Fusion proteins comprising MGF and IL-3

A fusion protein which comprises MGF and IL-3. Such fusion proteins have enhanced biological activity.

BACKGROUND OF THE INVENTION 
The present invention relates to fusion proteins for stimulating growth of 
hematopoietic cells, and more particularly to the construction of fusion 
proteins comprising MGF and IL-3. 
Hematopoietic growth factors (or hematopoietins) regulate the growth and 
maturation of various lineages of blood cells. All blood cells are 
believed to develop from a single class of precursor cells called stem 
cells. Each hematopoietin causes specific classes of blood cells to 
differentiate and proliferate. When a stem cell divides in the bone 
marrow, it can replicate itself as a stem cell or become committed to a 
particular developmental pathway. As a result of commitment to a 
particular developmental pathway, a stem cell displays receptors on its 
cell surface that enables it to respond to certain hormonal signals. Such 
signals push the cell further down a pathway leading to terminal 
differentiation. 
A number of hematopoietins have been identified which regulate cell 
development at various levels within the hematopoietic stem and progenitor 
cell hierarchy. The majority of growth factors that have been identified 
influence relatively late stages of differentiation and regulate the 
number and function of mature differentiated hematopoietic elements. 
Interleukin-3 ("IL-3" or "multi-CSF)), for example, stimulates formation 
of a broad range of hematopoietic cells, including granulocytes, 
macrophages, eosinophils, mast cells, megakaryocytes and erythroid cells. 
IL-3 has been identified, isolated and molecularly cloned (EP Publ. Nos. 
275,598 and 282,185). Recently, a Mast Cell Growth Factor "MGF"), which 
controls very early progenitors in the hematopoietic hierarchy, has been 
identified, isolated and molecularly cloned (Williams et al., Cell 63:167, 
1991; Anderson et al., Cell 63:235, 1991). 
Preclinical studies indicate that such hematopoietins may be useful in 
treating various cytopenias, potentiating immune responsiveness to 
infectious pathogens, and assisting in reconstituting normal blood cell 
populations following viral infection or radiation or chemotherapy-induced 
hematopoietic cell suppression. MGF, in particular, because of its early 
effect on hematopoietic cells, is likely to be useful for treating a 
plastic anemia. In order to determine their optimal therapeutic potential, 
the effect of various combinations of such proteins on hematopoietic cells 
has been the subject of considerable study. 
SUMMARY OF THE INVENTION 
The present invention relates to fusion proteins comprising MGF and IL-3. 
The fusion proteins preferably have a formula selected from the group 
consisting of 
EQU R.sub.1 -R.sub.2, R.sub.2 -R.sub.1, R.sub.1 -L-R.sub.2 and R.sub.2 
-L-R.sub.1 
wherein R.sub.1 is MGF; R.sub.2 is IL-3; and L is a linker peptide 
sequence. In the most preferred aspects of the present invention, MGF and 
IL-3 are linked together via a linker sequence which permits folding of 
the MGF or IL-3 domains in such a manner as to preserve the ability of 
each domain to bind to its respective cell surface receptor molecule. 
The fusion proteins of the present invention show enhanced ability to 
stimulate human bone marrow cells than MGF or IL-3 alone or in combination 
.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to fusion proteins comprising MGF and 
IL-3. The following terms are defined as follows: 
"Recombinant," as used herein, means that a protein is derived from 
recombinant (e.g., microbial or mammalian) expression systems. "Microbial" 
refers to recombinant proteins made in bacterial or fungal (e.g., yeast) 
expression systems. As a product. "recombinant microbial" defines a 
protein produced in a microbial expression system which is essentially 
free of native endogenous substances. Protein expressed in most bacterial 
cultures. e.g., E. coli, will be free of glycan. Protein expressed in 
yeast may have a glycosylation pattern different from that expressed in 
mammalian cells. 
"Biologically active," as used throughout the specification means that a 
particular molecule shares sufficient amino acid sequence similarity with 
native forms so as to be capable of binding to native receptor, 
transmitting a stimulus to a cell, or cross-reacting antibodies raised 
against the particular molecule. 
"DNA sequence" refers to a DNA polymer, in the form of a separate fragment 
or as a component of a larger DNA construct. Preferably, the DNA sequences 
are in a quantity or concentration enabling identification, manipulation, 
and recovery of the sequence and its component nucleotide sequences by 
standard biochemical methods, for example, using a cloning vector. Such 
sequences are preferably provided in the form of an open reading frame 
uninterrupted by internal nontranslated sequences, or introns, which are 
typically present in eukaryotic genes. Genomic DNA containing the relevant 
sequences could also be used. Sequences of non-translated DNA may be 
present 5' or 3' from the open reading frame, where the same do not 
interfere with manipulation or expression of the coding regions. 
"Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. 
DNA sequences encoding the proteins provided of this invention can be 
assembled from cDNA fragments and short oligonucleotide linkers, or from a 
series of oligonucleotides, to provide a synthetic gene which is capable 
of being expressed in a recombinant transcriptional unit. 
"Recombinant expression vector" refers to a replicable DNA construct used 
either to amplify or to express DNA which encodes the fusion proteins of 
the present invention and which includes a transcriptional unit comprising 
an assembly of (1) a genetic element or elements having a regulatory role 
in gene expression, for example, promoters or enhancers, (2) a structural 
or coding sequence which is transcribed into mRNA and translated into 
protein, and (3) appropriate transcription and translation initiation and 
termination sequences. Structural elements intended for use in yeast 
expression systems preferably include a leader sequence enabling 
extracellular secretion of translated protein by a host cell. 
Alternatively, where recombinant protein is expressed without a leader or 
transport sequence, it may include an N-terminal methionine residue. This 
residue may optionally be subsequently cleaved from the expressed 
recombinant protein to provide a final product. "Recombinant microbial 
expression system" means a substantially homogeneous monoculture of 
suitable host microorganisms, for example, bacteria such as E. coli or 
yeast such as S. cerevisiae, which have stably integrated a recombinant 
transcriptional unit into chromosomal DNA or carry the recombinant 
transcriptional unit as a component of a resident plasmid. Generally, 
cells constituting the system are the progeny of a single ancestral 
transformant. Recombinant expression systems as defined herein will 
express heterologous protein upon induction of the regulatory elements 
linked to the DNA sequence or synthetic gene to be expressed. 
MAST CELL GROWTH FACTOR 
The term mast cell growth factor ("MGF") refers to proteins having 
substantially the same characteristics of MGF in that they are capable of 
binding to receptors for MGF or transducing a biological signal initiated 
by binding to MGF receptors, or cross-reacting with anti-MGF antibodies 
raised against MGF. MGF includes a family of mammalian polypeptides which 
are capable of stimulating IL-3 dependent mast cell lines and 
hematopoietic progenitor cells, and serve as a ligand for the gene product 
of the c-kit proto-oncogene. MGF polypeptides and DNA sequences encoding 
MGF polypeptides are disclosed, for example, in Anderson et al., Cell 
63:235, 1991; Martin et al., Cell 65:203, 1991; and EP-A-0 423 980. As 
used herein, the term MGF includes analogs or subunits of native mammalian 
polypeptides with substantially identical or substantially similar amino 
acid polypeptide sequences which bind to the protein expressed by the 
c-kit proto-oncogene and which induce proliferation of mast cells, for 
example, the IL-3 dependent murine mast cell line MC6 or human cell line 
TF1. Although native forms of MGF are membrane bound and consist of an 
extracellular, transmembrane and cytoplasmic domains, the MGF used in the 
fusion proteins of the present invention preferably consists solely of its 
extracellular region or a fragment thereof including all four Cys residues 
of the extracellular domain and lacks a transmembrane region and 
intracellular domain. The extracellular region of MGF or fragment thereof 
is a soluble polypeptide which is capable of binding the gene product of 
the c-kit proto-oncogene. Both human and murine native sequence MGF have 
been found in two variations. One human variant is set forth in FIG. 42 of 
EP-A-0 423 980. The other variant has a 28 amino acid deletion (of amino 
acids 149-177) and is referred to as .DELTA.28 hMGF. 
Human MGF polypeptides have a 185 amino acid extracellular domain. This 
polypeptide has five glycosylation sites and four Cys residues, with 
Cys.sup.3 binding to Cys.sup.89 and Cys.sup.43 binding to Cys.sup.138. 
.DELTA.28 hMGF retains all four Cys residues, but eliminates the fifth 
glycosylation site. Several amino acids can be removed from the C-terminus 
of hMGF extracellular domain, up to Cys.sup.138, while retaining 
biological activity. Similarly, the first three N-terminal amino acids can 
be removed, up to Cys.sup.3 of mature human MGF, while retaining 
biological activity. Therefore, a human MGF polypeptide having only 136 
amino acid residues, but retaining all four Cys residues, retains 
significant biological activity compared to full length 185 amino acid 
form of hMGF or .DELTA.28 hMGF. .DELTA.28 hMGF has significant biological 
activity and is the preferred MGF fragment for use in fusion proteins 
comprising MGF and IL-3. 
Various other analogs of the hMGF molecule have been shown to retain MGF 
biological activity. For example, Lys.sup.91 confers human species 
specificity to MGF and Glu.sup.91 confers murine species specificity. 
Glycosylation sites can be altered to facilitate expression in yeast or 
mammalian cell systems. The region surrounding Cys.sup.89 is important for 
receptor binding. However, the Val.sup.90 can be substituted with any 
other amino acid without affecting biological activity. Human MGF analogs 
can vary in length from about 135 amino acids to about 185 amino acids 
constituting the extracellular domain of the hMGF polypeptide. 
