US20030125519A1 - Ligand for the c-kit receptor and methods of use thereof - Google Patents

Ligand for the c-kit receptor and methods of use thereof Download PDF

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US20030125519A1
US20030125519A1 US07/594,306 US59430690A US2003125519A1 US 20030125519 A1 US20030125519 A1 US 20030125519A1 US 59430690 A US59430690 A US 59430690A US 2003125519 A1 US2003125519 A1 US 2003125519A1
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kit
kit ligand
polypeptide
cells
patient
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Peter Besmer
Karl Nocka
Jochen Buck
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Memorial Sloan Kettering Cancer Center
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Priority to US07/594,306 priority Critical patent/US20030125519A1/en
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Assigned to SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH, A CORP. OF NY reassignment SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH, A CORP. OF NY ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: BESMER, PETER, NOCKA, KARL, BUCK, JOCHEN
Priority to AU85106/91A priority patent/AU654502B2/en
Priority to JP3515137A priority patent/JPH06504185A/ja
Priority to PCT/US1991/006130 priority patent/WO1992003459A1/en
Priority to CA002090469A priority patent/CA2090469A1/en
Priority to EP91916054A priority patent/EP0546054A1/en
Priority to HU9300541A priority patent/HUT64368A/hu
Priority to KR1019930700578A priority patent/KR930703339A/ko
Priority to US08/341,456 priority patent/US5767074A/en
Priority to US08/478,414 priority patent/US5935565A/en
Priority to US09/371,261 priority patent/US6403559B1/en
Priority to US10/132,345 priority patent/US20030103937A1/en
Publication of US20030125519A1 publication Critical patent/US20030125519A1/en
Priority to US10/988,476 priority patent/US20050276784A1/en
Priority to US11/890,212 priority patent/US20080152652A1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: SLOAN-KETTERING INSTITUTE FOR CANCER R
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Definitions

  • the c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor for an unidentified ligand and is a member of the colony stimulating factor-1 (CSF-1)—platelet-derived growth factor (PDGF)—kit receptor subfamily (Besmer et al., 1986, Qiu et al., 1988; Yarden et al., 1987; Majumder et al., 1988). c-kit was recently shown to be allelic with the white-spotting (W) locus of the mouse (Chabot et al., 1988; Geissler et al., 1988; Nocka et al., 1989).
  • CSF-1 colony stimulating factor-1
  • PDGF platelet-derived growth factor
  • W white-spotting
  • Mutations at the W locus affect proliferation and/or migration and differentiation of germ cells, pigment cells and distinct cell populations of the hematopoietic system during development and in adult life (Russell, 1979; Silvers, 1979).
  • the effects on hematopoiesis are on the erythroid and mast cell lineages as well as on stem cells, resulting in a macrocytic anemia which is lethal for homozygotes of the most severe W alleles (Russell, 1970), and a complete absence of connective tissue and mucosal mast cells (Kitamura and Go, 1978).
  • W mutations exert their effects in a cell autonomous manner (Mayer and Green, 1968; Russell, 1970), and in agreement with this property, c-kit RNA transcripts were shown to be expressed in targets of W mutations (Nocka et al., 1989). High levels of c-kit RNA transcripts were found in primary bone marrow derived mast cells and mast cell lines. Somewhat lower levels were found in melanocytes and erythroid cell lines.
  • BMMC bone marrow
  • IL-3 interleukin 3
  • MMC gastrointestinal mucosa
  • CTMC peritoneal cavity
  • the interleukins IL-3 and IL-4 are well characterized hematopoietic growth factors which are produced by activated T-cells and by activated mast cells (Yung et al., 1981; Schrader, 1981; Smith and Rennick, 1986; Brown et al., 1987; Plaut et al., 1989).
  • An additional mast cell growth factor has been predicted which is produced by fibroblasts (Levi-Schaffer et al., 1985).
  • BMMC and CTMC derived from the peritoneal cavity can be maintained by co-culture with 3T3 fibroblasts (Levi-Schaffer et al., 1986; Dayton et al., 1988).
  • BMMC from W/W V mice as well as mice homozygous for a number of other W alleles are unable to proliferate in the fibroblast co-culture system in the absence of IL-3 (Fujita et al., 1988; Tan et al., 1990; Nocka et al., 1990).
  • a short term mast cell proliferation assay was developed which means to purify a fibroblast derived activity (designated KL) which, in the absence of IL-3, supports the proliferation of normal BMMC's and peritoneal mast cells, but not W/W V BMMC's .
  • KL was shown to facilitate the formation of erythroid bursts (BFU-E).
  • KL The biological properties of KL are in agreement with those expected of the c-kit ligand with regard to mast cell biology and aspects of erythropoiesis.
  • the defect W mutations exert is cell autonomous; in agreement with this property, there is evidence for c-kit RNA expression in cellular targets of W mutations (Nocka et al., 1989; Orr-Urtreger et al., 1990).
  • SIISI homozygotes are deficient in germ cells, are devoid of coat pigment, and die perinatally of macrocytic anemia (Bennett, 1956; Sarvella and Russell, 1956). Mice homozygous for the Sl allele, although viable, have severe macrocytic anemia, lack coat pigment, and are sterile. Both SII + and Sl d /+heterozygotes have a diluted coat color and a moderate macrocytic anemia but are fertile, although their gonads are reduced in size.
  • Sl mutations are not cell autonomous and are thought to be caused by a defect in the micro-environment of the targets of these mutations (Mayer and Green, 1968; McCulloch et al., 1965; Dexter and Moore, 1977). Because of the parallel and complementary characteristics of mice carrying Sl and W mutations, it had been hypothesized that the Sl gene product is the ligand of the c-kit receptor (Russell, 1979; Chabot et al., 1988).
  • This invention provides an isolated nucleic acid molecule which encodes an amino acid sequence corresponding to a c-kit ligand (KL) and a purified c-kit ligand (KL) polypeptide.
  • a pharmaceutical compositions which comprise the c-kit ligand (KL) purified by applicants or produced by applicants' recombinant methods and a pharmaceutically acceptable carrier is further provided as well as methods of treating patients which comprise administering to the patient the pharmaceutical compositions of this invention.
  • KL c-kit ligand
  • FIG. 1 Proliferative response of +/+ and W/W V BMMC to fibroblast conditioned medium and IL-3.
  • Mast cells derived from +/+ or W/W V bone marrow were cultured in the presence of 1% 3 CM, 10% FCM (20 ⁇ concentrated), or medium alone. Incorporation of 3 H-thymidine was determined from 24-30 hours of culture.
  • FIG. 2 Chromatographic profiles of the purification of KL.
  • the NaCl gradient is indicated by a dotted line.
  • the 1-propanol gradient is indicated by a dotted line.
  • FIG. 3 Electrophoretic analysis of KL. Material from individual fractions was separated by SDS/PAGE (12%) and stained with silver. The position of KL (28-30 kD) is indicated by an arrow. KL activity of corresponding fractions is shown below.
  • FIG. 4 Proliferation of W* mutant mast cells in response to KL.
  • Mast cells were derived from individual fetal livers from W/+ ⁇ W/+ mating, or bone marrow of wildtype, W V and W 41 heterozygotes and homozygoses. The proliferation characteristics of mutant mast cells was determined by using increasing concentrations of KL in a proliferation assay. Homozygous mutant mast cells are indicated by a solid line, heterozygotes mutant mast cells by a broken line and wildtype mast cells by a dotted line, except for W where normal fetuses may be either +/+ or W/+.
  • FIG. 5 Comparison of c-kit expression and growth factor responsiveness in BMMC and peritoneal mast cells (CTMC/PMC).
  • Anti-c-kit serum is indicated by a solid line and non-immune control serum by a dotted line.
  • C Determination of the proliferation potential of PMC to KL. 5000 cells were plated in 0.5 ml, in the presence of 1000 U/ml of KL, 10% Wehi-3CM or RPMI-C alone and the number of viable cells was determined two weeks later.
  • FIG. 6 Determination of burst promoting activity of KL. Bone marrow and spleen cells were plated in the presence of erythropoietin (2U/ml) and pure KL was added at the concentrations shown. The number of BFU-E was determined on day 7 of culture. This data represents the mean of two separate experiments, each with two replicates per concentration of KL.
