WO1990002183A1

WO1990002183A1 - Production of a novel lymphokine exhibiting differentiation inhibitory activity

Info

Publication number: WO1990002183A1
Application number: PCT/US1989/003560
Authority: WO
Original assignee: Genetics Institute, Inc.; Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 1988-08-18
Filing date: 1989-09-18
Publication date: 1990-03-08

Abstract

The purification, cloning and expression of a novel lymphokine having differentiation inhibitory activity are presented.

Description

PRODUCTION OF A NOVEL LYMPHOKINE EXHIBITING DIFFERENTIATION INHIBITORY ACTIVITY

Background

The production of a novel lymphokine having differentiation inhibitory activity is disclosed.

When transplanted organs are rejected by the host organism the organ becomes perfused with lymphocytes. This lymphokine was isolated from T cell clones of lymphocytes obtained from rejected human kidney allografts, and subsequently characterized. Moreau et al. Ann. Inst. Pasteur/Immunol. 137:25-37 (1986) ; Moreau et al. J. Clin. Invest. 78:874-879 (Oct. 1986); Moreau et al. J. Immunol. 138: 3844-3849 (1987) ; and Godard et al. Blood 71:1618-1623 at 1622 (June 1988). Based upon its ability to enhance the proliferation of the murine IL-3 dependent cell line DA-1, which cell line was described by Ihle et al. Adv. Viral Oncol. 4:95 (1985), the factor has been termed HILDA (Human-Interleukin-DA) . Subsequently, a variation of the DA-1 cell line, termed DA-2, was discovered having improved responsiveness to the factors produced by the clones of interest. Moreau et al. J. Immunol. supra.

HILDA has been found to have an activating potential on eosinophils, and thus may play a role in eosinophilia. Eosinophilia has been related to allergic conditions and parasitic infections. Several reports describe graft-infiltrating eosinophils as well as blood eosinophilia early in the course of rejection crisis. Blood eosinophilia also has been reported to be under T-cell control. Thus, on a fundamental level, HILDA may be used in further elucidating the host- defense mechanisms against parasites, as well as the role of T-lymphocyte activation in triggering eosinophils in various i munopathologic models.

More recently, HILDA has been found to have Differentiation Inhibitory Activity (DIA) . DIA refers to an assay, which measures the ability of a protein to block the differentiation of embryonic stem cells. Such inhibitory activity prevents maturation and thus, sustains the cells at the totipotent stage indefinitely. Totipotent embryonic stem cells are cells capable of being (programmed) initiated into all possible paths of differentiation. Embryonic stem cells cultured under standard conditions will differentiate into embroid bodies and thus lose their totipotent properties. It is contemplated that the DIA properties of HILDA will provide a vehicle for genetically altering all members of a particular cell type in an animal. Thus, one may introduce alterations into an animal to produce a chimeric strain that was constructed in vitro.

HILDA may also be used as a contraceptive, i.e., by blocking implantation of a blastocyst (embryo) . HILDA may be used to help bone marrow transplants engraft. The DIA properties of HILDA further suggest that HILDA may be used to sustain human hematopoietic stem cells, and as such allow the culturing of human hematopoietic stem cells in vitro, e.g., so that their numbers can be increased. It is further contemplated that HILDA may be used to culture hematopoietic stem cells, e.g., for gene transfer, and other physical, chemical and biological manipulations and selection.

The Invention

The present invention provides a method for the purification of natural HILDA. The invention also provides cDNA sequences coding on expression for HILDA, and methods for its production. Also provided are methods for engineering chimeric animals.

