Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
• • 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
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • 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
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1816—Erythropoietin [EPO]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • 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
  • A61K38/193—Colony stimulating factors [CSF]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
• • 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
  • 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
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0641—Erythrocytes
• • 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
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
• • 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
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
  • C12N2500/62—DMSO
• • 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
  • C12N2501/14—Erythropoietin [EPO]
• • 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
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)

Definitions

• EPO erythropoietin
• Colony-forming unit- erythroid is a cell that gives rise to a small colony of 8 to 50 mature, hemoglobin-containing erythroblasts in 2 days (mouse) to 7 days (human) .
• CFU-E Colony-forming unit- erythroid
• Burst-forming unit- erythroid (BFU-E) is a less mature erythroid progenitor cell that gives rise to a large cluster of colonies of mature erythroblasts (several hundred to several thousand cells) in 8 days (mouse) to 14 days (human). (Gregory and Eaves Blood. 49:855-864 (1977)). CFU-E are absolutely dependent on EPO for development in tissue culture, whereas BFU-E can survive for several doublings in the absence of EPO if other hematopoietic growth factors are present. (Sawada et al.. J. Cell Physiol. 142:219-230 (1990)) .
• BPA burst promoting activity
• GM-CSF granulocyte-macrophage colony stimulating activity
• T. M. Dexter Science. 88:1 (1987)
• IL-3 interleukin 3
• Y.C. Yang et al. - Cell. 47:3 (1986)
• C . Sieff J. Clin. Invest.
• B-BPA B-lymphocyte derived burst promoting activity
• B-lymphocyte derived burst promoting activity (B-BPA) , a lineage specific regulator of erythroid pro ⁇ genitors, was first described by Dainiak and Cohen as a component of serum-free lymphocyte conditioned medium (N. Dainiak and CM. Cohen, Blood. 60:583 (1982)).
• B-BPA is an integral membrane protein of 28 kDa, and its activity recently has been localized to the plasma membrane of normal resting B lymphocytes (L. Feldman, et al. , Proc. Natl. Acad. Sci. USA. 84:6775 (1987); L. Feldman and N. Dainiak, Blood, 73:1814 (1990)).
• B-BPA is a potent stimulator of BFU-E proliferation, increasing the number of BFU-E derived colonies up to 600% above control while having no apparent effect on the proliferation of granulocyte-macrophage (CFU-GM) , megakaryocyte (CFU-Meg) ⁇ , or mixed (CFU-GEMM) hematopoietic colonies (N. Dainiak et al.. EXP. Hematol.. 18:1073 (1985); L. Feldman, et al.. Proc. Natl. Acad. Sci. USA f 84:6775 (1987)).
• CFU-GM granulocyte-macrophage
• CFU-Meg megakaryocyte
• CFU-GEMM mixed
• B-BPA appears to be biochemically and immunochemically distinct from other growth factors with BFU-E directed growth promoting activities (L. Feldman, et al.. Proc. Natl. Acad. Sci. USA, 84:6775 (1987); L. Feldman and N. Dainiak, Blood. 73:1814 (1990)).
• EPO In vertebrates, EPO is synthesized in response to hypoxia. Increased secretion of EPO into the serum is observed after blood loss and other causes of hypoxia such as high-altitudes.
• Administration of exogenous EPO has proven useful in treating patients suffering from severe anemia, resulting, for example, from chronic renal fail ⁇ ure.
• Administration of EPO has advantages over trans ⁇ fusions, which can transmit infectious diseases including hepatitis and AIDS and cause liver toxicity resulting from iron overload.
• Administration of EPO is, however, costly.
• the present invention relates to Applicants' finding that certain agents potentiate (i.e., increase) the effect of erythropoietin (EPO) on the growth of EPO-responsive erythroid precursor cells and on their differentiation into red blood cells.
• EPO erythropoietin
• the invention comprises, in one embodiment, a method of enhancing, or increasing, the effect of EPO in a vertebrate (particularly humans and other mammals) by administering to the vertebrate an effective amount of a potentiating agent.
• the amount of EPO required for growth and differentiation of erythroid precursor cells is decreased (i.e. less than it would be in the absence of the potentiating agent) .
• the invention comprises, in another embodiment, a method of promoting erythropoiesis in a vertebrate by administering to the vertebrate an appropriate amount of a potentiating agent.
• the potentiating agent is administered alone in a manner such that it potentiates the vertebrate's endogenous supply of EPO.
• the potentiating agent can be administered alone, followed by administration of exogenous EPO.
• This method known as "priming" results in a large amplification of the erythr ⁇ poietin biologic response characterized by an increased percentage of EPO-responsive cells, an increased rate of EPO responsiveness and a profound increase in sensitivity to EPO, as manifested by a left shifted EPO dose-response curve.