  • FIG. 7 Determination of KL dependent BFU-E formation from W/W fetal livers. Fetuses from mating W/+animals were collected at day 16.5 of gestation. One fetus out of four was a W/W homozygote. Liver cells were plated at 10 5 cells/ml in the presence of either control medium, IL-3 (50 U/ml) or KL (2.5 ng/ml). All cultures contained erythropoietin (2U/ml). Data is expressed as the number of BFU-E/liver and is the mean of 2 replicate plates. The data for +/+ or W/+fetuses is the mean from the three normal fetuses in the liver.
  • FIG. 8 N-terminal amino acid sequence of KL and deduction of the corresponding nucleic acid sequence by PCR.
  • Top line N-terminal amino acid sequence (residues 10-36) of KL.
  • Middle Line Nucleotide sequences of three cDNAs obtained by cloning the 101 bp PCR product (see FIG. 10) into M13 and subsequent sequence determination.
  • Bottom Line sequences of the degenerate sense and antisense primers used for first-strand cDNA synthesis and PCR. The amino acid sequence also is identified as SEQ ID:NO: 2.
  • FIG. 9 Northern blot analysis using the PCR generated oligonucleotide probes corresponding to the isolated c-kit ligand polypeptide. A 6.5 kb mRNA was isolated with labelled probes.
  • FIG. 10 Derivation of cDNAs corresponding to the N-terminal amino acids 10-36 of KL by RT-PCR.
  • One microgram of poly(A) + RNA from BALB/c 3T3 cells was used as template for cDNA synthesis and subsequent PCR amplification in combination with the two degenerate oligonucleotide primers. Electrophoretic analysis of the 101 bp PCR product in agarose is shown.
  • FIG. 11 Nucleotide Sequence and Predicted Amino Acid Sequence of the 1.4 kb KL cDNA clone.
  • the predicted amino acid sequence of the long open reading frame is shown above and the nucleotide sequence using the single-letter amino acid code.
  • the numbers at right refer to amino acids, with methionine (nucleotides 16-18) being number 1.
  • the potential N-terminal signal sequence (SP) and the transmembrane domain (TMS) are indicated with dashed lines above the sequence, and cysteine residues in the extracellular domain are circled.
  • SP N-terminal signal sequence
  • TMS transmembrane domain
  • a schematic of the predicted protein structure is indicated below. N-linked glycosylation sites and the location of the N-terminal peptide sequence (Pep. Seq.) are indicated.
  • the nucleic acid sequence is also identified as SEQ ID:NO: 1.
  • FIG. 12 Identification of KL-Specific RNA Transcripts in BALB/c 3T3 Cell RNA by Northern Blot Analysis. Poly(A) + RNA (4 ⁇ g) from BALB/c 3T3 cells was electro-phoretically separated, transferred to nitrocellulose, and hybridized with 32 P. labeled 1.4 kb KL cDNA. The migration of 18S and 28S ribosomal RNAs is indicated.
  • FIG. 13 SDS-PAGE Analysis of KL.
  • FIG. 14 Binding of 125 I-K to Mast Cells and c-kit-Expressing ⁇ 2 Cells.
  • FIG. 15 Coprecipitation and Cross-Linking of 125 I-KL with the c-kit receptor on mast cells.
  • FIG. 16 RFLP analysis of Taql-digested DNA from Sl/+ and SIISI mice.
  • the Sl allele from C3HeB/Fej a/a CaJ Sl Hm mice was introduced into a C57BL/6J background, and progeny of a C57BL/6J Sl C3H ⁇ Sl C3H cross were evaluated.
  • KL is the ligand of c-kit based on binding and cross-linking experiments. N-terminal protein sequence of KL was used to derive KL-specific cDNA clones. These cDNA clones were used to investigate the relationship of the KL gene to the Sl locus, and it was demonstrated that KL is encoded by the Sl locus.
  • the hematopoietic growth factor was recently purified, i.e., KL, from conditioned medium of BALB/c 3T3 fibroblasts, and it has the biological properties expected of the c-kit ligand (Nocka et al, 1990b). KL was purified based on its ability to stimulate the proliferation of BMMC from normal mice but not from W mutant mice in the absence of IL-3. The purified factor stimulates the proliferation of BMMC and CTMC in the absence of IL-3 and therefore appears to play an important role in mature mast cells.
  • KL was shown to facilitate the formation of erythroid bursts (day 7-14 BFU-E) in combination with erythropoietin.
  • KL has a molecular mass of 30 kd and a pI of 3.8; it is not a disulfide-linked dimer, although the characteristics of KL upon gel filtration indicate the formation of noncovalently linked dimers under physiological conditions.
  • This invention provides an isolated nucleic acid molecule which encodes an amino acid sequence corresponding to a c-kit ligand (KL).
  • KL c-kit ligand
  • the invention also encompasses nucleic acids molecules which differ from that of the nucleic acid molecule which encodes the amino acid sequence isolated by applicants, but which produce the same phenotypic effect. These altered, but phenotypically equivalent nucleic acid molecules are referred to as “equivalent nucleic acids”.
  • this invention also encompasses nucleic acid molecules characterized by changes in non-coding regions that do not alter the phenotype of the polypeptide produced therefrom when compared to the nucleic acid molecule described hereinabove.
  • nucleic acid molecules which hybridize to the nucleic acid molecule of the subject invention.
  • nucleic acid encompasses RNA as well as single and double-stranded DNA and cDNA.
  • polypeptide encompasses any naturally occurring allelic variant thereof as well as man-made recombinant forms.
  • the c-kit ligand (KL) is a human c-kit ligand (KL) or a murine c-kit ligand (KL).
  • a vector which comprises the nucleic acid molecule which encodes an amino acid sequence corresponding to a c-kit ligand (KL).
  • This vector may include, but is not limited to a plasmid, viral or cosmid vector.
  • This invention also provides the isolated nucleic acid molecule operatively linked to a promoter of RNA transcription, as well as other regulatory sequences.
  • operatively linked means positioned in such a manner that the promoter will direct the transcription of RNA off of the nucleic acid molecule. Examples of such promoters are SP6, T4 and T7.
  • Vectors which contain both a promoter and a cloning site into which an inserted piece of DNA is operatively linked to that promoter are well known in the art. Preferable, these vectors are capable of transcribing RNA in vitro. Examples of such vectors are the pGEM series [Promega Biotec, Madison, Wis.].
  • a host vector system for the production the c-kit ligand (KL) polypeptide is further provided by this invention which comprises one of the vectors described hereinabove in a suitable host.
  • a suitable host may include, but is not limited to an eucaryotic cell, e.g., a mammalian cell, or an insect cell for baculovirus expression.
  • the suitable host may also comprise a bacteria cell such as E. coli , or a yeast cell.
  • a purified c-kit ligand (KL) polypeptide as well as a fragment of the purified c-kit ligand (KL) polypeptide is further provided by this invention.
  • the soluble, c-kit ligand (KL) polypeptide is conjugated to an imageable agent.
  • Imageable agents are well known to those of ordinary skill in the art and may be, but are not limited to radioisotopes, dyes or enzymes such as peroxidase or alkaline phosphate. Suitable radioisotopes include, but are not limited to 125 I, 32 P and 35 S.
  • conjugated polypeptides are useful to detect the presence of cells, in vitro or in vivo, which express the c-kit receptor protein.
  • a sample of the cell or tissue to be tested is contacted with the conjugated polypeptide under suitable conditions such that the conjugated polypeptide binds to c-kit receptor present on the surface of the cell or tissue; then removing the unbound conjugated polypeptide, and detecting the presence of conjugated polypeptide, bound; thereby detecting cells or tissue which express the c-kit receptor protein.
  • the conjugated polypeptide may be administered to a patient, for example, by intravenous administration.
  • a sufficient amount of the conjugated polypeptide must be administered, and generally such amounts will vary depending upon the size, weight, and other characteristics of the patient. Persons skilled in the art will readily be able to determine such amounts.
  • the conjugated polypeptide which is bound to any c-kit receptor present on the surface of cells or tissue is detected by intracellular imaging.