A cDNA clone, C10-6R (pXM.6R), was isolated by cDNA expression cloning and screening with the DA2 assay (Moreau et al. J. Immunol, supra.) . The cDNA expression library was made from the cell line C10MJ2. The cDNA sequence of pXM.6R, coding on expression for a polypeptide having HILDA-like biological properties, is shown in Figure 1. The pXM.6R clone when transfected into COS cells generated conditioned medium active to a 1:4000 dilution in the DA2 assay. The pXM.6R conditioned medium was found to be active to a 1:4000 dilution in the DIA assay described infra. One skilled in the art may readily obtain DNA sequences that hybridize under stringent conditions to the DNA sequence of Figure 1 in order to produce functionally equivalent polypeptides. Such DNA sequences will code on expression for polypeptides having at least one of the following HILDA-like biological activities; (a) active on DA-2 cells; (b) differentiation inhibitory activity; (c) differentiation activity on Ml cells; (d) eosinophil activating activity; (e) erythroid burst promoting activity.

Thus, one aspect of the invention provides DNA sequences coding on expression for a polypeptide having HILDA-like biological properties. Such sequences include the sequence of nucleotides in a 5' to 3• direction illustrated in Figure 1. Alternatively, a DNA sequence which hybridizes under stringent conditions with the DNA sequence of Figure 1 or a DNA sequence which hybridizes under relaxed conditions with the illustrated DNA sequences and which codes on expression for a protein having at least one HILDA-like biological property are included in the present invention. Allelic variations of the sequences of Figure 1, whether such nucleotide changes result in changes in the peptide sequence or not, are also included in the present invention, as well as other analogues and derivatives thereof. The recombinant DNA of this invention may be cDNA, genomic DNA or wholly or partially synthetic DNA's.

Additionally, the present invention provides a polypeptide resulting from the expression of a DNA sequence selected from the group consisting of: a DNA sequence encoding the polypeptide of Figure 1; a DNA sequence that hybridizes with the DNA sequence of Figure 1 under stringent conditions; and a DNA sequence that hybridizes with the DNA sequence of Figure 1 under relaxed conditions or which codes on expression for a protein sufficiently duplicative of the polypeptide of Figure 1 to possess at least one HILDA-like biological property.

The present invention provides the additional alternative of obtaining the natural polypeptide by purification. One method of purifying HILDA to apparent homogeneity comprises a five-step procedure of ConA affinity chromatography; Ion exchange HPLC; Gel filtration HPLC; Reverse phase HPLC; and SDS-PAGE.

HILDA, e.g., the polypeptide having the a ino acid sequence of Figure 1, has the following physiochemical characteristics: it is a basic monomeric glycoprotein that binds to ConA; binds S-Sepharose at pH6; does not bind DEAE at pH7; has an apparent molecular weight of 38-46 kD by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) under nonreducing conditions; and a specific activity estimated at 10⁸ DA2 Units/mg.

Another aspect of the invention is a vector containing a recombinant DNA sequence as described above in operative association with an expression control sequence therefor. The presently preferred vector in which the recombinant DNA may be incorporated is the pXM vector (ATCC Ace. No. 40480) . Other suitable vectors are selected from the group consisting of pCD, pJL3, pJL4, pCSF-MLA, pSHIL-3-1, and other equivalent vectors. Such vector may be employed in a novel process for producing a HILDA-like polypeptide. Specifically, the invention provides a method for producing polypeptides having HILDA-like biological properties comprising the steps of: (1) producing a cell (or progeny thereof)

^{• ■}— containing and capable of expressing a recombinant DNA, which (a) has been operatively linked to an expression control sequence therefor, (b) hybridizes under stringent conditions to the DNA sequence of Figure 1, and (c) upon expression encodes a protein having at least one HILDA-like biological property; (2) culturing said cell, or the progeny thereof, under conditions permitting expression of the desired protein; and (3) recovering said protein from the culture. HILDA-like biological properties include the ability to promote the growth of DA-2 cells; exhibit differentiation inhibitory activity; (c) differentiation activity on Ml cells; (d) eosinophil activating activity; (e) erythroid burst promoting activity.

