SEQ ID NO: 8) is 79% identical with the mouse CT-l sequence (SEQ ID NO: 3), as evident from Figure 6, wherein the former is designated “hutnctl” and the latter is designated “chf.781.”
  • the mouse and human CT-l encoded by these clones have 80% amino acid identity and are about 200 amino acids in length corresponding to a calculated molecular mass of 21.5 kDA. Both human and mouse CT- 1 lack a conventional hydrophobic amino terminal secretion sequence, however, they are found in the medium of transfected mammalian cells. The coding regions of human and mouse CT-l are contained on three separate exons that span 6-7 kbp of genomic DNA. The human CT-l gene was localized by fluorescent in situ hybridization and by somatic cell hybridization to chromosome 16p 1 1.1 - p 11.2.
  • CT-l The expression pattern of mouse CT-l was determined by Northern blot analysis.
  • CT-l mRNA is widely (but not universally) expressed in adult mouse tissues including heart, kidney, skeletal muscle, and liver.
  • a single 1.4 kb CT-l mRNA species was detected in the adult mouse heart, skeletal muscle, liver, lung, and kidney.
  • Lower amounts of mRNA were seen in testis and brain, while no expression was observed in the spleen.
  • the CT-l transcript was also detected in seven-day embryoid body mRNA, which was the RNA used to prepare the cDNA expression library In a survey of human adult tissues ( Figure 20).
  • CT-l mRNA 1 7 kb mRNA
  • CT-l Like CNTF, CT-l lacks a conventional amino-terminal secretion signal sequence, it is. however, found in the medium of transfected mammalian cells
  • CT-l The predicted tertiary structure of CT-l is consistent with its containing four amphipathic helices that are features of a large number of cytokines and other proteins including growth hormone (For reference see Abdel-Meguid et al , Proc Natl Acad Sci USA, 84 6434-6437 (1987) and Bazan, Neuron, 1 197-208 (1991))
  • Human CT- l binds to the mouse LIF receptor.
  • human CT- 1 was expressed b> subclonmg the codmg region from plasmid pBSSK+ hu CT1 h.5, which contamed all ofthe cDNA protein codmg region, to give plasmid ⁇ RK5 hu CT1 Clarified conditioned medium was obtained from human 293 cells transfected with this plasmid and maintained in serum-free medium for four days
  • Bindmg to M 1 cells (ATCC TIB 192), Hela cells and WI-26 VA4 (ATCC CCL-95 1) cells was performed for 2 hours a 4 degrees C and analyzed as desc ⁇ bed herein
  • CM was concentrated 10 fold and added at a 3-fold dilution to the bmdmg assays
  • WI-26 bindmg the conditioned medium was used without concentration This conditioned medium competed for labeled human LIF (iodinated with IODO-B
  • CT-l does not bind the specific OSM Receptor Although oncostatin M binds and functions via the LIF receptor (Gearing et al (1992) New Biologist 4 61-65), but as shown herem CT-l is not a ligand for the OSM specific receptor, the oncostatin M receptor, which has been identified in and cloned from the human lung cell line WI-26 VA4 Both pu ⁇ fied mouse CT- 1 and the CM from 293 cells transfected with human CT- 1 cDNA failed to compete for labeled OSM binding ( Figure 19)
  • CT-l induces a distinct form of myocardial cell hypertrophy characterized bv sarcome ⁇ c assembly in series
  • the CT- 1 induced hypertrophic phenotype is distinct from the hypertrophic phenotype observed following G-protem dependent stimulation with ⁇ -adrenergic agonists (Knowlton et al (Journal of Biological Chemistry, 266 7759-7768 (1991), Knowlton et al, Journal of Biological Chemistry, 268 15374-15380 (1993), endothel ⁇ n-1 , Shubeita et al .
  • cardiomyocytes were dual-stained for thick ( ⁇ MHC) and thin (F-actin) myofilaments, and viewed by confocal laser microscopy (Messerli et ai, Histochemistry, 100: 193-202 (1993)). Cardiomyocytes stimulated with CT- 1 and LIF displayed a high degree of myofibrillar organization: myofibrils were organized in a strictly sarcomeric pattem, oriented along the longitudinal cell axis, and extended to the cell periphery.