  • the intracellular imaging may comprise any of the numerous methods of imaging, thus, the imaging may comprise detecting and visualizing radiation emitted by a radioactive isotope.
  • the imaging may comprise detecting and visualizing radiation emitted by a radioactive isotope.
  • the isotope is a radioactive isotope of iodine, e.g. 125 I
  • the detecting and visualizing of radiation may be effected using a gamma camera to detect gamma radiation emitted by the radioiodine.
  • the soluble, c-kit ligand (KL) polypeptide fragment may be conjugated to a therapeutic agent such as toxins, chemotherapeutic agents or radioisotopes.
  • a therapeutic agent such as toxins, chemotherapeutic agents or radioisotopes.
  • the conjugated molecule acts as a tissue specific delivery system to deliver the therapeutic agent to the cell expressing c-kit receptor.
  • a method for producing a c-kit ligand (KL) polypeptide comprises growing the host vector system described hereinabove under suitable conditions permitting production of the c-kit ligand (KL) polypeptide and recovering the resulting c-kit ligand (KL) polypeptide.
  • This invention also provides the c-kit ligand (KL) polypeptide produced by this method.
  • a soluble, mutated c-kit ligand (KL) polypeptide is also provided, wherein this mutated polypeptide retains its ability to bind to the c-kit receptor, but that the biological response which is mediated by the binding of a functional ligand to the receptor is destroyed.
  • these mutated c-kit ligand (KL) polypeptides act as antagonists to the biological function mediated by the ligand to the c-kit receptor by blocking the binding of normal, functioning ligands to the c-kit receptor.
  • a pharmaceutical composition which comprises the c-kit ligand (KL) purified by applicants or produced by applicants' recombinant methods and a pharmaceutically acceptable carrier is further provided.
  • the c-kit ligand may comprise the isolated soluble c-kit ligand of this invention, a fragment thereof, or the soluble, mutated c-kit ligand (KL) polypeptide described hereinabove.
  • pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • This invention further provides a substance capable of specifically forming a complex with the soluble, c-kit ligand (KL) polypeptide, or a fragment thereof, described hereinabove.
  • This invention also provides a substance capable of specifically forming a complex with the c-kit ligand (KL) receptor protein.
  • the substance is a monoclonal antibody, e.g., a human monoclonal antibody.
  • a method of modifying a biological function associated with c-kit cellular activity comprises contacting cells, whose function is to be modified, with an effective amount of a pharmaceutical composition described hereinabove, effective to modify the biological function of the cell.
  • Biological functions which may be modified by the practice of this method include, but are not limited to cell-cell interaction, propagation of a cell that expresses c-kit i.e., propagation of hemapoietic cells.
  • in vitro fertilization may also be facilitated by this method.
  • This method may be practiced in vitro or in vivo. When the method is practiced in vivo, an effective amount of the pharmaceutical composition described hereinabove is administered to a patient in an effective amount, effective to modify the biological function associated with c-kit function.
  • This invention also provides a method of stimulating the proliferation of mast cells in a patient which comprises administering to the patient the pharmaceutical composition described hereinabove in an amount which is effective to stimulate the proliferation of the mast cells in the patient.
  • Methods of administration are well known to those of ordinary skill in the art and include, but are not limited to administration orally, intravenously or parenterally.
  • Administration of the composition will be in such a dosage such that the proliferation of mast cells is stimulated.
  • Administration may be effected continuously or intermittently such that the amount of the composition in the patient is effective to stimulate the proliferation of mast cells.
  • a method of inducing differentiation of mast cells or erythroid progenitors in a patient which comprises administering to the patient the pharmaceutical composition described hereinabove in an amount which is effective to induce differentiation of the mast cells or erythroid progenitors is also provided by this invention.
  • Methods of administration are well known to those of ordinary skill in the art and include, but are not limited to administration orally, intravenously or parenterally.
  • Administration of the composition will be in such a dosage such that the differentiation of mast cells or erythroid progenitors is induced.
  • Administration may be effected continuously or intermittently such that the amount of the composition in the patient is effective to induce the differentiation of mast cells or erythroid progenitors.
  • This invention also provides a method of facilitating bone marrow transplantation or treating leukemia in a patient which comprises administering to the patient an effective amount of the pharmaceutical composition described hereinabove in an amount which is effective to facilitate bone marrow transplantation or treat leukemia.
  • Methods of administration are well known to those of ordinary skill in the art and include, but are not limited to administration orally, intravenously or parenterally. Administration of the composition will be in such a dosage such that bone marrow transplantation is facilitated or such that leukemia is treated. Administration may be effected continuously or intermittently such that the amount of the composition in the patient is effective. This method is particularly useful in the treatment of acute myelogenous leukemia and modifications of chronic myelogenous leukemia.
  • This invention also provides a method of treating melanoma in a patient which comprises administering to the patient an effective amount of a pharmaceutical composition described hereinabove in an amount which is effective to treat melanoma.
  • Methods of administration are well known to those of ordinary skill in the art and include, but are not limited to administration orally, intravenously or parenterally. Administration of the composition will be in such a dosage such that melanoma is treated. Administration may be effected continuously or intermittently such that the amount of the composition in the patient is effective.
  • the soluble, c-kit ligand (KL) polypeptide may also be mutated such that the biological activity of c-kit is destroyed while retaining its ability to bind to c-kit.
  • this invention provides a method of treating allergies in a patient which comprises administering to the patient an effective amount of the soluble, mutated c-kit ligand described hereinabove and a pharmaceutically acceptable carrier, in an amount which effective to treat the allergy.
  • the amount of the composition which is effective to treat the allergy will vary with each patient that is treated and with the allergy being treated. Administration may be effected continuously or intermittently such that the amount of the composition in the patient is effective.
  • this invention provides a method for measuring the biological activity of a c-kit (KL) polypeptide which comprises incubating normal bone-marrow mast cells with a sample of the c-kit ligand (KL) polypeptide under suitable conditions such that the proliferation of the normal bone-marrow mast cells are induced; incubating doubly mutant bone-marrow mast cells with a sample of the c-kit ligand (KL) polypeptide under suitable conditions; incubating each of the products thereof with 3 H-thymidine; determining the amount of thymidine incorporated into the DNA of the normal bone-marrow mast cells and the doubly mutant bone marrow mast cells; and comparing the amount of incorporation of thymidine into the normal bone-marrow mast cells against the amount of incorporation of thymidine into doubly mutant bone-marrow mast cells, thereby measuring the biological activity of c-kit ligand (KL) polypeptide.
  • references to specific nucleotides in DNA molecules are to nucleotides present on the coding strand of the DNA.
  • the following standard abbreviations are used throughout the specification to indicate specific nucleotides: C—cytosine A—adenosine T—thymidine G—guanosine U—uracil
  • WBB6 +/+ and W/k V , C57B16 W V /+ and WB W/+mice were obtained from the Jackson Laboratory (Bar Harbor, Me.). Heterozygous W 41 /+mice were kindly provided by Dr. J. Barker from the Jackson Laboratory and maintained in applicants' colony by brother sister mating. Livers were removed at day 14-15 of gestation from fetuses derived by mating W/+animals. W/W fetuses were identified by their pale color and small liver size relative to other W/+ and +/+fetuses in the litter. Their identity was confirmed by analysis of the c-kit protein in mast cells derived from each fetus (Nocka et al., 1990).
  • Mast cells were grown from bone marrow of adult mice and fetal liver cells of day 14-15 fetuses in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), conditioned medium from WEHI-3B cells, non-essential amino acids, sodium pyruvate, and 2-mercapto-ethanol (RPMI-Complete (C)) (Yung and Moore, 1982).
  • FCS fetal calf serum
  • Non-adherent cells were harvested, refed weekly and maintained at a cell density less than 7 ⁇ 10 5 cells/ml.
  • Mast cell content of cultures was determined weekly by staining cytospin preparations with 1% toluidine blue in methanol. After 4 weeks, cultures routinely contained greater than 95% mast cells and were used from proliferation assays.