The process for producing a HILDA-like polypeptide may employ a number of known cells as host cells for expression of polypeptide. Presently preferred cell lines are mammalian cell lines and bacterial cells. More specifically, preferred mammalian cell lines are the Chinese Hamster Ovary (CHO) cell, derivatives thereof (e.g., CHO CUKX Bll [DHFR_] , ENV-92 [Ada_] , and LDL [glycosylation mutant]) and the monkey COS cell line. Other mammalian cell lines that are suitable for use in the present invention are selected from the group consisting of HeLa, CV-1, mouse L-929, 3T3, BHK, HaK, 293, L cells, J558L, K562, KG1, ATT20, JM4, as well as a variety of other myeloma, fibroblast, and epithelial cell lines. Similarly, the various strains of E. coli, B. subtilis, Pseudomonas, other bacilli, as well as yeast and insect cells may also be used.

The HILDA and HILDA-like factors of the present invention include the natural proteins, recombinant versions thereof, and derivatives and analogues thereof, which may contain amino acid deletions, substitutions and/or insertions, but which retain the characteristic biological activity and are encoded by cDNAs capable of hybridizing under stringent conditions to cDNAs for the naturally occurring versions. Also

" s■^■"*r--τ"^!- ; ^•~r-^m _v ^{~ ~~}" ~~T* included are natually-occurring isotypes or allelic variations in the protein or its coding sequence resulting from expression of the protein in different members of a species. The polypeptides of the present invention may be used to engineer chimeric animals and strains of animals. When introduced into a blastocyst, embryonic stem cells are totipotent cells capable of being (programmed) initiated into all possible paths of differentiation. Thus, embryonic stem cells can be grown in vitro with a HILDA-like factor; manipulated genetically, e.g., transfected with retroviruses, DNAs encoding proteins, etc.; screened for the desired characteristic; and reintroduced into a blastocyst to generate a chimeric animal. When a chimeric animal with gonads derived from the transfected stem cell line is generated, animals may be crossed to create a strain which was genetically constructed in vitro. For background information regarding the engineering of such chimeric animals, see generally: Hogan, Nature 326:240-241 (March 1987); Gossler et al. Proc. Nat'l Acad. Sci. USA 83:9065-9069 (Dec 1986); Bradley et al. Nature 309:255-256 (May 1984); Evans et al. Nature 291:154-156 (1981); Hooper et al. Nature 326:292-295 (March 1987); Robertson et al. Nature 323:445-448 (Oct 1986); Kuehn et al. Nature 326:295-298 (March 1987); and Smith et al. Developmental Biology 121:1-9 (1987).

Additionally, the polypeptides of this invention may be used to culture relatively undifferentiated cells of various organs or tissue, e.g., muscle, nerve, epithelia, bone, spleen, liver, pancreas, thyroid, hematopoietic, or various other organs or tissues. In particular, the polypeptides may be especially useful for the in vitro and in vivo propagation and/or expansion and perhaps lineage-specific selection of such cells, especially bone marrow cells. Such use allows for the regeneration or repopulation of such tissues or organs. For example, bone marrow cells may be withdrawn from a subject prior to radiation treatment or other exposure deleterious to such cells, or even subsequent to such exposure prior to complete stem cell depletion. The withdrawn cells may then be cultured, and perhaps expanded with respect to the population of relatively undifferentiated stem cells therein, by culturing the withdrawn cells in the presence of the polypeptide of this invention. The resultant population of cells may then be reintroduced to the subject, with or without further manipulation. For example, such cells may be genetically engineered prior to reintroduction as a method for gene therapy. Alternatively, the cells may be further cultured in the presence of a differentiation or growth factor which directs the progeny to a different lineage-specific state of differentiation. For example, bone marrow cells withdrawn and cultured in the presence of the polypeptide of this invention, may then be cultured in the presence of primate IL-3 prior to reintroduction to expand the population of myeloid progenitor cells. Suitable concentrations of the polypeptide for use in culturing cells, whether for reintroduction into a subject or for the production of transgenic animals, comprise the range of 1-1000 picomolar, and preferably about 50-300 picomolar. By this method, it is contemplated that populations of various undifferentiated cells from any tissue or organ may be propagated, expanded and/or selected.