  • gpl 30 dependent stimulation and ⁇ -adrenergic stimulation result in distinct patterns of embryonic gene, MLC-2v, and immediate early gene expression.
  • the reactivation of an embryonic pattem of gene expression is a central feature of cardiomyocyte hypertrophy (Chien et ai, Faseb J, 5:5037-3046
  • ANF and skeletal ⁇ -actin are abundantly expressed in the ventricular myocardium during embryonic development, but their expression is down-regulated shortly after birth. Stimulation of cardiomyocytes with CT-l or LIF induced prepro-ANF mRNA expression, and perinuclear accumulation and secretion of immunoreactive ANF. However, in contrast to ⁇ -adrenergic stimulation, CT-l and LIF did not induce skeletal ⁇ -actin expression.
  • G- protein coupled receptors including ⁇ -adrenergic agonists, endothelin-1, and angiotensin 11, induce ANF and skeletal ⁇ -actin in a coordinate fashion (Knowlton et ai, Journal of Biological Chemistry, 266:7759-7768 (1991); Bishopric et ai. Journal of Clinical Investigation. 80:1194-1 199 (1987); Sadoshima et ai, Circ. Res., 73:413-423 ( 1993)).
  • Human IL-6 was from Genzyme
  • mouse LIF was from R & D Systems and Genentech manufacturing
  • rat CNTF and GDNF Pouisen et ai, Neuron, 13: 1245-1252 (1994) were produced by Genentech.
  • Mouse CT- l was expressed and purified as a fusion protein as described. This protein results in a 34 amino acid N- terminal extension that encodes a portion ofthe he ⁇ es simplex virus glycoprotein D and a factor Xa cleavage site. In some cases an alternative fusion protein was used that substitutes a different site for the Factor Xa cleavage site giving the amino acid sequence . . .
  • newborn rat sympathetic neurons were prepared as described (Hawrot et al, Meth. Enzymoi, 58:574-583 (1979)). Superior cervical ganglia were dissociated with trypsin (0.08%) and plated in serum free F- 12 medium containing nerve growth factor and additives as described (Davies et ai. Neuron, 1 1 :565-574 (1993)). Neurons were plated at 30,000 per well in 24 well plates precoated with poly-ornithine and ECL cell attachment matrix (Promega) and allowed to grow for ten days in the presence of indicated factors.
  • Tyrosine hydroxylase and choline acetyltransferase activities were assayed as described (Reinhard et ai. Life Sci. , 39:2185-2189 ( 1986); Fonnum, Biochem. J. , 1 15:465-472 ( 1969)).
  • rat dopaminergic neurons The survival of rat dopaminergic neurons was assayed as described (Poulsen et ai, Neuron. 13: 1245- 1252 (1994)). Ciliary neuron survival assays were performed with neurons isolated from E8 chick embryos as described (Manthorpe et al, (Rush, R., eds.) Vol. pp. 31-56, John Wiley & Sons (1989)). Survival was assessed by counting live neurons after staining with the vital dye MTT (Mosmann, J. Immunol. Meth., 65:55-63 (1983)). The data were fit to the four parameter equation described above.
  • ES.D3 Gossler et ai, Proc. Natl. Acad. Sci. USA, 83:9065-9069 (1986)
  • DMEM high glucose, no sodium pyruvate
  • HEPES high glucose, no sodium pyruvate
  • Cells were trypsinized, plated in duplicate at 1000 cells per well in 24-well tissue culture plates in the above culture medium with or without LIF or CT- 1 , and scored 9 days later. No change in colony numbers was observed except in the no addition group where the cells had flattened and differentiated. Cell binding and cross-linkine. Binding was performed in RPMI- 1640 containing 0.1 % bovine serum albumin with 7.5-10 million Ml cells (TIB 192, ATCC) in a volume of 250 ⁇ l for 2 h on ice with shaking.