  • Peritoneal mast cells were obtained from C57B1/6 mice by lavage of the peritoneal cavity with 7-10 ml of RPMI-C. Mast cells were purified by density gradient centrifugation on 22% Metrizamide (Nycomed, Oslo, Norway) in PBS without Ca ++ and Mg ++ , essentially as previously described (Yurt et al, 1977). Mast cells were stained with 1% toluidine blue in methanol for 5 minutes and washed for 5 minutes in H 2 O, and berberine sulfate by standard procedures (Enerback, 1974). Mast cells were labeled with c-kit specific rabbit antisera which recognizes extracellular determinants of c-kit as previously described and analyzed on a FACSCAN (Becton Dickinson) (Nocka et al. 1990).
  • Balb/3T3 cells (Aaronson and Todaro, 1968) were grown to confluence in Dulbecco's Modified MEM (DME) supplemented with 10% calf serum (CS), penicillin and streptomycin in roller bottles. Medium was removed and cells washed two times with phosphate buffered saline (PBS). DME without CS was added and conditioned medium was collected after three days. Cells were refed with serum containing medium for one to two days, then washed free of serum, and refed with serum free medium and a second batch of conditioned medium was collected after three days.
  • DME Dulbecco's Modified MEM
  • CS calf serum
  • PBS phosphate buffered saline
  • CM Conditioned medium
  • Blue Agarose chromatography (BRL, Gaithersburg, Md.) was performed by using column with a bed volume of 100 ml equilibrated with PBS. 50-80 ml of FCM concentrate was loaded onto the column and after equilibration for one hour the flow through which contained the active material was collected and concentrated to 15-20 ml in dialysis tubing with PEG 8000.
  • High performance liquid chromatography was performed using a Waters HPLC system (W600E Powerline controller, 490E programmable multiwavelength detector, and 810 Baseline Workstation, Waters, Bedford, Mass.). Active fractions from gel filtration were dialyzed in 0.05 M Tris-HCl pH 7.8 and loaded onto a Protein-Pak DEAETM DEAE-5PW HPLC column (7.5 mm ⁇ 7.5 cm, Waters), equilibrated with 0.05 M Tris-HCl pH 7.8. Bound proteins were eluted with a linear gradient from 0 to 0.05 M Tris-HCl pH 7.8. Bound proteins were eluted with a linear gradient from 0 to 0.4M NaCl in 0.02 M Tris-HCl pH 7.8. The flow rate was 1 ml/minute and 2 ml fractions were collected.
  • RP-HPLC was performed using a semi-preparative and an analytical size C 18 column from Vydac.
  • buffer A was 100 mM ammonium acetate pH 6.0
  • buffer B was 1-propanol.
  • the biologically active fractions from anion exchange were pooled and loaded onto the semi-preparative C 18 column. Bound proteins were eluted with a steep gradient of 0% -23% 1-propanol within the first 10 minutes and 23-33% 1-propanol in 70 minutes. The flow rate was adjusted to 2 ml/min and 2 ml fractions were collected. Biologically active fractions were pooled and diluted 1:1 with buffer A and loaded on the analytical C 18 reverse phase column.
  • Proteins were eluted with a steep gradient from 0% -26% 1-propanol in 10 minutes and then a shallow gradient from 26% -33% 1-propanol in 70 minutes. The flow rate was 1 ml/min and 1 ml fractions were collected. Separation on an analytical C4 reverse phase column was performed with a linear gradient of acetonitrile from 0-80% in aqueous 0.1% TFA.
  • BMMC were washed free of IL-3 containing medium, incubated with medium containing 20 fold concentrated fibroblast conditioned medium (FCM) or WEHI-3 CM (IL-3) and after 24 hours of incubation 3 H-thymidine incorporation was determined.
  • FCM fibroblast conditioned medium
  • IL-3 WEHI-3 CM
  • FCM Concentrated FCM was also tested for its ability to stimulate the proliferation of other IL-3 dependent cells.
  • the myeloid 32D cells are known to lack c-kit gene products (Nocka et al., 1989). No proliferation of the 32D cells was observed with FCM, although normal proliferation was obtained with WEHI-3 CM (not shown).
  • BMMC functional c-kit protein in mast cells
  • FCM activity was distinct from IL-3. Therefore, normal and W mutant mast cells provide a simple, specific assay system for the purification of the putative c-kit ligand (KL) from fibroblast conditioned medium.
  • the fractions of the main peak were pooled, dialyzed and fractionated by FPLC chromatography on a DEAE-5PW column with a NaCl gradient (FIG. 2B).
  • the activity eluted at 0.11 M NaCl from the FPLC column.
  • Peak fractions were pooled and subjected to HPLC chromatography with a semi-preparative C18 column and an ammonium acetate/n-propanol gradient (FIG. 2C).
  • the active material eluted at 30% n-propanol from the semi-preparative C18 column was diluted 1:1 with buffer A and rechromatographed by using an analytical C18 column (FIG. 2D).
  • Purified KL was tested for its ability to stimulate the proliferation of mast cells derived from wildtype animals as well as homozygotes and heterozygotes of W, W V , and W 41 alleles.
  • the original W allele specifies a nonfunctional c-kit receptor and animals homozygous for the W allele die perinatally, are severely anemic and mast cells derived from W/W fetuses do not proliferate when co-cultured with Balb/3T3 fibroblasts (deAeberle, 1927; Nocka et al., 1990).
  • the W V and W 41 alleles both specify a partially defective c-kit receptor and homozygous mutant animals are viable (Little and Cloudman, 1937; Geissler et al., 1981; Nocka et al., 1990).
  • Homozygous W V animals have severe macrocytic anemia and their mast cells display a minor response in the co-culture assay, and homozygotes for the less severe W 41 allele have a moderate anemia and their mast cells show an intermediate response in the co-culture assay.
  • Homozygous and heterozygous mutant and +/+mast cells were derived from the bone marrow for the W V and W 41 alleles and from day 14 fetal livers for the W allele as described previously (Nocka et al., 1990). Fetal liver derived W/W mast cells did not proliferate in response to KL whereas both heterozygous (W/+) and normal (+/+) mast cells displayed a similar proliferative response to KL (FIG. 4). Bone marrow derived mast cells from W V /W V mice were severely defective in their response to KL, although some proliferation, 10% of +/+values, was observed at 100 U/ml (FIG. 4).
  • W V /+mast cells in contrast to heterozygous W/+mast cells showed an intermediate response (40%) in agreement with the dominant characteristics of this mutation.
  • W 41 /W 41 and W 41 /+mast cells were also defective in their ability to proliferate with KL, although less pronounced than mast carrying the W and the W V alleles, which is consistent with the in vivo phenotype of this mutation (FIG. 4). These results indicate a correlation of the responsiveness of mast carrying the WI W V and W 41 alleles to KL with the severity and in vivo characteristics of these mutations. In contrast, the proliferative response of mutant mast cells to WEHI-3CM (IL-3) was not affected by the different W mutations.
  • WEHI-3CM IL-3CM
  • PMC peritoneal cavity
  • Peritoneal mast cells were purified by sedimentation in a metrizamide gradient and c-kit expression on the cell surface analyzed by immunofluorescence with anti-c-kit sera or normal rabbit sera.
  • the PMC preparation was 90-98% pure based on staining with toluidine blue and berberine sulfate.
  • Berberine sulfate stains heparin proteoglycans in granules of connective tissue mast cells and in addition the dye is also known to stain DNA (FIG. 5) (Enerback, 1974).
  • BMMC and mucosal mast cells contain predominantly chondroitin sulfate di-B/E proteoglycans rather than heparin proteoglycans (Stevens et al., 1986); berberine sulfate therefore did not stain the granules in BMMC (FIG. 5A).
  • Analysis of c-kit expression by flow-cytometry indicated that virtually all PMC expressed c-kit at levels similar to those observed in BMMC (FIG. 5B). KL was then examined to determine if it would effect the survival or stimulate the proliferation of PMC (FIG. 5C).
  • the number of BFU-E obtained by using spleen cells with KL+erythropoietin was similar to the number observed with WEHI-3 CM +erythropoietin.
  • KL +erythropoietin did not stimulate the proliferation of BFU-E from bone marrow cells
  • WEHI-3 CM +erythropoietin induced the formation of 18 BFU-E from 10 5 bone marrow cells.