Biologically active hMGF analog polypeptides comprise four Cys residues, 
but can vary at other positions according to the following sequence: 
__________________________________________________________________________ 
X.sub.n 
X.sub.n 
X.sub.n 
Cys 
Arg 
Asn 
Arg 
Val 
Thr 
Asn 
Asn 
Val 
Lys 
Asp 
Val 
Thr 
Lys 
Leu 
Val 
Ala 
Asn 
Leu 
Pro 
Lys 
Asp 
Tyr 
Met 
Ile 
Thr 
Leu 
Lys 
Tyr 
Val 
Pro 
Gly 
Met 
Asp 
Val 
Leu 
Pro 
Ser 
His 
Cys 
Trp 
Ile 
Ser 
Glu 
Met 
Val 
Val 
Gln 
Leu 
Ser 
Asp 
Ser 
Leu 
Thr 
Asp 
Leu 
Leu 
Asp 
Lys 
Phe 
Ser 
Asn 
Ile 
Ser 
Glu 
Gly 
Leu 
Ser 
Asn 
Tyr 
Ser 
Ile 
Ile 
Asp 
Lys 
Leu 
Val 
Asn 
Ile 
Val 
Asp 
Asp 
Leu 
Val 
R.sub.1 
Cys 
R.sub.2 
Q Glu 
Asn 
Ser 
Ser 
Lys 
Asp 
Leu 
Lys 
Lys 
Ser 
Phe 
Lys 
Ser 
Pro 
Glu 
Pro 
Arg 
Leu 
Phe 
Thr 
Pro 
Glu 
Glu 
Phe 
Phe 
Arg 
Ile 
Phe 
Asn 
Arg 
Ser 
Ile 
Asp 
Ala 
Phe 
Lys 
Asp 
Phe 
Val 
Val 
Ala 
Ser 
Glu 
Thr 
Ser 
Asp 
Cys 
X.sub.n 
X.sub.n 
X.sub.n 
X.sub.n 
X.sub.n 
X.sub.n 
X.sub.n 
X.sub.n 
X.sub.n 
X.sub.n 
__________________________________________________________________________ 
wherein n is 0 or 1, X is any naturally occurring amino acid; R.sub.1 is 
any amino acid except Glu; R.sub.2 is any amino acid except Val; and Q is 
Lys or Arg to provide human specificity, or is Glu to provide murine 
specificity. Such MGF proteins may be use to provide the MGF domains of 
the fusion proteins of the present invention. 
INTERLEUKIN-3 
The term "IL-3" refers to proteins having substantially the same 
characteristics of IL-3 in that they are capable of binding to receptors 
for IL-3 or transducing a biological signal initiated by binding to IL-3 
receptors, or cross-reacting with anti-IL-3 antibodies raised against 
IL-3. Such sequences are disclosed, for example, in EP Publ. Nos. 275,598 
and 282,185. The term "IL-3" specifically includes analogs or subunits of 
native mammalian IL-3 polypeptides with substantially identical or 
substantially similar amino acid polypeptide sequences which exhibit at 
least some biological activity in common with native IL-3. Exemplary 
analogs of IL-3 are also disclosed in EP Publ. No. 282,185. Particularly 
preferred forms of IL-3 which may be fused to MGF in accordance with the 
present invention include huIL-3[Pro.sup.8 Asp.sup.15 Asp.sup.70 ], 
huIL-3[Ser.sup.8 Asp.sup.15 Asp.sup.70 ], and huIL-3[Ser.sup.8 ]. A DNA 
sequence encoding another IL-3 protein suitable for incorporation into 
fusion proteins as described herein is on deposit with ATCC under 
accession number ATCC 67747. Other forms of IL-3 may also be used to 
provide the IL-3 domain of the fusion proteins of the present invention. 
FUSION PROTEINS COMPRISING MGF AND IL-3 
As used herein, the term "fusion protein" refers to a C-terminal to 
N-terminal fusion of MGF and IL-3. The fusion proteins of the present 
invention include constructs in which the C-terminal portion of MGF is 
fused to the N-terminal portion of IL-3, and also constructs in which the 
C-terminal portion of IL-3 is fused to the N-terminal portion of MGF. MGF 
is linked to IL-3 in such a manner as to produce a single protein which 
retains the biological activity of MGF and IL-3. In preferred aspects, MGF 
is linked to IL-3 via a linker sequence 
Examples of fusion proteins comprising MGF and IL-3 are shown in the 
accompanying Sequence Listing. SEQ ID NO:1 shows the nucleotide sequence 
and corresponding amino acid sequence of a human IL-3/MGF fusion protein, 
referred to as PIXY521. The fusion protein comprises human IL-3 (amino 
acids 1-133) linked to human MGF (amino acids 145-301) via a linker 
sequence (amino acids 134-144). SEQ ID NO:3 shows a nucleotide sequence 
and corresponding amino acid sequence of a human MGF/IL-3 fusion protein. 
The fusion protein comprises human MGF (amino acids 1-157) linked to human 
IL-3 (amino acids 171-303) via a linker sequence (amino acids 158-170). 
Equivalent fusion proteins may vary from the sequence of SEQ ID NO:1 and 
SEQ ID NO:3 by one or more substitutions, deletions, or additions, the net 
effect of which is to retain biological activity of the protein when 
derived as a fusion protein comprising MGF and IL-3. Alternatively, DNA 
analog sequences are equivalent to the specific DNA sequences disclosed 
herein if: (a) the DNA analog sequence comprises sequences derived from a 
biologically active fragments of the native IL-3 and MGF genes; or (b) the 
DNA analog sequence is capable of hybridization to DNA sequences of (a) 
under high or moderate stringent conditions and encodes biologically 
active MGF and IL-3 molecules; or (c) DNA analog sequence is degenerate as 
a result of the genetic code to the DNA analog sequences defined in (a) or 
(b) and which encode biologically active MGF and IL-3 molecules. 
Moderate stringency hybridization conditions, as defined herein and as 
known to those of skill in the art, refer to conditions described in, for 
example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 Ed. 
Vol. 1 pages 1.101-1.104 (Cold Spring Harbor Laboratory Press 1989). 
Exemplary conditions of moderate stringency are prewashing with 
5.times.SSC, 0.5% SDS, 1 mM EDTA (pH 8.0) and overnight hybridization at 
50.degree. C. in 2.times.SSC. Exemplary severe or high stringency 
conditions are overnight hybridization at about 68.degree. C. in a 
6.times.SSC solution, washing at room temperature with 6.times.SSC 
solution, followed by washing at about 68.degree. C. in a 0.6.times.SSC 
solution. 
CONSTRUCTION OF CDNA SEQUENCES ENCODING FUSION PROTEINS COMPRISING MGF AND 
IL-3 
A DNA sequence encoding a fusion protein is constructed using recombinant 
DNA techniques to assemble separate DNA fragments encoding MGF and IL-3 
into an appropriate expression vector. For example, the 3' end of a DNA 
fragment encoding MGF is ligated to the 5' end of the DNA fragment 
encoding IL-3, with the reading frames of the sequences in phase to permit 
mRNA translation of the sequences into a single biologically active fusion 
protein. The resulting protein is fusion protein comprising MGF and IL-3. 
Alternatively, the 3' end of a DNA fragment encoding IL-3 may be ligated 
to the 5' end of the DNA fragment encoding MGF, with the reading frames of 
the sequences in phase to permit mRNA translation of the sequences into a 
single biologically active fusion protein. The regulatory elements 
responsible for transcription of DNA into mRNA are retained on the first 
of the two DNA sequences, while binding signals or stop codons, which 
would prevent read-through to the second DNA sequence, are eliminated. 
Conversely, regulatory elements are removed from the second DNA sequence 
while stop codons required to end translation are retained. 
In preferred aspects of the present invention, means are provided for 
linking the MGF and IL-3 domains, preferably via a linker sequence. The 
linker sequence separates MGF and IL-3 domains by a distance sufficient to 
ensure that each domain properly folds into its secondary and tertiary 
structures. Suitable linker sequences (1) may adopt a flexible or a more 
rigid, extended conformation, (2) will not exhibit a propensity for 
developing an ordered secondary structure which could interact with the 
functional MGF and IL-3 domains, and (3) will have minimal hydrophobic or 
charged character which could promote interaction with the functional 
protein domains. Typical surface amino acids in flexible protein regions 
include Gly, Asn and Ser. Virtually any permutation of amino acid 
sequences containing Gly, Asn and Ser would be expected to satisfy the 
above criteria for a linker sequence. Other near neutral amino acids, such 
as Thr and Ala, may also be used in the linker sequence. 
The length of the linker sequence may vary without significantly affecting 
the biological activity of the fusion protein. For example, the MGF and 
IL-3 proteins may be directly fused without a linker sequence. Linker 
sequences are unnecessary where the proteins being fused have 
non-essential N- or C-terminal amino acid regions which can be used to 
separate the functional domains and prevent steric interference. In one 
preferred embodiment of the present invention, the C-terminus of MGF may 
be directly fused to the N-terminus of IL-2. Full-length hMGF has 47 and 
.DELTA.28 hMGF has 19 amino acids following the C-terminal cysteine 
residue in the extracellular region, which is involved in disulfide 
bonding and is essential for proper folding of the protein. IL-3 has 15 
amino acids preceding its N-terminal cysteine residue. The combined 
terminal regions may therefore provide sufficient separation to render the 
use of a linker sequence unnecessary. 
Generally, the two protein domains will be separated by a distance 
approximately equal to the small unit dimension of MGF or IL-3 (i.e., 
approximately 0.38 nm, as determined by analogy with other similar 
four-helix hormones). In a preferred aspect of the invention, a linker 
sequence length of about 11 amino acids is used to provide a suitable 
separation of functional protein domains, although longer linker sequences 
may also be used. The length of the linker sequence separating MGF and 
IL-3 is from 1 to 500 amino acids in length, or more preferably from 1 to 
100 amino acids in length. In the most preferred aspects of the present 
invention, the linker sequence is from about 1-20 amino acids in length. 
In the specific embodiments disclosed herein, the linker sequence is from 
about 5 to about 15 amino acids, and is advantageously from about 10 to 
about 15 amino acids. Preferred amino acid sequences for use as linkers of 
MGF and IL-3 include, for example, GlyAlaGlyGlyAlaGlySer(Gly).sub.5 Ser, 
(Gly.sub.4 Ser).sub. 3 Gly.sub.4 SerGly.sub.5 Ser, (Gly.sub.4 Ser).sub.2, 
and (GlyThrPro).sub.3. 
The linker sequence is incorporated into the fusion protein construct by 
well known standard methods of mutagenesis as described below. 
PROTEINS AND ANALOGS 
In preferred aspects, the present invention provides a fusion protein 
comprising human MGF and human IL-3. Derivatives of the fusion proteins of 
the present invention also include various structural forms of the primary 
protein which retain biological activity. Due to the presence of ionizable 
amino and carboxyl groups, for example, a fusion protein may be in the 
form of acidic or basic salts, or may be in neutral form. Individual amino 
acid residues may also be modified by oxidation or reduction. 
The primary amino acid structure may be modified by forming covalent or 
aggregative conjugates with other chemical moieties, such as glycosyl 
groups, lipids, phosphate, acetyl groups and the like, or by creating 
amino acid sequence mutants. Covalent derivatives are prepared by linking 
particular functional groups to amino acid side chains or at the N- or 
C-termini. Other derivatives of the fusion protein within the scope of 
this invention include covalent or aggregative conjugates of the fusion 
protein with other proteins or polypeptides, such as by synthesis in 
recombinant culture as N- or C-terminal fusions. For example, the 
conjugated peptide may be a signal (or leader) polypeptide sequence at the 
N-terminal region of the protein which co-translationally or 
postotranslationally directs transfer of the protein from its site of 
synthesis to its site of function inside or outside of the cell membrane 
or wall (e.g., the yeast .alpha.-factor leader). 