  • TIB 192, ATCC 7.5-10 million Ml cells
  • Anti-gpl30 antibody inhibition experiments were performed with a rat anti-mouse gpl30 monoclonal antibody (RX435) 2 or a rat anti-gpl20 control antibody (Genentech 6D8.1 E9) in a volume of 150 ⁇ l. Reactions were incubated on ice 2 h. centrifuged at 12,500 ⁇ m, and washed with 1 ml of cold phosphate buffered saline contammg 0.1 % albumin. The data were fit to the four parameter equation described above.
  • Binding to neonatal rat cardiac myocytes was performed as for M 1 cells, but cells isolated as described herein and plated for 16 h. Assays were performed with 1 million cells in a volume of 100 ⁇ l.
  • Cross-linking was performed with 10 million M l cells in phosphate buffered saline containing 0.1% albumin. 7.2 nM l25 I-mouse CT-l or 2.2 nM ,25 I-mouse LIF, with or without a 100 fold molar excess ofthe unlabeled ligands in a volume of 250 ⁇ l. After 1 h at room temperature, 10 mM l -ethyl-3-(3- dimethylaminopropyljcarbodiimide hydrochloride (EDC) and 5 mM /V-hydroxysulfosuccinimide (sulfo-NHS) (Pierce) were added and the incubation continued for 30 min at room temperature.
  • EDC dimethylaminopropyljcarbodiimide hydrochloride
  • sulfo-NHS 5 mM /V-hydroxysulfosuccinimide
  • binding reactions contained 10 mM Tris-HCl buffer (pH 7.5), 100 mM KCI, 5 mM MgCl 2 , 1 mM DTT, 6.7 % glycerol, 0.067 g 1 P oly(dIdC)(dIdC), 0.5 ng (25,000 cpm) "P-SIE DNA (5'- CTAGAGTCGACATTTCCCGTAAATCT and 5'-CTAGAGATTTACGGGAAATGTCGACT, high affinity m67 (Sadowski et ai.
  • DNA encoding the extracellular domain of the mouse LIF receptor (ammo acids 1-826) and mouse gpl 30 (1-617) was generated by PCR of M l cell (above) mRNA and of a mouse lung cDNA library (Clontech). These sequences were cloned with a C-terminal tag encoding 6 histidine residues in the mammalian expression vector, pRK5 (Suva et ai, Science, 237:893-896 (1987)) to give the piasmids, pRK5.mu.slifr and pRK5.mu.sgpl30.
  • DNA sequencing of the coding regions confirmed that these plasmids encode proteins that match the published amino acid sequence (Tomida et ai, J Biochem 1 15 557-562 (1994), Saito et al J Immunol . 148 4066-4071 ( 1992)), with the exception of the substitution of lysine for arginine at ammo acid 326 of gpl 30, a change that was found for three fragments from both sources
  • the plasmids were transfected into human 293 cells, and the proteins isolated from 4-day conditioned medium by N ⁇ "-NTA-agarose (Qiagen) affinity purification B ⁇ efly, the conditioned medium was concentrated ⁇ 18 fold (Cent ⁇ prep 10, Amicon), and the tagged protem purified by binding to the N ⁇ resin for 2 h at room temperature Following two washes with phosphate buffer saline containing 5 mM imidazole, the protems were eluted with phosphate buffer salme contammg 200
  • Binding to the soluble LIF receptor and soluble gpl 30 was performed in a manner similar to that previously descnbed (Layton et al , J Biol Chem , 269 17048-17055 (1994)) B ⁇ efly, assays were performed 96-well Multiscreen-HV filtration plates with 045 ⁇ m PVDF membranes (Millipore) in phosphate buffered salme contammg 0 1 % bovme serum albumin and mcludmg 25 ⁇ l of phosphate buffer saline-washed N ⁇ -NTA- Agarose (Qiagen) in a final volume of 175 ⁇ l Plates were incubated at room temperature overnight with agitation Following vacuum filtration and one wash with 200 ⁇ l of cold phosphate buffer saline, the individual assay wells were cut from the plate and counted The data were analyzed as desc ⁇ bed above for M 1 binding RESULTS
  • IL-6 promotes the proliferation and differentiation of B cells into antibody producing cells following antigen stimulation (Akira et al , Adv Immunol , 54 1 -78 (1993))
  • CT-l was tested on the mouse hybridoma cell line, B9 (Aarden et al , Eur J Immunol , 17 141 1-1416 (1987))
  • IL-6 stimulates the proliferation of B9 cells as indicated by an increase in 3H-thym ⁇ d ⁇ ne inco ⁇ oration
  • CT-l and LIF were inactive ( Figure 7A) even at concentrations as high as 2 uM (data not shown)
  • CT-l does not mimic the activity of IL-6 in promoting B cell expansion While IL-6 stimulates the growth of several B cell lymphomas.