  • the effect of KL on the day 14 fetal liver cells was also examined and similar results were observed as with spleen cells.
  • KL +erythropoietin did not stimulate the proliferation of BFU-E from bone marrow cells
  • WEHI-3 CM +erythropoietin induced the formation of; 18 BFU-E from bone marrow cells
  • WEHI-3 CM +erythropoietin induced the formation of 18 BFU-E from 10 5 bone marrow cells.
  • the effect of KL on day 14 fetal liver, cells was also examined and similar results were observed as with spleen cells. In the presence of WEHI-3 CM +erythropoietin 18 ⁇ 3 BFU-E were observed with fetal liver cells.
  • BFU-E from spleen cells were stimulated by KL in a dose dependent manner, from 12 BFU-E/10 6 with erythropoietin alone to 50 BFU-E/10 6 cells with maximal stimulation at 2.5 ng of KL/ml (FIG. 6).
  • the average size of the bursts was dramatically increased by KL.
  • the number of BFU-E obtained by using spleen cells with KL +erythropoietin was similar to the number observed with WEHI-3 CM +erythropoietin.
  • KL +erythropoietin did not stimulate the proliferation of BFU-E from bone marrow cells
  • WEHI-3 CM erythropoietin induced the formation of 18 BFU-E from 10 5 bone marrow cells.
  • the effect of KL on day 14 fetal liver cells was also examined and similar results were observed as with spleen cells.
  • a significant number of BFU-E from fetal liver cells were observed with erythropoietin alone; however, this number increased from 6 ⁇ 2 to 20 ⁇ 5 with 2.5 ng/ml of KL.
  • WEHI-3 CM +erythropoietin 18 ⁇ 3 BFU-E were observed with fetal liver cell.
  • KL facilitates the formation of erythroid bursts (BFU-E) from fetal liver cells of W/W mice.
  • W/W and W/+ or +/+liver cells were prepared from fetuses at day 16.5 of gestation from mating w/+mice.
  • the total number of nucleated cells was reduced eight fold in the liver of the W/W mutant embryo as compared to the healthy fetuses.
  • c-kit receptor protein was isolated as described hereinabove and the sequence of the protein was determined by methods well known to those of ordinary skill in the art.
  • the single letter amino acid sequence of the protein from the N-teminal is: K E I X G N P V T D N V K D I T K L V A N L P N D Y M I T L N Y V A G M X V L P,
  • KL has a molecular mass of 30 kD and an isoelectric point of 3.8. KL is not a disulfide linked dimer, in contrast to CSF-1, PDGF-A and PDGF-B which have this property (Das and Stanley, 1982; Betsholz et al., 1986). Although, the behavior of KL upon gel filtration in PBS indicated a size of 55 -70 kD which is consistent with the presence of non-covalently linked dimers under physiological conditions. KL is different from other hematopoietic growth factors with effects on mast cells, such as IL-3 and IL-4, based on its ability to stimulate the proliferation of BMMC and purified peritoneal mast cells (CTMC), but not BMMCs from W mutant mice.
  • CSF-1 CSF-1, PDGF-A and PDGF-B
  • Balb/3T3 fibroblasts are a source for the hematopoietic growth factors G-CSF, GM-CSF, CSF-1, LIF and IL-6; however, none of these have the biological activities of KL (Nicola, 1989; Gough and Williams, 1989). Furthermore, preliminary results from the determination of the protein sequence of KL indicate that KL is different from the known protein sequences.
  • mast cells derived in vitro from bone marrow, fetal liver, or spleen with IL-3 resemble mucosal mast cells (MMC), although they may represent a precursor of both types of terminally differentiated mast cells, MMC and CTMC (Stevens and Austin, 1989).
  • c-kit is not required for the generation of BMMC from hematopoietic precursors since IL-3 dependent mast cells can be generated with comparable efficiency from bone marrow or fetal liver of both normal and W mutant mice (Yung et al., 1982).
  • the demonstration of c-kit expression in BMMC and CTMC/PMC and the corresponding responsiveness of BMMC and mature CTMC/PMC to KL suggests a role for c-kit at multiple stages in mast cell differentiation.
  • KL may be a mast cell proliferation and differentiation activity which is independent from these immune responses for its production and action on target cells.
  • the defect W mutations exert on erythropoiesis indicates an essential role for c-kit in the maturation of erythroid cells (Russell, 79; Gregory and Eaves, 1978; Iscove, 1978b).
  • the analysis of erythroid progenitors in fetal livers of W/W fetuses compared with normal littermates suggested that in the absence c-kit function, maturation proceeds normally to the BFU-E stage, but that progression to the CFU-E stage is suppressed (Nocka et al., 1989).
  • KL may stimulate an earlier erythroid-multipotential precursor in bone marrow which appears at later times in culture (day 14-20).
  • experiments with purified progenitor populations need to be performed.
  • CFU-S spleen colony forming units
  • Mast cells were grown from the bone marrow of adult +/+, W v /W v and W/+mice and W/W fetal liver of day 14-15 fetuses in RPMI 1640 medium supplemented with 10% fetal cell serum (FCS), conditioned medium from WEHI-3B cells, nonessential amino acids, sodium pyruvate, and 2-mercaptoethanol (RPMI-Complete) (Nocka et al., 1990a; Yung and Moore, 1982).
  • FCS fetal cell serum
  • conditioned medium from WEHI-3B cells conditioned medium from WEHI-3B cells
  • nonessential amino acids sodium pyruvate
  • 2-mercaptoethanol RPMI-Complete
  • BALB/c 3T3 cells (Aaronson and Todaro, 1968) were obtained from Paul O'Donnell (Sloan-Kettering Institute, New York, New York) and were grown in Dulbecco's modified MEM supplemented with 10% calf serum, penicillin, and streptomycin.
  • KL was purified from conditioned medium of BALB/c 3T3 cells by using a mast cell proliferation assay as described elsewhere (Nocka et al., 1990b). Conditioned medium was then concentrated 100- to 200-fold with a Pellicon ultrafiltration apparatus followed by an Amicon stirred cell. The concentrate was then chromatographed on Blue Agarose (Bethesda Research Laboratories, Gaithersburg, Md.), and the flow-through, which contained the active material, was concentrated in dialysis tubing with polyethylene glycol 8000 and then fractionated by gel filtration chromatography on an ACA54 Ultrogel (LKB, Rockland, Md.) column.
  • ACA54 Ultrogel LLB, Rockland, Md.
  • the biological activity eluted as a major and a minor peak, corresponding to 55-70 kd and 30 kd, respectively.
  • the fractions of the main peak were pooled, dialyzed, and fractionated by FPLC on a DEAE-5PW column with an NaCl gradient.
  • the activity eluted at 0.11 M NaCl from the FPLC column.
  • Peak fractions were pooled and subjected to HPLC with a semi-preparative C18 column and an ammonium acetate-n-propanol gradient.
  • the active material eluted at 30% n-propanol from the semipreparative C18 column was diluted 1:1 and re-chromatographed by using an analytical C18 column.
  • KL was iodinated with chloramine T with modifications of the method of Stanley and Guilbert (1981). Briefly, the labeling reaction contained 200 ng of KL, 2 nmol of chloramine T, 10% dimethyl sulfoxide, and 0.02% polyethylene glycol 8000, in a total volume of 25 ⁇ l in 0.25 M phosphate buffer (pH 6.5). The reaction was carried out for 2 min. at 4° C. and stopped by the addition of 2 nmol of cysteine and 4 ⁇ M KI. KL was then separated from free NaI by gel filtration on a PD10 column (Pharmacia). Iodinated KL was stored for up to 2 weeks at 4° C.
  • Binding buffer contained RPMI 1640 medium, 5% BSA (Sigma), 20 mM HEPES (pH 7.5) and NaN 3 . Binding experiments with nonadherent cells were carried out in 96-well tissue culture dishes with 2 ⁇ 10 5 cells per well in a volume of 100 ⁇ l. Binding experiments with ⁇ 2 cells were carried out in 24-well well dishes in a volume of 300 ⁇ l. Cells were equilibrated in binding buffer 15 minutes prior to the addition of competitor or labeled KL. To determine nonspecific binding, unlabeled KL or anti-c-kit rabbit serum was added in a 10-fold excess 30 minutes prior to the addition of 125 I-KL. Cells were incubated with 125 1 -KL for 90 minutes, and nonadherent cells were pelleted through 150 ⁇ l of FCS. Cell pellets were frozen and counted.