• the potentiating agent is administered in conjunction with EPO administration. Co-administered EPO and potentiating agent synergize to affect the growth and differentiation of erythroid precursor cells.
• the invention therefore, also comprises a composition comprising a potentiating agent and erythropoietin.
• the invention comprises, in another embodiment, a method of repopulating progenitor cells in bone marrow of a vertebrate, comprising administering to the bone marrow an appropriate amount of a potentiating agent.
• the potentiating agents can be either biological or chemical in nature.
• An example of a biological potentiating agent is B-lymphocyte derived burst promoting activity (B-BPA) , which will be known herein as erythroid colony stimulating factor (E-CSF) .
• B-BPA B-lymphocyte derived burst promoting activity
• E-CSF erythroid colony stimulating factor
• E-CSF is a preferred potentiating agent, because it is present naturally in a vertebrate host, suggesting that administration of additional amounts will not result in adverse immune reactions by the host.
• the invention further relates to methods of obtaining E-CSF.
• Preferred chemical potentiating agents are polar/ planar compounds, such as dimethylsulfoxide (DMSO) and hexamethylene bisacetamide (HMBA) and cytodifferentiating chemicals such as sodium butyrate.
• DMSO dimethylsulfoxide
• HMBA hexamethylene bisacetamide
• cytodifferentiating chemicals such as sodium butyrate.
• a major advantage of using the methods and compo ⁇ sitions of the subject invention is that erythropoiesis can be induced in an individual with smaller quantities of EPO, which means that less exogenous EPO or no exogenous EPO is required to induce erythropoiesis in an individual, such as in treating a patient suffering from hypoxia, which results for example from severe anemia.
• the present invention is based on the finding that contacting erythroid precursor cells with a potentiating agent increases their response to erythropoietin (EPO) .
• EPO-responsive erythroid precursor cells include early burst forming unit-erythroid (BFU-E) and late colony-forming unit erythroid (CFU-E) .
• EPO potentiating agents can be biological or chemical in nature and can be obtained by a variety of methods from a variety of sources.
• An example of a biological potentiating agent is erythroid potentiating B-lymphocyte derived erythroid burst promoting activity (B-BPA) .
• B-BPA is a lineage specific but pleiotropic regulator of erythropoiesis.
• B-BPA has distinct effects on both early (BFU-E) and late (CFU-E) erythroid progenitors from normal human bone marrow. Given its pleiotropic effect, B-BPA is more appropriately called E-CSF, erythroid colony stimulating factor. Analogs and derivatives of E-CSF which have the same biological effects are also useful in the subject invention.
• E-CSF Human E-CSF has been purified from normal peripheral blood B-lymphocytes, as explained in detail in Example 1. In addition to normal B-lymphocytes, other transformed cell types may yield the protein. Two B-cell lines, Raji and Ramos (ATCC designation Nos. CCL86 and CRL1596, respectively) , both derived from patients with Burkitts lymphoma, have been found to yield particularly high levels of the protein. Therefore, these cell lines provide a preferred source of E-CSF.
• E-CSF activity has also been identified in medium conditioned by, and on the membranes of, lymphocytes from non-human sources, including mouse, rat, rabbit and bovine.
• E-CSF can be purified from non-human sources essentially as described for human E-CSF.
• E-CSF can be produced synthetically.
• E-CSF can be cloned by indirect expression. (Sambrook, J. e al. - eds. Molecular Cloning f Cold Spring Harbor Laboratory Press (1989)).
• E-CSF can be cloned by direct expression.
• a cDNA library can be prepared from B cells or another cell type secreting E-CSF. The library can then be introduced into a suitable expression vector such as CDMB (Seed, B. and A. Aruffo, Proc. Natl. Acad. Sci. USA 84:3365-3369 (1987)).
• the CDM8 library can be divided into pools and used to transfect COS cells, which are allowed to express E-CSF.
• the COS culture expressing the factor can be identified for example, by bioassaying the supernatant medium, or by antibody identification of either soluble factor (e.g.. using RIA or ELISA) or membrane bound factor (D'Andrea, A.D. et al.. Cell 57: 277-285 (1989)).
• the cDNA pool thus identified can then be subdivided and the process repeated until a pure clone of E-CSF cDNA is obtained.
• An alternative method of direct expression involves "panning" the transfected COS cells with an anti-E-CSF antibody, thereby enriching for cells containing the cDNA.
• the panned cells are lysed and the cDNA is harvested and used to transform bacteria where it is amplified.
• the amplified cDNA is used to transfect cells and the cycle is repeated until a pure cDNA clone is obtained.
• E-CSF acts on both early (BFU-E) and late (CFU-E) erythroid progenitors and that it has different effects on these two classes of progenitors.
• E-CSF stimulates the proliferation of BFU-E, resulting in an increase in the number of erythroid bursts over a wide range of EPO concentrations.