  • CT-l did share at least some of the growth inhibitory activities of the IL-6 family cytokines
  • LIF and CNTF can mduce a switch m the transmitter phenotype of sympathetic neurons from noradrenergic to cholmergic, a change that is accompanied by the induction of several neuropeptides mcludmg substance P, somatostatin, and vasoactive intestinal polypeptide (Rao, J Neurobiol , 24-215-232 (1992))
  • the ability of CT-l to induce this switch in the transmitter phenotype was determmed with cultured rat sympathetic neurons CT-l inhibited the tyrosine hydroxylase activity (a noradrenergic marker) and stimulated somewhat the choline acetyltransferase activity (a cholmergic marker) ofthese cells, effects that paralleled the actions of LIF (Fig 8 A)
  • Parkinson's disease is caused by the degeneration of dopammergic neurons ofthe midbrain (Hirsch et al , Nature, 334 345-348 (1988)) While protems ofthe neurotro ' phin family (bram-de ⁇ ved neurotrophic factor and neurotroph ⁇ n-4/5) as well as ofthe TGF- ⁇ family (GDNF, TGF- ⁇ 2 and TGF- ⁇ 3) promote the survival of cultured dopammergic neurons (Poulsen et al , Neuron, 13 1245- 1252 ( 1994)) many other growth factors and cytokmes. mcludmg CNTF, do not Unlike CNTF, CT-l was found to promote the survival of rat dopammergic neurons, although it was not as potent as GDNF (Fig 8B)
  • CT-l While inactive on dopaminergic neurons, CNTF does promotes the survival of ciliary neurons (Ip et al , Prog Growth Factor Res , 4 139-155 (1992)) CT-l was tested for its activity in promoting the survival of chick ciliary neurons (Fig 8C) While at maximal concentrations, CT-l was as active as CNTF, the potency of CT-l in promotmg ciiiary neuron survival was about 1000 fold less than that of CNTF (Fig 8C) Thus, CT-l shares some neuronal activities with the IL-6 family cytokines such as CNTF
  • Embryonic development assay The presence or absence of soluble factors plays a key role during embryonic and fetal development
  • embryonic stem cells require the continuous presence of soluble factors secreted by fibroblasts to maintain their undifferentiated, plu ⁇ potent phenotype LIF (Williams et al Nature 336 688-690 ( 1988), Smith et al , Nature, 336 688-690 ( 1988)), CNTF (Conover et al , Development, 119.559-565 (1993)), and OSM (Rose et al , Cytokine.6.48-54 (1994)) ⁇ but not IL-6 without the soluble IL-6 receptor (Yoshida et al .