  • BMMC were incubated with 125 I-KL under standard binding conditions and washed in FCS and then in PBS at 40° C.
  • Cells were lysed as previously described (Nocka et al., 1989) in 1% Triton X-100, 20 mM Tris (pH 7.4), 150 mM NaCl, 20 mM EDTA, 10% glycerol, and protease inhibitors phenylmethylsufonyl fluoride (1 mM) and leupeptin (20 ⁇ g/ml).
  • Lysates were immunoprecipitated with normal rabbit serum, or c-kit specific sera raised by immunization of rabbits with a fragment of the v-kit tyrosine kinase domain (Majumder et al., 1988); or the murine c-kit expressed from a cDNA in a recombinant vaccinia virus (Nocka et al., 1990a).
  • immunoprecipitates were washed three times with wash A (0.1% Triton X-100, 20 mM Tris [pH 7.4], 150 mM NaCl, 10% glycerol), solubilized in SDS sample buffer, and analyzed by SDS-PAGE and autoradiography.
  • RT-PCR amplification was carried out essentially as described (Tan et al., 1990).
  • 1 ⁇ g of poly(A)-RNA from confluent BALB/c 3T3 cells in 25 ⁇ l of 0.05 M Tris-HCl (pH 8.3), 0.075 M KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, 200 ⁇ M dNTPs and 25 U of RNAsin (Promega) was incubated with 50 pmol of antisense primer and 50 U of Moloney murine leukemia virus reverse transcriptase at 40° C. for 30 minutes.
  • the cDNA was amplified by bringing up the reaction volume to 50 ⁇ l with 25 ⁇ l of 50 mM KCl, 10 mM Tris-HCl(pH 8.3), 1.5 mM MgCl 2 , 0.01% (w/v) gelatin, and 200 ⁇ M dNTPs, adding 50 pmol of sense primer and 2.5 U of Tag DNA polymerase, and amplifying for 25-30 cycles in an automated thermal cycler (Perkin-Elmer Cetus). The amplified fragments were purified by agarose gel electrophoresis, digested with the appropriate restriction enzymes, and subcloned into M13 mp18 and M13 mp19 for sequence analysis (Sanger et al., 1977).
  • a mouse 3T3 fibroblast lambda g11 cDNA library obtained from Clontech was used in this work. Screening in duplicate was done with Escherichia coli Y1090 as a host bacterium (Sambrook et al, 1989); 5′ end-labeled oligonucleotide was used as a probe. Hybridization was in 6 ⁇ SSC at 63° C., and the final wash of the filters was in 2 ⁇ SSC, 0.2% SDS at 63° C. Recombinant phage were digested with EcoRI and the inserts subcloned into M13 for sequence analysis.
  • nucleotide sequence of these cDNAs was determined, on both strands and with overlaps, by the dideoxy chain termination method of Sanger et al. (1977) by using synthetic oligodeoxynucleotides (17-mers) as primers.
  • Genomic DNA was prepared from tail fragments, digested with restriction enzymes, electrophoretically fractionated, and transferred to nylon membranes as described elsewhere (D.R.B. and P.L., submitted).
  • the 1.4 kb KL cDNA and TIS Dra/SaI were used as probes.
  • mice For the isolation of human monoclonal antibodies, eight week old Balb/c mice are injected intraperitoneally with 50 micrograms of a purified human soluble c-kit ligand (KL) polypeptide, or a soluble fragment thereof, of the present invention (prepared as described above) in complete Freund's adjuvant, 1:1 by volume. Mice are then boosted, at monthly intervals, with the soluble ligand polypeptide or soluble ligand polypeptide fragment, mixed with incomplete Freund's adjuvant, and bled through the tail vein. On days 4, 3, and 2 prior to fusion, mice are boosted intravenously with 50 micrograms of polypeptide or fragment in saline.
  • KL human soluble c-kit ligand
  • Splenocytes are then fused with non-secreting myeloma cells according to procedures which have been described and are known in the art to which this invention pertains. Two weeks later, hybridoma supernatant are screened for binding activity against c-kit receptor protein as described hereinabove. Positive clones are then isolated and propagated.
  • Sprague-Dawley rats or Louis rats are injected with murine derived polypeptide and the resulting splenocydes are fused to rat myeloma (y3-Ag 1.2.3) cells.
  • the KL protein was purified from conditioned medium from BALB/c 3T3 cells by a series of chromatographic steps including anion exchange and reverse-phase HPLC as described hereinabove (Nocka et al, 1990b). As previously noted, the sequence of the N-terminal 40 amino acids of KL was determined to be: K E I X G N P V T D N V K D I T K L V A N L P N D Y M I T L N Y V A G M X V L P.
  • oligonucleotide primers corresponding to amino acids 10-16 (sense primer) and 31-36 (antisense is primer) provided with endonuclease recognition sequences at their 5′ ends were synthesized as indicated in FIG. 8.
  • a cDNA corresponding to the KL mRNA sequences that specify amino acids 10-36 of KL was obtained by using the reverse transcriptase modification of the polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase modification of the polymerase chain reaction
  • Poly (A) + RNA from BALB/c 3T3 cells was used as template for cDNA synthesis and PCR amplification in combination with the degenerate oligonucleotide primers.
  • the amplified DNA fragment was subcloned into M13, and the sequences for three inserts were determined. The sequence in between the primers was found to be unique and to specify the correct amino acid sequence (FIG. 8). An oligonucleotide (49 nucleotides) corresponding to the unique sequence of the PCR products was then used to screen a ⁇ gt11 mouse fibroblast library. A 1.4 kb clone was obtained that, in its 3′ half, specifies an open reading frame that extends to the 3′ end of the clone and encodes 270 amino acids (FIG. 11). The first 25 amino acids of the KL amino acid sequence have the characteristics of a signal sequence.
  • the N-terminal peptide sequence that had been derived from the purified protein follows the signal sequence.
  • a hydrophobic sequence of 21 amino acids (residues 217-237) followed at its carboxyl end by positively charged amino acids has the features of a transmembrane segment.
  • a C-terminal segment of 33 amino acids follows the transmembrane segment without reaching a termination signal (end of clone).
  • the KL amino acid sequence therefore has the features of a transmembrane protein: an N-terminal signal peptide, an extracellular domain, a transmembrane domain, and a C-terminal intracellular segment.
  • RNA blot analysis was performed to identify KL-specific RNA transcripts in BALB/c 3T3 cells (FIG. 12).
  • a major transcript of 6.5 kb and two minor transcripts of 4.6 and 3.5 kb were identified on a blot containing poly(A) + RNA by using the 1.4 kb KL cDNA as a probe.
  • Identical transcripts were detected by using an end-labeled oligonucleotide derived from the N-terminal protein sequence. This result then indicates that KL is encoded by a large mRNA that is abundantly expressed in BALB/c 3T3 cells.
  • the Soluble form of KL is a Ligand of the c-kit Receptor
  • the fibroblast-derived hematopoietic growth factor KL had been shown to facilitate the proliferation of primary bone marrow mast cells and peritoneal mast cells and to display erythroid burst-promoting activity.
  • KL is the ligand of the c-kit receptor
  • BMMC mast cells
  • NIH ⁇ 2 cells expressing the c-kit cDNA KL was labeled to high specific activity with 125 I by using the modified chloramine T method (Stanley and Guilbert, 1981). Analysis of the labeled material by SDS-PAGE showed a single band of 28-30 kd (FIG.
  • the W v mutation is the result of a missense mutation in the kinase domain of c-kit that impairs the in vitro kinase activity but does not affect the expression of the c-kit protein on the cell surface (Nocka et al., 1990a).
  • W results from a deletion due to a splicing defect that removes the transmembrane domain of the c-kit protein; the protein therefore is not expressed on the cell surface (Nocka et al., 1990a).