Peptides may also be added to facilitate purification or identification of 
MGF/IL-3 fusion proteins (e.g., poly-His). For example, in a preferred 
embodiment of the present invention, the amino acid sequence of the fusion 
protein is linked to the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys 
(DYKDDDDK) (Hopp et al., Bio/Technology 6:1204, 1988). The latter sequence 
is highly antigenic and provides an epitope reversibly bound by a specific 
monoclonal antibody, enabling rapid assay and facile purification of 
expressed recombinant protein. This sequence is also specifically cleaved 
by bovine mucosal enterokinase at the residue immediately following the 
Asp-Lys pairing. Fusion proteins capped with this peptide may also be 
resistant to intracellular degradation in E. coli. 
Fusion protein derivatives may also be used as immunogens, reagents in 
receptor-based immunoassays, or as binding agents for affinity 
purification procedures of binding ligands. Derivatives may also be 
obtained by cross-linking agents, such as M-maleimidobenzoyl succinimide 
ester and N-hydroxysuccinimide, at cysteine and lysine residues. Fusion 
proteins may also be covalently bound through reactive side groups to 
various insoluble substrates, such as cyanogen bromide-activated, 
bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activated 
agarose structures, or by adsorbing to polyolefin surfaces (with or 
without glutaraldehyde cross-linking). 
The present invention also includes proteins with or without associated 
native-pattern glycosylation. Expression of DNAs encoding the fusion 
proteins in bacteria such as E. coli provides non-glycosylated molecules. 
Functional mutant analogs having inactivated N-glycosylation sites can be 
produced by oligonucleotide synthesis and ligation or by site-specific 
mutagenesis techniques. These analog proteins can be produced in a 
homogeneous, reduced-carbohydrate form in good yield using yeast 
expression systems. N-glycosylation sites in eukaryotic proteins are 
characterized by the amino acid triplet Asn-A.sub.1 -Z, where A.sub.1 is 
any amino acid except Pro, and Z is Ser or Thr. In this sequence, 
asparagine provides a side chain amino group for covalent attachment of 
carbohydrate. Such a site can be eliminated by substituting another amino 
acid for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z 
amino acid between A.sub.1 and Z, or an amino acid other than Asn between 
Asn and A.sub.1. Human MGF has three possible glycosylation sites at amino 
acids 209-211, 216-218, 237-239 and 264-266 (SEQ ID NO:1) which may be 
removed. Examples of human IL-3 analogs in which glycosylation sites have 
been removed include huIL-3[Pro.sup.8 Asp.sup.15 Asp.sup.70 ], 
huIL-3[Asp.sup.70 ], huIL-3[Asp.sup.15 Asp.sup.70 ], huIL-3[Pro.sup.8 
Asp.sup.15 ], huIL-3[Pro.sup.8 Asp.sup.70 ], and huIL-3[Asp.sup.15 ](SEQ 
ID NO:1). 
Derivatives and analogs may also be obtained by mutations of the fusion 
protein. A derivative or analog, as referred to herein, is a polypeptide 
in which the MGF and IL-3 domains are substantially homologous to the 
extracellular region of MGF and the full-length IL-3 of the sequences 
disclosed in SEQ ID NO:1 but which has an amino acid sequence difference 
attributable to a deletion, insertion or substitution. 
Bioequivalent analogs of fusion proteins may be constructed by, for 
example, making various substitutions of residues or sequences. For 
example, cysteine residues can be deleted or replaced with other amino 
acids to prevent formation of incorrect intramolecular disulfide bridges 
upon renaturation. Other approaches to mutagenesis involve modification of 
adjacent dibasic amino acid residues to enhance expression in yeast 
systems in which KEX2 protease activity is present. Generally, 
substitutions should be made conservatively; i.e., the most preferred 
substitute amino acids are those having physicochemical characteristics 
resembling those of the residue to be replaced. Similarly, when a deletion 
or insertion strategy is adopted, the potential effect of the deletion or 
insertion on biological activity should be considered. 
Mutations in nucleotide sequences constructed for expression of analogs 
must, of course, preserve the reading frame phase of the coding sequences 
and preferably will not create complementary regions that could hybridize 
to produce secondary mRNA structures such as loops or hairpins which would 
adversely affect translation of the MGF/IL-3 receptor mRNA. Although a 
mutation site may be predetermined, it is not necessary that the nature of 
the mutation per se be predetermined. For example, in order to select for 
optimum characteristics of mutants at a given site, random mutagenesis may 
be conducted at the target codon and the expressed mutants screened for 
the desired activity. 
Not all mutations in nucleotide sequences which encode fusion proteins 
comprising MGF and IL-3 will be expressed in the final product, for 
example, nucleotide substitutions may be made to enhance expression, 
primarily to avoid secondary structure loops in the transcribed mRNA (see 
EPA 75,444A, incorporated herein by reference), or to provide codons that 
are more readily translated by the selected host, e.g., the well-known E. 
coli preference codons for E. coli expression. 
Mutations can be introduced at particular loci by synthesizing 
oligonucleotides containing a mutant sequence, flanked by restriction 
sites enabling ligation to fragments of the native sequence. Following 
ligation, the resulting reconstructed sequence encodes an analog having 
the desired amino acid insertion, substitution, or deletion. 
Alternatively, oligonucleotide-directed site-specific mutagenesis 
procedures can be employed to provide an altered gene having particular 
codons altered according to the substitution, deletion, or insertion 
required. Exemplary methods of making the alterations set forth above are 
disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 
1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic 
Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. 
Nos. 4,518,584 and 4,737,462, and are incorporated by reference herein. 
EXPRESSION OF RECOMBINANT FUSION PROTEINS COMPRISING MGF AND IL-3 
The present invention provides recombinant expression vectors which include 
synthetic or cDNA-derived DNA fragments encoding human fusion proteins 
comprising MGF and IL-3 or bioequivalent analogs operably linked to 
suitable transcriptional or translational regulatory elements derived from 
mammalian, microbial, viral or insect genes. Such regulatory elements 
include a transcriptional promoter, an optional operator sequence to 
control transcription, a sequence encoding suitable mRNA ribosomal binding 
sites, and sequences which control the termination of transcription and 
translation, as described in detail below. The ability to replicate in a 
host, usually conferred by an origin of replication, and a selection gene 
to facilitate recognition of transformants may additionally be 
incorporated. DNA regions are operably linked when they are functionally 
contiguous to each other. For example, DNA for a signal peptide (secretory 
leader) is operably linked to DNA for a polypeptide if it is expressed as 
a precursor which participates in the secretion of the polypeptide; a 
promoter is operably linked to a coding sequence if it controls the 
transcription of the sequence; or a ribosome binding site is operably 
linked to a coding sequence if it is positioned so as to permit 
translation. Generally, operably linked means contiguous and, in the case 
of secretory leaders, contiguous and in reading frame. 
Due to code degeneracy, there can be considerable variation in nucleotide 
sequences encoding the same MGF or IL-3 amino acid sequence; exemplary DNA 
embodiments are those corresponding to the nucleotide sequences shown in 
SEQ ID NO:1 or SEQ ID NO:3. Other embodiments within the scope of the 
present invention include nucleotide sequences which encode fusion 
proteins comprising MGF and IL-3, in which the MGF and IL-3 encoding 
regions are capable of hybridizing to the respective MGF and IL-3 
nucleotide regions of SEQ ID NO:1 or SEQ ID NO:3 under moderate or high 
stringency conditions and which encode biologically active fusion 
proteins. 
Transformed host cells are cells which have been transformed or transfected 
with fusion protein vectors constructed using recombinant DNA techniques. 
Transformed host cells ordinarily express the desired fusion protein, but 
host cells transformed for purposes of cloning or amplifying DNA do not 
need to express the protein. Expressed fusion protein will generally be 
secreted into the culture supernatant. Suitable host cells for expression 
of fusion protein include prokaryotes, yeast or higher eukaryotic cells 
under the control of appropriate promoters. Prokaryotes include gram 
negative or gram positive organisms, for example E. coli or bacilli. 
Higher eukaryotic cells include established cell lines of mammalian origin 
as described below. Cell-free translation systems could also be employed 
to produce fusion protein using RNAs derived from the DNA constructs of 
the present invention. Appropriate cloning and expression vectors for use 
with bacterial, fungal, yeast, and mammalian cellular hosts are described 
by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 
1985), the relevant disclosure of which is hereby incorporated by 
reference. 
Prokaryotic expression hosts may be used for expression of fusion protein 
that do not require extensive proteolytic and disulfide processing. 
Prokaryotic expression vectors generally comprise one or more phenotypic 
selectable markers, for example a gene encoding proteins conferring 
antibiotic resistance or supplying an autotrophic requirement, and an 
origin of replication recognized by the host to ensure amplification 
within the host. Suitable prokaryotic hosts for transformation include E. 
coli, Bacillus subtilis, Salmonella typhimurium, and various species 
within the genera Pseudomonas, Streptomyces, and Staphyolococcus, although 
others may also be employed as a matter of choice. 
Useful expression vectors for bacterial use can comprise a selectable 
marker and bacterial origin of replication derived from commercially 
available plasmids comprising genetic elements of the well-known cloning 
vector pBR322 (ATCC 37017). Such commercial vectors include, for example, 
pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega 
Biotec, Madison, Wis., USA). These pBR322 "backbone" sections are combined 
with an appropriate promoter and the structural sequence to be expressed. 
E. coli is typically transformed using derivatives of pBR322, a plasmid 
derived from an E. coli species (Bolivar et al., Gene 2:95, 1977). pBR322 
contains genes for ampicillin and tetracycline resistance and thus 
provides simple means for identifying transformed cells. 
Promoters commonly used in recombinant microbial expression vectors include 
the .beta.-lactamase (penicillinase) and lactose promoter system (Chang et 
al., Nature 275:6 15, 1978; and Goeddel et al., Nature 281:544, 1979), the 
tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 
1980; and EPA 36,776) and tac promoter (Maniatis, Molecular Cloning: A 
Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A 
particularly useful bacterial expression system employs the phage 
.lambda.P.sub.L promoter and cI857ts thermoinducible repressor. Plasmid 
vectors available from the American Type Culture Collection which 
incorporate derivatives of the .lambda.P.sub.L promoter include plasmid 
pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident 
in E. coli RR1 (ATCC 53082). 
