A NOVEL FAMILY OF PRIMATE HEMATOPOIETIC GROWTH FACTORS

Abstract: A novel family of primate IL-3-like polypeptides is provided via recombinant techniques.

Description: A NOVEL FAMILY OF PRIMATE HEMATOPOIETIC GROWTH FACTORS The present invention relates to a novel family of primate IL-3-like hematopoietic growth factors, and a process for producing them by recombinant genetic engineering techniques. Background Hematopoietins, i.e., hematopoietic growth factors, are proteins that promote the survival, growth and differ¬ entiation of hematopoietic cells. Colony stimulating factors (CSFs) are a subset of these hematopoietic growth factors that are characterized by the ability to support the growth, in. vitro, of colonies of hematopoietic cells arising from progenitor cells of bone marrow, fetal liver, and other hematopoietic organs. The biochemical and biological identification and characterization of certain hematopoietins was hampered by the small quantities of the naturally occurring factors available from natural sources, e.g., blood and urine. Some of these hematopoietins have recently been molecularly cloned, heterologously expressed and purified to homogeneity. [D. Metcalf, "The Molecular Biology and Functions of the Granulocyte-Macrophage Colony Stimulating Factors," Blood. 67_(2) :257-267 (1986).] Among these hematopoietins are human and murine GM-CSF, human G-CSF, human CSF-1 and murine IL-3. Human GM-CSF [R. Donahue et al., Nature. 321: 872-875 (1986)], murine IL-3 [J. Kindler et al, Proc. Natl. Acad. Sci. U.S.A.. £1:1001-1005 (1986); Metcalf et al., Blood. 68:46-57 (1986)] and human G-CSF [K. Welte et al, J\. EXP. Med.. 165.941-948 (1987)] have demonstrated effects on hematopoiesis in vivo. Despite extensive research with the murine protein IL-3, no human counterpart had heretofore been found. [D. R. Cohenet l, Nucl. AcidsRes., 14:3641 (1986)]. Brief Summary of the Invention The present invention provides a family of primate IL-3-like growth factors substantially free from other primate proteins and characterized by peptide sequences the same as or substantially homologous to the a ino acid sequences illustrated in Tables I and II below. These primate proteins, including especially the human proteins, are also referred to hereinafter simply as IL-3. These proteins are encoded by the DNA sequences depicted in the Tables and by sequences capable of hybridizing thereto, or capable of hybridizing thereto but for the degeneracy of the genetic code, which encode polypeptides with IL-3-like biological properties, and by other variously modified sequences demonstrating such properties. These polypeptides are also characterized by IL-3-like biological properties. As one example, the invention provides a human polypeptide substantially free from association with other human polypeptides characterized by a peptide sequence comprising the same or substantially the same sequence as depicted in Table II and having at least one IL-3-like biological property. As another example of the polypeptides of the present invention a gibbon polypeptide substantially free of association with other primate polypeptides is characterized by a peptide sequence comprising the same or substantially the same sequence as shown in Table I and having at least one IL-3-like biological property. The polypeptides of the present invention are characterized by the ability to stimulate at 10 to 100 picomolar concentrations the formation of multiple types of hematopoietic colonies of various lineages in the standard human bone marrow assay, other IL-3-like properties which may be shared by the proteins of the present invention include the following: (a) an apparent molecular weight of between about 14 to about 35 kilodaltons as determined by reducing SDS polyacrylamide gel electrophoresis; (b) the ability to stimulate CML cell proliferation at 1 to 100 picomolar concentrations in the CML assay; (c) an isoelectric point between pH6.0 and 7.6; (d) a single, preferably sharp, peak at 43 kd on a Superos 6 gel filtration column; (e) a single band of 20.5kd on a gel following N-glycanas treatment; and (f) a level of contaminants no more than about 2% upon N terminal analysis using Edman degradation. Another aspect of the invention provides pharmaceutica compositions containing a therapeutically effective amount o one or more polypeptides according to the invention. Thes compositions may be employed in methods for treating a numbe of disease states characterized by a deficiency in the level o hematopoietic cells. These methods according to the inventio entail administering to a patient an effective amount of a least one polypeptide as described herein. Such therapeutic methods may be directed to treat variou pathological states resulting from disease, exposure to radiatio and/or drugs, including e.g., leukopenia, thrombocytopenia anemia, B cell deficiency, T cell deficiency, bacterial infection and viral infection, including immune cell or hematopoieti cell deficiency following a bone marrow transplantation. Thes methods of the present invention may also include administerin simultaneously or sequentially with the IL-3-like polypeptides an effective amount of at least one other hematopoietin interleukin, or growth factor. Exemplery hematopoietins fo such use include GM-CSF, G-CSF, CSF-1 or erythropoietin Exemplary interleukins are IL-1, IL-2, IL-4 or IL-6. Th methods may also employ growth factors like a B cell growt factor, a B cell differen-tiation factor, or an eosinophi differentiation factor. Still a further aspect of the invention are DNA sequences, including cDNA sequences, coding on expression for a primat polypeptide having at least one IL-3-like biological property. Such sequences include the sequence of nucleotides in a 5' to 3' direction illustrated in Table I or II. Alternatively a DNA sequence which hybridizes under stringent conditions with the DNA sequence of Tables I or II or a DNA sequence which hybridizes under non-stringent conditions with the illustrated DNA sequences and which codes on expression for a protein having at least one IL-3-like biological property is included in the present invention. Allelic variations of the sequences of Tables I and II, whether such nucleotide changes result in changes in the peptide sequence or not, are also included in the present invention, as well as other analogs and derivatives thereof. Still a further aspect of the invention are vectors containing, and cells transformed with, a DNA sequence as described above in operative association with an expression control sequence. Such vectors and transformed cells may be employed in a novel process for producing a primate IL-3-like polypeptide in which a cell line transformed with a DNA sequence encoding expression of an IL-3-like polypeptide in operative association with an expression control sequence therefor is cultured. This claimed process may employ a number of known cells as host cells for expression of the polypeptide. Presently preferred cell lines are mammalian cell lines and bacterial cells. Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of preferred embodiments thereof. Detailed Description of the Invention The family of primate IL-3-like growth factors provided by the present invention are substantially free of association with other primate proteinaceous materials and are characterized by amino acid sequences the same as or substantially homologous to the sequences shown in Tables I and II below. The members of this novel family of growth factors are also characterized by having at least one biological property of an IL-3-lik growth factor, as described below. Preferably more than on IL-3-like biological property is demonstrated by any one membe of the family of growth factors of the present invention. The term "IL-3-like biological property" is defined herei to include one or more of the following biological characteristic and in vivo and n vitro activities. One such property is th support of the growth and differentiation of progenitor cell committed to erythroid, lymphoid, and myeloid lineages. Fo example, in a standard human bone marrow assay, an IL-3-lik biological property is the stimulation of granulocytic typ colonies and erythroid bursts. Another such property is th interaction with early multipotential stem cells. Another IL-3-like biological property is the sustaining o the growth of pluripotent precursor cells. Another property i the ability to stimulate chronic myelogenous leukemia (CML) cel proliferation. An IL-3-like biological property also is th stimulation of proliferation of mast cells. IL-3-like growt factors may also support the growth of various factor-dependen cell lines and/or induce the expression of 20-alpha-steroi dehydrogenase (20-alpha-SPH) and Thy-1 antigen. Further IL-3 like biological properties are the stimulation of colony formatio on KG-1 cells and/or the stimulation of increased histamin synthesis in spleen and bone marrow cultures. Yet another IL-3 biological property is an apparent molecular weight of betwee about 14 to about 35 kd by reducing sodium dodecyl sulfate polyacryla ide gel electrophoresis. Other biological properties of IL-3-like proteins have been disclosed in the art with reference to murine IL-3. The specific peptide sequences illustrated in Tables I and II are two exemplary members of the growth factor family of the present invention. The 865bp DNA sequence of Table I was isolated from a cDNA expression library of the gibbon ape leukemia virus-infected gibbon T-cell line UCD-144-MLA [T.G. Kuwakami et al. Nature. 