The HILDA-like polypeptides of the present invention may be used in a therapeutic pharmaceutical composition for treating a number of disease states characterized by a deficiency in the level of hematopoietic cells, such as macrophages, monocytes or stem cells of hematopoietic or other lineages. Such a composition comprises a therapeutically effective amount of the HILDA-like protein in admixture with a pharmaceutically acceptable carrier. A therapeutically effective amount of HILDA-like protein is between about 0.101000 ug/kg/day, and more preferably between about l- 500 ug/kg/day. The composition may be administered systemically, either parenterally, e.g., intravenously.

~~ '"~'~~" ^•"— — or subcutaneously. When systemically administered, the therapeutic composition for use in this invention may be in the form of a non-pyrogenic, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is presently thought to be within the skill of the art.

The use of the present invention may also include the simultaneous or sequential administration of an effective amount of at least one other hematopoietin, interleukin, or growth factor with the HILDA-like polypeptide. As such the invention comprises the use of a therapeutically effective amount of a HILDA-like polypeptide, with an effective amount of at least one other hematopoietin, interleukin or growth factor, for the preparation of a pharmaceutical product for treating a patient suffering from a disease characterized by a deficiency in the level of hematopoietic or other lineages comprising. Exemplary hematopoietins for such use include GM-CSF, G-CSF, CSF-

1 and erythropoietin. Exemplary interleukins are IL-1,

IL-2, IL-3, IL-4, IL-5, IL-6 and IL-7. Growth factors, such as B cell growth factor, a B cell differentiation factor, and an eosinophil differentiation factor, may also be employed.

The therapeutically effective amount of HILDA should be insufficient to cause an unwarranted systemic toxic reaction, but sufficient to elicit the desired therapeutic response. The actual dosing regimen for such formulations will be determined by the attending physician considering various factors which modify the action of drugs, for example, the condition, body weight, sex and diet of the patient, time of administration, and the degree of onset of the disease. The following examples are illustrative of the purification of natural HILDA, and the cloning and expression of a cDNA encoding HILDA. Example II utilizes the expression cloning technique of Clark et al. U.S. Pat. No. 4,675,285, which is incorporated herein by reference.

EXAMPLE I Purification of Natural HILDA

A. Cell Cultures Supernatants of human alloreactive T-lymphocyte clones (ATLCs) were harvested 4 days after specific antigenic stimulation in RPMI 1640 culture medium supplemented with 0.94 nmol/L pure recombinant IL-2 and 5% human agammaglobulinemic sera as already described in detail (Moreau, J. Clin. Invest. 78:874. 1986). Supernatants were kept sterile and stored at 4°C until used. The HILDA biologic activity of all batches was assessed as indicated below and is given in units per milliliter. Only supernatants containing >100 U/mL were further used.

B. Assays of HILDA and Recombinant Cytokines

This assay, previously reported in detail (Moreau, J. Immunol. 138: 3844, 1987), is based on the proliferation of the murine DA2 cell line induced by HILDA. In the proliferation assay, 20 x 10³ DA2 cells (in 0.05mL) were added in duplicates to 0.05mL of the threefold dilutions of the HILDA containing supernatants to be tested or in IL-3 WEHI-3 conditioned medium (CM) for positive control in 96-well microtiter plates (Falcon, Oxnard, CA) . After 36-hour incubation (37°C, 5% C0₂), 0.01 ml. phophate-buffered saline (PBS)- MTT solution (MTT [Sigma, St. Louis] 5mg/mL of PBS) was added to the wells. Four hours later, 0.15 mL propanol- 1-HC1 0.04 N was added according to the colorimetric assay previously described (Mosmann, J. Immunol. 65:55. 1983) . Microplates were then read in a Multiskan Titertek (Flow) . Results are reported in optical density at 570nm. The standard ATLC-CM was arbitrarily designated lOOU/ L (aliquoted and kept frozen at -80°C) and titrated in each experiment to determine maximum of absorbance arbitrary units. The dilution giving a

~ response equal to 50% of the maximum was then determined (optical density 50% max) . Each unknown sample was also titrated, and the dilution corresponding to the optical density 50% max was similarly determined. The ratio of dilutions of sample divided by standard and multiplied by 100 equals the number of units of HILDA per milliliter.

