IE913426A1 - Hybrid growth factors - Google Patents

Abstract

The present invention provides recombinant hematopoietic molecules comprising at least a portion of a first hematopoietic molecule having early myeloid differentiation activity and at least a portion of a second hematopoietic molecule having late myeloid differentiation activity. Nucleic acid molecules encoding such recombinant molecules, as well as pharmaceutical compositions comprising such recombinant factors are also disclosed.

Description

Within this application several publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

A variety of factors can influence the activity of a cell. Frequently a factor exerts its influence by interacting with a receptor on the surface of a cell.

After binding to the receptor, the singal which determines the cellular response to the factor can be mediated through a number of different events, including internalization of the factor or alterations of the receptor caused by ligand binding. During the course of hematopoietic differentiation, a number of different factors are involved in the maturation of a pluripotent stem cell into a fully differentiated cell. The activities of these factors during the course of hematopoietic differentiation have resulted in these factors being characterized as early factors or late factors. For example, factors such as interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) are considered early factors, while erythropoietin (Epo), macrophage colony stimulating factor (M-CSF), and granulocyte colony stimulating factor (G-CSF) are considered late factors.

BCI-14 -2Based upon studies performed with purified factors and in vitro colony forming unit assays, it appears that both IL-3 and GM-CSF act on pluripotent cells before they become committed to a particular hematopoietic pathway.

After the events stimulated by these factors are underway, such lineage restricted cells become receptive to further differentiation mediated by such late factors as Epo, (which leads to the maturation of erythrocytes), G-CSF (which leads cells into the granulocytic pathway), and M-CSF (which leads to the maturation of macrophages).

Experiments described in recent publications (1,2,3) have demonstrated in vitro that early or late factors alone are poor stimuli of colony formation. However, when an early factor such as IL-3 or GM-CSF is combined with a late factor, levels of colony formation equivalent to that seen with conditioned media having full activity is observed. Thus, differentiation appears to be dependent upon the dual activities of early and late factors.

Despite a clear requirement for both IL-3 or GM-CSF and Epo for the formation of erythroid colony forming units, published results indicate that IL-3 can down-modulate high affinity Epo receptors (4). Because the amount of IL-3 required to demonstrate down-modulation of the Epo receptor was higher than that reported by others who demonstrated functional full IL-3 activity in the presence of Epo, it is unclear whether this phenomenon is relevant in vivo.

SUMMARY OF THE INVENTION The present invention concerns hybrid molecules comprising early and late differentiation factors produced by genetic manipulation. By covalently linking such factors the local concentration of the late factor is very high at the BCI-14 -3surface of a cell to which the early factor is bound. Additionally, if down-modulation is relevant in vivo. binding of late factors to any remaining low-affinity receptors, e.g. Epo receptors, could be enhanced, thus reducing the amount of late factor required to stimulate the cell. Furthermore, by linking an early factor with a late factor, such early factor may act more specifically to stimulate only the desired lineage, thus reducing any undesirable effects mediated by the early factor.

Finally, it is considerably easier to produce and administer to a patient a single factor with two activities rather it would to produce and administer two separate factors.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the amino acid sequence of human IL-3 and a single-stranded DNA sequence encoding IL-3. Also shown are various restriction enzyme cleavage sites utilized to construct the recombinant hybrid molecules of the present invention. (*) indicates a stop codon.

Figure 2 shows the amino acid sequence of human Epo and a single-stranded DNA sequence encoding Epo. Also shown are various restriction enzyme cleavage sites utilized to construct the recombinant hybrid molecules of the present invention. (*) indicates a stop codon.

Figure 3 shows the amino acid sequence of human G-CSF and a single-stranded DNA sequence encoding G-CSF. Also shown are various restriction enzyme cleavage sites utilized to construct the recombinant hybrid molecules of the present invention. (*) indicates a stop codon.

BCI-14 -4Figure 4 shows the amino acid sequence and the DNA sequence encoding an IL-3:Epo construct of the present invention, including synthetic leader and junction sequences. (*) indicates a stop codon.

Figure 5 shows the amino acid sequence and the DNA sequence encoding an Epo:lL-3 construct of the present invention, including synthetic leader and junction sequences. (*) indicates a stop codon.

Figure 6 shows the amino acid sequence and the DNA sequence encoding an IL-3:G-CSF construct of the present invention, including synthetic leader and junction sequences. (*) indicates a stop codon.

