Compositions for co-administration of interleukin-3 mutants and other cytokines and hematopoietic factors

The invention provides compositions comprising a human interleukin-3 (hIL-3) variant or mutant protein (mutein) and another colony stimulating factor, cytokine, lymphokine, interleukin, or hematopoietic growth factor. The compositions are useful for the therapeutic co-administration of the proteins, as for example in hematopoietic reconstitution. factors or IL-3 variants.

FIELD OF THE INVENTION 
The present invention relates to the coadministration or sequential 
treatment using mutants or variants of human interieukin-3 (hIL-3) and 
other colony stimulating factors (CSFs), cytokines, lymphokines, 
interleukins, hematopoietic growth factors or IL-3 variants. 
BACKGROUND OF THE INVENTION 
Colony stimulating factors (CSFs) which stimulate the differentiation 
and/or proliferation of bone marrow cells have generated much interest 
because of their therapeutic potential for restoring depressed levels of 
hematopoietic stem cell-derived cells. CSFs in both human and murine 
systems have been identified and distinguished according to their 
activities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF 
(M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and 
macrophage colonies, respectively while GM-CSF and interleukin-3 (IL-3) 
have broader activities and stimulate the formation of both macrophage, 
neutrophilic and eosinophilic granulocyte colonies. IL-3 also stimulates 
the formation of mast, megakaryocyte and pure and mixedSerythroid colonies 
(when erythropoietin is also added). 
Because of its ability to stimulate the proliferation of a number of 
different cell types and to support the growth and proliferation of 
progenitor cells, IL-3 has potential for therapeutic use in restoring 
hematopoietic cells to normal amounts in those cases where the number of 
cells has been reduced due to diseases or to tnerapeutic treatments such 
as radiation and chemotherapy. 
Interleukin-3 (IL-3) is a hema copoietic growth factor which has the 
property oL being able Lc promote the survival, growth and differentiation 
of hematopoietic cells. Among the biological properties of IL-3 are the 
ability (a) to support the growth and differentiation of progenitor cells 
committed to all, or virtually all, blood cell lineages; (b) to interact 
with early multipotential stem cells; (c) to sustain the growth of 
pluripotent precursor cells; (d) to stimulate proliferation of chronic 
myelogenous leukemia (CML) cells; (e) to stimulate proliferation of mast 
cells, eosinophils and basophils; (f) to stimulate DNA synthesis by human 
acute myelogenous leukemia (AML) cells; (g) to prime cells for production 
of leukotrienes and histamines; (h) to induce leukocyte chemotaxis; and 
(i) to induce cell surface molecules needed for leukocyte adhesion. 
Mature human interleukin-3 (hIL-3) consists of 133 amino acids. It has one 
disulfide bridge and two potential glycosylation sites (Yang, et al., CELL 
47:3 (1986)). 
Murine IL-3 (mIL-3) was first identified by Ihle, et al., J. IMMUNOL. 
126:2184 (1981) as a factor which induced expression of a T cell 
associated enzyme, 20-hydroxsteroid dehydrogenase. The factor was purified 
to homogeneity and shown to regulate the growth and differentiation of 
numerous subclasses of early hematopoietic and lymphoid progenitor cells. 
In 1984, CDNA clones coding for murine IL-3 were isolated (Fung, et al., 
NATURE 307:233 (1984) and Yokota, et al., PROC. NATL. ACAD. SCI. USA 
81:1070 (1984)). The murine DNA sequence coded for a polypeptide of 166 
amino acids including a putative signal peptide. 
The gibbon IL-3 sequence was obtained using a gibbon CDNA expression 
library. The gibbon IL-3 sequence was then used as a probe against a human 
genomic librar s to obtain a human IL-3 sequence. 
Gibbon and human genomic DNA homologues of the murine IL-3 sequence were 
disclosed by Yang, et al., CELL 47:3 (1986). The human sequence reported 
by Yang, et al. included a serine residue at position 8 of the mature 
protein sequence. Following this finding, others reported isolation of 
Pro.sup.8 hIL-3 cDNAs having proline at position 8 of the protein 
sequence. Thus it appears that there may be two ailelic forms of hIL-3. 