Recombinant fusion proteins may also be expressed in yeast hosts, 
preferably from the Saccharomyces species, such as S. cerevisiae. Yeast of 
other genera such as Pichia or Kluyveromyces may also be employed. Yeast 
vectors will generally contain an origin of replication from the 2.mu. 
yeast plasmid or an autonomously replicating sequence (ARS), promoter, DNA 
encoding the fusion protein, sequences for polyadenylation and 
transcription termination and a selection gene. Preferably, yeast vectors 
will include an origin of replication and selectable marker permitting 
transformation of both yeast and E. coli, e.g., the ampicillin resistance 
gene of E. coli and S. cerevisiae trp 1 gene, which provides a selection 
marker for a mutant strain of yeast lacking the ability to grow in 
tryptophan, and a promoter derived from a highly expressed yeast gene to 
induce transcription of a structural sequence downstream. The presence of 
the trp 1 lesion in the yeast host cell genome then provides an effective 
environment for detecting transformation by growth in the absence of 
tryptophan. 
Suitable promoter sequences in yeast vectors include the promoters for 
metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. 
Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. 
Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such 
as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate 
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, 
phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters 
for use in yeast expression are further described in R. Hitzeman et al., 
EP-A-0 073 657. 
Preferred yeast vectors can be assembled using DNA sequences from pBR322 
for selection and replication in E. coli (Amp.sup.r gene and origin of 
replication) and yeast DNA sequences including a glucose-repressible ADH2 
promoter and .alpha.-factor secretion leader. The ADH2 promoter has been 
described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et 
al. (Nature 300:724, 1982). The yeast .alpha.-factor leader, which directs 
secretion of heterologous proteins, can be inserted between the promoter 
and the structural gene to be expressed. See, e.g., Kurjan et al., Cell 
30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984. 
The leader sequence may be modified to contain, near its 3' end, one or 
more useful restriction sites to facilitate fusion of the leader sequence 
to foreign genes. Exemplary yeast expression vectors are PIXY521 and 
PIXY523, described in Examples 1 and 2 below. 
Suitable yeast transformation protocols are known to those of skill in the 
art; an exemplary technique is described by Hinnen et al., Proc. Natl. 
Acad. Sci. USA 75: 1929, 1978, selecting for Trp.sup.+ transformants in a 
selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino 
acids, 2% glucose, 10 .mu.g/ml adenine and 20 .mu.g/ml uracil. 
Host strains transformed by vectors comprising the ADH2 promoter may be 
grown for expression in a rich nutrient medium consisting of 1% yeast 
extract, 2% peptone, and 1% glucose supplemented with 80 .mu.g/ml adenine 
and 80 .mu.g/ml uracil. Derepression of the ADH2 promoter occurs upon 
exhaustion of medium glucose. Crude yeast supernatants are collected by 
filtration or centrifugation and held at 4.degree. C. prior to further 
purification. In the most preferred embodiments of the present invention, 
the yeast host cells are cultured in a high cell density fermentation 
process in which the nutrient medium is added at a continuous rate during 
fermentation to permit high cell density growth. An exemplary high cell 
density fermentation process is described below in Example 3. 
Various mammalian or insect cell culture systems can be employed to express 
recombinant protein. Baculovirus systems for production of heterologous 
proteins in insect cells are reviewed by Luckow and Summers, 
Bio/Technology 6:47 (1988). Examples of suitable mammalian host cell lines 
include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 
23:175, 1981), and other cell lines capable of expressing an appropriate 
vector including, for example, L cells, C127, 3T3, Chinese hamster ovary 
(CHO), HeLa and BHK cell lines. Mammalian expression vectors may comprise 
non-transcribed elements such as an origin of replication, a suitable 
promoter and enhancer linked to the gene to be expressed, and other 5' or 
3' flanking nontranscribed sequences, and 5' or 3' nontranslated 
sequences, such as necessary ribosome binding sites, a poly-adenylation 
site, splice donor and acceptor sites, and transcriptional termination 
sequences. 
The transcriptional and translational control sequences in expression 
vectors to be used in transforming vertebrate cells may be provided by 
vital sources. For example, commonly used promoters and enhancers are 
derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human 
cytomegalovirus. DNA sequences derived from the SV40 viral genome, for 
example, SV40 origin, early and late promoter, enhancer, splice, and 
polyadenylation sites may be used to provide the other genetic elements 
required for expression of a heterologous DNA sequence. The early and late 
promoters are particularly useful because both are obtained easily from 
the virus as a fragment which also contains the SV40 viral origin of 
replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 
fragments may also be used, provided the approximately 250 bp sequence 
extending from the Hind III site toward the BglI site located in the viral 
origin of replication is included. Exemplary vectors can be constructed as 
disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). 
A useful system for stable high level expression of mammalian receptor 
cDNAs in C127 murine mammary epithelial cells can be constructed 
substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). 
Particularly preferred eukaryotic vectors for expression of MGF/IL-3 DNA 
include pIXY521 and pIXY523, both of which are yeast expression vectors 
derived from pBC102.K22 (ATCC 67,255) and contain DNA sequences from 
pBR322 for selection and replication in E. coli (Apr gene and origin of 
replication) and yeast, as described below in Examples 1 and 2. 
Purified mammalian fusion proteins or analogs are prepared by culturing 
suitable host/vector systems to express the recombinant translation 
products of the DNAs of the present invention, which are then purified 
from culture media or cell extracts. 
For example, supernatants from systems which secrete recombinant protein 
into culture media can be first concentrated using a commercially 
available protein concentration filter, for example, an Amicon or 
Millipore Pellicon ultrafiltration unit. Following the concentration step, 
the concentrate can be applied to a suitable purification matrix. For 
example, a suitable affinity matrix can comprise a MGF or IL-3 receptor or 
leetin or antibody molecule bound to a suitable support. Alternatively, an 
anion exchange resin can be employed, for example, a matrix or substrate 
having pendant diethylaminoethyl (DEAE) groups. The matrices can be 
acrylamide, agarose, dextran, cellulose or other types commonly employed 
in protein purification. Alternatively, a cation exchange step can be 
employed. Suitable cation exchangers include various insoluble matrices 
comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are 
preferred. 
Finally, one or more reversed-phase high performance liquid chromatography 
(RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel 
having pendant methyl or other aliphatic groups, can be employed to 
further purify a fusion protein composition. Some or all of the foregoing 
purification steps, in various combinations, can also be employed to 
provide a homogeneous recombinant protein. 
Recombinant protein produced in bacterial culture is usually isolated by 
initial extraction from cell pellets, followed by one or more 
concentration, salting-out, aqueous ion exchange or size exclusion 
chromatography steps. Finally, high performance liquid chromatography 
(HPLC) can be employed for final purification steps. Microbial cells 
employed in expression of recombinant fusion proteins can be disrupted by 
any convenient method, including freeze-thaw cycling, sonication, 
mechanical disruption, or use of cell lysing agents. 
Fermentation of yeast which express fusion proteins as a secreted protein 
greatly simplifies purification. Secreted recombinant protein resulting 
from a large-scale fermentation can be purified by methods analogous to 
those disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). This 
reference describes two sequential, reversed-phase HPLC steps for 
purification of recombinant murine GM-CSF on a preparative HPLC column. 
Fusion protein synthesized in recombinant culture is characterized by the 
presence of non-human cell components, including proteins, in amounts and 
of a character which depend upon the purification steps taken to recover 
the fusion protein from the culture. These components ordinarily will be 
of yeast, prokaryotic or non-human higher eukaryotic origin and preferably 
are present in innocuous contaminant quantities, on the order of less than 
about 5 percent by scanning densitometry or chromatography. Further, 
recombinant cell culture enables the production of the fusion protein free 
of proteins which may be normally associated with MGF or IL-3 as they are 
found in nature in their respective species of origin, e.g., in cells, 
cell exudates or body fluids. 
Fusion protein compositions are prepared for administration by mixing 
fusion protein having the desired degree of purity with physiologically 
acceptable carders. Such careers will be nontoxic to recipients at the 
dosages and concentrations employed. Ordinarily, the preparation of such 
compositions entails combining the fusion protein with buffers, 
antioxidants such as ascorbic acid, low molecular weight (less than about 
ten residues) polypeptides, proteins, amino acids, carbohydrates including 
glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione 
and other stabilizers and excipients. 
Fusion protein compositions may be used to enhance proliferation, 
differentiation and functional activation of hematopoietic progenitor 
cells, such as bone marrow cells. Specifically, compositions containing 
the fusion protein may be used to increase peripheral blood leukocyte 
numbers and increase circulating granulocyte counts in myelosuppressed 
patients. To achieve this result, a therapeutically effective quantity of 
a fusion protein composition is administered to a mammal, preferably a 
human, in association with a pharmaceutical carder or diluent. 
The following examples are offered by way of illustration, and not by way 
of limitation. 
EXAMPLES 
Example 1 
Synthesis of Expression Vectors Encoding an MGF/IL-3 Fusion Protein 
A. Construction of PIXY321 Intermediate Plasmid. Peripheral blood 
lymphocytes were isolated from buffy coats prepared from whole blood 
(Portland Red Cross, Portland, Oreg., USA) by Ficoll hypaque density 
centrifugation. T cells were isolated by rosetting with 
2-amino-ethylthiouronium bromide-treated sheep red blood cells. Cells were 
cultured in 175 cm.sup.2 flasks at 5.times.10.sup.6 cells/ml for 18 hour 
in 100 ml RPMI, 10% fetal calf serum, 50 .mu.M b-mercaptoethanol, 1% 
phytohemagglutinin (PHA) and 10 ng/ml phorbol 12-myristate 13-acetate 
(PMA). RNA was extracted by the guanidinium CsCl method and poly A.sup.+ 
RNA prepared by oligo-dT cellulose chromatography (Maniatis et al., 
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982). cDNA 
was prepared from poly A.sup.+ RNA essentially as described by Gubler and 
Hoffman, Gene 25:263-269 (1983). The cDNA was rendered double-stranded 
using DNA polymerase I, blunt-ended with T4 DNA polymerase, methylated 
with EcoR1 methylase to protect EcoR1 cleavage sites within the cDNA, and 
ligated to EcoR1 linkers. These constructs were digested with EcoR1 to 
remove all but one copy of the linkers at each end of the cDNA, ligated to 
EcoR 1-cut and dephosphorylated arms of phage .lambda.gt10 (Huynh et al., 
DNA Cloning: A Practical Approach, Glover, ed., IRL Press, pp. 49-78) and 
packaged into .lambda. phage extracts (Stratagene, San Diego, Calif., USA) 
according to the manufacturer's instructions. 500,000 recombinants were 
plated on E. coli strain C600hfl.sup.- and screened by standard plaque 
hybridization techniques using the following probes. 
Two oligonucleotides were synthesized, with sequences complementary to 
selected 5' and 3' sequences of the huIL-3 gene. The 5' probe, 
complementary to a sequence encoding part of the huIL-3 leader, had the 
sequence 5'-GAGTTGGAGCAGGAGCAGGAC-3'. The 3' probe, corresponding to a 
region encoding amino acids 123-130 of the mature protein, had the 
sequence 5'-GATCGCGAGGCTCAAAGTCGT-3'. The method of synthesis was a 
standard automated triester method substantially similar to that disclosed 
by Sood et at., Nucl. Acids Res. 4:2557 (1977) and Hirose et al., Tet. 