Figure 7 shows the amino acid sequence and the DNA sequence encoding another IL-3:Epo construct of the present invention, including synthetic leader and junction sequences. (*) indicates a stop codon.

Figure 8 shows the amino acid sequence and the DNA sequence encoding another Epo:IL-3 construct of the present invention, including synthetic leader and junction sequences. (*) indicates a stop codon.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a recombinant hematopoietic molecule comprising at least a portion of a first hematopoietic molecule having early myeloid differentiation activity and at least a portion of a second hematopoietic molecule having late myeloid differentiation activity. This recombinant molecule has early myeloid differentiation activity associated with the first hematopoietic molecule and late myeloid BCI-14 -5differentiation activity associated with the second hematopoietic molecule. Within this application, hematopoietic molecule means a molecule which promotes and/or regulates hematopoiesis. Hematopoietic molecules exert such promotional or regulatory activities at different stages during hematopoiesis, such stages being referred to herein as early myeloid differentiation and late myeloid differentiation. Also within this application, early myeloid differentiation activity means the ability to promote the differentiation, self-renewal, or proliferation of pluripotent myeloid cells, i.e., stem cells or colony forming unit, granulocyte-erythrocyte-monocyte-megacaryocyte, cells. Moreover, within this application, late myeloid differentiation activity means the ability to promote the maturation or differentiation of a lineage restricted myeloid cell, i.e., a myeloid precursor cell committed to a specific cell lineage such as erythrocytes, megakaryocytes, monocytes, neutrophils, eosinophils, and basophils.

In one embodiment of the invention, the first hematopoietic molecule is selected from the group consisting of IL-3 and GM-CSF. In another embodiment of the invention, the second hemopoietic molecule is selected from the group consisting of Epo, G-CSF, IL-5 and M-CSF.

In a preferred embodiment of the invention, the portion of the first hematopoietic molecule is linked to the portion of the second hematopoietic molecule by an amino acid linker sequence comprising at least two amino acid residues.

Within the context of the present invention, it is understood that variations in proteins and nucleic acids exist among individuals, e.g. amino acid or nucleotide BCI-14 -6substitutions, deletions, insertions, and degree or location of glycosylation, and that functional derivatives resulting resulting therefrom are included within the scope of the present invention.

In a preferred embodiment of the invention, the recombinant molecule comprises the entire amino acid sequence of human IL-3. Moreover, the recombinant hematopoietic molecule may preferably comprise an amino acid sequence derived from human IL-3 and contained within the amino acid sequence from amino acid 1 to amino acid 79 in Figure 1.

Further still, in yet another preferred embodiment of the invention, the recombinant molecule comprises the entire amino acid sequence of human erythropoietin. In still a further embodiment of the invention, the hemapoietic molecule comprises an amino acid sequence derived from human erythropoietin and contained within the amino acid sequence from amino acid 7 to amino acid 161 in Figure 2.

In another preferred embodiment of the invention, the recombinant hematopoietic molecule comprises the entire amino acid sequence of human G-CSF.

In one embodiment of the invention, the first hematopoietic molecule is IL-3 and the second hematopoietic molecule is erythropoietin. The first hematopoietic molecule, i.e. IL-3, may comprise the amino portion and the second hematopoietic molecule, i.e. Epo, may comprise the carboxy portion of the recombinant molecule. Preferably, the recombinant hematopoietic molecule comprises the amino acid sequence shown in Figure 4 from amino acid 1 to amino acid 302. Also preferably, the recombinant hematopoietic molecule comprises the amino BCI-14 -7acid sequence shown in Figure 7 from amino acid 1 to amino acid 321. However, in another embodiment of the invention, the first hematopoietic molecule, i.e. IL-3, may comprise the carboxy portion and the second hemapoietic molecule, i.e. Epo, may comprise the amino portion of the recombinant molecule. In a preferred embodiment of the invention, the recombinant molecule comprises the amino acid sequence shown in Figure 5 from amino acid 1 to amino acid 303. In yet another preferred embodiment, the recombinant molecule comprises the amino acid sequence shown in Figure 8 from amino acid 1 to amino acid 322.

In still a further embodiment of the invention, the first hematopoietic molecule is IL-3 and the second hematopoietic molecule is G-CSF. In one such embodiment, the first hematopoietic molecule comprises the amino portion and the second hematopoietic molecule comprises the carboxy portion of the recombinant molecule. In yet a more specific embodiment, the recombinant molecule comprises the amino acid sequence shown in Figure 6 from amino acid 1 to amino acid 317.