  • binding of 125 I-KL could be completed with unlabeled KL and with two different anti-c-kit antisera.
  • the cells Upon washing to remove free 125 I-KL, the cells were solubilized by using the Triton X-100 lysis procedure and precipitated with anti-v-kit and anti-c-kit rabbit sera conjugated to protein A-Sepharose. 125 I-KL was retained in immunoprecipitates obtained by incubation with anti-kit sera but not with nonimmune controls, as shown by the analysis of the immune complexes by SDS-PAGE (FIG. 15A), where recovery of intact 125 I-KL was demonstrated from the samples containing the immune complexes prepared with anti-kit sera.
  • the locus identified by KL was also examined in mice that carry the original Sl mutation (Sarvella and Russell, 1956). For this purpose, the observation that the transgene insertion site locus is polymorphic in inbred strains was taken advantage of, and was utilized to determine the genotype at Sl during fetal development (D.R.B. and P.L., submitted). C57BL/6J mice that carry the S1 mutation maintained in the C3HeB/FeJ strain were generated by mating, and F1 progeny carrying the Sl allele were intercrossed (C57BL/6J Sl 3CH /+Sl C3H /+).
  • Nonanemic mice are either heterozygous SlI+ or wild type, and are heterozygous for the C3HeB/FeJ- and C57BL/6J-derived polymorphism or are homozygous for the C57BL/6J polymorphism, respectively.
  • genomic DNA from SII+ and SIISI mice was analyzed using the 1.4 kb KL cDNA probe, no hybridization to the homozygous SIISI DNA was observed (FIG. 16). It thus appears that the locus that encodes the KL protein is deleted in the Sl mutation. This finding further supports the notion that KL is the product of the Sl gene.
  • KL sequences are deleted in the genome of the Sl mouse. Taken together, these results suggest that KL is encoded by the Sl locus and is the ligand of the c-kit receptor, thus providing a molecular basis for the Sl defect.
  • KL is synthesized as an integral transmembrane protein.
  • the structural features of the primary translation product of KL therefore are akin to those of CSF-1.
  • CSF-1 is synthesized as a transmembrane molecule, which is processed by proteolytic cleavage to form a soluble product that is secreted (Kawasaki et al., 1985; Rettenmier and Roussel, 1988). Presumable, like CSF-1, KL is also synthesized as a cell surface molecule that may be processed to form a soluble protein.
  • the protein purified from conditioned medium of BALB/c 3T3 cells then would represent the soluble form of KL that was released from the cell membrane form by proteolytic cleavage.
  • a cell surface-bound form of KL may mediate the cell-cell interactions proposed for the proliferative and migratory functions of the c-kit/W receptor system.
  • a soluble c-kit receptor-alkaline phosphatase fusion protein has been shown to bind to the cell surface of BALB/c 3T3 cells but not to fibroblasts derived from SIISI mice (Flanagan and Leder, 1990).
  • a most significant aspect of the identification of the ligand of the c-kit receptor lies in the fact that it will facilitate the investigation of the pleiotropic functions of c-kit.
  • c-kit/W mutations affect the erythroid and mast cell lineages, and an effect on the stem cell compartment has been inferred as well.
  • c-kit/KL plays an essential role, and this is best seen by the anemia of mutant animals.
  • Applicants' demonstration of proliferation of BMMC and connective tissue-type mast cells in response to KL indicates a role for c-kit/KL at multiple stages in mast cell proliferation and differentiation independent of IL-3 and IL-4, which are thought to be mediators of allergic and inflammatory responses (Stevens and Austen, 1989).
  • the affected populations possibly include the spleen colony-forming units (CFU-S), which produce myeloid colonies in the spleen of lethally irradiated mice, as well as cells with long-term repopulation potential for the various cell lineages (McCulloch et al., 1964; Russell, 1979; Harrison, 1980; Barker and McFarland, 1988).
  • SIISI d adherent cells are defective but the nonadherent hematopoietic cells are not, and in the mast cell-fibroblast coculture system SIISI d fibroblasts are defective but the mast cells are not (Dexter and Moore, 1977; Fujita et al., 1989).
  • the results from these in vitro systems then would suggest that hematopoietic stromal cells and embryonic and connective tissue fibroblasts produce KL.
  • the BALB/c 3T3 cell line which is of embryonic origin, expresses significant levels of KL and was the source for its purification.
  • KL-expressing cell types may help to evaluate if there is a function for c-kit in the digestive tract, the nervous system, the placenta, and certain craniofacial structures, sites where c-kit expression has been documented (Nocka et al., 1989; Orr-Urtreger et al., 1990). No Sl or W phenotypes are known to be associated with these cell systems.
  • the original W mutation is an example of a c-kit null mutation (Nocka et al., 1990a).
  • WI+mice When heterozygous with the normal allele, WI+mice typically have a ventral spot but no coat dilution and no effects on hematopoiesis and gametogenesis.
  • the weak heterozygous phenotype of WI + mice is in contrast to the phenotype of heterozygous SII + mice, which have moderate macrocytic anemia and a diluted coat pigment in addition to a ventral spot and gonads that are reduced in size.
  • 50% gene dosage of KL is limiting and is not sufficient for normal function of the c-kit receptor, yet 50% dosage of the c-kit receptor does not appear to be limiting in most situations.
  • the c-kit receptor system functions in immature progenitor cell populations as well as in more mature cell types in hematopoiesis, gametogenesis, and melanogenesis. Severe Sl or W mutations may block the development of these cell W mutations in which c-kit/KL function is only partially impaired often reveal effects in more mature cell populations. Numerous weak Si alleles are known. Their phenotypes, e.g., in gametogenesis and melanogenesis, will be of great value in the elucidation of the pleiotropic functions of the c-kit receptor system.

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US07/594,306 US20030125519A1 (en) 1990-08-27 1990-10-05 Ligand for the c-kit receptor and methods of use thereof
AU85106/91A AU654502B2 (en) 1990-08-27 1991-08-27 Ligand for the c-kit receptor and methods of use thereof
JP3515137A JPH06504185A (ja) 1990-08-27 1991-08-27 Ｃ・ｋｉｔ受容体に対するリガンド及びその使用法
PCT/US1991/006130 WO1992003459A1 (en) 1990-08-27 1991-08-27 LIGAND FOR THE c-KIT RECEPTOR AND METHODS OF USE THEREOF
CA002090469A CA2090469A1 (en) 1990-08-27 1991-08-27 Ligand for the c-kit receptor and methods of use thereof
EP91916054A EP0546054A1 (en) 1990-08-27 1991-08-27 LIGAND FOR THE c-KIT RECEPTOR AND METHODS OF USE THEREOF
HU9300541A HUT64368A (en) 1990-08-27 1991-08-27 Ligand to receptor c-kit and method for its utilization
KR1019930700578A KR930703339A (ko) 1990-08-27 1991-08-27 씨(c)-키트 수용체의 리간드 및 그의 이용방법
US08/341,456 US5767074A (en) 1990-08-27 1994-11-17 Compositions of soluble C-kit ligand and hematopoietic factors
US08/478,414 US5935565A (en) 1990-08-27 1995-06-07 Method for increasing the level of stem cells in peripheral blood
US09/371,261 US6403559B1 (en) 1990-08-27 1999-08-10 C-kit ligand-based method for expanding peripheral blood cell levels
US10/132,345 US20030103937A1 (en) 1990-08-27 2002-04-24 Ligand for the c-kit receptor and methods of use thereof
US10/988,476 US20050276784A1 (en) 1990-08-27 2004-11-12 Ligand for the c-kit receptor and methods of use thereof