  • CT-l is active in assays where LIF is active and vice versa
  • these assays systems can be used to screen for and identify CT-l agonists and antagonists useful for treating disorders dependent upon or resultinf from the biological activity (or loss reduction or ove ⁇ roducction ofthe activity) demonstrated these assays
  • CT-l is active in assays where CNTF is active, but that the converse is not always the case, and that CT- 1 is inactive in IL-6 specific assays, assays m which LIF is
  • CT-l bmdmg to Ml cells
  • LIF bindmg has been previously characterized (Hilton et al , Proc Natl Acad Sci USA, 85 5971-5975 (1988)) Both CT-l and LIF inhibit the growth of this cell line (see above) Labeled CT-l was specifically bound to Ml cells (Fig 10A), and this binding was completely competed by unlabeled LIF (Fig 10B) Similarly, labeled LIF bmdmg was competed by both unlabeled LIF and CT-l (Fig IOC and 10D) These data suggest that CT- 1 and LIF bind to the same receptor on M 1 cells Scatchard analysis yields a single class of binding sites in ail cases, the binding parameters are similar regardless ofthe labeled l ⁇ gand ⁇ K d (CT-l] - 0 7 nM, K d [LIF] ⁇ 0
  • CT-l and LIF were bound to Ml cells, chemically cross-linked, and the solubilized proteins analyzed by SDS gel electrophoresis (Fig 1 1) Both ligands gave one specific band with a mobility of- 200 kDa. and in both cases this cross-linked band was competed by either unlabeled ligand
  • CT-l and LIF interact with a protein of the same size on the surface of Ml cells, this protein has a mobility expected for the LIF receptor (Davis et ai , Science, 260-1805-1808 (1993); Gearing et ai , EMBOJ , 10 2839-2848 (1991))
  • CT-l induction of DNA bmdmg activity in Ml cells To show that CT- 1 induces intracellular signaling events like those found for other cytokmes that signal via gpl30 (Yin et al , Exp Hematol , 22 467-472 ( 1994) Narazaki et al . Proc Natl Acad USA, 91 2285-2289 (1994), Zhong et al . Science. 264 95-98 ( 1994), Akira et ai .
  • CT- 1 bmdmg to cardiac mvocvtes The bmdmg of labeled CT-l and LIF was also determined for rat cardiac myocytes, the cells used for the original assay and isolation of CT-l Both ligands specifically bound and cross-competed for binding to these cells (Fig 13A and 13B), as was the case for M 1 cells.
  • LIF binds directly to the LIF binding protein, a naturally occurring soluble form of the LIF receptor (K d ⁇ 2 nM for the mouse proteins) (Layton et al , J Biol Chem , 269 17048-17055 (1994) Layton et al , Proc Natl Acad Sci USA, 89 8616-8620 (1992))
  • the soluble mouse LIF receptor and gpl30 were expressed in mammalian cells, purified by N ⁇ chelate chromatography, and judged to be at least 90 % pure by SDS gel electrophoresis (data not shown).
  • CT-l is more potent than LIF in inhibiting the growth ofthe myeioid leukemia cell line, Ml, it mduces a phenotypic switch sympathetic neutrons, it promotes the survival of dopaminergic neurons from the central nervous system and ciliary neurons from the periphery, and it maintains the undifferentiated phenotype of embryonic stem cells
  • CT-l and LIF share a common activity profile— both inhibit the growth of M l cells, induce the switch in sympathetic neuron phenotype, inhibit the differentiation of embryonic stem cells, and mduce cardiac myocyte hypertrophy
  • CT-l is active in assays where CNTF is active- both induce the switch in sympathetic neuron phenotype (Saadat et ai , J Cell
  • the receptors for cytokines ofthe IL-6 family are composed of related subunits some of which are cytokine specific and some of which are shared (Davis et al Curr Opin Cell Biol.. 5:281-285 (1993); Stahl et ai. Cell, 74:587-590 (1993); Kishimoto et ai. Cell. 76:253-262 ( 1994); Hilton et al., EMBO J., 13:4765-4775 (1994)). All the receptors in this family have in common the transmembrane signaling subunit, gpl30.
  • the binding of IL-6 to the 80 kDa IL-6 receptor ⁇ subunit leads to the dimerization of gpl 30 as the first step in signal transduction.
  • the binding of IL-1 1 to the IL-1 1 receptor also leads to gpl 30 dimerization.
  • LIF, OSM. and CNTF induce the heterodimeriztion of gpl 30 and with another signaling subunit, the LIF receptor.
  • LIF and OSM bind directly to the LIF receptor or gpl30 and induce dimerization without a ligand-specific ⁇ subunit, while CNTF binds first to the GPI-linked CNTF receptor.