• E-CSF increases the sensitivity of CFU-E to their primary regulator, EPO.
• E-CSF potentiates the effect of EPO for growth and differ ⁇ entiation of erythroid precursor cells, resulting in a left shift in the EPO dose response curve.
• E-CSF plus EPO in contact with erythroid precursor cells results in a synergistic. effect, wherein erythroid colonies produced are dramatically larger and hemoglobinized to a much greater extent than is the case with either growth factor alone.
• E-CSF alone in the absence of EPO is able to effect proliferation of erythroleukemia cells.
• the presence of E-CSF and EPO synergize to affect the growth and differentiation of cells.
• EPO-responsive erythroid precursor cells include E-CSF, some chemical agents have been shown to induce potentiation of EPO on growth and differ ⁇ entiation of EPO-responsive erythroid precursor cells.
• polar/planar compounds such as dimethyl sulfoxide (DMSO) and polymethylene bisacetamides such as hexamethylene bisacetamide (HMBA) (Reuben, R.C et al.. Biochimica et. Biophysica Acta 605:325-346 (1980)
• cytodifferentiating chemicals such as sodium butyrate are preferred chemical potentiating agents.
• DMSO potentiates the biologic response of erythroid precursor cells to EPO in three ways: First, the total percentage of EPO responsive cells is increased by DMSO. Second, the rate of response to EPO is increased, i.e., the time required for appearance of maximum numbers of hemoglobinized (Hb + ) cells is reduced. Third, the EPO sensitivity of cells is increased approximately 20-fold, as manifested by a pronounced leftward shift of the EPO dose-response curve. These changes are accompanied by an almost seven-fold increase in the erythropoietin receptor density.
• Hb + cells are differentiated cells and are evidenced by staining with benzidine. Hb + values were determined by subtracting the % Hb + cells detected in replicate cultures pretreated with chemical inducers but incubated in the absence of EPO from that obtained in cultures incubated in the presence of EPO.
• Table 1 also shows that in identical experiments performed on Friend erythroleukemia cells, which do not hemoglobinize in response to erythropoietin, the presence of chemical potentiating agents do not induce differentiation. A small effect on receptor density was observed in the Friend cells, but it did not approach the magnitude seen with Rauscher cells.
• the binding curve of DMSO-primed PAN-4 cells was analyzed by the method of Scatchard.
• K d 0.8 nM
• DMSO priming was found to result in an initial decrease in receptor message followed by an increase of approximately 2- to 3-fold after 24-48 hours. No change was seen in glyceraldehyde-3-phosphate dehydrogenase, a control, housekeeping gene. This observed increase in receptor message as a result of DMSO priming does not appear to account for the magnitude of the receptor up-regulation.
• EPO potentiating agents In addition to an induced increase in receptor transcript levels, whether due to an effect on gene transcription or on mRNA processing, other possibilities exist that might explain the increase in receptor binding induced by EPO potentiating agents. Post-translational processing of receptor protein leading to stabilization within the cell and/or increased efficiency of transport to the plasma membrane surface may play a role. Ad ⁇ ditionally, an EPO potentiating agent may exert a direct effect on the plasma membrane itself, thereby "unmasking" . otherwise "buried" receptor binding units. Utilitv
• EPO potentiating agents can be administered to individuals suffering from any disease or condition which results in an inadequate supply of hemoglobin containing red blood cells.
• EPO potentiating agents can be administered to patients recovering from chemotherapy or to patients with chronic renal failure.
• EPO potentiating agents can be administered alone or in conjunction with EPO to vertebrates to promote erythropoiesis (i.e. the development of red blood cells from undifferentiated progenitor cells, for example, BFU-E and CFU-E) .
• Administration can be by any route appropriate to the condition being treated.
• administration of compounds containing E-CSF is via a parenteral route, such as by injection into the blood stream.
• compositions of this invention can be employed in admixture with conventional excipients, (i.e., pharmaceutically accept ⁇ able organic or inorganic carrier substances) .
• excipients i.e., pharmaceutically accept ⁇ able organic or inorganic carrier substances.
• the carrier must be "acceptable” in that it does not inactivate or otherwise adversely affect the EPO potentiating agent and is compatible with the other ingredients of the formulation and not deleterious to the recipient.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
• an effective amount of the potentiating agent i.e., an amount effective to increase the effect of EPO on the growth of erythroid precursor cells and their differentiation into red blood cells will depend, for example, upon the severity of the condition being treated, the route of administration chosen and the amount- of EPO present in the vertebrate (i.e., the vertebrate's endogenous EPO supply).