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US07/873,692 Continuation-In-Part US5316883A (en) 1989-11-04 1992-04-21 Method for controlling pressure during image development
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Cited By (1)

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US20050276784A1 (en) * 1990-08-27 2005-12-15 Solan-Kettering Institute For Cancer Research Ligand for the c-kit receptor and methods of use thereof

Families Citing this family (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7144731B2 (en) * 1989-10-16 2006-12-05 Amgen Inc. SCF antibody compositions and methods of using the same
AU6541090A (en) * 1989-10-16 1991-05-16 Amgen, Inc. Stem cell factor
US20040181044A1 (en) * 1989-10-16 2004-09-16 Zsebo Krisztina M. Method of stimulating growth of epithelial cells by administering stem cell factor
US6852313B1 (en) 1989-10-16 2005-02-08 Amgen Inc. Method of stimulating growth of melanocyte cells by administering stem cell factor
US20030125519A1 (en) 1990-08-27 2003-07-03 Peter Besmer Ligand for the c-kit receptor and methods of use thereof
AU675429B2 (en) * 1992-04-23 1997-02-06 Sloan-Kettering Institute For Cancer Research Ligand for the c-kit receptor and methods of use thereof
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US5792771A (en) * 1992-11-13 1998-08-11 Sugen, Inc. Quinazoline compounds and compositions thereof for the treatment of disease
US5981569A (en) * 1992-11-13 1999-11-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Substituted phenylacrylonitrile compounds and compositions thereof for the treatment of disease
US5763441A (en) * 1992-11-13 1998-06-09 Sugen, Inc. Compounds for the treatment of disorders related to vasculogenesis and/or angiogenesis
US6177401B1 (en) 1992-11-13 2001-01-23 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften Use of organic compounds for the inhibition of Flk-1 mediated vasculogenesis and angiogenesis
US5712395A (en) * 1992-11-13 1998-01-27 Yissum Research Development Corp. Compounds for the treatment of disorders related to vasculogenesis and/or angiogenesis
US6872699B2 (en) 1992-11-13 2005-03-29 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften, E.V. Truncated Flk-1 receptor protein, methods of use and a recombinant vector containing a nucleotide encoding the truncated Flk-1 protein
AU6117294A (en) * 1993-02-16 1994-09-14 Auckland Uniservices Limited Developmental tyrosine kinases and their ligands
US5525708A (en) * 1994-03-28 1996-06-11 Cytomed, Inc. Covalent dimer of kit ligand
US6024734A (en) * 1994-03-31 2000-02-15 Brewitt; Barbara A. Treatment methods using homeopathic preparations of growth factors
GB9515839D0 (en) * 1995-08-02 1995-10-04 Q One Biotech Ltd Feline stem cell factor
US5885962A (en) * 1996-04-05 1999-03-23 Amgen Inc. Stem cell factor analog compositions and method
US7294755B1 (en) 1997-11-14 2007-11-13 Cedars-Sinai Medical Center Genetic modification of male germ cells for generation of transgenic species and genetic therapies
US6734338B1 (en) 1997-11-14 2004-05-11 Cedars-Sinai Medical Center Transfection, storage and transfer of male germ cells for generation of transgenic species and genetic therapies
EP1030929B1 (en) * 1997-11-14 2009-03-18 Cedars-Sinai Medical Center Transfection and transfer of non-human male germ cells for generation of transgenic non-human mammals
AU3563499A (en) * 1998-11-13 2000-06-05 Cedars-Sinai Medical Center Transfection of male germ cells for generation of selectable transgenic stem cells
WO2000029601A1 (en) * 1998-11-13 2000-05-25 Cedars-Sinai Medical Center A method for depopulating of vertebrate testis and for generation of transgenic species
FR2853663B1 (fr) * 2003-04-14 2007-08-31 Aventis Pharma Sa Procede d'obtention de lignees de mastocytes a partir de tissus de porcs et procede de production de molecules de type heparine
US20060153799A1 (en) 2004-11-05 2006-07-13 Northwestern University Use of SCF and G-CSF in the treatment of cerebral ischemia and neurological disorders
JP2008539700A (ja) 2005-05-04 2008-11-20 オーストラリアン ステム セル センター リミテッド 系統拘束された造血幹細胞の選択、培養および作出法
CA2629775A1 (en) * 2005-11-14 2007-05-24 Enterprise Partners Venture Capital Stem cell factor therapy for tissue injury
US8227437B2 (en) * 2006-06-22 2012-07-24 Tai June Yoo Restoration of hearing loss
MX2009008413A (es) * 2007-02-06 2009-11-02 Tai June Yoo Tratamiento y prevencion de enfermedades neurodegenerativas usando terapia genica.
US20120189633A1 (en) 2011-01-26 2012-07-26 Kolltan Pharmaceuticals, Inc. Anti-kit antibodies and uses thereof
KR101384360B1 (ko) * 2012-05-04 2014-04-14 아주대학교산학협력단 Ｓｃｆ 또는 이의 수용체를 억제하는 물질을 포함하는 혈관 투과성 관련 질환의 치료 또는 예방용 조성물
KR102268351B1 (ko) 2012-07-25 2021-06-22 셀덱스 쎄라퓨틱스, 인크. 항-kit 항체 및 그의 용도
US10239943B2 (en) 2014-05-23 2019-03-26 Celldex Therapeutics, Inc. Treatment of eosinophil or mast cell related disorders
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EP4511127A1 (en) 2022-04-21 2025-02-26 Gilead Sciences, Inc. Kras g12d modulating compounds
US20240116928A1 (en) 2022-07-01 2024-04-11 Gilead Sciences, Inc. Cd73 compounds
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WO2024215754A1 (en) 2023-04-11 2024-10-17 Gilead Sciences, Inc. Kras modulating compounds
WO2024220917A1 (en) 2023-04-21 2024-10-24 Gilead Sciences, Inc. Prmt5 inhibitors and uses thereof
WO2025006720A1 (en) 2023-06-30 2025-01-02 Gilead Sciences, Inc. Kras modulating compounds
US20250100998A1 (en) 2023-07-26 2025-03-27 Gilead Sciences, Inc. Parp7 inhibitors
US20250066328A1 (en) 2023-07-26 2025-02-27 Gilead Sciences, Inc. Parp7 inhibitors
EP4516807A1 (en) 2023-08-28 2025-03-05 Consorcio Centro de Investigación Biomédica en Red Compositions for use in a method of providing improved hematopoietic stem cell engraftment
US20250109147A1 (en) 2023-09-08 2025-04-03 Gilead Sciences, Inc. Kras g12d modulating compounds
WO2025054347A1 (en) 2023-09-08 2025-03-13 Gilead Sciences, Inc. Kras g12d modulating compounds
WO2025096589A1 (en) 2023-11-03 2025-05-08 Gilead Sciences, Inc. Prmt5 inhibitors and uses thereof
US20250230168A1 (en) 2023-12-22 2025-07-17 Gilead Sciences, Inc. Azaspiro wrn inhibitors

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5399493A (en) 1989-06-15 1995-03-21 The Regents Of The University Of Michigan Methods and compositions for the optimization of human hematopoietic progenitor cell cultures
ATE403713T1 (de) * 1989-10-16 2008-08-15 Amgen Inc Stamzellfaktor
AU7552691A (en) 1990-03-23 1991-10-21 GSF-Institut Fur Experimentalle Hamatologie Method for enhanced growth and proliferation of mast cells
WO1992000376A1 (en) * 1990-06-25 1992-01-09 Immunex Corporation Mast cell growth factor
US20030125519A1 (en) 1990-08-27 2003-07-03 Peter Besmer Ligand for the c-kit receptor and methods of use thereof
US5767074A (en) 1990-08-27 1998-06-16 Sloan-Kettering Institute For Cancer Research Compositions of soluble C-kit ligand and hematopoietic factors
WO1992006220A1 (en) 1990-10-05 1992-04-16 President And Fellows Of Harvard College Detection and isolation of ligands
CA2107897A1 (en) 1991-04-09 1992-10-10 Ronald Hoffman System and process for supporting hematopoietic cells
US5199942A (en) 1991-06-07 1993-04-06 Immunex Corporation Method for improving autologous transplantation
US6001803A (en) 1992-04-23 1999-12-14 Sloan-Kettering Institute For Cancer Research Composition of c-kit ligand, GM-CSF, and TNF-α and method of use

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050276784A1 (en) * 1990-08-27 2005-12-15 Solan-Kettering Institute For Cancer Research Ligand for the c-kit receptor and methods of use thereof
US20080152652A1 (en) * 1990-08-27 2008-06-26 Sloan-Kettering Institute For Cancer Research Ligand for the c-kit receptor and methods of use thereof

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