  • the ligand-induced dimerization of gpl30 or gpl30 and LIF receptor leads to the tyrosine phosphorylation and activation of associated tyrosine kinases ofthe Jak family (Jakl. Jak2, and Tyk2) followed by the activation of transcription factors ofthe STAT family (STAT1 and STAT3) (L ⁇ tticken et al. Science, 263:89-92 (1994); Stahl et al, Science, 263:92-95 (1994); Yin et al, Exp. Hematol. 22:467-472 (1994); Narazaki et ai, Proc. Natl Acad. USA, 91:2285-2289 (1994); Zhong et al.
  • cross-linking data show that CT-l and LIF specifically interact with a protein of - 200 kDa, a protein about the size expected for the LIF receptor (Davis et ai, Science, 260:1805-1808 (1993); Gearing et ai. EMBOJ.. 10:2839-2848 (1991)).
  • an anti-gpl30 monoclonal antibody specifically inhibits the binding of labeled CT-l to M l cells, showing that gpl30 is a component of the CT- l receptor complex.
  • CT-l induces the activation of a DNA binding activity, an intracellular signaling event induced by LIF and other members ofthe IL-6 cytokine family in the course of activation of the Jak/STAT pathway (Lutticken et al. Science, 263:89-92 (1994); Yin et al, Exp. Hematol. 22:467-472 (1994); Zhong et ai, Science. 264:95-98 (1994); Akira et al.. Cell, 77:63-71 (1994)).
  • CT-l and LIF also cross-compete for binding to rat cardiac myocytes. This finding is consistent with the hypothesis that these two ligands act on these cells via the LIF receptor, as established herein for M 1 cells.
  • LIF and OSM induce the heterodimerization of the same receptor subunits.
  • LIF receptor and gpl 30 the affinity ofthese two ligands for the individual receptor components differs.
  • LIF binds to the LIF receptor (K d ⁇ 2 nM (Gearing et al, EMBO J., 10:2839-2848 (1991)) but does not interact with gpl30 in the absence of the LIF receptor.
  • OSM binds to gpl30 (K d - 1 nM (Liu et al, J. Biol.
  • CT-l binds to the soluble LIF receptor with about the same affinity as LIF (K d - 2 nM, for the mouse proteins) and in the absence of other proteins. CT-l does not bind to soluble mouse g ⁇ l30 even at high concentrations.
  • soluble gpl 30 does increase the binding of CT-l to the soluble LIF receptor, however. presumably by the formation of a heterotrimeric complex.
  • concentration of soluble gp 130 required for this effect ( ⁇ 100 nM), while high by solution binding standards, is readily attainable on the surface of a cell. For example. 500 molecules of gpl30 expressed on the surface of a cell of 10 ⁇ m diameter would have an effective concentration of- 300 nM in a 100 A shell su wnding the cell, see (Ward et at, J. Biol. Chem.. 269:23286- 23289 (1994)).
  • CT-l binds to the LIF receptor in the same manner as LIF, by first binding with low affinity to the LIF receptor subunit, an interaction that does not require additional components, and second by recruiting gpl 30 to form a high affinity signaling complex.
  • CT-l was isolated based on its ability to induce cardiac myocyte hypertrophy, it clearly has a much wider range of activities, as is found for the other cytokines ofthe IL-6 family (Kishimoto et at. Science.
  • CT- 1 will mimic the many effects of LIF in vitro and in vivo
  • AGGCCATTCA CCTTAGGACC TTCCCACCAG TTCCTTTGTA GGCAAATCCC 1200
  • AAGAGCAAAA CAGAGAGAGG TCAGAACTTG TGAAAACAGA GGCTCTCCAG 850 AGAAAAACAA AGGAACAGAG AACCAAGAAA GAAACGAACG AACGAACGAA 900
  • GCCACCGCCT CAGCCGCCTC CGCCACCGGG GTCTTCCCCG CCAAGGTGCT 550

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Abstract

CT-1 isolée, ADN isolé codant cette CT-1 et méthodes d'élaboration par recombinaison ou par synthèse de cette CT-1. La CT-1 est connue pour se lier au récepteur LIFRβ et pour l'activer. Ces molécules CT-1 sont connues pour exercer une influence sur l'activité hypertrophique, l'activité neurologique et d'autres activités liées au récepteur LIFRβ. En conséquence, ces composés ou leurs antagonistes peuvent être utilisés pour le traitement de l'insuffisance cardiaque, des troubles arythmiques, inotropes et neurologiques ou autres associés au récepteur LIFRβ.