The subject invention also provides a nucleic acid molecules which encode the recombinant hematopoietic molecules of the subject invention. Examples of such nucleic acid molecules are depicted in Figures 4, 5 and 6. Moreover, vectors which comprise the nucleic acid molecules of the subject invention are also disclosed. In one embodiment of the invention, the vector comprises a plasmid. Moreover, host vector systems for the production of a recombinant hematopoietic molecule of the present invention are provided which comprise a vector of the present invention in a suitable host, preferably a mammalian cell such as a CHO or COS cell. This host BCI-14 -8vector system may be grown under suitable conditions which permit the expression of the recombinant hematopoietic molecule, which may be recovered by purification techniques known in the art, e.g. ion exchange chromatography, affinity chromatography, and size excision chromatography.

The present invention further provides pharmaceutical compositions useful for treating patients suffering from anemias of various origins, e.g. renal failure, and AIDS. Moreover, these pharmaceutical compositions are useful for administering to patients for preoperative autologous blood donations, patients receiving or donating bone marrow for transplantation purposes, and patients undergoing cancer chemotherapy. These pharmaceutical compositions comprise effective hematopoiesis-promoting amounts of a recombinant molecule of the present invention and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are known in the art and are disclosed in The Pharmacopeia of the United States and the National Formulary. Depending on the specific application contemplated, the pharmaceutical composition may be fromulated as a solution, suspension, parenteral preparation, or spray. Parenteral preparations may include a vehicle such as specially distilled, pyrogen-free water, phosphate buffer, or normal saline. Oral and/or transmucosal dosage forms may comprise phospholipids, often in the form of liposomes.

Also provided is a method for treating a patient to promote hematopoiesis which comprises administering to the patient an effective hematopoiesis-promoting amount of a pharmaceutical composition of the present invention.

BCI-14 -9The recombinant hematopoietic molecules, nucleic acid molecules, pharmaceutical compositions and methods of the present invention will be better understood by reference to the following experiments and examples, which are provided for purposes of illustration and are not to be construed as in any way limiting the scope of the invention, which is defined by the claims appended hereto.

Examples Construction of the hybrid protein genes: Genes encoding IL-3, Epo and G-CSF were purchased from British Biotech. Ltd. These genes were utilized to construct three different hybrid hematopoietic proteins, i.e., IL-3:Epo, Epo:IL-3 and IL-3:G-CSF. In these hybrids the first named gene forms the amino portion and the second named gene the carboxy portion of the hybrid protein.

Example I IL-3:Epo was constructed as follows: CSF, the native leader sequence of IL-3 was synthesized as 4 oligonucleotides (oligonucleotides 1-4 in Table I) which represents both strands of the leader sequence. In addition, the 5* end of the leader (oligonucleotide 1) encoded a convenient restriction enzyme overhang (EcoRl), although the EcoRl site was not regenerated, in front of the ATG start codon. The 3* end of the leader (oligonucleotide 3) included the first several amino acid codons of IL-3 and an Spel overhang so that the annealed leader sequence could be easily ligated to IL-3, which was altered by British Biotech to include an Spel site. The leader sequence was annealed and ligated to pKS (Stratagene Cloning Systems, Inc., San Diego, CA) cleaved with EcoRl and Spel. The resulting plasmid was designated BCI-14 -10pKSO. The IL-3 containing pUC18 plasmid obtained from British Biotech was cleaved with Spel and Nhel, then ligated to a linker oligonucleotide (complimentary oligonucleotides 5 and 6) which contained the following three restriction sites: Nhel, Xbal and Ncol. Cleavage was then performed with Spel and Xbal. The resulting 379 base pair fragment was then ligated to pKSO cleaved with Spel and Xbal. The resulting plasmid (pKSOIL-a) contained 10 the IL-3 leader, the IL-3 gene and a small linker fragment The Epo gene was inserted into pEE6 (Celltech, Ltd., Slough, U.K.), a mammalian expression vector which contains the human Cytomegalovirus promoter, a polylinker region and a poly-A addition site in addition to ampicillin resistance and a bacterial origin of replication, by cleaving the Epo containing plasmid obtained from British Biotech with Hindlll and BamHl. Epo was then cleaved with Ncol. The same linker comprising oligonucleotides 5 and 6 as described earlier was ligated to Epo and then cleaved with Xbal to yield the entire Epo gene. This was then ligated to Xbal and Bell cleaved pEE6 to yield pEE6 containing the Epo gene (pEepo). pKSOIL-a was cleaved with EcoRV and an Xbal linker was ligated to the blunt ends followed by cleavage with Xbal, which released the IL-3 gene with the leader sequence. This was then ligated to Xbal cleaved pEepo to yield a plasmid containing an entire hybrid protein gene (pEepie-a) (see Figure 4 for the structure of the inserted gene). The glutamine synthetase (gs) gene was then inserted into the BamHl site of pEepie-a to yield pEepogs-a or pEpogs-b, depending upon the orientation of the gs gene. Glutamine synthetase confers resistance to methionine sulphoximine (MSX) in order to select cells which have taken up the plasmid after transfection. After the plasmid was BCI-14 -11constructed a large batch was grown, purified by CsCl ultracentrifugation, and used for transfection. At each step in this process all ligation joints between fragments were analzyed by DNA sequence analysis in order to assure that there were no changes that would cause frameshifts and prevent the hybrid gene from being expressed.