235:170 (1972)]. This sequence contains a single long open reading frame of 456 nucleotides which encodes an approximately 152 amino acid protein, called CSF-80, and includes a conventional leader secretory sequence indicated by the highly hydrophobic sequence (leu leu leu leu gin leu leu) . The mature protein begins at amino acid number 20, alanine, in Table I. The coding region contains three cysteines, two in the mature protein, thereby suggesting one disulfide bond. There are two potential asparagine-linked glycosylation sites illustrated by the characteristic sequences, Asn-X-Ser or Asn-X-Thr. Both the size and glycosylation pattern revealed by the coding sequence are typical of lymphokine-like proteins. The remaining non-coding portions of the 865bp region may have a regulatory role in transcription in the natural host. The 3' end of the sequence also contains an AT-rich segment including several repeats of the sequence ATTTA, which is believed to be related to the RNA message stability [See, G. Shaw and R. Kamen, Cell. 46 (5): 659-677 (1986)]. Table I 10 20 30 49 CTCGAGCTAC GTCAACGAAA AATAAAftTCC AAAC ATG AGC TGC CTG COC GTC C G CTC MET Ser Cys Leu Pro Val Leu Leu 64 79 94 109 CTG CTC CAA CTC CTG GTC AGC CCC GGA CTC CAA GCT CCC ATG ACC CAG ACA ACG Leu Leu Gin Leu Leu Val Ser Pro Gly Leu Gin Ala Pro MET Thr Gin Thr Thr 124 139 154 TCC TIG AAG ACA AGC TGG GTT AAC TGT TCT AAC ATG ATC GAT GAA ATT ATA ACA Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn MET lie Asp Glu lie lie Thr 169 184 199 214 CAC TTA AAG CAG CX-AOCT TTG < CTIG CTGGACTTCAAC AAC CTC AAT GGG GAA His Leu Lys Gin Pro Pro Leu Pro leu Leu Asp Phe Asn Asn Leu Asn Gly Glu 229 244 259 274 GAC CAA GAC ATT CTG ATG GAA AAT AAC CT CGA AGG CCA AAC CTG GAG GCA TTC Asp Gin Asp lie Leu MET Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 289 304 319 AAC AAG GCT GTC AAG ACT TTA CAG AAT GCA TCA GCA ATC GAG AGC ATT CTT AAG Asn Lys Ala Val Lys Ser Leu Gin Asn Ala Ser Ala lie Glu Ser lie Leu Lys 334 349 364 379 AAT CTC CCC CCA TGC CTG CCC ATG GCC ACA GCC GCA CCC ACG CGA CAT CCA ATC Asn Leu Pro Pro Cys Leu Pro MET Ala Thr Ala Ala Pro Thr Arg His Pro lie 394 409 424 CGT ATC AAG GAC GGT GAC TGG AAT GAA TTC CGG AGG AAA CTG AAG TTC TAT CTG Arg lie Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Lys Phe Tyr Leu 439 454 469 484 AAA ACC CTΓ GAG AAT GAG C-AA GCT CAA CAG ATG ACT TIG AGC COT ATC TCΓ Lys Thr Leu Glu Asn Glu Gin Ala Gin Gin MET Thr Leu Ser Leu Glu lie Ser 500 510 520 530 540 550 560 TGAGTCCAAC GTCCAGCTCT CTCTCTGGGC CGTCTCACCG CAGAGCCTCA GGACATCAAA AACAGCAGAA 570 580 590 600 610 620 630 C TCTGAAAC CTCTGGGTCG TCTCTCACAC AGTCCAGGAC CAGAAGCATT TCACCTTTTC CTGCGGCATC 640 650 660 670 680 690 700 AGATGAATTG TTAATTATCT AATTTCTGAA ATGTGCAGCT CCCATTTGGC CITGTGTGGT TGTGTTCTCA Table I (cont'd) 710 720 730 740 750 760 770 TΓΓΓΓATCCC ATTGAGACA TITATGTATG TCTGTATΓTA UALL IAT TATΓTATΓGC CTCTGGAGC 780 790 800 810 820 830 840 GTGAΆGTGTATITATΓTCAG CAGAGGAGCC ATGTCATGCT GCTTCGCAAAAAACΓCAAG AGTGGGGTGG 850 860 GGAGCATGTT CATITGTACC TCGAG The 674bp DNA sequence of Table II was obtained from human genomic library [J. J. Toole et al. Nature. 312:342-34 (1984)] by employing the sequence of Table I as a probe. The DN sequence of Table II was initially constructed by splicing togethe the exons of the human genomic sequence, which were identified b comparison with the DNA sequence of the gibbon IL-3-like polypeptid of Table I. This human sequence confirmed by mRNA analysis o the human cDNA clone also codes for a polypeptide of approximatel 152 amino acids, which is a member of this family of primat proteins. This human polypeptide includes a conventional leade secretory sequence indicated by the highly hydrophobic sequenc (leu leu leu gin leu leu) . The mature polypeptide begins a amino acid number 20, alanine, in Table II. The coding regio contains two cysteines in the mature protein, suggesting on disulfide bond. There are two potential asparagine-linke glycosylation sites illustrated by the characteristic sequence Asn-X-Ser. The remaining non-coding portions of the 674bp sequenc may have a regulatory role in transcription in the natural host The nucleotide sequences of the exons of the human genomi gene [Table II] were more than 96% homologous with the DNA sequenc of the gibbon gene [Table I] . Changes in the nucleotide sequence in 11 codons result in amino acid differences in the gibbon an human proteins. The nucleotides appearing above the sequence o Table II indicate the sites where the gibbon sequence differ from the related human sequence. Similarly, the amino acid appearing below the human amino acid sequence indicate where th gibbon sequence differs. Table II A 9 T 24 39 A54 GATCCAAAC ATG AGC CGC CTG CCC GTC CSG CTC CTG CTC CAA C3C CXG GTC CGC MET Ser Arg Leu Pro Val Leu Leu eu Leu Gin Leu Leu Val Arg Cys Ser 20 (27) 69 84 [C] 99 CCC GGA CTC CAA GCT CCC ATG ACC CAG ACA ACG TGC TTG AAG ACA AGC TGG GTT Pro Gly Leu Gin Ala Pro MET Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val 114 T 129 144 159 AAC TGC TCT AAC ATG ATC GAT GAA ATT ATA ACA CAC TTA AAG CAG CCA CCT TTG Asn Cys Ser Asn MET lie Asp Glu lie He Thr His Leu Lys Gin Pro Pro Leu 50 C 174 189 204 CCT TTG CTG GAC TTC AAC AAC CTC AAT GGG GAA GAC CAA GAC ATT CTG ATG GAA Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp He Leu MET Glu 219 234 249 A 264 AAT AAC CTT CGA AGG CCA AAC CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Lys T 279 C 294 G 309 CC C 324 CAG AAC GCA TCA GCA ATT GAG AGC ATT C T AAA AAT CTC CTG OCA TGT CTG CCC Gin Asn Ala Ser Ala lie Glu Ser lie Leu Lys Asn Leu Leu Pro Cys Leu Pro 100 Pro A A 339 354 G 369 CTG GCC ACG GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG Leu Ala Thr Ala Ala Pro Thr Arg His Pro lie His lie Lys Asp Gly Asp Trp MET Arg 429 384 399 A 414 A A AAT GAA TTC CGG AGG AAA CTG ACG TTC TAT CTG AAA ACC CIT GAG AAT GCG CAG Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gin 130 Lys Glu 444 459 485 T T A C G 475 TC 495 GCT CAA CAG ACG ACT TTG AGC CTC GCG ATC TTT T-AGTCCAACG TCCAGCTCGT TCTCTGGGCC Ala Gin Gin Thr Thr Leu Ser Leu Ala lie Phe MET 147 Glu Ser Table 21 (cont'd) 505 515 555 C G A A 525 535 545 G 565 TΓCΓCACCAC AGCGCCTCGG GACATCAAAAACAGCAGAAC TΓCTGAAACC TCTGGGTCAT CTCTCACACA G 575 585 595 605 615 625 635 TΓCCAGGACC AGAAGCATΓT CACLTΓΓICC TGCGGCATCA GATGAATΓGT TAATΓATCΓA ATΓΓCΓGAAA 645 655 T 665 TGTGCAGCTC CX30TTGGCC TTGTGCGGTT GTGrTTCTCA A computer search by National Biomedical Services of Washington, D.C. revealed that the gibbon and human IL-3-like sequences have approximately 29% homology at the amino acid level and 45% homology at the nucleotide level to the murine IL-3 DNA sequence, as published by M. C. Fung et al.. Nature. 