Dorssers, et al., GENE 55:115 (1987), found a clone from a human cDNA 
library which hybridized with mIL-3. This hybridization was the result of 
the high degree of homology between the 3' noncoding regions of mIL-3 and 
hIL-3. This cDNA coded for an hIL-3 (Pro.sup.8) sequence. 
U.S. Pat. No. 4,877,729 and U.S. Pat. No. 4,959,454 disclose human IL-3 and 
gibbon IL-3 cDNAs and the protein sequences for which they code. The hIL-3 
disclosed has serine rather than proline at position 8 in the protein 
sequence. 
Clark-Lewis, et al., SCIENCE 231:134 (1986) performed a functional analysis 
of murine IL-3 analogues synthesized with an automated peptide 
synthesizer. The authors concluded that the stable tertiary structure of 
the complete molecule was required for full activity. A study on the role 
of the disulfide bridges showed that replacement of all four cysteines by 
alanine gave a molecule with 1/500th the activity as the native molecule. 
Replacement of two of the four Cys residues by Ala(Cys.sup.79, 
Cysl.sup.140 .fwdarw.Ala.sup.79, Ala.sup.140) resulted in an increased 
activity. The authors concluded that in murine IL-3 a single disulfide 
bridge is required between cysteines 17 and 80 to get biological activity 
that approximates physiological levels and that this structure probably 
stabilizes the tertiary structure of the protein to give a conformation 
that is optimal for function. (Clark-Lewis, et al., PROC. I TATL. aCAD. 
SCI. USA 85:7897 (1988) 
International Patent Application (PCT) WO 88/00598 discloses gibbon- and 
human-like IL-3. The hIL-3 contains a Ser.sup.8 .fwdarw.Pro.sup.8 
replacement. Suggestions are ade to replace Cys by Ser, thereby breaking 
the disulfide bridge, and to replace one or more amino acids at the 
glycosylation sires. 
EP-A-0275598 (WO 88/04691) illustrates that Alal can be deleted while 
retaining biological activity. Some mutant hIL-3 sequences are provided, 
e.g., two double mutants,Ala.sup.1 .fwdarw.Asp.sup.1, Trp.sup.13 
.fwdarw.Argl.sup.13 (pGB/IL-302) and Ala.sup.1 .fwdarw.Asp.sup.1, Met 
.sup.3 .fwdarw.Thr.sup.3 (pGB/IL-304) and one triple mutant Ala.sup.1 
.fwdarw.Asp.sup.1, Leu.sup.9 .fwdarw.Pro.sup.9, Trp.sup.13 
.fwdarw.Arg.sup.13 (pGB/IL-303). 
Wo 88/05469 describes how deglycosylation mutants can be obtained and 
suggests mutants of Arg.sup.54 Arg.sup.55 and Arg.sup.108 Arg.sup.109 
Lys.sup.110 might avoid proteolysis upon expression in Saccharomvces 
cerevisiae by KEX2 protease. No mutated proteins are disclosed. 
Glycosylation and the KEX2 protease activity are only important, in this 
context, upon expression in yeast. 
Wo 88/06161 mentions various mutants which theoretically may be 
conformationally and antigenically neutral. The only actually performed 
mutations are Met.sup.2 .fwdarw.Ile.sup.2 and Ile.sup.131 
.fwdarw.Leu.sup.131. It is not disclosed whether the contemplated 
neutralities were obtained for these two mutations. 
Wo 91/00350 discloses nonglycosylated hIL-3 analog proteins, for example, 
hIL-3 (Pro.sup.8 Asp.sup.15 Asp.sup.70), Met.sup.3 rhul-3 (Pro.sup.8 
Asp.sup.15 Asp.sup.70); Thr.sup.4 rhuL-3 (Pro8Aspl5Asp.sup.70)and 
Thr.sup.6 rhuIL-3 (Pro.sup.8 Asp.sup.15 Asp.sup.70). It is said that these 
protein compositions do not exhibit certain adverse side effects 
associated with native hIL-3 such as urticaria resulting from infiltration 
of mast cells and lymphocytes into the dermis. The disclosed analog hIL-3 
proteins may have N termini at Met.sup.3, Thr.sup.4, or Thr.sup.6. 