To construct IL-3:Epo with a longer linker sequence separating IL-3 and Epo, pEepie-a was cleaved with Nhel and annealed oligonucleotides 21 and 22 were ligated into the cleaved plasmid. This linker encodes the flexible amino acid sequence (Gly-Gly-Gly-Gly-Ser). Clones with the insert in the proper orientation were selected by probing colonies with the junction oligonucleotide 23 (see Figure 7 for the structure of the inserted gene). The glutamine synthetase gene was then added to the construct as described above.

Example 2 IL-3:G-CSF was constructed as follows: pUC18 containing G-CSF (British Biotech) was cleaved with Hindlll. A linker composed of an overhanging Xbal site, a Notl site and an overhanging Hindlll site (oligonucleotides 7 and 8) was ligated to the pUC18:G-CSF. This was then cleaved with Xbal and BamHl which released the entire G-CSF gene. The G-CSF fragment was then inserted into Xbal and Bell cleaved pEE6 (pEE6:G-CSF). IL-3 with its signal sequence was removed from the IL-3:Epo plasmid pEepogs-a as an Xbal fragment. This IL-3 fragment was then inserted into Xbal cleaved pEE6-G-CSF. After restriction analysis, a plasmid containing the IL-3 gene in the proper orientation was obtained (pEGll), this plasmid encoded a gene capable of expressing IL-3 and G-CSF as a hybrid protein (see Figure 6 for the structure of the inserted gene). The gs gene BCI-14 -12was inserted into this plasmid as described in Example 1 above to yield plasmids pEG13 and pEG14, depending upon the orientation of the gs gene.

Example 3 Epo:IL-3 was constructed by first synthesizing the native Epo signal sequence as oligonucleotides (9-14). These were annealed to yield an overhanging 5' Xhol sequence and a 3' Pstl sequence. These were then ligated and subcloned as an Xhol/Pstl fragment (pEpol) . In order to obtain the proper reading frame and signal sequence processing site, the plasmid containing the signal sequence was cleaved with Pstl and the 3' overhang left by Pstl was enzymatically removed with T4 polymerase. This was then cleaved with BamHl. The Epo gene was then amplified by PCR as a fragment with a 5* blunt end using oligonucleotide 15 as a primer and a 3' BamHl end using oligonucleotide 16 as a primer. This fragment was then ligated into pEpol to yield a complete Epo gene with its leader sequence. PCR was used to amplify the Epo gene with its signal sequence as an (S’) Xbal and (3*) Notl fragment using oligonucleotides 17 and 18 as primers.

This was then digested with Xbal and Notl. At the same time, a purified IL-3 fragment was amplified by PCR as a (5') Notl and (3') BamHl fragment using oligonucleotides 19 and 20, followed by digestion with Notl and BamHl.

These two fragments were ligated to pEE6 cleaved with Xbal and Bell to yield a full length hybrid gene encoding both Epo and IL-3 (pEG16) (see Figure 5 for the structure of the inserted gene). The gs gene was inserted as described in Example 1 above to yield pEG17 and pEG18, depending upon the orientation of the gs gene.

BCI-14 -13A flexible linker is inserted into Epo:IL-3 by cleaving pEG17 or pEG18 with Notl. Annealed oligonucleotides 24 and 25 are then ligated into the cleaved plasmid. Clones with the insert in the proper orientation are selected by probing colonies with a junction oligonucleotide as described above. See Figure 8 for the structure of the inserted gene.