307:233-237 (1984). Exon structures of the human IL-3-like gene compared similarly with the coding regions of the murine IL-3. The novel 865bp cDNA sequence illustrated in Table I included in a plasmid in E. coli HB101, has been deposited in the American Type Culture Collection, 12301 Parklawn Dr. , Rockville, MD on July 11, 1986 and given accession number ATCC 67154. The novel genomic sequence, for which the cDNA sequence is illustrated in Table II below, included in bacteriophage lambda, has been similarly deposited on August 7, 1986 and given accession number ATCC 40246. The human IL-3 DNA included in plasmid pSHIL-3-1 in E. coli HB101 has been deposited in the ATCC on February 24, 1987 and given accession number ATCC 67326. The exemplary human and gibbon IL-3-like polypeptides have been further characterized by SDS polyacrylamide gel analysis of the 35 S-labeled proteins from transfected COS cells, as described below in the examples. Both polypeptides are heterogenous in size with molecular species having a range of apparent molecular weight of between about 14kd-35kd, and more specifically, 18kd-30kd. This range of molecular weights for these exemplary IL-3-like factors is believed to result from variations in glycosylation of the purified COS cell produced molecules. The purified proteins, at 10 to 100 picomolar concentrations, cause the formation of small granulocytic-type colonies in in vitro human bone marrow assays. Additionally, in the presence of erythropoietin in these human bone marrow assays both polypeptides support the growth of erythroid and myeloid progenitor cells at comparable levels of activity. Thus these IL-3-like factors are multi-CSFs. These IL-3-like factors also cause the proliferation of leukemic blast cell from patients with CML. These polypeptides may also be capabl of stimulating accessory and mature cells, e.g. onocytes, t produce other hematopoietic-like factors, which in turn stimulat the formation of colonies of other hematopoietic cells, as wel as other hematopoietic-type activities. Also included in the present invention are syntheti polypeptides which wholly or partially duplicate continuou sequences of the amino acid residues of Tables I and II. Method for constructing the polypeptides of the present invention b synthetic means are known to those of skill in the art. Th synthetically-constructed sequences, by virtue of sharin primary, secondary, or tertiary structural and conformationa characteristics with IL-3-like polypeptides of Tables I and I may possess IL-3-like biological properties in common therewith Thus, they may be employed as biologically active or immunologica substitutes for natural, purified primate IL-3-like polypeptide in therapeutic and immunological processes. The family of IL-3-like growth factors provided herei also includes factors encoded by the sequences similar to thos of Tables I and II, but into which modifications are naturall provided or deliberately engineered. For example, one suc modified human protein has the mature peptide sequence depicte in Table II except that Ser-27 is replaced by Proline. Tha protein is encoded by a human cDNA sequence as illustrated i Table II with the modificiation that the proline codon CCC is present at amino acid position #27 instead of the serine codo TCC which appears in the Table at that position. This exemplary modified IL-3-like DNA sequence, which produces an active human IL-3-like factor, included in E. coli HB101 as pHucIL3-2, was deposited in the ATCC on February 13, 1987 under accession number ATCC 67319. Other modifications in the peptide or sequences can be made by one skilled in the art using known techniques. Specific modifications of interest in these IL-3-like related sequences may include the replacement of one or both of the two cysteine residues in each coding sequence with other amino acids. Preferably both cysteines are replaced with another amino acid, e.g. serine, to eliminate the disulfide bridge. Mutagenic techniques for such replacement are well known to one skilled in the art. [See, e.g.. United States patent 4,518,584.] Other specific mutations of the sequences of the IL-3-like factors described herein involve modifications of one or both of the glycosylation sites. The absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at one or both of the asparagine-linked glycosylation recognition sites present in the sequences of the IL-3-like factors shown in Tables I and II. The asparagine-linked glycosylation recognition sites comprise tripeptide sequences which are specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either asparagine-X-threonine or asparagine-X-serine, where X is usually any amino acid. A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptide sequence. For example, Asn3 of the sequence of Table I can be replaced with glutamine in one such modified IL-3-like factor. The resulting factor (Gln34) should contain only one asparagine- linked carbohydrate moiety (at Asngg) , rather than two such moieties. Those skilled in the artwill appreciate that analogous glycoproteins having the same Asngg monoglycosylation may be prepared by substituting another amino acid at position 34, and/or by substituting another amino acid at the other positions within the glycosylation recognitions site, e.g., inserting valine at Ser 36 . Similarly, the Asn codon corresponding to position 89 and/or the serine codon corresponding to position 91 may be altered by a mutagenic technique to codons for other amino acids. Expression of such altered nucleotide sequences produces variants which are not glycosylated at that site Alternatively, both sites may be altered as above. Suc modifications to the glycosylation sites may also be made t create modifications of the sequence of Table II. [See, e.g A. Miyajima et al., EMBO J.. 5(6) :1993-1197 (1986) and Exampl IV below.] Other analogs and derivatives of the sequences o Tables I and II which would be expected to retain IL-3-lik acitivity in whole or in part may also be easily made by one o skill in the art given the disclosures herein. Such obviou modifications are believed to be encompassed by this invention The present invention also encompasses the novel DN sequences, free of association with DNA sequences encodin other primate proteins, and coding on expression for primat IL-3-like polypeptides or growth factors. These DNA sequence include those depicted in Table I and Table II in a 5' to 3 direction and those sequences which hybridize under stringen hybridization conditions [see, T. Maniatis et al, Molecula Cloning (A Laboratory Manual) . Cold Spring Harbor Laborator (1982) , pages 387 to 389] to the DNA sequences of Tables I an II. An example of one such stringent hybridization conditio is hybridization at 4XSSC at 65°C, followed by a washing i 0.1XSSC at 65°C for an hour. Alternatively an exemplary stringen hybridization condition is in 50% formamide, 4XSSC at 42°C. DNA sequences which hybridize to the sequences of Tables or II under relaxed hybridization conditions and which code o expression for growth factors having primate IL-3-like biological properties also encode members of this family of novel growth factors. Examples of such non-stringent hybridization conditions are 4XSSC at 50°C or hybridization with 30-40% formamide at 42°C. For example, a DNA sequence which shares regions of significant homology, e.g. , sites of glycosylation or disulfide linkages, with the sequences of Tables I and/or II and encodes a primate protein having one or more IL-3-like biological properties clearly encodes a member of this novel family of growth factors, even if such a DNA sequence would not stringently hybridize to the sequence of Table I or II. Similarly, DNA sequences which code for primate IL-3-like polypeptides coded for by the sequence of Table I or II, but which differ in codon sequence due to the degeneracies of the genetic code or allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change) also encode the novel growth factors of this family described herein. Variations in the DNA sequences of Tables I and II which are caused by point mutations or by induced modifications to enhance the activity, half-life or p.roduction of the polypeptides encoded thereby are also encompassed in the invention. Another aspect of the present invention provides a novel method for producing the novel family of primate IL-3-like growth factors. The method of the present invention involves culturing a suitable cell or cell line, which has been transformed with a DNA sequence coding on expression for a novel primate IL-3-like polypeptide under the control of known regulatory sequences. Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO) or 3T3 cells. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol.. 5 . (7) :1750-1759 (1985) or Howley et al, U.S. Patent 4,419,446. Another suitable mammalian cell line, which is described in the accompanying examples, is the monkey COS-1 cell line. A similarly useful mammalian cell line is the CV-1 cell line. Similarly useful as host cells suitable for the present invention are bacterial cells. For example, the various strains of E_. coli (e.g., HB101, MC1061 and strains used in the following examples) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis. Pseudo onas. other bacilli and the like may also be employed in this method. Many strains of yeast cells known to those skilled in t art are also available as host cells for expression of th polypeptides of the present invention. Additionally, wher desired, insect cells may be utilized as host cells in th method of the present invention. See, e.g. Miller et al Genetic Engineering. 