Lett. 28:2449 (1978). Following synthesis, oligonucleotides were deblocked 
and purified by preparative gel electrophoresis. For use as screening 
probes, the oligonucleotides were terminally radiolabeled with .sup.32 
P-ATP and T4 polynucleotide kinase using techniques similar to those 
disclosed by Maniatis et al. The E. coli strain used for library screening 
was C600hfl.sup.- (Huynh et al., 1985, supra). 
Thirteen positive plaques were purified and re-probed separately with the 
two hybridization probes. Eleven clones hybridized to both 
oligonucleotides. The cDNA inserts from several positive recombinant phage 
were subcloned into an EcoR1-cut derivative (pGEMBL18) of the standard 
cloning vector pBR322 containing a polylinker having a unique EcoR1 site, 
a BamH1 site and numerous other unique restriction sites. An exemplary 
vector of this type, pGEMBL, is described by Dente et al., Nucl. Acids 
Res. 11:1645 (1983), in which the promoters for SP6 and T7 polymerases 
flank the multiple cloning sites. The nucleotide sequences of selected 
clones were determined by the chain termination method. Specifically, 
partial EcoR1 digestion of .lambda.GT10:IL-3 clones 2, 3, 4 and 5 yielded 
fragments ranging from 850 bp to 1,000 bp in size which were separately 
subcloned into the EcoR1 site of pGEMBL18. The inserts of the 
pGEMBL:rhuIL-3 subclones were sequenced using a universal primer that 
binds adjacent to the multiple cloning site of pGEMBL18, and synthetic 
oligonucleotide primers derived from the huIL-3 sequence. 
The two asparagine-linked glycosylation sites present in the natural 
protein (Asn.sup.15 and Asn.sup.70) were altered by changing the codons at 
these positions to ones that encode aspartic acid. This prevents N-linked 
glycosylation (often hyperglycosylation) of the secreted protein by the 
yeast cells, and a more homogeneous product is obtained. These changes 
were made as described below upon subcloning the huIL-3 cDNA into the 
yeast expression vector pIXY120. 
The yeast expression vector pIXY120 is substantially identical to 
pBC102-K22, described in EPA 243,153, except that the following synthetic 
oligonucleotide containing multiple cloning sites was inserted from the 
Asp718 site (amino acid 79) near the 3' end of the .alpha.-factor signal 
peptide to the Spel site contained in the 2.mu. sequences: 
##STR1## 
In addition, a 514-bp DNA fragment derived from the single-stranded 
bacteriophage f1 containing the origin of replication and intergenic 
region was inserted at the Nru1 site in the pBR322 DNA sequences. The 
presence of the f1 origin of replication enables generation of 
single-stranded copies of the vector when transformed into appropriate 
(male) strains of E. coli and superinfected with bacteriophage f.sub.1. 
This capability facilitates DNA sequencing of the vector and allows the 
possibility of in vitro mutagenesis. 
The yeast expression vector pIXY120 was digested with the restriction 
enzymes Asp718, which cleaves near the 3' end of the .alpha.-factor leader 
peptide (nucleotide 237), and BamH1, which cleaves in the polylinker. The 
large vector fragment was purified and ligated to the following DNA 
fragments: (1) a huIL-3 cDNA fragment derived from plasmid GEMBL18:huIL-3 
from the Clal site (nucleotide 58 of mature huIL-3) to the BamH1 site (3' 
to the huIL-3 cDNA in a polylinker); and (2) the following synthetic 
oligonucleotide linker A: 
##STR2## 
Oligonucleotide A regenerates the sequence encoding the C-terminus of the 
.alpha.-factor leader peptide and fusing it in-frame to the octapeptide 
DYKDDDDK, which is, in turn, fused to the N-terminus of mature rhuIL-3. 
This fusion to the rhuIL-3 protein allows detection with antibody specific 
for the octapeptide and was used initially for monitoring the expression 
and purification of rhuIL-3. This oligonucleotide also encodes an amino 
acid change at position 15 (Asn.sup.15 to Asp.sup.15) to alter this 
N-linked glycosylation site. The underlined nucleotides in oligonucleotide 
A represent changes from the wild type cDNA sequence. Only the A to G and 
C to T changes at nucleotides 43 and 45, respectively (counting from the 
codon corresponding to the N-terminal alanine of the mature huIL-3 
molecule), result in an amino acid change (Asp.sup.15). The other base 
changes introduce convenient restriction sites (AhaII and PvuII) without 
altering the amino acid sequence. The resulting plasmid was designated 
pIXY 139 and contains a rhuIL-3 cDNA with one remaining N-linked 
glycosylation consensus sequence (Asn.sup.70). 
Plasmid pIXY 139 was used to perform oligonucleotide-directed mutagenesis 
to remove the second N-linked glycosylation consensus sequence by changing 
Asn.sup.70 to Asp.sup.70. The in vitro mutagenesis was conducted by a 
method similar to that described by Walder and Walder, Gene 42:133 (1986). 
The yeast vector, pIXY139, contains the origin of replication for the 
single-stranded bacteriophage fl and is capable of generating 
single-stranded DNA when present in a suitable (male) strain of E. coli 
and superinfected with helper phage. 
Single-stranded DNA was generated by transforming E. coli strain JM107 and 
superinfecting with helper phage IR1. Single-stranded DNA was isolated and 
annealed to the following mutagenic oligonucleotide B, GTC AAG AGT TTA CAG 
GAC GCA TCA GCA AAT G, which provides a codon switch substituting Asp for 
Asn at position 70 of mature huIL-3. Annealing and yeast transformation 
conditions were done as described by Walder and Walder, supra. Yeast 
transformants were selected by growth on medium lacking tryptophan, 
pooled, and DNA extracted as described by Holm et al., Gene 42:169 (1986). 
This DNA, containing a mixture of wild type and mutant plasmid DNA, was 
used to transform E. coli RR1 to ampicillin resistance. The resulting 
colonies were screened by hybridization to radiolabeled oligonucleotide B 
using standard techniques. Plasmids comprising DNA encoding huIL-3 
Asp.sup.70 were identified by the hybridization to radiolabeled 
oligonucleotide B under stringent conditions and verified by nucleotide 
sequencing. 
The resulting yeast expression plasmid was designated pIXY138, and 
contained the huIL-3 gene encoding the Asp.sup.15 Asp.sup.70 amino acid 
changes and the octapeptide DYKDDDDK at the N-terminus. The final yeast 
expression plasmid is identical to pIXY138 except that it lacks the 
nucleotide sequences coding for the octapeptide, thus generating mature 
rhuIL-3 as the product. 
The final yeast expression plasmid encoding IL-3 was constructed as 
described below. The yeast expression vector pIXY120 was cleaved with the 
restriction enzymes Asp718 and BamH1 as described above. The large vector 
fragment was ligated together with (1) a huIL-3 cDNA fragment derived from 
plasmid pIXY138 that extended from the Aha2 site (which cleaves at 
nucleotide 19 of mature huIL-3) to the BamH1 site 3' to the cDNA, and (2) 
the following synthetic oligonucleotide C: 
##STR3## 
Oligonucleotide C regenerates the 3' end of the a-factor leader peptide 
from the Asp718 site (the amino acids Pro-Leu-Asp-Lys-Arg) and the 
N-terminal seven amino acids of huIL-3 to the AhaII site. The resulting 
plasmid was designated pIXY151. This vector, when present in yeast, allows 
glucose-regulated expression and secretion of rhuIL-3 (Pro.sup.8 
Asp.sup.15 Asp.sup.70). 
The plasmid PIXY321 (encoding a GM-CSF/IL-3 fusion protein) was constructed 
as follows and used as an intermediate in constructing the MGF/IL-3 fusion 
protein. The wild-type gene coding for human GM-CSF, resident on plasmid 
priG23, has been deposited with the American Type Culture Collection 
(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, USA, under accession 
number 39900. The wild-type gene inserted in a yeast expression vector, 
pY.alpha.fHuGM, has also been deposited with the ATCC under accession 
number 53157. In order to provide a non-glycosylated analog of human 
GM-CSF, oligonucleotide-directed site-specific mutagenesis procedures were 
employed to eliminate potential N-glycosylation sites, as described in PCT 
publication WO 89/03881. A plasmid encoding this analog, huGM-CSF 
(Leu.sup.23 Asp.sup.27 Glu.sup.39), was deposited with the ATCC as plasmid 
L207-3 in E. coli strain RR1 under accession number 67231. 
DNAs encoding GM-CSF and IL-3 were first ligated together without regard to 
reading frame or intervening sequences. A cDNA fragment encoding 
nonglycosylated human GM-CSF was excised from plasmid L207-3 as a 977bp 
restriction fragment (Sph1 to Ssp1). The IL-3 cDNA was excised from 
pIXY151 by digestion with Asp718, which was then blunt ended using the T4 
polymerase reaction of Maniatas et al. (Molecular Cloning: A Laboratory 
Manual, Cold Spring Harbor, 1982, p. 118) and further digested with Xhol 
giving an 803 bp fragment. These two fragments were then directly ligated 
to a pIXY151 vector fragment cut with Sph1 and Xho1. This plasmid was 
called GM/IL-3 direct fusion. 
The GM/IL-3 direct fusion plasmid was used as a template in 
oligonucleotide-directed mutagenesis using methods similar to those 
described by Walder and Walder, supra. The following oligonucleotide was 
then synthesized 
##STR4## 
This oligonucleotide overlaps the 3' end of GM-CSF by 13 bp but does not 
include the stop codon, contains the Gly Ser linker, and overlaps the 5' 
end of IL-3 by 13 bp. The linker sequence was a modified version of the 
linker described by Huston et al. (Proc. Natl. Acad. Sci. USA 
85:5879-5883, 1988) but was optimized for codon usage in yeast as per 
Bennetzen et al. (J. Biol. Chem. 257:3026, 1982). 
Single stranded plasmid DNA was made from the GM/IL-3 direct fusion using 
R408 helper phage (Stratagene) and the methods of Russel et al. (Gene 
45:333-338, 1986). Oligonucleotide directed mutagenesis was then carried 
out by annealing the above oligonucleotide to the single stranded plasmid 
DNA and transforming yeast strain XV2181 with annealed DNA as described by 
Walder and Walder, supra. The yeast vector contains the origin of 
replication for the single stranded bacteriophage f.sub.1 and is capable 
of sponsoring single stranded DNA production when present in a suitable 
(male) strain of E. coli and superinfected with helper phage. Yeast 
transformants were selected by growth on medium lacking tryptophan, 
pooled, and DNA was extracted as described by Holm et at. (Gene 42:169, 
1986). This DNA, containing a mixture of mutant and wild type plasmid DNA, 
was used to transform E. coli RR1 to ampicillin resistance. The resulting 
colonies were screened by hybridization to radiolabeled oligonucleotide 
using standard techniques. Plasmids comprising DNA encoding 
GM-CSF/linker/IL-3 were identified by their hybridization to radiolabeled 
oligonucleotide containing the linker under stringent conditions and 
verified by nucleotide sequencing. 