C. Supernatant Filtration-Concentration

Crude supernatants (2L) of ATLCs were first filtrated through a PTHK cassette (100,000 mol wt retention, Millipore Minitan System) to remove large molecular complexes and then concentrated -60-fold using a PTGC cassette (10,000 mol wt retention).

D. ConA Ultrogen Chomatography All chomatographic procedures were carried out at room temperature. ConA ultrogel (IBF) was packed in a 25-mL chomatographic column (IBF 25, ID 2.5 cm) and equilibrated with 40 mmol/L Tris-HCl buffer, pH 7.4 containing 110 mmol/L NaCl, 1 mmol/L MnC12, 1 mmol/L MgC12, and 1 mmol/L CaC12 (buffer A). The concentrated HILDA containing material (40 mL) was loaded on the column at a flow rate of 15 mL/h. The column was then washed with 100 mL buffer A before being eluted at a flow rate of 25 mL/h with different concentrations of a- methyl D-glucopyranoside (10 to 100 mmol/L) and a-methyl D-mannopyranoside (300 mmol/L) . Fractions of 4mL were collected and assayed for protein content and HILDA activity.

E. Ion-Exchange High-Performance Liquid Chromatography (HPLC)

Concentrated material (4mL) eluted by a-methyl D- glucose or a-methyl D-mannose from the ConA ultrogel column was applied after extensive dialysis (against 20 mmol/L Tris-HCl buffer, pH 7.4) on an anion exchange column [LKB ultropac diethylamino ethanol (DEAE) 5 PW, 7.5 x 75 mm. Beckman apparatus]. The elution was performed with 25 mL 20 MMol/L Tris-HCl buffer, pH 7.4 followed by a gradient of 0 to 0.5 mol/L NaCl in 20 mmol/L Tris-HCl, pH 7.4. Fractions of 1 mL were collected and assayed for HILDA bioactivity.

F. Gel-filtration HPLC

HILDA-active material not bound on the DEAE column and eluted in the void volume was concentrated and further applied to a preparative gel-filtration column (LKB ultropac TSK G 2000 SWG, 21.5 x 600 mm. Waters apparatus) . Samples of 2 mL were run at 4 mL/min and eluted at the same rate with 0.12 mol/L NaCl in 20 mmol/L phosphate buffer, pH 7.2. Fractions of 4 mL were collected and assayed for HILDA activity. The column was calibrated with bovine serum albumin (BSA) (67 kD) chymotrypsinogen (25 kD) , and ribonuclease (13.7 kD) .

G. Reverse-Phase HPLC

Fractions of gel filtration with the highest HILDA activity were pooled, concentrated with polyethylene glycol (PEG) 35,000 (FLUKA) , dialyzed against 0.1% trifluoroactic acid (TFA) in water, pH 2.5, and applied to the column.

Reverse-phase chomatography was performed with an LKB System and a TMS 250 column (4.6 x 75mm), with 0.1% TFA in water as starting solvent. The column was eluted with increasing concentrations of acetonitrile in 0.1% TFA using the following gradient: 0 to 20% acetonitrile for 5 minutes, 20% to 60% for 40 minutes, and 60% to 100% for 5 minutes. The flow rate was 0.8 mL/min.

Fractions of 0.8 mL were automatically collected, directly evaporated to dryness in a Speed Vac Concentrator-Evaporator system (Savant, Hicksville, NY), resuspended in a PBS buffer, pH 7.4 (lmL) , and assayed for HILDA activity.