BCI-14 -14TABLE I OLIGONUCLEOTIDES All oligonucleotides are listed in the 5' to 3* 5 orientation: 1. AATTGCCGCCACCATGAGCCGCCTGCCCGTCCTGCTCCT 2. GCTCCAACTCCTGGTCCGCCCCGGACTCCAAGCTCCCATGACCCAGACAA 3. CTAGTTGTCTGGGTCATGGGAGCTTGGAGTCCGGGGCGG . ACCAGGAGTTGGAGCAGGAGCAGGACGGGCAGGCGGCTCATGGTGGCGGC . CTAGCGATCTTTCTAGA 6. CATGTCTAGAAAGATCG 7. CTAGAAGCGGCCGCA 8. TTCGCCGGCGTTCGA 9. TCGAGCCATGGGGGTGCACGAATGTCCT . GCCTGGCTGTGGCTTCTCCTGTCCCTGCTGTC 11. GCTCCCTCTGGGCCTCCCAGTCCTGGGCTGCA 12. GCCCAGGACTGGGAGGCCCAGAGGGA . GCGACAGCAGGGACAGGAGAAGCCACAGCCAGGCAGGACATT 14. CGTGCACCCCCATGGC . GCCCCACCACGCCTCATCTGT 16. GAATTCGGATCCTTATCATCT 17. CTAGTCTCTAGAATGGGGGTCCACGAATGT . AGCCATGGCGGCCGCTCTGTCCCCTGTCCT . GACAGAGCGGCCGCCATGGCTCCCATGACC . GAATTCGGATCCTTACTAAAAGATCGCTAG 21. CTAGCGTCCGGAGGCGGTGGCTCGGGCGGTGGCGGCTCGGGTGGCGGCGGCTCTGCG 2 2 . CTAGCGCAGAGCCGCCGCCACCGCAGCCGCCACCGCCCGAGCCACCGCCTCCGGACG 23. TTGTCGCTAGCGTCCGGAGGC . GGCCGCTTCCGGAGGCGGTGGCTCGGGCGGTGGCGGCTCGGGTGGCGGCGGCTCTGC . GGCCGCAGAGCCGCCGCCACCCGAGCCGCCACCGCCCGAGCCACCGCCTCCGGCAGC BCI-14 -15Example 4 Transfection of the hybrid gene containing plasmids. All transfections were performed using the Lipofectin”* transfection kit (Bethesda Research Labs, Gaithersburg, MD) using 15-30 pg. of purified plasmid DNA (pEepogs-a, pEepogs-b, pEG13, pEG14, pEG17, and pEG18). The following alterations were made to the protocol provided by the company: the growth medium in these experiments was GMEM-S and the CHO-K1 cells were incubated in the presence of 10% c°2: a^ter addition of the lipofectin:DNA complex, cells were incubated without selection for 24 hours. The cells were transferred to GMEM-S supplemented with 25 pM MSX after 24 hours. The MSX concentration was subsequently increased to 50 pM after one week. Cloning rings were used to subclone MSX resistant colonies and each of these colonies was placed into an individual well of a 24 well plate. Selected clones were incubated in the absence of MSX to insure that the hybrid protein gene was stably integrated. Strongly positive clones were grown in large cultures to provide larger amounts of hybrid proteins for further analysis.

Example 5 Assays for hybrid protein production. Cell supernatants from transfected or control cells were assayed using several different assays. In order to demonstrate Epo production, an RIA kit for Epo was used (Incstar Corp., Stillwater, MN). The presence of IL-3 was determined using an ELISA assay in which the capture antibody was a polyclonal goat anti-IL-3 (RRD Systems, Minneapolis, MN) and the probe antibody was a murine anti-IL-3 monoclonal. Goat anti-mouse conjugated to horseradish peroxidase followed by suitable substrate was used to detect the BCI-14 -16presence of the monoclonal anti-IL-3. A very similar assay was used to demonstrate the presence of the hybrid proteins except that a murine anti-Epo monoclonal or anti G-CSF monoclonal was used in place of anti IL-3 monoclonal. Additionally, IL-3:Epo was analyzed by Western blot analysis. The blot was probed with antibody 125 to Epo and then with I goat anti-mouse. A single broad band appeared on the autoradiogram with a molecular weight of slightly more than 50,000 daltons.