During nucleotide sequencing it was discovered that a mutation had occurred 
within the linker region. The nucleotide sequence TGGTGGATCTGG was deleted 
(see sequence), resulting in the expression of a protein in which the 
sequence of amino acids GlyGlySerGly were deleted. This mutation did not 
change the reading frame or prevent expression of a biologically active 
protein. The resulting plasmid was designated pIXY321 and expressed the 
fusion protein huGM-CSF[Leu.sup.23 Asp.sup.27 Glu.sup.39 ]/Gly.sub.4 
SerGly.sub.5 Ser/huIL-3[Pro.sup.8 Asp.sup.15 Asp.sup.70 ]. This plasmid 
was use as described below as an intermediate plasmid in constructing 
fusion proteins comprising MGF and IL-3. 
B. Construction of Vector Encoding MGF. A yeast expression vector encoding 
MGF was constructed by inserting the huMGF-2D cDNA sequence into the 
pIXY-120 vector, described above. The huMGF-2D cDNA also contains the ADH2 
promoter sequence, an factor leader sequence, and a sequence encoding a 
FLAG.RTM. identification peptide (AspLysArgAspAspAspAspLys) contiguous and 
in reading frame with MGF to facilitate purification of the expressed 
protein. The resulting vector was designated pIXY490. 
C. Construction of Vector Encoding Fusion Protein Comprising MGF and IL-3. 
A vector encoding a fusion protein comprising MGF and IL-3 was constructed 
by combining four cDNA fragments containing regulatory sequences for yeast 
expression, and DNA sequences encoding a FLAG.RTM. identification peptide, 
MGF and IL-3. The first cDNA fragment contained the plasmid backbone and a 
portion of the ADH2 promoter and was excised from PIXY490 as a SpiH1 to 
BamH1 fragment. 
The second fragment contained the remaining portion of the ADH2 promoter, 
the .alpha.-factor leader, sequences encoding the FLAG.RTM. identification 
peptide (GACTACAAGGACGAC GATGACAAG) and the 5' end of the extracellular 
region of human MGF and was excised as a SpHI to EcoRI restfiction 
fragment of pIXY490, described above. 
The third fragment contained sequences encoding the Y end of the 
extracellular region of human MGF. The linker sequence joining the MGF 
coding region and the IL-3 coding region was generated by polymerase chain 
reaction (PCR) using the following 5' PCR oligonucleotide primer which 
corresponds to the coding region of MGF (nucleotides 334-357 of SEQ ID 
NO:3) and includes an EcoRI restriction site: 
##STR5## 
and the following 3' PCR oligonucleotide antisense primer of which a 
portion is complementary to the 3' end of MGF (nucleotides 460-471 of SEQ 
ID NO:3) and introduces part of a linker sequence (nucleotides 472-492 of 
SEQ ID NO:3) containing SfiI and BamHI restriction sites contiguous to the 
3' end of MGF: 
##STR6## 
Following synthesis, the oligonucleotides were deblocked and purified by 
preparative gel electrophoresis. For use as screening probes, the 
oligonucleotides were kinased with T4 polynucleotide kinase using 
techniques similar to those disclosed by Maniatis et al. A cDNA sequence 
encoding human MGF-2D which encodes the extracellular region of .lambda.28 
human MGF (amino acids 1-157, described in EP-A-0 423 980, FIG. 44) was 
subcloned into a pBluescript SK(-) cloning vector (Stratagene, La Jolla, 
Calif.). Using the resulting pBluescript:huMGF2-D plasmid as a template, 
the above primers were used to amplify the 3' end of human MGF and to add 
the linker sequence joining human MGF and IL-3. Fifty pM of each primer 
and 50 ng template were combined in a reaction buffer containing 29.75 
.mu.l water, 5 .mu.l 10x standard PCR buffer (500 mM KCl, 100 mM Tris-C1 
(pH 8.3), 15 mM MgCl.sub.2, 0.1% (w/v) gelatin), 5 .mu.l 10X low magnesium 
buffer (500 mM KCl, 100 mM Tris-Cl (pH8.3), 5 mM MgCl.sub.2, 0.1% gelatin) 
(for control) or 5 .mu.l 10X low potassium buffer (50 mM KCl, 100 mM 
Tris-Cl, 15 mM MgCl.sub.2, 0.1% gelatin), 8 .mu.l 1.25 mM dNTPs, and 0.25 
ml Taq polymerase. Reactions were performed on an Ericomp TwinBlock.RTM. 
temperature cycler (Ericomp, San Diego, Calif.) for 6 cycles of 94.degree. 
C. for 30 seconds, 37.degree. C. for 45 seconds and 72.degree. C. for 30 
seconds, followed by approximately 25 cycles of 94.degree. C. for 30 
seconds, 60.degree. C. for 45 seconds and 72.degree. C. for 30 seconds and 
an additional 5 minutes of extension at 72.degree. C. Agarose gel 
electrophoresis of the products of PCR amplification showed a band 
corresponding to the 3' end of human MGF. 
The fourth fragment contained sequences encoding IL-3 and part of the 
linker sequence and was excised as a BamHI restriction fragment from 
pIXY321, described above. This fragment was treated with calf intestinal 
alkaline phosphatase (Boehringer Mannheim) to remove 5' phosphates. 
The four cDNA fragments described above were combined in a four-way 
ligation to generate the yeast expression vector pIXY523, which contains 
sequences encoding an N-terminal FLAG.RTM. identification peptide linked 
to a fusion protein comprising MGF and IL-3. The sequence of the MGF/IL-3 
fusion protein (the N-terminal FLAG.RTM. identification peptide is not 
shown) is set forth SEQ ID NO:3. 
Example 2 
Construction of IL-3/MGF Fusion Protein 
A fusion protein comprising IL-3 followed by MGF was constructed by 
combining fragments containing regulatory sequences for yeast expression 
and coding sequences for IL-3 and MGF. The first cDNA fragment was 
obtained by excising a Pst1 to SnaB1 restriction fragment from pIXY490. 
This fragment contained most of the 5' end of the extracellular region of 
human MGF. 
The second fragment was obtained by excising a SnaB1 to BamH1 restriction 
fragment from the plasmid PIXY344 and contained sequences encoding IL-3 
and a portion of the linker sequence. The intermediate plasmid PIXY344 
encodes an IL-3/GM-CSF fusion protein was constructed as follows. The 
yeast expression vector pIXY120 (described in Example 1, above) was 
digested with the restriction enzymes Asp718, which cleaves near the 3' 
end of the .alpha.-factor leader peptide (nucleotide 237), and Nco1, which 
cleaves in the polylinker. The large vector fragment was purified and 
ligated to an approximately 500bp Asp718-Nco1 fragment (encoding 
GM-CSF(Leu.sup.23 Asp.sup.27 Glu.sup.39)) from a partial digest of L207-3 
(ATCC 67231), to yield pIXY273. A 9kb Asp718-Bg12 fragment of piXY273 
(still containing the GM-CSF(Leu.sup.23 Asp.sup.27 Glu.sup.39)cDNA) was 
then ligated to an Asp718-Nru1 fragment encoding human IL-3 (Pro.sup.8 
Asp.sup.15 Asp.sup.70) from pIXY151 (described in Example 1B) and the 
following double stranded oligonucloetide: 
##STR7## 
This oligonucleotide overlaps the 3' end of IL-3 by 8bp but does not 
include the stop codon, contains the Gly-Ser linker, and overlaps the 5' 
end of GM-CSF by 10bp. The resulting vector was designated pIXY344 and 
contains sequences encoding an N-terminal IL-3 and a C-terminal GM-CSF. 
The third fragment comprised the linker sequence and was generated by 
synthesizing, kinasing and annealing the following two oligonucleotides: 
##STR8## 
The three cDNA fragments described above were combined in a three-way 
ligation to generate the yeast expression vector pIXY521, which encodes a 
fusion protein comprising IL-3 and MGF. The sequence of this IL-3/MGF 
fusion protein is set forth SEQ ID NO:1. 
Example 3 
Expression and Purification of MGF/IL-3 Fusion Protein 
The host strain, YNN281, a haploid S. cerevisiae strain [a, trpl-.DELTA., 
his3-.DELTA.200, ura 3-52, lys 2-801.sub.a, ade 2-1.sub.o ] was obtained 
from the Yeast Genetic Stock Center, University of California, Berkeley, 
Calif., USA. The host strain was transformed with the expression plasmid 
by the method of Sherman et al., Laboratory Course Manual for Methods in 
Yeast Genetics, Cold Spring Harbor Laboratory, 1986. 
Yeast containing the expression plasmid PIXY523 (encoding the MGF/IL-3 
fusion protein) was maintained on YNB-trp agar plates stored at 4.degree. 
C. A preculture was started by inoculating several isolated recombinant 
yeast colonies into 50 ml of YNB-trp growth medium (Difco Laboratories, 
Detroit, Mich.) (6.7 g/L Yeast Nitrogen Base, 5 g/L casamino acids 
(Hy-Case SF.RTM.), 40 mg/L adenine, 160 mg/L uracil, and 200 mg/L 
tyrosine) and was grown for 21 hours in a shake flask at 30.degree. C. 
with vigorous shaking. By morning the culture was saturated, in stationary 
phase, at an OD.sub.550 of 1.2. 
The following reagents were prepared to the following concentrations: 
glucose (500 g/L), Hy-Case.RTM. SF (Sheffield Products, Norwich, N.Y.) (60 
g/L), yeast extract (Difco Laboratories, Detroit, Mich.) (200 g/L), 
peptone (Difco Laboratories, Detroit, Mich.) (200 g/L), yeast feed salts 
(250 g/L ammonium sulfate, 125 g/L monobasic potassium phosphate, 
anhydrous, 28.5 g/L magnesium sulfate), ethanol (95%), vitamins (0.02 g/L 
biotin, 2 g/L calcium pantothenate, 25 g/L myo-inositol, 5 g/L niacin, 0.4 
g/L pyridoxine HCl, 0.1 g/L folic acid, 0.5 g/L choline chloride), trace 
elements (5 g/L boric acid, 2 g/L cupric sulfate, 10 g/L ferric chloride, 
10 g/L manganese sulfate, 0.5 g/L sodium molybdate, 10 g/L, zinc sulfate, 
0.5 g/L cobalt chloride), adenine (10 g/L), thiamine (10 g/L), uracil (10 
g/L), histadine (10 g/L), lysine (15 g/L). 