H. Radiolabeling of Purified HILDA The most active fraction from reverse-phase chromatography (2,000 U) was dialyzed, evaporated to dryness, and resuspended in 0.05 mL 50 mmol/L sodium- phosphate buffer pH 7.5. This 0.05 mL was transferred to a glass tube coated with iodogen as described by Fraker fBiochem. Biophvs. Res. Commun. 80:849. 1978), and 0.002 mL (100 uCi) of (¹²⁵I)-Na (C.E.A. , Saclay, France) were added. After 20 minutes of agitation at room temperature, the reaction was quenched by addition of 0.45 mL PBS. The mixture was then transferred to another glass tube, and 0.05 L PBS containing 1% BSA was added to stabilize the protein. Radiolabeled proteins were separated from free iodine by chomatography on a Dowex AX1 (Biorad) column. Radioactive fractions were pooled, dialyzed against 10 mmol/L Tris-HCL buffer, pH 7.2, and chomatographed on a lOmL anion-exchange column (DEAE Ultragel, LKB) to separate radioactive HILDA, which eluted in the void volume, from contaminating radiolabeled BSA, which eluted when 1 mol/L NaCl was applied. The peak HILDA fraction (0.045 L - 10,000 cpm) was analyzed by sodium dodecyl sulfate polyaery1amide gel electrophoresis (SDS- PAGE) and autoradiographed for 4 days at -70°C. The same material (0.40 mL) was concentrated and run on the same gel for detection of HILDA activity after elution from the gel.

I. SDS-PAGE

SDS-PAGE was performed according to the method of Laemmli (Laemmli, Nature (London) 227:680, 1970) on linear 5% to 15% acrylamide gradient slab gels (1.5 mm thick) . Before loading, lyophilized samples were heated for 30 minutes at 56°C in sample buffer under reducing or nonreducing conditions (Lane, Anal. Biochem. 86:655, 1978). After electrophoresis (3 hours at 45 mV) , the acrylamide gels were either stained by the silver-nitrate method of Merril (Merril, Science 211:1437, 1981) or cut in thin slices of 1 mm that were each eluted overnight at 4°C in RPMI 1640 containing 10% human serum (agammaglobulinemic) and 10 mg/mL BSA and assayed after centrifugation for HILDA activity. Apparent mol wt was determined with protein standards: phosphorylase b (94,000), BSA (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,000), and lactalbumin (14,400).

J. Protein Assays

For all steps up to HPLC gel filtration, protein content of fractions was estimated by the method of Lowry (Lowry, J. Biol. Chem. 193:265, 1951), with BSA as standard. After this step, the protein content was too low to be determined with accuracy by any common protein assay method, so that the protein concentration was estimated either from the absorbance profile at 280 n , assuming an average OD₂80 ^{of 1 for a} protein sample of 10 mg.mL, or from the intensity of silver-stained bands on polyacrylamide gels using BSA as standard. These estimates constitute approximate values.

The purification protocol gave a hydrophobic glycoprotein of 38-46 kD with specific activity of -10⁸-10⁹ U/mg (see Godard et al. supra. at 1618 for the definition of U) with an 85,000-fold purification and a recovery of 15% of the biologic activity of the starting material.

EXAMPLE II

Production of Recombinant HILDA

A. Assay for HILDA activity:

T-lymphocyte clones from rejected human kidney allografts have been shown to produce a novel lymphokine which supports the growth of a murine cell line, DA2. Moreau et al. Ann. Inst. Pasteur 137:25-27 (1986) . Conditioned medium obtained from the human lung carcinoma cell line H23 following induction with TPA also supports the proliferation of the DA2 line. DA2 cultures were grown in RPMI medium (5% D02) containing 10% H23 post-induction conditioned medium. 5% heat inactivated Fetal Calf Serum, 2mM glutamine, lOOU/mL streptomycin and lOOug/ml penicillin. Cells were subcultured every 3-4 days by seeding into 10 ml cultures at IO5 cells/ml. Post-induction H23 conditioned medium was obtained by growing cells in RPMI as described above for 24 hours in the presence of 40ng/ml TPA. Cells were washed and conditioned medium collected after three days.