• an effective amount of EPO will depend, for example, upon the severity of the condition being treated, the route of administration chosen, the concentration of endogenous EPO and the amount of potentiating agent present in the vertebrate and the amount of potentiating agent being administered. The more potentiating agent present in the vertebrate, the less EPO need be administered. Effective amounts can be determined by attending physicians or veterinarians using conventional considerations, (e.g., by means of an appropriate, conventional pharmacological protocol) .
• EPO potentiating agents can also be administered to repopulate progenitor cells.
• at least one potentiating agent can be administered to individuals, who are in need of bone marrow transplants (e.g., patients with aplastic anemia, acute leukemias, recurrent lymphomas or solid tumors) .
• a recipient Prior to receiving a bone marrow transplant, a recipient is prepared by ablating or removing endogenous hematopoietic stem cells. This preparation is usually carried out by total body irradiation or delivery of a high dose of an alkylating agent or other chemotherapeutic, cytotoxic agents (Greenberger, J.S., Br. J. He atol.
• EPO potentiating agents can be administered in conjunction with the transplanted bone marrow, or can be administered separately (e.g., subsequent to bone marrow transplantation.)
• E-CSF In serum-free culture of human bone marrow pro ⁇ genitors, E-CSF was found to increase the number of day 12 burst forming unit erythroid tBFU-E) derived erythroid bursts in a concentration dependent manner. In the absence of added E-CSF, control cultures produced a maximum of 11 +/- 1.5 bursts at 2 U EPO/ml. Addition of 100 ul of a 1/50 dilution of E-CSF increased the maximum number of day 12 bursts to 33 +/- 2, while addition of a 1/5 dilution of E-CSF increased the maximum number of bursts to 38 +/- 1.
• E-CSF appears saturable, since the ten-fold increase in amount of E-CSF added (from 1/50 to 1/5 dilution of concentrated column-purified E-CSF) resulted in only a small, but significant, increase in the number of day 12 BFU-E.
• E-CSF In addition to increasing the number of BFU-E derived bursts, E-CSF also appears to lower the requirement for EPO during burst formation. To determine the magnitude of this "EPO sparing" effect, E-CSF was administered to early erythroid progenitors over an expanded range of EPO concentrations. Serum-free cultures were established in the presence of 0.00 - 2.0 U EPO/ml, in the absence or presence of a specified amount of E-CSF.
• E-CSF was found to significantly increase the number of BFU-E over a wide range of EPO concentrations. At all EPO concentrations greater than 0.1 U/ml, E-CSF signifi ⁇ cantly stimulates the proliferation of BFU-E, resulting in a two to four fold increase in the number of day 12 erythroid bursts.
• E-CSF colony forming unit erythroid
• E-CSF acts on CFU-E and increases their sensitivity to EPO dramatically.
• E-CSF acts in concert with EPO to stimulate growth and differentiation of CFU-E.
• E-CSF Early log phase cells were plated at limiting dilution in the absence or presence of E-CSF, incubated under specified conditions, and colony-containing wells were enumerated from replicate 96-well plates. E-CSF increased the plating efficiency of both murine and human erythroleukemia cells. Proliferation of three Rauscher clones; R404, R28 and EMSIII was increased by 51%, 166% and 50%, respectively; and that of K562 cells was in ⁇ creased by 52%. E-CSF had no apparent effect on HEL cell growth.
• E-CSF's effect on the prolif ⁇ eration of Rauscher erythroleukemia cells was demonstrated by plating R28 cells at limiting dilution in the presence of either 10% E-CSF, 10 - 100 U rmIL-3, or 10 100 U rm GM-CSF/ml. Under conditions where E-CSF markedly increased the plating efficiency of these Rauscher cells, the addition of either IL-3 or GM-CSF was without effect on the proliferation of these cells.
• the simultaneous addition of E-CSF plus IL-3 or of E-CSF plus GM-CSF provided no proliferative effect above that which was seen with E-CSF alone.
• the growth promoting activity of E-CSF on these cells is not affected by co-addition of EPO, consistent with the hypothesis that the action of E-CSF on cell proliferation is erythropoietin. independent.
• the growth promoting effect of E-CSF on Rauscher erythroleukemia cells was also determined by plating the cells in serum-free fibrin clots (semi-solid medium) in the absence of added growth factor (i.e., "control" conditions) , in the presence of 10% E-CSF, in the presence of 2 U recombinant human erythropoietin/ml (rhEpo/ml) , or in the presence of both E-CSF and EPO. In the absence of any added growth factor, the cells formed very small diffuse colonies in semi-solid medium. The addition of E-CSF to the cultures resulted in colonies that were more compact and significantly larger than the control colonies.
• Rauscher cells were plated in serum-substituted fibrin clot. Size of the resultant colonies was determined by counting individual cells in a gridded field. > 10 colonies were counted for each culture condition in a given experiment.