The growth medium for a 1 liter fermentation tank was prepared by combining 
5.0 g potassium phosphate, 20.0 g ammonium sulfate, 1.0 g magnesium 
sulfate, 0.1 g calcium chloride, 0.2 ml L61 antifoam and water to a total 
volume of 650 ml. This medium was then sterilized by autoclaving in a 
fermentation tank at 121.degree. C. for 30 min. and allowed to cool to 
room temperature. Following sterilization, a nutrient feed was prepared by 
combining the following nutrients (prepared as described above): 10 ml 
glucose, 83.3 ml Hycase.RTM. SF, 3.5 ml thiamine HCl, 2.5 ml vitamins, 2.5 
ml trace elements, 25 ml adenine, 15 ml uracil, 12.5 ml yeast extract, and 
12.5 ml peptone. The yeast seed was then added to the fermentation tank 
and cultured for a period of 5 hours. The yeast was then cultured at a 
temperature of 30.degree. C. while a nutrient feed comprising 180 ml 
glucose, 75 ml Hycase.RTM. SF, 22.5 ml yeast extract, 22.5 ml peptone, 
31.25 ml yeast feed salts, 22.5 ml ethanol, 1.25 ml vitamins, 1.25 ml 
trace elements, 25 ml adenine, 3.0 ml thiamine, 10 ml uracil, 10 ml 
hisadine and 10 ml lysine (prepared as described above) was then added 
continuously at a rate of 0.11 ml/min for 20 hours, followed by a rate of 
0.2 ml/min for an additional 24 hours. The final production medium 
composition was as follows: 
The resulting yeast broth was centrifuged at 9,000 rpm for 10 minutes. The 
centrifuged supernatant was filtered through a 0.45 g filter and the 
clarified yeast broth was collected. 
The MGF/IL-3 fusion protein was purified to homogeneity from the clarified 
yeast broth on a FLAG.RTM.affinity column as instructed by the 
manufacturer (International Biotechnologies, Inc., New Haven, Conn.). 
Briefly, a FLAG.RTM. affinity column was prepared and equilibrated with 1 
mM CaCl.sub.2, 0.1M HEPES, pH 7.5. The clarified crude yeast broth was 
pumped over the column, followed by a wash with equilibration buffer. The 
FLAG-MGF/IL-3 fusion protein was eluted from the affinity column with 0.1M 
sodium titrate, pH 3.0. 
Example 4 
Activity of MGF/IL-3 Fusion Proteins in Human Bone Marrow Colony Assay 
The ability of MGF, IL-3 and fusion proteins comprising MGF and IL-3 to 
induce colony formation were compared in the following assays. In the 
first assay, granulocyte-macrophage colony- and cluster-forming cells 
(CFU-GM) were assayed by a procedure substantially similar to that 
described in Williams et al. Exp. Hematol. 15:243 (1987). Briefly, bone 
marrow cells were suspended in 0.5 ml of 0.3% agar (Difco, Detroit Mich.) 
or 0.4% agarose (FMC, Rockland, Me.) culture medium containing McCoy's 5A 
medium supplemented with essential and nonessential amino acids, 
glutamine, serine, asparagine, and sodium pyruvate (Gibco) with 20% fetal 
bovine serum. Quadruplicate cultures were incubated for seven days in a 
fully humidified atmosphere of 5% CO.sub.2 in air. Colonies (&gt;50 cells) 
and clusters (3-49 cells) were counted with an inverted microscope at 
32.times.. 
In the second assay, erythroid burst-forming units (BFU-E) and 
multipotential colony-forming cells (CFU-GEMM) were assayed by a procedure 
described in Williams et al., supra. Briefly, duplicate 35.times.10 mm 
cultures were stimulated with 2 units per ml of recombinant human 
erythropoietin (Hyclone), 0.1 mM hemin (Kodak), and 1000 U/ml IL-3 as a 
source of burst promoting activity. Cultures were incubated for seven days 
in a fully humidified atmosphere of 5% CO.sub.2, 5% O.sub.2 and 90% 
N.sub.2. Maximal colony formation by murine BFU-E and CFU-GEMM was 
observed on day 14 using the foregoing culture conditions. BFU-E and 
CFU-GEMM were scored using an inverted stage microscope at 80.times. on 
the basis of hemoglobinization of erythroid elements, producing a 
characteristic red color, and the absence or presence of myeloid elements 
for the former and latter, respectively. 
Using the foregoing assay, we compared the colony-forming activity of MGF, 
IL-3 MGF plus IL-3 and fusion proteins comprising MGF and IL-3 to control 
medium. The results are set forth in the following table: 
TABLE A 
__________________________________________________________________________ 
Colonies/culture (Mean .+-. S.D.) 
Cytokine Dose (ng/ml) 
CFU-GM 
BFU-E CFU-GEMM 
__________________________________________________________________________ 
Control Medium 0 64 .+-. 4 
0.5 .+-. 0.3 
MGF 3 0 62 .+-. 2 
0.8 .+-. 0.5 
IL-3 2 8 .+-. 1 
104 .+-. 2 
5.3 .+-. 0.6 
MGF + IL-3 
2 + 3 14 .+-. 1 
98 .+-. 4 
6 .+-. 0.5 
PIXY523 4 10 .+-. 2 
136 .+-. 4* 
14 .+-. 1* 
12 36 .+-. 2 
158 .+-. 5* 
31 .+-. 2* 
__________________________________________________________________________ 
= value equal to media control 
*p&lt;0.05 compared to media control 
These data indicate that MGF/IL-3 fusion proteins stimulate erythroid and 
primitive mixed colony formation. PIXY523 had significant BFU-E and 
CFU-GEMM stimulatory activity over controls and over MGF and IL-3 alone 
and MGF and IL-3 combined. 
Example 5 
Activity of MGF/IL-3 Fusion Proteins in Human Peripheral Blood Expansion 
Assay 
An experiment was conducted to compare the expansion ratios for human 
hematopoietic progenitor cells expanded ex vivo with progenitor expansion 
media comprising different growth factors or growth factor combinations or 
media without added growth factors. 
Human peripheral blood was obtained from normal, healthy, volunteers via 
veinipuncture and collected in a heparinized tube. Mononuclear cells were 
obtained from peripheral blood by density gradient centrifugation on 
Histopaque.RTM. (Sigma, St. Louis). The mononuclear cells, containing a 
population of human hematopoietic progenitor cells were washed twice in 
phosphate buffered saline (PBS) and viable cells counted by trypan blue 
dye exclusion. 
Ex vivo cultures were made from approximately 10.sup.7 viable cells in 10 
ml of Super McCoys medium supplemented with 20% fetal bovine serum. Cells 
were cultured and expanded in petri dishes incubated at 37.degree. C. in 
an atmosphere of 7% CO.sub.2, 8% O.sub.2, 85% air. Culture media were 
replaced on day 4 with new growth factor(s). 
Growth factors were added to media at the following concentrations: PIXY321 
(100 ng/ml), MGF (1 .mu.g/ml), IL-3 (100 ng/ml), PIXY 523 (1 .mu.g/ml). 
Progenitor cells in culture tend to be nonadherent. For each colony assay, 
50% of nonadherent cells in each culture were obtained. Cells were 
separated from media by centrifugation, washed twice and viable cells 
counted by trypan blue due exclusion. 
A CFU-GM assay (Lu et al., Exp. Hematol. 13:989, 1985) measured a myeloid 
component of the progenitor cell population. Viable cells were plated in a 
methyl cellulose cloning media (Terry Fox Labs, Vancouver, B.C.) in the 
presence of PIXY32 1 (GM-CFU). The number of myeloid colonies were counted 
and this number was divided by the number of cells plated into each well 
to determine a colony-forming capacity (CFC) incidence. CFC incidence was 
multiplied by total cell number to determine CFC number per culture. Each 
CFC number was compared to a day 0 CFC number to determine an expansion 
ratio for each progenitor expansion media tested. 
Myeloid component cell expansion was determined after 4 and 8 days of 
incubation with growth factors MGF, IL-3, PIXY321, PIXY523 and 
combinations of these growth factors. An expansion number of 1 means that 
there was no expansion of colony number, whereas an expansion number of 2 
means that the number of colonies doubled from the day 0 number. 