For the proliferation assay, cells were washed once and seeded at 10₄ cells/10Oul/well in Costar U- bottomed 96-well tissue culture plates. Three-fold serial dilutions of experimental samples were applied in duplicate and at 72 hours, proliferation was measured by the MTT colorimetriσ assay. Mosmann, Journal of Immunol. Methods 65:55-63 (1983) . Assay medium was growth medium without H23 post-induction conditioned medium.

B. Identification of the T-σell line C10-mj2 as a source of HILDA: Conditioned media from various human cell lines were assayed for HILDA using the DA2 proliferation assay. PMA/PHA-M post-induction conditioned medium from the human T-cell line C10-mj2 was identified as a source of HILDA. Yang et al. Cell 47:3-10 (1986).

c. Identification of pA+ RNA encoding HILDA:

Total RNA was isolated from C10-mj2 cells either 24 hours after induction with TPA/PHA. Chirgwin et al. Biochemistry, 18:5294-5299 (1979). Superinduction of RNA was achieved by the addition of cycloheximide to the C10-mj2 culture at 50 micrograms/ml 24 hours after induction with TPA/PHA after 4 hours. pA+ RNAs selected by affinity σhromatography using oligo(dT)- cellulose from either the TPA/PHA induced or superinduced total RNA were injected into Xenopus oocytes. Gurdon et al. Nature. 233:177-182 (1971). Forty-eight hour oocyte conditioned medium was collected and tested for DA2 proliferation activity. Conditioned medium from oocytes injected with TPA/PHA induced PA+ RNA caused proliferation of DA2 cells.

D. Expression cloning of HILDA:

A cDNA library was constructed using superinduced C10-mj2 pA+ RNA and the expression cloning system described by Clark et al., (U.S.P. 4,675,285) with the following changes. The cDNA was not methylated and semi-Xho adapters were ligated to the cDNA after the blunting reaction. The vector pXM was digested with Xho I and made complementary to the semi-Xho adapters by a fill-in reaction using dTTP and the large fragment of DNA polymerase I. Y.C. Yang et al. Cell. 47:3-10 (1986) . The conditioned medium from each COS transfection was assayed for HILDA using the DA2 assay. A single clone, pXM.6R, (ATCC Ace. No. 40480) was isolated which directed the expression of HILDA by COS cells.

EXAMPLE III Assay for Differentiation Inhibitory Activity

HILDA produced in accordance with the procedure of Example II was assayed for differentiation inhibitory activity according to the procedure of Smith et al. Dev. Bio. 121:1-9 (1987).

BRL cells are a clonal isolate from the liver of an adult Buffalo rat. Coon J. Cell Biol. 39: 29a

(1968) . The embryonal carcinoma feeder dependent line

PSA4 was isolated from teratocarcinoma OTT-5568 by

Martin and Evans. Martin, G.R. , and Evans, M.J. "The formation of embryoid bodies in vitro by homogenous embryonal carcinoma cell cultures derived from isolated single cells." Teratomas and Differentiation (M.I. Sherman and D. Solter, Eds.), pp. 169-187. Academic Press, New York (1975) . BRL cells were grown in Dulbecco's modified Eagles/Ham's F12 medium (1:1) supplemented with 10% fetal bovine serum and 1 mM glutamine and incubated under 5% carbon dioxide at 37°C. BRL cell-conditioned medium was obtained by incubating 30 mL of the above medium on a 175 cm. flask of BRL cells and harvesting every 3 days for up to 1 week. The harvested BRL conditioned medium was sterilized by filtration and stored at -20°C. The embryonal carcinoma cells were grown in BRL conditioned medium diluted to 70% (v/v) with fresh DME/Ham's F-12 medium supplemented with 1 mM glutamine, 10% fetal bovine serum, and 0.1 mM B-mercaptoethanol. In addition, the final fetal bovine serum concentration was adjusted to 20% (v/v) . All flasks used for culturing EC cells were precoated with 0.1% gelatin just prior to use. Bernstine et al. Proc. Nat'l Acad. Sci. USA 70:3899-3903 (1973). HILDA samples screened for DIA activity were serially diluted in assay medium in 24-well assay dishes. Assay medium contained DME/Ham's F-12 medium supplemented with 20% fetal bovine serum, 1 mM glutamine, and 0.1 mM B-mercaptoethanol. Each well was then seeded with 10,000 freshly trypsinized and washed EC cells. After 4 days incubation the cells were fixed in methanol/acetic acid (3:1) and stained with Leishman's stain. The existence of DIA in the samples was indicated by the normal growth of undifferentiated EC cells. The undifferentiated EC cells were small and grew in "nests" or tightly packed colonies and stained intensely blue. Griep et al. Exp. Cell Res. 164:223-231 (1986) .