8.:277-298 (Plenum Press 1986) and reference cited therein. Another aspect of the present invention provides vector for use in the method of expression of these novel primat polypeptides. These vectors contain the novel DNA sequence described above which code for the novel primate polypeptide of the invention. Alternatively, vectors incorporating modifie sequences as described above are also embodiments of the presen invention and useful in the production of these IL-3-lik polypeptides. The vector employed in the method also contain selected regulatory sequences in operative association with th DNA coding sequences of the invention and capable of directin the replication and expression thereof in selected host cells. The members of the novel family of primate IL-3-lik growth factors herein disclosed may be used in the treatment o a number of pathological or disease states, particularly thos characterized by a decreased level of either myeloid, erythroid lymphoid, or megakaryocyte cells of the hematopoietic system o combinations thereof. In addition, they may be used to activat mature myeloid and/or lymphoid cells. Among condition susceptible to treatment with the polypeptides of the presen invention is leukopenia, a reduction in the number of circulatin leukocytes (white cells) in the peripheral blood. Leukopeni may be induced by exposure to certain viruses or to radiation It is often a side effect of various forms of cancer therapy e.g., exposure to chemotherapeutic drugs. Therapeutic treatmen of leukopenia with these IL-3-like polypeptide compositions ma avoid undesirable side effects caused by treatment with presentl available drugs. Various immunodeficiencies e.g., in T and/or B lympho cytes, or immune disorders, e.g., rheumatoid arthritis, may also be beneficially effected by treatment with the polypeptides of the present invention. These factors, alone or in combination with other treatment regimens, may be useful in treating or correcting immunodeficiencies which are the result of viral infections e.g. HTLVI, HTLVII, HIV, severe exposure to radiation, cancer therapy or the result of other medical treatment. The polypeptides of the present invention may also be employed, alone or in combination with other hematopoietins, in the treatment of other blood cell deficiencies, including thrombocytopenia (platelet deficiency) , or anemia (red cell deficiency) . Other uses for these novel polypeptides are in the treatment of patients recovering from bone marrow transplants, and in the development of monoclonal and polyclonal antibodies generated by standard methods for diagnostic or therapeutic use. Therefore, as yet another aspect of the invention are methods and therapeutic compositions for treating the conditions referred to above. Such compositions comprise a therapeutically effective amount of one or more of the members of the family of primate IL-3-like polypeptides of the present invention in admixture with a pharmaceutically acceptable carrier. This composition can be systematically administered parenterally. Alternatively, the compositionmay be administered intravenously. If desirable, the composition may be administered subcutaneously. When systematically administered, the therapeutic composition for use in this invention is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a pharmaceutically acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art. The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regime should be in the range of 200-1000 micrograms of polypeptide o 50 to 5000 units (ie, a unit being the concentration o polypeptide which leads to half maximal stimulation in a standar human bone marrow assay) of polypeptide per kilogram of bod weight. The therapeutic method and compositions of the presen invention may also include co-administration with other huma factors. A non-exclusive list of other appropriat hematopoietins, CSFs and interleukins for simultaneous or seria co-administration -with he polypeptides of the present inventio includes GM-CSF, CSF-1, G-CSF, Meg-CSF, erythropoietin (EPO) , IL-1, IL-4, IL-2, B-cell growth factor, B-cell. differentiatio factor and ep.sinopr.il differentiation factor. Of particula interest for amplifying hematopoietic progenitor cells an their descendants in vivo is a combination of IL-3 with IL- (also known in the art as B cell stimulatory factor 2) , whic demonstrate the ability in human blast cell assays to cause th proliferation of early stem cell colonies. Additionally, th IL-3-like polypeptides may be administered with, or chemicall attached to, monoclonal or polyclonal antibodies in a therapeuti use. Alternatively, -these growth factors may be attached t certain toxins, e.g., ricin, in a therapeutic regimen. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated -pa ient -can ~be monitored by periodic assessment of the hematological profile, e.g. white cell count and the like. The following examples illustratively describe members of the novel family of primate IL-3-like polypeptides and the methods of the present invention. EXAMPLE I Isolation of Gibbon IL-3-like Gene A gibbon T-cell line infected with gibbon-ape leukemia virus, UCD-144-MLA, and available from the National Institute of Health Laboratories was induced with phytohemagglutinin and phorbol myristate acetate (PHA/PMA) . Total RNA was prepared from these cells by the procedures of J.M. Chirgwin et al., Biochem. 18:5294 (1979). Poly A + RNA was selected and fractionated on a 10% to 30% sucrose gradient. To identify the mRNA encoding this novel hematopoietic factor, sixteen aliquots of sucrose gradient-fractionated mRNA from the UCD-144-MLA cell line were micro-injected into Xenopus laevis oocytes and the resulting conditioned medium tested for the ability to stimulate the proliferation of leukemic blast cells in the presence of antibody to human GM-CSF as illustrated in the CML assay of Example V. mRNA from the sucrose gradient fractions identified as containing the message encoding IL-3-like growth factor activity was converted to double stranded cDNA by the procedure of U. Gubler and B.J. Hoffman, Gene. Z5:263 (1983). A COS cell expression vector, pXM, containing the SV40 enhancer, major adenovirus late promoter, DHFR coding sequence, SV40 late message poly A addition site and Val gene was linearized with the endonuclease enzyme Xhol, treated with DNA polymerase I large fragment in the presence of dTTP and ligated to equimolar amounts of cDNA, at a final DNA concentration of 100 ug/ml. The ligation products resulting from the Xhol digestion of pXM and the insertion of the Xhol adapted cDNA sequence were transformed into E_ s _ coli strain HB101 and plated on L+ Amp plates to generate a library of approximately 30xl0 3 colonies. [Other functionally similar expression vectors known in the art can also be used in this procedure as alternatives to pXM.] The cDNA library in pXM was replica plated onto nitrocellulose filters. Colonies from each filter were scraped into L. broth, and plasmid DNA was isolated. Each DNA sample was prepared from a pool of 200-300 bacterial colonies. The DNA was purified by the method of J. A. Meyers et al., J. Bacteriol. 127:1529 (1976). Monkey COS cells (ATCC CRL 1650) were transfected with approximately 5 ug plasmid DNA per 10 6 COS cells by DEAE mediated DNA transfection and treated wit chloroquine according to the procedures described in G. G. Won et al.. Science: 228:810-815 (1985) and R. J. Kaufman et al. Mol. Cell Biol.. 2:1304 (1982). 72 hours following transfection, medium is harvested an assayed in the human CML assay, as described in Example below. One pool produced conditioned medium with colon stimulating activity and CML proliferation activity completel resistant to neutralizing antiserum to GMCSF, and was selecte for further analysis. Plasmid DNA from individual colonie picked from the original active pool was prepared and transfecte to produce conditioned medium. This conditioned medium wa assayed for CSF and CML proliferation activity. A single clon responsible for such activity was isolated. The cDNA insert o this clone was subcloned into M13 and was sequenced by th Sanger dideoxy chain termination method. [See Table I] EXAMPLE II Isolation of a Human IL-3-like Gene Using the sequence of Table I as a probe, lxlO 6 clone from a human genomic library (Sau 3AI partial digest of huma DNA cloned into the Bam HI site of the lambda vector Jl) [J. J. Toole et al, supra] were screened. Three plaques wer identified which contained sequences which hybridized strongl with the cDNA probe. The DNAs from two of these phages were digested to completion with the endonuclease enzyme Sau 3AI and subcloned into the Bam HI site of the bacteriophage lambda M13 cloning vector mp9. Subclones containing exon sequences were identified by hybridization with the gibbon cDNA. One subclone, lambda CSF-16, containing the human genomic DNA sequence as an approximately lOkb Bgl II insert, was deposited with the ATCC as described above. The complete sequences of all of the exons of the human gene were determined using dideoxy chain termination DNA sequencing with a battery of oligonucleotide