TABLE B 
______________________________________ 
Colony Forming Activity of Cytokines Including MGF 
CFU-GM 
Expansion Index 
Cytokine Day 4 Day 8 
______________________________________ 
Medium 0.5 0.7 
MGF 1.2 1.1 
IL-3 2.0 3.6 
PIXY321 2.2 4.8 
PIXY523 3.2 9.2 
IL-3 + MGF 2.6 4.2 
PIXY321 + MGF 4.0 7.0 
______________________________________ 
These data indicate that PIXY523 stimulates the production of 
granulocyte-macrophage colony forming cells to a significantly greater 
degree than the medium control or either MGF or IL-3 alone or in 
combination. At day 8, PIXY523 also had a greater expansion index relative 
to PIXY321 plus MGF. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 906 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: PIXY521 
(ix) FEATURE: 
(A) NAME/KEY: matpeptide 
(B) LOCATION: 1..903 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..906 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GCTCCCATGACCCAGACGACGCCCTTGAAGACCAGCTGGGTTGAT TGC48 
AlaProMetThrGlnThrThrProLeuLysThrSerTrpValAspCys 
151015 
TCTAACATGATCGATGAAATTATAACACACTTAAAGCAGCCACCT TTG96 
SerAsnMetIleAspGluIleIleThrHisLeuLysGlnProProLeu 
202530 
CCTTTGCTGGACTTCAACAACCTCAATGGGGAAGACCAAGACATTCT G144 
ProLeuLeuAspPheAsnAsnLeuAsnGlyGluAspGlnAspIleLeu 
354045 
ATGGAAAATAACCTTCGAAGGCCAAACCTGGAGGCATTCAACAGGGCT 192 
MetGluAsnAsnLeuArgArgProAsnLeuGluAlaPheAsnArgAla 
505560 
GTCAAGAGTTTACAGGACGCATCAGCAATTGAGAGCATTCTTAAAAAT240 
ValLy sSerLeuGlnAspAlaSerAlaIleGluSerIleLeuLysAsn 
65707580 
CTCCTGCCATGTCTGCCCCTGGCCACGGCCGCACCCACGCGACATCCA288 
L euLeuProCysLeuProLeuAlaThrAlaAlaProThrArgHisPro 
859095 
ATCCATATCAAGGACGGTGACTGGAATGAATTCCGGAGGAAACTGACG336 
IleHisIleLysAspGlyAspTrpAsnGluPheArgArgLysLeuThr 
100105110 
TTCTATCTGAAAACCCTTGAGAATGCGCAGGCTCAACAGACGACTTTG384 
Phe TyrLeuLysThrLeuGluAsnAlaGlnAlaGlnGlnThrThrLeu 
115120125 
AGCCTCGCGATCTTTGGTGGCGGTGGATCCGGCGGTGGCGGCGGCTCA432 
SerLeuAl aIlePheGlyGlyGlyGlySerGlyGlyGlyGlyGlySer 
130135140 
GAAGGGATCTGCAGGAATCGTGTGACTAATAACGTAAAAGACGTCACT480 
GluGlyIleCysArgA snArgValThrAsnAsnValLysAspValThr 
145150155160 
AAATTGGTGGCAAATCTTCCAAAAGACTACATGATAACCCTCAAATAT528 
LysLeuValAla AsnLeuProLysAspTyrMetIleThrLeuLysTyr 
165170175 
GTCCCCGGGATGGATGTTTTGCCAAGTCATTGTTGGATAAGCGAGATG576 
ValProGlyMet AspValLeuProSerHisCysTrpIleSerGluMet 
180185190 
GTAGTACAATTGTCAGACAGCTTGACTGATCTTCTGGACAAGTTTTCA624 
ValValGlnLeuSe rAspSerLeuThrAspLeuLeuAspLysPheSer 
195200205 
AATATTTCTGAAGGCTTGAGTAATTATTCCATCATAGACAAACTTGTG672 
AsnIleSerGluGlyLeuS erAsnTyrSerIleIleAspLysLeuVal 
210215220 
AATATAGTGGATGACCTTGTGGAGTGCGTGAAAGAAAACTCATCTAAG720 
AsnIleValAspAspLeuValGluCys ValLysGluAsnSerSerLys 
225230235240 
GATCTAAAAAAATCATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCT768 
AspLeuLysLysSerPheLysSer ProGluProArgLeuPheThrPro 
245250255 
GAAGAATTCTTTAGAATTTTTAATAGATCCATTGATGCCTTCAAGGAC816 
GluGluPhePheArgIlePheAs nArgSerIleAspAlaPheLysAsp 
260265270 
TTTGTAGTGGCATCTGAAACTAGTGATTGTGTGGTTTCTTCAACATTA864 
PheValValAlaSerGluThrSerA spCysValValSerSerThrLeu 
275280285 
AGTCCTGAGAAAGGGAAGGCCAAAAATCCCCCTGGAGACTAA906 
SerProGluLysGlyLysAlaLysAsnPro ProGlyAsp 
290295300 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 301 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
AlaProMetThrGlnThrThr ProLeuLysThrSerTrpValAspCys 
151015 
SerAsnMetIleAspGluIleIleThrHisLeuLysGlnProProLeu 
2025 30 
ProLeuLeuAspPheAsnAsnLeuAsnGlyGluAspGlnAspIleLeu 
354045 
MetGluAsnAsnLeuArgArgProAsnLeuGluAlaPheAsnArg Ala 
505560 
ValLysSerLeuGlnAspAlaSerAlaIleGluSerIleLeuLysAsn 
65707580 
LeuLeuProC ysLeuProLeuAlaThrAlaAlaProThrArgHisPro 
859095 
IleHisIleLysAspGlyAspTrpAsnGluPheArgArgLysLeuThr 
100 105110 
PheTyrLeuLysThrLeuGluAsnAlaGlnAlaGlnGlnThrThrLeu 
115120125 
SerLeuAlaIlePheGlyGlyGlyGlySerGly GlyGlyGlyGlySer 
130135140 
GluGlyIleCysArgAsnArgValThrAsnAsnValLysAspValThr 
145150155160 
LysLeuValAlaAsnLeuProLysAspTyrMetIleThrLeuLysTyr 
165170175 
ValProGlyMetAspValLeuProSerHisCysTrpIleSerGluMet 
180185190 
ValValGlnLeuSerAspSerLeuThrAspLeuLeuAspLysPheSer 
195200205 
AsnIleSerGluGlyLeuSerA snTyrSerIleIleAspLysLeuVal 
210215220 
AsnIleValAspAspLeuValGluCysValLysGluAsnSerSerLys 
225230235 240 
AspLeuLysLysSerPheLysSerProGluProArgLeuPheThrPro 
245250255 
GluGluPhePheArgIlePheAsnArgSerIleAspAlaPhe LysAsp 
260265270 
PheValValAlaSerGluThrSerAspCysValValSerSerThrLeu 
275280285 
SerProGluLy sGlyLysAlaLysAsnProProGlyAsp 
290295300 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 912 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapiens 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: PIXY523 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..912 
(ix) FEATURE: 
(A) NAME/KEY: matpeptide 
(B) LOCATION: 1..909 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GAAGG GATCTGCAGGAATCGTGTGACTAATAATGTAAAAGACGTCACT48 
GluGlyIleCysArgAsnArgValThrAsnAsnValLysAspValThr 
151015 
AAAT TGGTGGCAAATCTTCCAAAAGACTACATGATAACCCTCAAATAT96 
LysLeuValAlaAsnLeuProLysAspTyrMetIleThrLeuLysTyr 
202530 
GTCCCC GGGATGGATGTTTTGCCAAGTCATTGTTGGATAAGCGAGATG144 
ValProGlyMetAspValLeuProSerHisCysTrpIleSerGluMet 
354045 
GTAGTACAATTG TCAGACAGCTTGACTGATCTTCTGGACAAGTTTTCA192 
ValValGlnLeuSerAspSerLeuThrAspLeuLeuAspLysPheSer 
505560 
AATATTTCTGAAGGCTTGAG TAATTATTCCATCATAGACAAACTTGTG240 
AsnIleSerGluGlyLeuSerAsnTyrSerIleIleAspLysLeuVal 
65707580 
AATATAGTGGATGACC TTGTGGAGTGCGTGAAAGAAAACTCATCTAAG288 
AsnIleValAspAspLeuValGluCysValLysGluAsnSerSerLys 
859095 
GATCTAAAAAAATCA TTCAAGAGCCCAGAACCCAGGCTCTTTACTCCT336 
AspLeuLysLysSerPheLysSerProGluProArgLeuPheThrPro 
100105110 
GAAGAATTCTTTAGAATT TTTAATAGATCCATTGATGCCTTCAAGGAC384 
GluGluPhePheArgIlePheAsnArgSerIleAspAlaPheLysAsp 
115120125 
TTTGTAGTGGCATCTGAAACTAG TGATTGTGTGGTTTCTTCAACATTA432 
PheValValAlaSerGluThrSerAspCysValValSerSerThrLeu 
130135140 
AGTCCTGAGAAAGGGAAGGCCAAAAATCCCC CTGGAGACGGGGCCGGC480 
SerProGluLysGlyLysAlaLysAsnProProGlyAspGlyAlaGly 
145150155160 
GGGGCCGGATCCGGGGGTGGCGGCGGC TCAGCTCCCATGACCCAGACG528 
GlyAlaGlySerGlyGlyGlyGlyGlySerAlaProMetThrGlnThr 
165170175 
ACGCCCTTGAAGACCAGCTGGGTTGAT TGCTCTAACATGATCGATGAA576 
ThrProLeuLysThrSerTrpValAspCysSerAsnMetIleAspGlu 
180185190 
ATTATAACACACTTAAAGCAGCCACCTTT GCCTTTGCTGGACTTCAAC624 
IleIleThrHisLeuLysGlnProProLeuProLeuLeuAspPheAsn 
195200205 
AACCTCAATGGGGAAGACCAAGACATTCTGATGG AAAATAACCTTCGA672 
AsnLeuAsnGlyGluAspGlnAspIleLeuMetGluAsnAsnLeuArg 
210215220 
AGGCCAAACCTGGAGGCATTCAACAGGGCTGTCAAGAGTTTA CAGGAC720 
ArgProAsnLeuGluAlaPheAsnArgAlaValLysSerLeuGlnAsp 
225230235240 
GCATCAGCAATTGAGAGCATTCTTAAAAATCTCCTGCCA TGTCTGCCC768 
AlaSerAlaIleGluSerIleLeuLysAsnLeuLeuProCysLeuPro 
245250255 
CTGGCCACGGCCGCACCCACGCGACATCCAATCCATAT CAAGGACGGT816 
LeuAlaThrAlaAlaProThrArgHisProIleHisIleLysAspGly 
260265270 
GACTGGAATGAATTCCGGAGGAAACTGACGTTCTATCTGA AAACCCTT864 
AspTrpAsnGluPheArgArgLysLeuThrPheTyrLeuLysThrLeu 
275280285 
GAGAATGCGCAGGCTCAACAGACGACTTTGAGCCTCGCGATCTTT TGA912 
GluAsnAlaGlnAlaGlnGlnThrThrLeuSerLeuAlaIlePhe 
290295300 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 303 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GluGlyIleCysArgAsnArgValThrAsnAsnValLysAspValThr 
151015 
LysLeuValAlaAsnLeuProLysAspTyr MetIleThrLeuLysTyr 
202530 
ValProGlyMetAspValLeuProSerHisCysTrpIleSerGluMet 
354045 
ValValGlnLeuSerAspSerLeuThrAspLeuLeuAspLysPheSer 
505560 
AsnIleSerGluGlyLeuSerAsnTyrSerIleIleAspLysLeuVal 
65 707580 
AsnIleValAspAspLeuValGluCysValLysGluAsnSerSerLys 
859095 
AspLeuLysLysSerPhe LysSerProGluProArgLeuPheThrPro 
100105110 
GluGluPhePheArgIlePheAsnArgSerIleAspAlaPheLysAsp 
115120 125 
PheValValAlaSerGluThrSerAspCysValValSerSerThrLeu 
130135140 
SerProGluLysGlyLysAlaLysAsnProProGlyAspGlyAlaGly 
14 5150155160 
GlyAlaGlySerGlyGlyGlyGlyGlySerAlaProMetThrGlnThr 
165170175 
ThrProL euLysThrSerTrpValAspCysSerAsnMetIleAspGlu 
180185190 
IleIleThrHisLeuLysGlnProProLeuProLeuLeuAspPheAsn 
195 200205 
AsnLeuAsnGlyGluAspGlnAspIleLeuMetGluAsnAsnLeuArg 
210215220 
ArgProAsnLeuGluAlaPheAsnArgAlaValLysSer LeuGlnAsp 
225230235240 
AlaSerAlaIleGluSerIleLeuLysAsnLeuLeuProCysLeuPro 
245250 255 
LeuAlaThrAlaAlaProThrArgHisProIleHisIleLysAspGly 
260265270 
AspTrpAsnGluPheArgArgLysLeuThrPheTyrLeuLysThrLeu 
275280285 
GluAsnAlaGlnAlaGlnGlnThrThrLeuSerLeuAlaIlePhe 
290295300