Example 6 Cellular assays. Epo and/or IL-3 dependent and responsive cell lines were used to test the biological activities of the hybrid proteins. B6SUtA (5) is a multipotential hematopoietic progenitor cell line established from nonadherent cell populations removed from continuous B6.S mouse bone marrow culture. This cell line demonstrates absolute dependence upon a source of growth factor(s). In response to Epo a population of the cells synthesize hemoglobin. Studies of globin expression indicated that the globin programs of B6SUtA cells are similar to those of erythroid progenitors at the period of transition from the yolk sac to fetal liver erythropoiesis. TF-1 (6) it is a cell line of immature erythroid origin established from a patient with erythroleukemia. The cell line shows complete dependency on GM-CSF or IL-3. Epo sustains short-term growth of TF-1 and will induce hemoglobin synthesis in a very small population of cells (8%). Hemin and 6-aminolevulinic acid induce hemoglobin synthesis in most of the cells.

Human IL-3 will not bind the murine IL-3 receptor, therefore experiments that were done with B6SUtA cells measured only the functionality of the Epo moiety of the BCI-14 -17hybrid. B6SUtA cells are carried in murine IL-3. In each experiment, they are washed thoroughly and set up with 5 growth factors at 10 cells/ml. Cell growth and hemoglobin content were monitored on days 3 and 6 of each experiment. Cells grown in the presence of concentrated (10X) CHO conditioned medium (CM) containing IL-3:Epo at a final concentration equivalent to 4.8 units/ml of Epo grew as well as cells grown in an equivalent amount of recombinant human (rHu) Epo. The percentage of cells which synthesized hemoglobin in response to the CHO-IL-3:Epo CM was always four times that of cells exposed to rHu Epo. B5SUtA cells grown in the presence of rHu IL-3 and rHu Epo grew as well as cells grown in the presence of IL-3:Epo and induced hemoglobin synthesis in the same percentage of cells as did rHu Epo. Cells exposed to recombinant murine (rMu) IL-3 and rHu Epo grew similarly to cells exposed to rMu IL-3 alone and neither effectively induced cells to synthesize hemoglobin. Concentrated control CHO CM did not support the growth of B6SUtA cells nor did it induce hemoglobin synthesis. CHO CM plus rHu Epo supported cell growth and hemoglobinization as well as CHO-IL-3:Epo CM.

CHO-IL-3:Epo CM supported growth of human TF-1 cells as well as rHu Epo. rHu IL-3 was twice as good at supporting growth of the TF-1 cells as was CHO-IL-3:Epo CM. Control CHO CM supported only limited growth of the TF-1 cells. &j^cu?si.Qn These results demonstrate that a hybrid protein comprising two growth factors can be expressed in mammalian cell culture systems. In vitro assays of IL-3:Epo indicate that this hybrid protein has the activities of both IL-3 and Epo. The therapeutic application of such hybrid BCI-14 -18factors has advantages over using two factors separately simply in terms of patient administration, and moreover since the production, purification and formulation of one factor is less labor intensive than for two separate factors.

References 1. Sonoda, et al., Proc. Natl. Acad. Sci. USA, ££:4630-4364 (1988). 2. Migliaccio, et al., Blood, Vol. 72, No. 3, 844-851 (1988). 3. Sieff, et al., Blood, Vol.73, No. 3, 688-693 (1989). 4. Fraser, et al., Exp. Hematol., 16: 769-775 (1988) . Enver, et al, Proc.Natl. Acad. Sci. USA, ££:9091-9095 (1988). 6. Kitamura, et al., J. Physiol., 140:323-334 (1989) BCI-14

Claims (26)

What is claimed is:

1. A recombinant hematopoietic molecule comprising at least a portion of a first hematopoietic molecule having 5 early myeloid differentiation activity and at least a portion of a second hematopoietic molecule having late myeloid differentiation activity, said recombinant hematopoietic molecule having early myeloid differentiation activity associated with said first 10 hematopoietic molecule and late myeloid differentiation activity associated with said second hematopoietic molecule.

2. A recombinant hematopoietic molecule of claim 1 15 wherein the first hematopoietic molecule is selected from the group consisting of IL-3 and GM-CSF.

3. A recombinant hematopoietic molecule of claim 1 wherein the second hematopoietic molecule is selected from 20 the group consisting of Epo, G-CSF, IL-5 and M-CSF.