primers whose sequences were based upon the sequence of the gibbon gene described in Example I. Because the nucleotide sequences of the exons of the human gene were more than 96% homologous with the sequence of the gibbon cDNA, the nucleotide sequence of the corresponding human cDNA was reconstructed. Changes in the nucleotide sequences in 11 codons result in amino aciddifferences in the polypeptides from the two species. [See Table II]. The human genomic sequence can be excised from lambda CSF- 16 and inserted into an expression vector, numerous types of which are known in the art for mammalian, insect, yeast, fungal, and bacterial expression. For example, the human genomic sequencewas excised from the depositedbacteriophageby digestion with the endonucleases Smal and Xhol which cleave the human gene region at nucleotides 629 and 3183 respectively. The resulting 2.5kb contains the entire human IL-3 gene coding sequences and includes the "TATAA-related" sequence in the promoter region but lacks the "CAT-related" sequences and the polyadenylation signal at the 3' end of the gene. The Smal end of this fragment was converted to Xhol with a commercially available linker sequence. This fragment was sub-cloned by standard molecular biology techniques [see, e.g., Y-C. Yang et al. Cell.47.:3-10 (1986)] into a Xhol-digested plasmid expression vector pXM, yielding plasmid pY3. pY3 was then amplified in bacteria and transfected into monkey COS-1 cells, where the human gene is transcribed and the RNA spliced. Media from the transfected cells is positive in assays for IL-3-like biological activity in the human bone marrow assay and the CML assay as described below. Northern blot analysis with a gibbon cDNA probe indicates the presence of a single 1 kb mRNA which is obtained from these cells and is the same size as RNA obtained from peripheral blood lymphocytes, as described below. cDNA is synthesized from the mRNA by standard procedures and a clone identified which has CML activity. cDNA for a novel IL-3-like growth factor is isolated therefrom. EXAMPLE III A Human IL-3-Like Growth Factor The cDNA sequence of Table II encoding a human IL-3-lik polypeptide may also be obtained in ways other than that describe in Example II. For example, the sequence of Table II may b chemically synthesized according to procedures well known t those skilled in the art. One such chemical synthetic metho involves reconstructing the gibbon IL-3 gene to provide th human IL-3 coding sequence. The first amino acid differenc between the mature forms of the gibbon and human IL-3 protei occurs at amino acid 82. The coding sequence from amino aci 82 in the gibbon IL-3 gene to the 3'-end of the gene can b replaced by chemically synthesized DNA sequences encoding fo human IL-3, thereby yielding a functional gene capable o producing human IL-3 in a suitable expression system. Two unique restriction sites in the gibbon sequence can b used for cloning synthetic sections of DNA: an Asu II site a amino acid 73 and EcoRI at amino acid 125. A DNA "cassette" for insertion into the gibbon gene was synthesized from the As II site to the EcoRI by enzymatically providing a DNA duplex o approximately 160 bp from two oligonucleotides which ar complementary to each other over 21 base pairs. The complet duplex was formed by extending the complementary region to th ends of the oligonucleotides using deoxynucleoside triphosphates and DNA polymerase I, Klenow fragment. The complete duplex was digested with Asu II and/or EcoRI to yield cohesive ends for subsequent cloning. A second synthetic DNA "cassette" consists of two oligonucleotides, complementary to each other throughout their length, from the EcoRI site at amino acid 125 to, and including, the termination codon following amino acid 152 at the end of the gene. These oligos were designed with an EcoRI cohesive end and suitable restriction sites or cohesive ends at or just after the termination codon for cloning into different expression vectors. Two sets of these cassettes were synthesized, one set for mammalian expression, the other for bacterial expression responsive to differences in codon usage between prokaryotes and eukaryotes, the low incidence of CpG doublets in eukaryotic genes and retention of, or the need for, different restriction- sites for cloning. Such codon preferences are known to those skilled in the art. See, e.g., T. Maruyama et al., Nucl. Acids Res. f .I4_:rl51 (1986). Exemplary codon changes and cohesive ends synthesized for these cassettes appear in Table III below. The cassettes are then cloned into the vector, pCSF-MLA, to transform it into a vector bearing the gene encoding the human IL-3-like factor. This resulting vector is then used to express the human factor. TABLE III I. CODON CHANGES Amino acid # Bacterial expression Mammalian expression 73 CGT 74 CGT 75 CCG 82 CGT 86 AGC 87 CTG CTG 88 CAA CAA 89 AAT 91 AGC 97 CTG CTG 100 CTG CTG 102 CCG 105 CCG 108 ACC ACA 109 GCT 111 CCG 112 ACC ACC 113 CGT AGG 115 CCG 120 GAT 127 CGC AGG 128 CGC 131 ACC ACC 137 CTG CTG 140 GCT GCT 143 CAG CAG 145 ACC ACC 146 ACC ACC 147 CTG CTG 152 TTC TTC TABLE III (cont ' d) II. CASSETTE TERMINI Cassette # Bacterial expression I 73 125 arg arg...glu phe arg T CGT...GAA TTC CGT A GCA...CTT AAG GCA Cassette # Mammalian Expression I 71 125 asn leu arg arg...glu phe arg AAC CTT CGA AGG...GAA TCC CGTC TTG GAA GCT TCC...CTT AGG GCAG Cassette # Bacterial expression II 126 152 phe arg phe AA TTC CGC. TTC TAG AACTCGAGACTGCA G GCG. ...AAG ATC TTGAGCTCTG Cassette # Mammalian expression II 126 152 phe arg phe AA TTC AGG TTC TAG AACTCGAGA G TCC AAG ATC TTGAGCTCTTTAA Alternatively, obtaining the cDNA sequence of a human IL 3-like growth factor can involve cloning the cDNA from a tissu source. The gibbon cDNA sequence of Table I was employed as probe according to T. Maniatis et al., supra and identifie peripheral blood lymphocytes as a human source for isolating mRN encoding this human IL-3-like polypeptide. Poly A + RNA i prepared from the peripheral blood lymphocyte source, converte to cDNA and cloned as either a phage or plasmid cDNA library. A human cDNA clone can be identified by hybridization with th gibbon coding sequence of Table I as a DNA probe and determination of IL-3-like biological properties. Additional tissue sources which may also be screened fo human IL-3-like cDNA include spleen, liver, thymus, tonsils, kidney, and other fresh tissues available from biopsies an cadavers. Of special interest are cases where the tumor may b responsible for elevated hematopoietic cell counts, e.g., leukemia. Additional sources are the cell lines deposited fo public use in depositories, e.g. ATCC, or available throug government agencies and certain private sources. Exemplar cell lines include transformed T and B cell lines, and cell lines which are not of hematopoietic origin, but generat hematopoietins. In order to express this human IL-3-like polypeptide, the cDNA encoding it is transferred into an appropriate expression vector, e.g. pCD or pXM, and introduced into selected host cells by conventional genetic engineering techniques as described above. One mammalian expression system for a biologically active recombinant human IL-3-like polypeptide is stably transformed CHO cells. However, an active polypeptide can be produced intracellularly or extracellularly from E. coli, and other bacteria. Yeast or insect cells may also be employed as expression systems, as described in Example IV. Another alternative method for expressing this human IL-3- like polypeptide is to employ the Bgl II fragment from lambda CSF-16 containing the complete human genomic gene to construct mammalian cell lines expressing thepolypeptide, e.g. as described by PCT WO85/20610 for human erythropoietin. In addition, this human genomic gene can be engineered with the appropriate promoter and processing signals for expression in some other heterologous system, e.g. insect promoters for constructing insect cell culture lines. Similarly, this genomic gene may be expressed in yeast or other eukaryotic systems. EXAMPLE IV Expression of IL-3-like Growth Factors A plasmid, pCSF-MLA, is simply constructed by inserting the gibbon sequence of Table I into Xhol-digestedpXM as described above. A plasmid with the human sequence, pSHIL-3-1 is constructed synthetically for mammalian expression as described above in Example III. Another plasmid pY3 is made as described above. Each of these plasmids carrying a primate IL-3-like growth factor is then transformed by conventional techniques into a selected host cell for expression of a polypeptide. A. Mammalian Cell Expression: To obtain expression of the IL-3-like factors for use in the assays described below, pSHIL-3-1 andpCSF-MLAaretransfected onto COS