Claims

What is claimed is:

1. A DNA sequence coding on expression for a polypeptide having at least one of the following HILDA- like biological activities: a. active on DA-2 cells; and b. differentiation inhibitory activity

2. A vector comprising the DNA sequence of claim 1.

3. A cell transfected with the vector of claim 2.

4. The cell of claim 3 selected from the group consisting of Chinese Hamster Ovary cells (CHO) , derivatives thereof, and monkey COS cells.

5. A polypeptide encoded by the DNA sequence of claim 1.

6. The polypeptide having the a ino acid sequence of Figure 1.

7. The polypeptide resulting from the expression of an isolated DNA sequence selected from the group consisting of: a DNA sequence homologous with the DNA sequence of Figure 1; a DNA sequence that hybridizes with the DNA sequence of Figure 1; a DNA sequence that hybridizes with the DNA sequence of Figure 1 under stringent conditions; and a DNA sequence that hybridizes with the DNA sequence of Figure 1 under relaxed conditions and codes on expression for a protein having at least one HILDA-like biological property.

8. A method for producing polypeptides having HILDA and HILDA-like biological properties comprising the steps of: (1) culturing an isolated cell transformed with a vector that comprises DNA, which

BS ii έESHEET (a) has been operatively linked to an expression control sequence therefor,

(b) hybridizes under stringent conditions to a DNA sequence of claim 1, and (c) upon expression encodes a protein having at least one HILDA-like biological property; and (2) recovering said protein from the culture.

9. Use of a therapeutically effective amount of a polypeptide having HILDA-like biological properties in admixture with a pharmaceutically acceptable carrier for the preparation of a pharmaceutical composition for treating a disease state characterized by a deficiency in the level of hematopoietic cells, such as macrophages, monocytes or stem cells of hematopoietic or other lineages.

10. A method for producing chimeric animals comprising culturing genetically manipulated embryonic stem cells in vitro with a HILDA-like factor, screening for the desired characteristic; and reintroducing the stem cells into a blastocyst.

11. The use of the polypeptide of claim 7 in a therapeutically effective amount, with an effective amount of at least one other hematopoietin, interleukin or growth factor, for the preparation of a pharmaceutical product for treating a patient suffering from a disease characterized by a deficiency in the level of hematopoietic cells, such as macrophages, monocytes or stem cells of hematopoietic or other lineages.

12. The use according to claim 11, wherein said hematopoietin is selected from the group consisting of M-CSF, GM-CSF, G-CSF, CSF-1 and erythropoietin.

13. The use according to claim 11, wherein said interleukin is selected from the group consisting of IL-1, IL-2, IL-3, IL-4 , IL-5, IL-6 and IL-7.

14. The use according to claim 11, wherein said growth factor is a B cell growth factor, a B cell differentiation factor, or an eosinophil differentiation factor.

15. The vector pXM.6R.

16. Purified HILDA having an activity of at least about 10⁸-10⁹ DA2 Units/mg.

SUBSTITUTE SHEET