• Lymphopheresis of hematologically normal volunteer donors was performed at the New England Deaconess Hospital Blood Bank. Informed consent and approval of the Institutional Review Board was obtained for each donor.
• Cells were fractionated by sedimentation in Ficoll-Paque (Pharmacia, Piscataway, NJ) as described (L. Feldman, et al.. Proc. Natl. Acad. Sci. USA. 84:6775 (1987)). Mononuclear cells layering at the interface were washed in minimal essential medium, alpha modification (MEM ⁇ ; Gibco, Grand Island, NY) and depleted of platelets by the method of R.J. Perper, et al. , J. Lab. Clin. Med. 72:5 (1968) .
• Monocytes were removed by adherence to plastic tissue culture flasks. These procedures have been demonstrated previously to result in a nonadherent cell population which is greater than 97% lymphocytes by cytochemical and histochemical identification, (L. Feldman and N. Dainiak, Blood. 73:1814 (1990)). In some cases, B-lymphocytes were further enriched by separation of lymphocytes over nylon wool columns (S.A. Eisen, et al. f Immunol. Commun. , 1:571 (1972)). Nylon wool "adherent" cells were recovered, mixed with AET-treated sheep red blood cells (SRBC) and, following rosette formation, were separated over Ficoll-Paque (A. Saxon, et al.
• SRBC sheep red blood cells
• Bone marrow was aspirated from the posterior ileac crest of hematologically normal volunteers. Informed consent and approval of the Institutional Review Board were obtained for each donor. Cells were placed in MEM ⁇ containing 20 U preservative-free heparin/ml and separated over Ficoll-Paque. Light-density mononuclear cells were washed in MEM ⁇ and depleted of monocytes by plastic adherence.
• CM Conditioned Medium
• PM Lymphocyte Plasma Membranes
• Unfractioned lymphocytes, enriched B-cells, or highly purified B-cells were suspended at 5 x 10 6 cells/ml in MEM ⁇ supplemented with L-glutamine, penicillin and streptomycin (all Gibco) and were incubated overnight 14-18 hours) at 37°C, 5% C0 2 humidified air.
• CM was harvested by centrifugation at 00xg for 10 minutes, and cell-free.CM was fractionated into supernatants (S) and plasma membrane vesicle rich pellets (P) as described previously (L. Feldman, et al.. Proc. Natl. Acad. Sci. USA, 84:6775 (1987)).
• CM(S) were sterilized through 0.2u filters and stored at 4°C CM(P) were stored at -70°C in phosphate buffered saline (5mM sodium phosphate - 150mM sodium chloride, pH 7.6; PBS).
• phosphate buffered saline 5mM sodium phosphate - 150mM sodium chloride, pH 7.6; PBS.
• Lymphocytes recovered from the above CM were washed twice with PBS, lysed by Dounce homogenization, and plasma membranes (PM) were isolated by differential centrifugation and sucrose gradient fractionation by modifications of the methods of L. Feldman, et al. , Proc. Natl. Acad. Sci. USA. 84:6775 (1987); M. Jett, et a , J ⁇ . Biol. Chem.. 252:2134 (1977). Purified PM were stored at -70°C in PBS.
• E-CSF was solubilized from PM and CM(P) by sequential extraction with O.IN NaOH and 30mM octyl yff-D-glucopyranoside (Calbiochem, La Jolla, CA) (L. Feldman, et al. - Proc. Natl. Acad. Sci. USA. 84 : 6775 (1987); L. Feldman and N. Dainiak Blood. 73:1814 (1990)). Protein in solubilized conditioned medium (CM(S)) was concentrated by (NH 4 ) 2 S0 4 fractionation.
• Solubilized membrane proteins and concentrated proteins from CM(S) were fractionated on Sephacryl S-300 (Pharmacia) , DE-52 DEAE cellulose (Whatman, Clifton, NJ) , and hydroxylapatite (Calbiochem) columns as described to generate "column purified E-CSF", (L. Feldman, et al.. Proc. Natl. Acad. Sci. USA. 84: 6775 (1987)).
• Partially purified E-CSF was designated as that material with E-CSF activity which was recovered following S-300 chromatography. Partially purified or column purified E-CSF also can be electrophoresed on SDS-PAGE and electroeluted to generate "gel purified E-CSF".
• the electrophoresed protein(s) can be transferred to a PVDF membrane, such as Immobilon P (Millipore, Bedford, MA) .
• the band corresponding to E-CSF can be excised and used either to immunize animals for antibody production and/or to generate the amino acid sequence of the gel purified protein. (Matsudaira, P., Ed Practical Guide to Protein and Peptide Puri ication for Microsequencing Acad. Press (1989)) .
• E-CSF also has been solubilized from PM and CM(P) prepared from transformed ⁇ -lymphocytes (Raji, Ramos cells) by the same method of sequential extraction described above.