PCT/US1997/002675 1996-02-14 1997-02-11 Cardiotrophine et ses utilisations WO1997030146A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043790A2 (fr) * 1999-01-21 2000-07-27 Genentech, Inc. Compositions et procedes de traitement de tumeurs
US6472585B1 (en) 1994-04-25 2002-10-29 Genentech, Inc. Cardiotrophin-1 defective mouse
WO2005054449A1 (fr) * 2003-11-26 2005-06-16 Applied Research Systems Ars Holding N.V. Utilisation de cytokines du type il-6 pour la maturation d'oocytes
US7258983B2 (en) 1994-04-25 2007-08-21 Genentech, Inc. Cardiotrophin-1 compositions and methods for the treatment of tumor
US9962427B2 (en) 2016-07-11 2018-05-08 Ottawa Hospital Research Institute Treatment of right ventricular dysfunction due to pressure overload

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EP1856147A4 (fr) * 2005-02-15 2008-07-02 Apollo Life Sciences Ltd Molécule et molécules chimères de celle-ci
JP6754966B2 (ja) * 2016-02-25 2020-09-16 国立大学法人広島大学 始原生殖細胞の培養方法及び始原生殖細胞の培養用培地添加物

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WO1995029237A1 (fr) * 1994-04-25 1995-11-02 Genentech, Inc. Cardiotrophine et ses utilisations

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* Cited by examiner, † Cited by third party
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WO1995029237A1 (fr) * 1994-04-25 1995-11-02 Genentech, Inc. Cardiotrophine et ses utilisations

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7258983B2 (en) 1994-04-25 2007-08-21 Genentech, Inc. Cardiotrophin-1 compositions and methods for the treatment of tumor
US6472585B1 (en) 1994-04-25 2002-10-29 Genentech, Inc. Cardiotrophin-1 defective mouse
WO2000043790A3 (fr) * 1999-01-21 2000-11-30 Genentech Inc Compositions et procedes de traitement de tumeurs
JP2002534987A (ja) * 1999-01-21 2002-10-22 ジェネンテック・インコーポレーテッド 腫瘍の治療のための組成物と方法
AU776063B2 (en) * 1999-01-21 2004-08-26 Genentech Inc. Compositions and methods for the treatment of tumor
AU776063C (en) * 1999-01-21 2005-05-19 Genentech Inc. Compositions and methods for the treatment of tumor
WO2000043790A2 (fr) * 1999-01-21 2000-07-27 Genentech, Inc. Compositions et procedes de traitement de tumeurs
WO2005054449A1 (fr) * 2003-11-26 2005-06-16 Applied Research Systems Ars Holding N.V. Utilisation de cytokines du type il-6 pour la maturation d'oocytes
EP2325201A1 (fr) 2003-11-26 2011-05-25 Merck Serono SA Usage de cytokines du type il-6 pour la maturation des oocytes
US8071375B2 (en) 2003-11-26 2011-12-06 Merck Serono Sa Use of IL-6-type cytokines for maturation of oocytes
EP2325201B1 (fr) 2003-11-26 2016-11-09 Merck Serono SA Usage de cytokines du type il-6 pour la maturation des oocytes
NO341221B1 (no) * 2003-11-26 2017-09-18 Merck Serono Sa Anvendelse av cytokiner av IL-6-typen for modning av oocytter
US9962427B2 (en) 2016-07-11 2018-05-08 Ottawa Hospital Research Institute Treatment of right ventricular dysfunction due to pressure overload

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