4. A recombinant hematopoietic molecule of claim 1 wherein the portion of the first hematopoietic molecule is linked to the portion of the second hematopoietic molecule 25 by an amino acid linker sequence of at least two amino acid residues.

5. A recombinant hematopoietic molecule of claim 1 comprising the entire amino acid sequence of human IL-3.

6. A recombinant hematopoietic molecule of claim 1 comprising an amino acid sequence contained within the amino acid sequence from amino acid 1 to amino acid 79 in Figure 1. BCI-14 -207. A recombinant hematopoietic molecule of claim 1 comprising the entire amino acid sequence of human Epo.

7. 8. A recombinant hematopoietic molecule of claim 1 5 comprising an amino acid sequence contained within the amino acid sequence from amino acid 7 to amino acid 161 in Figure 2.

8. 9. A recombinant hematopoietic molecule of claim 1

9. 10 comprising the entire amino acid sequence of human G-CSF. 10. A recombinant hematopoietic molecule of claim 1 wherein the first hematopoietic molecule is IL-3 and the second hematopoietic molecule is Epo.

10. 11. A recombinant hematopoietic molecule of claim 10 wherein the first hematopoietic molecule comprises the amino portion and the second hematopoietic molecule comprises the carboxy portion of the recombinant 20 hematopoietic molecule.

11. 12. A recombinant hematopoietic molecule of claim 11 which comprises the amino acid sequence shown in Figure 4 from amino acid 1 to amino acid 302.

12. 13. A recombinant hematopoietic molecule of claim 11 which comprises the amino acid sequence shown in Figure 7 from amino acid 1 to amino acid 321. 30

13. 14. A recombinant hematopoietic molecule of claim 10 wherein the first hematopoietic molecule comprises the carboxy portion and the second hematopoietic molecule comprises the amino portion of the recombinant hematopoietic molecule. BCI-14 -2115. A recombinant hematopoietic molecule of claim 14 which comprises the amino acid sequence shown in Figure 5 from amino acid 1 to amino acid 303. 5 16. A recombinant hematopoietic molecule of claim 14 which comprises the amino acid sequence shown in Figure 8 from amino acid 1 to amino acid 322. 17. A recombinant hematopoietic molecule of claim 1 10 wherein the first hematopoietic molecule is IL-3 and the second hematopoietic molecule is G-CSF 18. A recombinant hematopoietic molecule of claim 17 wherein the first hematopoietic molecule comprises the

14. 15 amino portion and the second hematopoietic molecule comprises the carboxy portion of the recombinant hematopoietic molecule.

15. 19. A recombinant hematopoietic molecule of claim 18

16. 20 which comprises the amino acid sequence shown in Figure 6 from amino acid 1 to amino acid 317. 20. A nucleic acid molecule which encodes the recombinant hematopoietic molecule of claim 1.

17. 21. An expression vector which comprises the nucleic acid molecule of claim 20.

18. 22. A host cell transformed with the expression 30 vector of claim 19.

19. 23. A host cell of claim 22 which comprises a mammalian cell. BCI-14 -2224. A method for producing a recombinant hematopoietic molecule comprising at least a portion of a first hematopoietic molecule having early myeloid differentiation activity and at least a portion of a 5 second hematopoietic molecule having late myeloid differentiation activity, which comprises culturing a host cell of claim 22 under suitable conditions so as to allow the expression of such recombinant hematopoietic molecule, and recovering such recombinant hematopoietic molecule.

20. 25. A pharmaceutical composition which comprises a recombinant hematopoietic molecule of claim 1 and a pharmaceutically acceptable carrier. 15 26. A method for promoting hematopoiesis in a patient which comprises administering to such patient a pharmaceutical composition of claim 25. [6862R] BCI-14 -2327. A recombinant hematopoietic molecule according to claim 1, substantially as hereinbefore described and exemplified.

21. 28. A nucleic acid molecule according to claim 20, substantially as hereinbefore described and exemplified.

22. 29. An expression vector according to claim 21, substantially as hereinbefore described and exemplified.

23. 30. A host cell according to claim 22, substantially as hereinbefore described and exemplified.

24. 31. A method according to claim 24 for producing a recombinant hematopoietic molecule, substantially as hereinbefore described and exemplified.

25. 32. A recombinant hematopoietic molecule, whenever produced by a method claimed in claim 24 or 31 .

26. 33. A pharmaceutical composition according to claim 25, substantially as hereinbefore described.