• Transformed ⁇ -lymphocyte cell lines, Ramos and Raji are maintained in RPMI 1640 - 3% fetal calf serum. Twenty-four hours prior to use, cells are washed extensively in serum-free RMPl 1640 and resuspended in the same serum-free medium for CM production (incubation overnight at 37°C, 5% C0 2 in humidified air) .
• Harvesting of CM and preparation of PM from the recovered cells is precisely the same as for the normal peripheral blood lymphocyte populations.
• E-CSF can be purified from non-human sources essentially as described for human E-CSF.
• Lymphocytes can be purified from non-human blood by the same procedures used to purify lymphocytes from human lymphopheresis products. In the cases where organs, such as spleens, are . used as a source of lymphocytes, the organs can be minced/disaggregated and single-cell suspensions can be prepared. Lymphocytes then are purified from the single-cell suspensions in the same manner as they are purified from whole blood or blood products.
• Conditioned media from non-human lymphocytes are harvested and fractionated into supernatants (CM(S)) and membrane-vesicle containing pellets (CM(P)) the same as for human-derived material.
• PM lymphocyte plasma membranes
• E-CSF has not yet been further purified from non-human sources, it is anticipated that the same chromatographic and electrophoretic manipulations can be used for non-human as for human E-CSF.
• the membranes of these non-human cells may differ from those of normal human cells (e.g., in protein and/or lipid composition) , it is expected that the presence of these differences may alter the behavior of E-CSF during purification. In this event, additional steps can be employed for the purification as needed.
• Clones R404, R28 and EMSIII are independently derived from primary Rauscher murine erythroleukemia line by limiting dilution, (N.J. DeBoth, et al.. Nature, 272:626 (London, 1978) .
• Rauscher cells, as well as human K256 and HEL cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 10% heat inactivated (56°C, 30 min) fetal bovine serum (FBS; HyClone, Logan, UT) , (C.B. Lozzio and B.B. Lozzio, Blood. 45:321 (1975); P. Martin and T.H. Papayannopoulou, Science. 216:1233 (1982) . Serum-free Culture of Bone Marrow Progenitors
• Adherent cell-depleted, human bone marrow progenitors were cultured in serum-free fibrin clots by modifications of the method of, E. Bruno, et al.. Exp. Hematol. , 16:371 (1988) ; and of the modifications of N. Dainiak, et al.. Hematol. 18:1073 (1985).
• Cultures contained highly purified recombinant human erythropoietin (rhEPO; 2 x 10 5 U/mg) (Elanex Pharmaceuticals, Bothell, WA) at 0 - 2.0 U/ml and 10% (vol/vol) column purified or partially purified E-CSF.
• Control cultures contained 10% (vol/vol) NCTC-109 (GIBCO) in place of E-CSF. Cultures were incubated at 37°C, 6% C0 2 in humidified air and were harvested on day 7 (CFU-E) and day 12 (for BFU-E) . Fixed clots were stained with benzidine and counterstained with hematoxylin. CFU-E containing >8 cells and BFU-E containing >50 cells were counted by light microscopy. Data are expressed as the mean +/- SEM for quadruplicate determinations for each test point. Each experiment was repeated a minimum of three times.
• rmGM-CSF and rmIL-3 were obtained from Genzyme Corp., Cambridge, MA. The experimental plates were incubated at 37°C, 5% C0 2 in humidified air and were examined under an inverted microscope on days 3, 7 and 10. Wells containing colonies of greater than 4 cells were counted.
• E-CSF The proliferative effect of E-CSF also can be assayed by measuring the uptake of [ 3 H]thymidine into responsive target cells. Several variations of this assay may be used, depending on the particular target cell employed.
• Derivative from this "Krystal-type" assay is the potential assay, using PHZ cells or enriched CFU-E from thiamphenicol-treated mice (J. Cell Biol 96:386 (1983)), where a specified number of cells are plated in the presence of a single sub-saturating dose of EPO minus or plus E-CSF.
• EPO plus E-CSF which potentiate the effect of EPO for CFU-E, will incorporate more [ 3 H]thymidine at a given dose of EPO than cells plated in the absence of E-CSF.
• erythroleukemia cells such as murine Rauscher or human K562 cells, which are EPO independent for their in vitro growth, at specified concentrations, are plated in serum- free on other suitable media, in the absence or presence of E-CSF for 12-96 hours and then pulse labeled with [ 3 H]thymidine.
• This variation of the assay measures the direct proliferative effect of E-CSF on cells in the absence of EPO.
• Protein was estimated by the method of O.H. Lowry, et al.. J. Biol. Chem.. 193:265 (1951) using bovine serum albumin (BSA) as a standard.