cells. The conditioned medium for the transfected COS cells contained high levels of growth factor activity as described. The mammalian cell expression vectors described herein may be synthesized by techniques well known to those skilled in this art. The components of the vectors, e.g. replicons, selection genes, enhancers, promoters, and the like, may be obtained from natural sources or synthesized by known procedures. See, Kaufman et al, J. Mol. Biol.. 159:511-521 (1982); and Kaufman, Proc. Natl. Acad. Sci.. U.S.A.. 81:689-693 (1985). Exemplary mammalian host cells include particularly primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from i vitro culture of primary tissue, as well as primary explants are also suitable. Candidate cells need not be genotypicall deficient in the selection gene so long as the selection gene i dominantly acting. For stable integration of the vector DNA and for subsequent amplification of the integrated vector DNA both by conventional methods, CHO cells may be employed Alternatively, the vector DNA may include all or part of th bovine papillo a virus genome [Lusky et al. Cell. 36:391-40 (1984)] and be carried in cell lines such as C127 mouse cell as a stable episomal element. Other suitable mammalian cel lines include but are not limited to, HeLa, COS-1 monkey cells mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NI mice, BHK or HaK hamster cell lines. Stable transformants are then screened for expression o the product by standard immunological or enzymatic assays. Th presence of the DNA encoding the variant proteins may be detecte by standard procedures such as Southern blotting. Transien expression of the DNA encoding the variants during the severa days after introduction of the expression vector DNA int suitable host cells such as COS-1 monkey cells is measure without selection by activity or immunologic assay of th proteins in the culture medium. One skilled in the art can also construct other mammalia expression vectors comparable to pCSF-MLA and pSHIL-3-1 by e.g., cutting the DNA sequence of Table I or Table II from th respective plasmids with Xhol and employing well-known recombinan genetic engineering techniques and other known vectors, such a pJL3 and pJL4 [Gough et al., EMBO J.. 4.:645-653 (1985)] an pMT2 (starting with pMT2-VWF, ATCC #67122; see PCT applicatio PCT/US87/00033) . The transformation of these vectors int appropriate host cells can result in expression of the IL 3-like growth factors. B. Bacterial Expression Systems: Similarly, one skilled in the art could manipulate th sequences of Tables I and II by eliminating or replacing the mammalian regulatory sequences flanking the coding sequences with bacterial sequences to create bacterial vectors for intracellular or extracellular expression of the IL-3-like factors of the invention by bacterial cells. The DNA encoding the factor may be further modified as in Example III to contain different codons for bacterial expression as is known in the art. Preferably the sequence is operatively linked in-frame to a nucleotide sequence encoding a secretory leader polypeptide permitting bacterial expression, secretion and processing of the mature variant protein, also as is known in the art. The compounds expressed in bacterial host cells may then be recovered, purified, and/or characterized with respect to physiochemical, biochemical and/or clinical parameters, all by known methods. To construct one such bacterial vector for bacterial expression, a partially synthetic human IL-3-like DNA sequence was constructed from the gibbon cDNA and the synthetic bacterial cassettes described in Example III. This sequence was placed into a Ndel/Xbal digested vector pal181 which is deposited wit the ATCC under accession number 40134. The resulting vector pPLHIL-3-181 was transfected into E. coli GL400 and cultured according to the conditions described for GM-CSF in publishe PCT application 86/00639. The IL-3-like factor in the E. coli is produced in insoluble inclusion bodies. To solubilize and obtain the proteintherefrom, the following procedure is followed. Approximately 50 g of cells in a frozen paste are resuspended in 120 ml of 50 mM Tris-HCl, pH7.5, 0.1 mM phenyl-methylsulphonylflouride [PMSF] and 2 mM dithiothreotol [DTT] (Buffer A) . The cells are disrupted by passage through a French press or a Matin Gaulin valve at 10,000 psi or greater. These disrupted cells are then centrifuged at 20,000 x G for 30 minutes to pellet the cell debris, which includes the inclusion bodies. The pellet is resuspended in 55% sucrose in Buffer A by passage through the French press, and again centrifuged at 30,000 x G for 30 minutes. The pellet containing IL-3-like facto inclusion bodies is resuspended in 55% sucrose in Buffer A an layered onto a step gradient of 60, 65 and 70% sucrose. Th gradient is centrifuged at 150,000 x G for two hours. IL-3 like factor inclusion bodies sediment in to the 65% layer which is collected. To reconstitute and refold the now approximately 80% pur IL-3-like factor, these inclusion bodies are resuspended at 2 3 mgs protein per milliliter in 8M urea. The urea solutio containing the protein is diluted with 50mM TrisHCl, pH 8.0, 0.1 mM PMSF, 2mM DTT and 0.1 mM ethylenediaminetetraacetic aci [EDTA] (Buffer B) to a final concentration of 3M urea (in whic the IL-3 concentration is approximately 1 ug/ml) . A reducing/ oxidizing buffer containing 6mM reduced glutathioneand 6 m oxidized glutathione is added to the urea solution and incubate at 20 degrees centigrade for two hours. The 3M urea solution i dialyzed against Buffer B overnight to remove the urea. Th IL-3 protein solution is then centrifuged to remove an precipitate. At this stage the IL-3-like factor is refolde and about 85% pure. Refolded IL-3-like factor is dialyzed against 50 mM MES buffer at pH6.0, containing 0.1 M EDTA and applied onto column of DEAE-Sepharose equilibrated in the same buffer. IL 3-like factor is in the flow-through of the DEAE at approximatel 99% purity. This step also removes any pyrogens. The pH of IL-3 from DEAE flow-through is adjusted to 5.0 with 200 mM sodium acetate buffer at pH4.0. The IL-3-like factor is applied to a sulphonyl propyl-Sepharose column and IL3-like factor binds to the SP-Sepharose and is eluted with the sodium acetate containing buffer. At this stage the IL-3-like factor is pure and refolded correctly. In the CML assay, this human IL-3-like factor has a specific activity of between 1 to 3 X 10 7 CML units/mg. Similarly, the coding sequence of Table I or that of Table II could be cut from pCSF-MLA or pSHIL-3-1, with Xhol and further manipulated (e.g., ligated to other known linkers or modified by deleting non-coding sequences therefrom or altering nucleotides therein by other known techniques) . The modified IL-3-like coding sequence could then be inserted into a known bacterial vector using procedures such as described in T. Taniguchi et al., Proc. Natl Acad. Sci. USA. 77:5230-5233 (1980) . This exemplary bacterial vectorcouldthenbe transformed into bacterial host cells and the IL-3-like factor expressed thereby. For a strategy for producing extracellular expression of IL-3-like factors in bacterial cells, see, e.g. European patent application EPA 177,343. C. Insect Cell Expression: Similar manipulations can be performed for the construction of an insect vector [See, e.g., procedures described in published European patent application 155,476] for expression in insect cells. A yeast vector could also be constructed employing yeast regulatory sequences for intracellular or extracellular expression of the proteins of the present invention by yeast cells. [See, e.g., procedures described in published PCT application WO 86 00639 and European patent application EP 123,289.] EXAMPLE V Construction of CHO cell lines expressing high levels of Primate IL-3-like Growth Factor One method for producing high levels of the novel primate family of IL-3-like polypeptides of the invention from mammalian cells involves the construction of cells containing multiple copies of the heterologous IL-3-like gene. The heterologous gene can be linked to an amplifiable marker, e.g., the dihydrofolate reductase (DHFR) gene for which cells containing increased gene copies can be selected for propagation in increasing concentrations of methotrexate (MTX) according to the procedures of Kaufman & Sharp, J. Mol.Biol.. (1982) supra. This approach can be employed with a number of different cell types. For example, pY3 contains a human IL-3-like gene in operativ association with other plasmid sequences enabling expressio thereof. pY3 and the DHFR expression plasmid pAdA26SV(A) (Kaufman & Sharp, Mol. Cell Biol.. 2(9) :1598-1608 (1983) can b co-introduced into DHFR-deficient CHO