We 91/12874 discloses cysteine added variants (CAVs) of IL-3 which have at 
least one Cys residue substituted for a naturally occurring amino acid 
residue. 
U.S. Pat. No. 4,810,643 discloses the DNA sequence encoding human G-CSF. 
WO 91/07988 discloses a method to increase megakaryocyte production 
comprised of administration of G-CSF with IL-3 or GM-CSF. Also disclosed 
is a method for increasing platelet production comprised of administration 
of IL-6 with IL-3, G-CSF or GM-CSF. 
SUMMARY OF THE INVENTION 
The present invention encompasses recombinant human interleukin-3 (hIL-3) 
variant or mutant proteins (muteins) These hIL-3 muteins contain one to 
three amino acid substitutions and may also have amino acid deletions at 
either/or both the N- and C- termini. This invention encompasses 
coadministration or sequential treatment using IL-3 variants of the 
present invention with other colony stimulating factors (CSFs), cytokines, 
lymphokines, interleukins, hematopoietic growth factors (herein after 
collectively referred to as "colony stimulating factors") which may have 
the potential for therapeutic use in restoring hematopoietic cells to 
normal amounts in those cases where the number of cells has been reduced 
due to diseases or to therapeutic treatments such as radiation and/or 
chemotherapy. Coadministration or sequential treatment using IL-3 variants 
of the present invention with other colony stimulating factors may enhance 
therapeutic value due to the synergistic effects of the proteins that make 
up the treatment. The use o f multiple factors may also have the potential 
advantage by lowering the demands placed on factor-producing cells and 
their induction systems. If there are limitations in the ability of a cell 
to produce a factor then by lowering the required concentrations of each 
of the factors by using them in combination may usefully reduce demands on 
the factor-producing cells. The use of multiple factors may lower the 
amount of the factors that would be needed, probably reducing the 
likelihood of adverse responses. 
Coadministration or sequentiaL treatment may have the usua L activit v c f 
the peptides forming the mixture or it ma y be furthe characterized by 
having a bioloaicaL or physiological activity greater than simply the 
additive function of the presence of IL-3 or the other growth factor 
alone. Coadmiristration or sequential treatment may also unexpectedly 
provide an enhanced effect on the activity or an activity different from 
that expected by the presence of IL-3 or the other colony stimulating 
factors, The IL-3 variants of the present invention may also have an 
improved activity profile which may include reduction of undesirable 
biological activities associated with native hIL-3. 
The present invention includes mutants of hIL-3 in which from 1 to 14 amino 
acids have been deleted from the N-terminus and/or from 1 to 15 amino 
acids have been deleted from the C-terminus, containing one to three amino 
acid substitutions, which are used with other colony stimulating factors 
or IL-3 variant Preferred IL-3 variants of the present invention include 
variants in which amino acids 1 to 14 have been deleted from the 
N-terminus, amino acids 126 to 133 have been deleted from the C-terminus 
and contain from one to three amino acid substitutions in the polypeptide 
sequence. 
The present invention also provides IL-3 variants which may function as 
IL-3 antagonists or as discrete antigenic fragments for the production of 
antibodies useful in immunoassay and immunotherapy protocols. Antagonists 
of hIL-3 would be particularly useful in blocking the growth of certain 
cancer cells like AML, CML and certain types of B lymphoid cancers. Other 
conditions where antagonists would be useful include those in which 
certain blood cells are produced at abnormally high numbers or are being 
activated by endogenous ligands. Antagonists would effectively compete for 
ligands, presumably naturally occurring hemopoietins including and not 
limited to IL-3, GM-CSF and IL-5, which might trigger or augment the 
growth of cancer cells b ar virtue of their abilit t bind to the receptor 
complex while intrinsic activation properties of the ligand are 
diminished. IL-3, GM-CSF and/or IL-5 also play a role in certain asthmatic 
responses. An antagonist of the IL-3 receptor may have the utility in this 
disease by blocking receptor-mediated activation and recruitment of 
inflammatory cells. 
In addition to the use of the IL-3 variants of the present invention with 
other colony stimulating factors in vivo, it is envisioned that in vitro 
uses would include the ability to stimulate bone marrow and blood cell 
activation and growth before infusion into patients.