Methods of ex-vivo expansion of hematopoietic cells using...

Details Methods of ex-vivo expansion of hematopoietic cells using... Methods of ex-vivo expansion of hematopoietic cells using...

1996-12-09

2002-07-02

Kunz, Gary L. (Department: 1647)

Drug, bio-affecting and body treating compositions

Lymphokine

C424S085200, C424S085400, C435S372000

Reexamination Certificate

active

06413509

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods of ex-vivo expansion of hematopoietic cells by culturing hematopoietic cells in a growth medium comprising a variant of human interleukin-3 (hIL-3) and another colony stimulating factors, cytokines, lymphokines, interleukins, hematopoietic growth factors or IL-3 variants and uses for the expanded cells for treating patients having a hematopoietic disorders.
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 mixed erythroid colonies (when erythropoietin is 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 therapeutic treatments such as radiation and/or chemotherapy.
Interleukin-3 (IL-3) is a hematopoietic growth factor which has the property of being able to 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-hydroxysteroid 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 library 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
8
hIL-3 cDNAs having proline at position 8 of the protein sequence. Thus it appears that there may be two allelic 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
8
) sequence.
U.S. Pat. No. 4,877,729 and U.S. Pat. No. 4,959,455 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 analogs 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 {fraction (1/500)}th the activity as the native molecule. Replacement of two of the four Cys residues by Ala(Cys
79
, Cys
140
→Ala
79
, Ala
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. NATL. 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
8
→Pro
8
replacement. Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites.
EP-A-0275598 (WO 88/04691) illustrates that Ala
1
can be deleted while retaining biological activity. Some mutant hIL-3 sequences are provided, e.g., two double mutants, Ala
1
→Asp
1
, Trp
13
→Arg
13
(pGB/IL-302) and Ala
1
→Asp
1
, Met
3
→Thr
3
(pGB/IL-304) and one triple mutant Ala
1
→Asp
1
, Leu
9
→Pro
9
, Trp
13
→Arg
13
(pGB/IL-303).
WO 88/05469 describes how deglycosylation mutants can be obtained and suggests mutants of Arg
54
Arg
55
and Arg
108
Arg
109
Lys
110
might avoid proteolysis upon expression in
Saccharomyces 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
2
→Ile
2
and Ile
131
→Leu
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
8
Asp
15
Asp
70
), Met
3
rhuIL-3 (Pro
8
Asp
15
Asp
70
); Thr
4
rhuIL-3 (Pro
8
Asp
15
Asp
70
)and Thr
6
rhuIL-3 (Pro
8
Asp
15
Asp
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
3
, Thr
4
, or Thr
6
.
WO 90/12874 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.
Hematopoietic growth factors, such as hIL-3, have been administered alone, co-administered with other hematopoietic growth factors, or in combination with bone marrow transplants subsequent to high dose chemotherapy to treat the neutropenia and thrombocytopenia which are often the result of such treatment. However the period of severe neutropenia and thrombocytopenia may not be totally eliminated. The myeloid lin

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