cells, DUKX-BII, by calciu phosphate coprecipitation and transfection. Alternatively, th gene may be introduced into pMT2 as previously mentioned an the resultant vector used in place of pY3 and pAdA26SV(A)3. DHFR expressing transformants are selected for growth in alph media with dialyzed fetal calf serum, and subsequently selecte for amplification by growth in increasing concentrations of MT (sequential steps in 0.02, 0.2, 1.0 and 5uM MTX) as describe in Kaufman et al. , Mol. Cell Biol. 5_:1750 (1983) . Transformants are cloned, and biologically active IL-3-like polypeptide expression is monitored by CML assays. IL-3-like polypeptide expression should increase with increasing levels of MTX resistance. Similar procedures can be followed to produce other members of this family of IL-3-like polypeptides, including the gibbon IL-3-like polypeptides. EXAMPLE VI Biological Activities of an IL-3-like Polypeptide The following assays were performed using both the gibbon polypeptide and the human polypeptide as representative members of the novel family of primate IL-3-like polypeptides of the present invention. However, other members of the family will exhibit IL-3-like biological properties in these same assays or in other assays depending on the number of IL-3-like biological properties displayed by the individual polypeptide. A. CML Assay The CML assay was performed essentially according to procedures described in Blood. j6.(4) :904-lll (1984). A stock of cells were obtained from a frozen bag of peripheral blood from a CML patient in stable phase. This bag was thawed and refrozen into 500 aliquots of 15 x 10 6 cells/vial. These cells, "CML 8-3", were used to test for the IL-3-like activity of the IL-3-like polypeptides. One vial is thawed quickly at 37*C the day before the assay is „set up. The contents of the vial are the/i transferred to a 15 ml tube and washed 2 times with 5% Hi Human. AB Serum in RPMI (GIBCO,RPMI 1640) [HAB/RPMI]. The cells are incubated overnight in 5% HiHAB/RPMI at 5% C0 2 and 37°C. The following day the cells are removed from culture, ficolled, washed, recounted and set aside. 100 ul of 10% HIFCS2/RPMI medium containing the material to be assayed is plated in each well of a microtiter plate. The cells prepared above are spun down and resuspended at a concentration of 1.3 to 2 xlO 5 cells/ul in 10% HIFCS/RPMI. 100 uls of cells are plated in each well and incubated in the presence or absence of anti-human GMCSF antibodies at 37"C in 5% CO 2 for 48 or 72 hours. Thereafter 0.5 uCi 3 H-thymidine is added per well and the wells are incubated for 6 hours at 37 β c. Cells are harvested using a filtration manifold device onto GFC Type C filter paper (Schleicher-Schuller) , washed with phosphate buffered saline and dried. Filters are then immersed in scintillation fluid and counted for 3 H uptake. Based on the thymidine uptake measurement, both the gibbon IL-3-like growth factor and the human IL-3-like growth factor are active in this assay in stimulating the proliferation leukemic blast cells. B. Bone Marrow Assays Human bone marrow assays, employing non-adherent bone marrow cells, were performed as described in G.G. Wong, et al., supra. Conditioned media for both the gibbon and human factors was found to be active in this assay, producing small colonies of apparently granulocytic-type lineage. Also produced upon morphological examination of stained agar cultures were macrophage, granulocyte-macrophage and eosinophil colonies. When this assay is performed in the presence of erythropoietin the ability of conditioned medium to support the growth o erythroid progenitor cells is demonstrated by the production o red blood cell colonies. When GM-CSF was compared with th IL-3-like polypeptide in the human bone marrow assay, IL-3-lik polypeptide supported the formation of more colonies than GM CSF, when both polypeptides were in the presence o erythropoietin. The majority of colonies supported by GM-CS were single lineage; while the polypeptide of the presen invention supported the formation of multi-lineage colonies. Similarly, in blast cell colony formation assays, the IL-3-lik polypeptide produced greater numbers of blast cell colonies o multiple lineages. GM-CSF in the same assay produced very fe secondary colonies. C. KG-1 Cell Assay The KG-1 assay was performed as described in G. G. Wong et al, supra. The gibbon IL-3-like polypeptide member o the novel primate family of IL-3-like growth factors produce according to the present invention was active in this assay. D. Miscellaneous Assays In an antibody-dependent cell-mediated cytotoxicit assay, the IL-3 like polypeptide of the present inventio stimulated eosinophils to kill antibody-coated tumor targe cells in a dose-dependent manner. The polypeptide additionall stimulated eosinophils to phagocytose serum-opsonized baker's yeast, and to directly stimulate superoxide anion production by eosinophils. In preliminary studies in which IL-3-like factors of the invention are infused in o Wealthy monkeys and the white counts observed, reproducible increases in both platelet count and number of eosinophils have been observed. These preliminary results have been observed for both the human and gibbon IL- 3-like factors. EXAMPLE VII Purification of IL-3-Like Polypeptide from COS Cell Conditioned Medium The following procedures are presently employed to obtain homogeneous IL-3-like protein from COS cells, as described in Example IV above. A. Ion Exchange COS cell conditioned media [DMEM, 0.5%FBS in roller bottles at a total protein concentration of 200 ug/ml] contained the human IL-3-like polypeptide at a concentration of approximately 2-3 ug/ml. The media is diluted with water until the conductivity is less than 8.0 ms/cm 2 . An ion exchange cartridge [QAE Zeta Prep] is equilibrated at 4 degrees centigrade with approximately 500 mis 0.1M Tris-Cl, pH8.0 and then two liters of 40 mM Tris- Cl, pH7.4. Media was loaded at 40 ml/minute, and the unbound fraction collected. The cartridge was washed with 40mM Tris-Cl until no further activity washed off. To obtain bench-scale amounts of IL-3-like protein, the unbound fraction was concentrated on a diafiltration unit membrane [Amicon YM-10] . An alternative to this concentration step for larger scale purification of an IL-3-like protein is to acidify the QAE-Zeta Prep unbound fraction with 1M glacial acetic acid to a pH of 4.5. Media is loaded at 40ml/minute -onto a sulfonylpropyl (SP)-Zeta Prep, which is equilibrated at 4 degrees centigrade with approximately 500 mis of 20mM sodium acetate, at pH4.5. The cartridge is washed with 20mM sodium acetate and the bound fraction eluted with 20mM sodium acetate and 0.25 to 0.5M sodium chloride. This fraction is neutralized with 1M Tris-Cl, pH8.0 to pH7.4 and Tween-20 is added to 0.05% final concentration. B. Lentil Lectin Column A lentil lectin column was equilibrated in 20 mM Tris, pH7.4, 0.05% Tween-20 at 4 degrees centigrade [Buffer I] and then loaded at 1 column volume per hour. The column was washed with Buffer I to remove non-specifically-bound protein and the bound protein was eluted with Buffer I plus 0.2M alpha-methyl mannopyranoside. The elution fractions were pooled. C. Reverse Phase HPLC This preparation of IL-3-like polypeptide was subjected t reverse phase HPLC at room temperature as described below. Th IL-3-like polypeptide preparation was injected onto a RP HPL column [C4 Vydac] equilibrated in 100% Buffer A. Buffer A wa 0.1% trifluoroacetic acid [TFA] in water and Buffer B was 0.1 TFA in 95% acetonitrile. The gradient was 0.2%/minute from 4 to 70% Buffer B. The fractions pooled from this gradient wer 46.8% B to 47.5% Buffer B. These fractions were speed vacuume to remove the acetonitrile. In a second reverse phase HPL step. Buffer A was 0.15%HFBA in water and Buffer B was 0.15 heptafluorobutyric acid [HFBA] in 95% acetonitrile. The gradien was 0.2%/minute from 45 to 70% Buffer B. The fractions poole from this step were 49% to 51 % Buffer B. This fraction elutin from the HPLC was pyrogen free. EXAMPLE VIII Analyses of IL-3-Like Polypeptides A. SPS-PAGE Following the procedure of R.J. Kaufman and P.A Sharp., J. Mol. Biol.159:601-621 (1982) , 35 S methionine is metabolicall incorporated into the polypeptides made by COS cells transfected with pCSF-MLA and COS cells transfected with pY3. SDS polyacrylamide gel electrophoresis (reducing conditions) [U.K. Laemmli, Nature 222:680-685 (1970)] of labeled proteins secreted by the transfected COS-1 cells revealed a distributio of polypeptides with apparent molecular masses ranging between 14kd and 35kd for both the gibbon and human factors. This distribution was absent in the mock transfected control sample. More specifically, CHO-produced human IL-3-like factor revealed