• BSA bovine serum albumin
• R28 was subcloned from a primary Rauscher murine erythroleukemia cell line by limiting dilution. N.J. DeBoth et al.. Nature. 272:626 (1978); A. Hagemeijer, J. Natl. Cancer Inst.. 69:945 (1982). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Grand Island, NY). To induce differentiation, DMSO (Sigma, St. Louis, MO) or highly purified recombinant human EPO (Elanex Pharmaceuticals, Inc. Bothell, WA) was added to the medium.
• DMEM Dulbecco's modified Eagle's medium
• FBS fetal bovine serum
• DMSO was "removed" by repeating washing and centrifugation of cells in DMSO-free medium followed by replating in the absence of DMSO.
• the effect of DMSO treatment on cell proliferation was examined by performing daily cell counts on replicate cultures grown for specified times in the absence or presence of DMSO.
• Hemoglobinized cells Hb + were assayed using benzidine staining. (R.A. Rifkind et al.. In Vitro Aspects of Erythropoiesis. New York, NY, p226 (1978)).
• Assays were performed by adding 10 ML freshly prepared benzidine reagent containing 0.6% H 2 0 2 , 0.5 mol/L CH j COOH, and 0.2% benzidine dihydrochloride to 50 ⁇ L of cells (0.5 to 1 x 10 cell/mL) in culture medium.
• the proportion of benzidine positive cells (blue cells) was scored out of 200 cells counted and is expressed as "Hb + cells, %". Variation between duplicate counts and repeat experiments was usually less than 5%.
• Rauscher cells were pretreated with 1% DMSO for 24 hours, washed twice, and then incubated in the absence (EPO " ) or presence (EPO + ) of EPO (10 U/mL) .
• EPO EPO
• the EPO " cells were 28% Hb +
• the EPO ⁇ cells were 56% Hb + (28% EPO-specific Hb + cells) .
• the EPO " cells were only 6% Hb + while the EPO + cells were 45% Hb + (35% EPO-specific Hb + ) .
• DMSO priming To characterize "DMSO priming" further, cells were pretreated with 1% DMSO for various periods of time. DMSO was removed, and EPO was added to the medium for 24 hours. The percentage of hemoglobinized cells was then assessed. A slight amplification of the EPO response was seen after only 6 hours of priming. A clear effect was observed after 12 hours. Maximal amplification occurred after 48 hours of priming.
• the dose-response of "DMSO priming" was examined next by pretreating R28 cells with different concentrations of DMSO for 24 hours. After removing DMSO, EPO was added to the cells for 24 hours. An almost linear relationship was observed between the EPO specific hemoglobinization and the concentration of DMSO used for priming.
• the EPO dose-response relationships in DMSO-primed and unprimed cells was examined. There is a marked left-shift of the dose-response curve.
• the EPO activation constant (K act ) defined as the concentration of agonist that causes 50% of the maximal response (M.E. Maguire et al.. Adv. Cyclic Nucl. Res.. 8:1, (1977)) is 0.1 U/mL and 2 U/mL for DMSO-primed and unprimed cells, respectively, representing an increased sensitivity of approximately 20-fold.
• the length of EPO exposure had no significant effect in the K act in either cell group under each condition tested.
• EPO priming amplified the DMSO response
• cells were incubated in the absence or presence of 10 U EPO/mL for 24 hours. The cells were washed and plated in the presence of 1% DMSO. The percent of Hb + cells in each culture was determined after an additional 16 hours and 48 hours of growth in DMSO. The unprimed cultures contained 0% and 23% ⁇ 3% Hb + cells, whereas the EPO-primed cultures contained 3% ⁇ 2% and 26% ⁇ 3% Hb + cells after 16 hours and 48 hours of DMSO treatment, respectively. EPO priming therefore has no significant effect on the DMSO response.
• Recombinant human EPO (Elanex) was labeled using IODOGEN (Pierce) (Fraker, P.J., and Speck, J.C, Biochem. Biophys. Res. Commun. 80:849 (1978); K. Sawada et al. ⁇ J. Clin. Invest.. 80:357 (1987)) and carrier-free (Amersham; 174 mCi/ ⁇ g iodine; 664 MBq/ ⁇ g) .
• 125 I-labeled EPO was purified using BioGel P6DG (BioRad) gel filtration in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (Sigma) and 0.02% Tween-20 (Sigma).
• PBS phosphate-buffered saline
• the specific radioactivity of 125 I-labeled EPO ranged from 0.1 to 0.4 gram atom/mol for different preparations.
• Iodinated EPO prepared using this method retained full biologic activity when assayed by an in vitro bioassay. (Krystal, G., EXP. Hematol. 11:649 (1983)).