a distribution of between 21 to 32kd, illustrating more glycosylation than the COS cell produced human factor with a distribution primarily between 21 and 28kd. With silver stain after the purification procedures of Example VI, two major bands of average molecular weights 21, 000 and 25 , 000 appeared in approximately equal amounts for both factors . The differences in the two bands is presently attributed to differences in N-linked glycosylation. B. Isoelectric Focusing Native isoelectric focusing of the purified polypeptides of Example VII reveal four species for both the gibbon and human polypeptides, which range in Pi value between pH 6.0 and pH7.6. C. Superose 6 Fast Protein Liquid Chromatography The purified fraction from HPLC was run in a gel filtration column [Superose 6] in 20mM Tris, pH7.4, 200 mM NaCl, and 0.05% Tween-20. This column run revealed one sharp peak of apparent molecular weight of 43kd for both factors. D. Specific Activity in CML Assay The specific activity of an exemplary gibbon IL-3-like factor in the CML assay described above falls within the range of 2X10 6 to 1X10 7 dilution units/ g of polypeptide, with an average of 8 10 6 dilution unit/ g. The bacterially-produced human polypeptide described in Example IV (B) was found to have a specific activity of 1 to 3X10 7 dilution units per mg of polypeptide. CHO-produced human IL-3-like factor has a specific activity of about 2 to 3X10 6 units per mg protein in this assay. COS-produced human IL-3-like factor has a specific activity of about 1 to 2X10 7 units per mg. A dilution unit (or CML unit) is defined as that dilution of the factor which gives one-half maximal stimulation in the CML assay. E. N-Terminal Analysis Analysis of the N-terminal sequence of the gibbon polypeptide was made using automated Edman degradation which demonstrated level of purity of the factor of 98%. F. N-Glycanase Treatment The human and gibbon factors produced in COS cells wer treated with the enzyme N-glycanase, which digests the N-1inke carbohydrate moieties. Each factor was shown to be purified b this method. The 14-35kd smear of each factor on the gels wa reduced to a single band of 15kd for the gibbon factor and 20.5k for the human factor. Numerous modifications and variations in practice of thi invention are expected to occur to those skilled in the ar upon consideration of the foregoing descriptions of preferre embodiments thereof. Such modifications and variations ar believed to be encompassed in the appended claims. O 88/00598 GI 5080-E-PCT International Application No: PCT/ MICROORGANISMS Optional Sheet in connection wan the microorganism referred to on sage , llne- - «4 the description < A. IDENTIFICATION OF DEPOSIT • Further depoafta are Identified on on additional aheet Q» Name of depositary Inatitution * American Type Culture Collection Addreea of depositary inatitution (including poital coda and country) * 12301 Parklawn Drive Rockville, Maryland 20852 USA Name of Referred to on Date of Deposit ATCC No. page/line Deposit pCSF-MLA 67154 12/13 July 11, 1986 CSF-16 40246 - 12/16 August 5, 1986 pHucIL3-2 67319 13/32 February 13, 1' 87 pSHIL-3-1 67326 12/19 February 24, 1 87 C. OESISNATED STATES FOR WHICH INDICATIONS ARE MADE > (if the indications are not for all designated States) O. SEPARATE FURNISHING OF INDICATIONS • (lea.e blank if not applicable) Tne indications hated below will be eubm ed to the International Bureau later* (Specify the general nature of the Indications e.g., " Accession Numoer of Deposit ") E. PH This eneet was recened with tne international application when filed (to be checked by the reeoniing Office) (Authorised Officer) !~~1 The date of receipt (from the applicant) by the International Bureau '• waa - - — (Authorlted Officer) Form PCT,R0.134 (January 1M1)

Claims: 40 What is claimed is: 1. An IL-3 protein substantially free from association wit other proteinaceous materials and characterized by a peptid sequence comprising substantially the same sequence as depicte in Table II and having the ability to stimulate at 10 to 10 pico olar concentrations the formation of multiple types o hematopoietic colonies of various lineages in the standar human bone marrow assay. 2. An IL-3 protein characterized by the peptide sequence 5' (MET) a Ser Arg Leu Pro Val Leu Leu Leu Leu Gin Leu Leu Va Arg Pro Gly Leu Gin Ala Pro MET Thr Gin Thr Thr Ser Leu Lys Th Ser Trp Val Asn Cys Ser Asn MET lie Asp Glu lie lie Thr His Le Lys Gin Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Gl Asp Gin Asp lie Leu MET Glu Asn Asn Leu Arg Arg Pro Asn Leu Gl Ala Phe Asn Arg Ala Val Lys Ser Leu Gin Asn Ala Ser Ala lie Gl Ser lie Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro lie His lie Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu Ser Leu Ala lie Phe 3' ; or 5' (MET) a Ser Arg Leu Pro Val Leu Leu Leu Leu Gin Leu Leu Val Arg Pro Gly Leu Gin Ala Pro MET Thr Gin Thr Thr Pro Leu Lys Thr Ser Trp Val Asn Cys Ser Asn MET lie Asp Glu lie lie Thr His Leu Lys Gin Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp lie Leu MET Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gin Asn Ala Ser Ala lie Glu Ser lie Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro lie His lie Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu Ser Leu Ala lie Phe 3•; wherein a is 0 or 1, and alleles thereof. 3. The protein according to claim 1, further characterized by at least one property selected from the group consisting of: 41 a. an apparent molecular weight of between about 14 to about 35 ilodaltons as determined by reducing SDS polyacryl- amide gel electrophoresis; b. the ability to stimulate CML cell proliferation at 10 to 100 picomolar concentrations in the CML assay; c. an isoelectric point between pH6.0 and 7.6; d. a single sharp peak at 43 kd on a Superose 6 gel filtration column; e. a single band of 20.5kd on a gel following N-glycanase treatment; and f. a level of contaminants no more than about 2% upon N- terminal analysis using Edman degradation. 4. A protein according to claim 2 having a level of contaminants no more than about 2% upon N-terminal analysis using Edman degradation. 5. A pharmaceutical composition comprising a therapeutically effective amount of an IL-3 protein characterized by a peptide sequence comprising substantially the sequence as depicted in Table II in a pharmaceutically acceptable vehicle. 6. A method for treating a disease state characterized by a deficiency in the level of hematopoietic cells comprising administering to a patient an effective amount of at least one polypeptide according to claim 1 or 2. 7. The method according to claim 6 wherein said disease state is selected from the group consisting of leukopenia, thrombocyto- penia, anemia, B cell deficiency, T cell deficiency, bacterial infection, viral infection. 8. The method according to claim 6 further comprising administering simultaneously or sequentially with said protein an effective amount of at least one other hematopoietin, 42 interleukin, or growth factor. 9. The method according to claim 8 wherein said hematopoieti is GM-CSF, G-CSF, CSF-1 or erythropoietin. 10. The method according to claim 9 , wherein said interleuki is IL-1, IL-2, IL-4 or IL-6. 11. The method according to claim 10 wherein said growt factor is a B cell growth factor, a B cell differentiatio factor, or an eosinophil differentiation factor. 12. The cDNA sequence coding for IL-3 protein and comprisin the sequence Of nucleotides in a 5' to 3' direction selecte from the group consisting of: a. "the sequence of Table I; b. the sequence of Table II; c. a DNA sequence which hybridizes under stringen conditions with the DNA sequence of (a) or (b) ; d. allelic variations of the sequences of (a) or (b) . 13. A cell transformed with a cDNA sequence according to clai 12 in operative association with an expression control sequen—. 14. A process for producing an IL-3 protein comprisin culturing a cell line transformed with a cDNA sequence encodin expression of an IL-3 protein in operative association iwth an expression control sequence therefor. 15. The process according to claim 14 wherein said cell is a mammalian cell line. 16. The process according to claim 14 wherein said cell is a bacterial cell line. 43 17. Homogeneous IL-3 protein characterized by a peptide sequence substantially the same as the sequence of Table II, and having a speci ic activity in the CML assay of at least 2 X 10 6 dilution units per mg polypeptide.

Inventors: CIARLETTA AGNES B, CLARK STEVEN C, YANG YU-CHUNG, CIARLETTA, AGNES, B., CLARK, STEVEN, C., YANG, YU-CHUNG
Applicants: GENETICS INST, GENETICS INSTITUTE, INC.
Application year: 1987
Main classification: C07K13
IPC codes: A61K38, A61P7, A61P31, A61P37, C07H15, C07K14, C12N1, C12N5, C12N15, C12P21, C12R1