• Cells were harvested by centrifugation, washed twice in Dulbecco's PBS, and incubated in DMEM containing 10% FBS, and 0.2% sodium azide (binding medium) for 30 minutes. Triplicate samples of 5 x 10 6 cells in 200 ⁇ h of binding medium containing 125 I-EPO at specified concentrations were incubated in the absence or presence of 100-fold unlabeled EPO at 37°C for 30 minutes. At the end of the incubation, cells were transferred to microfuge tubes containing 200 ⁇ L of FBS, centrifugated through the cushion of FBS for 5 minutes in a Beckman Microfuge 12, and frozen at -80°C The tip of the tube containing the cell pellet was cut off.
• Radioactivity in the cell pellet was measured in a Beckman Gamma 5500 counter. Results were corrected for nonspecific binding of 125 I-EPO. The nonspecific binding was in all cases less than 10% of the total. The binding data were analyzed by the method of Scatchard. (Scatchard, G., Ann. NY Acad Sci.. 51:660 (1949) .
• Rauscher murine erythroleukemia were the generous gift of N.J. DeBoth (Erasmus University, Rotterdam) .
• Clone R28 was derived by limiting dilution from its parent line.
• Clone PAN-4 was derived by sequential "panning" (Wysocki and Sato, Proc. Natl. Acad. Sci. USA. 75:2844-2848 (1978)) on Petri dishes coated with streptavidin and biotinylated erythropoietin as follows.
• Petri dishes (Falcon 1024) were coated with 0.01 ⁇ g of streptavidin per ml (10 ml) in coating buffer (1.6 g of Na 2 -C0 3 and 2.9 g of NaHC0 3 per liter) for 16 hours at 23°C The dishes were washed four times with sterile Dulbecco's PBS. Biotinylated, biologically active recombinant human erythropoietin (rhEPO) (10 ⁇ g) in 10 ml of PBS/1% bovine serum albumin was added for 2 hours at 23°C The solution was removed, and the dishes were washed five times with culture medium containing 1% FBS.
• coating buffer 1.6 g of Na 2 -C0 3 and 2.9 g of NaHC0 3 per liter
• R28 and PAN-4 appeared identical by detection of hemoglobin before or after treatment with erythropoietin and/or by staining with benzidine (Rifkind, supra) .
• DMSO, HMBA and sodium butyrate were purchased from Sigma.
• Cells were collected by centrifugation, washed twice with PBS, resuspended in DMEM/10% FBS/0.2% sodium azide (binding buffer) , and incubated at 0°C for 30 min to inhibit energy-dependent receptor-mediated endocytosis.
• each cell suspension was transferred onto a 200 ⁇ l cushion of FBS in a polypropylene centrifuge tube.
• the cells were sedimented by centrifugation at 9000 rp for 3 minutes (Beckman Microfuge) .
• the tube contents were frozen and the tips were cut off for measurement of bound radioactivity by ⁇ scintillation spectrometry.
• Specific binding at a given erythropoietin concentration was defined as the difference in bound radioactivity between the mean of samples incubated in the absence or presence of a 100-fold excess of unlabeled erythropoietin.
• Cytoplasmic RNA was prepared using guanidinium isothiocynate (Chirgwin, et al.. Biochemistry, 18:5294- 5299 (1979)). Forty micrograms of total RNA was subjected to electrophoresis in 1.2% agarose containing 5.5% formaldehyde and transferred to GeneScreen (DuPont) . The filters were hybridized sequentially with a 3 P-labeled synthetic oligomer complementary to nucleotides 256-305 of the Friend murine erythroleukemia cell erythropoietin receptor cDNA (D'Andrea et a . / Cell.
• the radiolabeled plasmid probe was generated by nick-translation (Rigby et al.. J. Mol. Biol.. 113:237-242 (1977)). Relative molecular mass of the mRNA that ⁇ hybridized to each of the probes agreed with previously published values.
• Log phase Rauscher cells are pretreated by incubation in serum-free ⁇ MEM medium, or other suitable medium, at a concentration of 2 x 10 6 /ml for 5 hours at 37°C, 5% C0 2 in humidified air.
• a "bioassay medium stock” is made from ⁇ MEM containing 1% deionized bovine serum albumin and 10% "synthetic serum” (see Dainiak et al.. Exp. Hematol. 18:1073, 1985 for composition of synthetic serum).
• a small snap-cap tube is made up containing 0.8 ml bioassay medium, 0.1 ml cells (5 x 10 4 /ml) , and 0.1 ml sample to be tested (i.e., E-SCF containing sample or ⁇ MEM for control tubes) .
• Replicate (typically 3-6) lOO ⁇ l aliquots from each control or experimental tube are plated in sterile 96-well tissue culture plates.
• Plates are incubated for up to 96 hrs. at 37°C, 5% C0 2 in humidified air.
• cells are pulsed for two hours with [ 3 H]thymidine (typically 25 ⁇ l of 20 ⁇ Ci/ml [ 3 H]thymidine in ⁇ MEM is added to each well) .

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