Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1993-07-27
2001-03-20
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
Reexamination Certificate
active
06204364
ABSTRACT:
TECHNICAL FIELD
The invention relates to the field of cell proliferation and differentiation and to factors which regulate the composition of the blood and the viability of other tissues. More specifically, the invention concerns hybrid cytokines which have unique physiological properties derived from the cytokine family members leukemia inhibitory factor (LIF); granulocyte colony stimulating factor (G-CSF); interleukin-6 (IL-6); and oncostatin-M (OSM).
BACKGROUND ART
More than two dozen cytokines that regulate blood composition by controlling the growth and differentiation of hematopoietic stem cells have been identified. The interferons, tumor necrosis factor, stem cell factor, the numbered interleukins and the various colony stimulating factors are exemplary of these proteins and glycoproteins. The invention described below focuses on four closely related cytokines whose structural similarity has been discovered by applicants.
One of these factors, interleukin-6 (IL-6) was originally identified as a B-cell differentiation factor, but has subsequently been shown to induce acute phase proteins in liver cells, to inhibit growth of certain myeloid leukemia cell lines and induce their differentiation into macrophage cells, to promote IL-3 dependent colony formation of primitive blast colony forming cells, to cause differentiation of neuronal cells, to enhance keratinocyte and mesangial cell growth, to promote the maturation of megakaryocytes, and to induce the proliferation and differentiation of T cells. In vivo, IL-6 increases the hematopoietic cell count of the erythroid, myeloid, and thrombocytic lineages. Other former names for IL-6 are beta2-interferon, B-cell stimulatory factor-2, hybridoma/plasmacytoma growth factor, and monocyte granulocyte inducer type 2. The spectrum of activities attributable to IL-6 indicates that it is useful in tumor inhibition, bone remodeling, kidney development, and T- and B-cell proliferation and stimulation.
Leukemia inhibitory factor (LIF) has been demonstrated to inhibit the growth of certain myeloid leukemia cells and to induce their differentiation into macrophage cells; to enhance interleukin-3 dependent colony formation of primitive blast cells; to promote megakaryocyte growth and differentiation; to induce neuronal differentiation; to stimulate the production of acute phase proteins and hepatocytes (all properties it shares with IL-6) and to inhibit the differentiation of embryonic stem cells and kidney cells and to induce bone resorption.
Oncostatin-M (OSM) is known to be a tumor inhibitor for melanoma and certain carcinoma cells and inhibits the growth of human A375 melanoma cells but not normal human fibroblasts. It is also an inhibitor of the growth of M1 myeloid leukemic cells and induces their differentiation into macrophage-like cells as well as stimulating megakaryocyte production in the spleen. This factor was first isolated from conditioned medium of U937 human histolytic leukemia cells that had been induced with phorbol myristate acetate (PMA) and is also present in the supernatants of activated human T-cells.
Granulocyte colony stimulating factor (G-CSF) stimulates neutrophil proliferation and differentiation and induces the differentiation of M1 murine myeloid leukemic cells into macrophage-like cells as well as enhancing interleukin-3 dependent colony formation of primitive blast cells. It appears to have little effect on the hematopoietic cell lineages of megakaryocytes or platelets but enhances cytosine arabinoside-mediated cytoxicity in human myeloid leukemia cells.
The reported biological activities of the foregoing cytokine family members is summarized in the following table:
TABLE 1
Reported Biological Activities of Cytokine Family Members
LIF
OSM
G-CSF
IL-6
Endothelial Cell Proliferation
NR
+
NR
NR
Tumor Inhibition
+
+
NR
+
Embryonic Stem Cell Maintenance
+
+
NR
NR
Hematopoietic Leukemic Cell
+
+
+
+
Differentiation
Melanoma Cell Inhibition
−
+
−
+
Neutrophil Proliferation/Stimulation
NR
NR
+
+
Myoblast Proliferation
+
NR
NR
NR
Bone Remodeling
+
NR
NR
+
Kidney Development
+
NR
NR
NR
Neuronal Differentiation
+
NR
NR
+
Hepatocyte Stimulation
+
NR
NR
+
Megakaryocyte Augmentation
+
NR
−
+
T-Cell Proliferation
NR
NR
NR
+
Keratinocyte Proliferation
NR
NR
NR
+
B-Cell Proliferation/Stimulation
NR
NR
NR
+
Binding to Human Placental Cell Receptor
+
+
−
−
These data were obtained from multiple assay systems and few direct comparisons of the growth factors have been made. The table lists several biological systems in which one of the four growth factors shows activity, while the role of other family members is not yet known. NR = not reported.
As shown in the foregoing table, the four related factors that are the subject of the present invention do not display identical activity patterns. Although a number of characteristics have not yet been reported for each of these factors, it is clear that at least one difference in activity spectrum exists between any two of them. For example, OSM and IL-6 inhibit the growth of melanoma cells; LIF and G-CSF do not. However, LIF and G-CSF differ in that LIF is capable of augmenting megakaryocytes; G-CSF is not. OSM binds to human placental cell receptor; IL-6 does not.
While there have been excellent clinical successes, especially with the use of G-CSF in enhancing the health of the immune system and white blood cell replacement in patients with depleted lymphocyte populations, such as patients undergoing radiation or chemotherapy, no ideal pharmaceutical which has the desired effects, free of complications, has been discovered. Clearly this is not surprising, since normally the composition of the blood is regulated by controlling the differentiation of cells originating in the bone marrow through the interaction of a multiplicity of indigenous factors whose levels are in turn presumably controlled by mechanisms not yet understood. Thus, it is desirable to augment the repertoire of available therapeutic agents which participate in the control of blood composition. The present invention augments this repertoire by providing hybrid cytokines with unique properties characteristic of these previously unavailable therapeutic agents.
DISCLOSURE OF THE INVENTION
The invention is directed to therapeutic hybrid cytokines which are composed of portions of the closely related cytokines leukemia inhibitory factor (LIF), granulocyte-colony stimulating factor (G-CSF), interleukin-6 (IL-6) and oncostatin-M (OSM). These cytokines have a high degree of sequence homology (as well as a similar genetic organization. It is disclosed in the art that IL-6 and G-CSF are comprised of four &agr;-helical regions; applicants herein have deduced that LIF and OSM also are comprised of four &agr;-helical regions organized in a similar manner. In each case, the four helical regions are linked by non-&agr; helical sequences of about 5-100 amino acids, and in some cases the &agr;-helices are maintained in the proper conformation and geometry with respect to each other through disulfide bridges. The hybrid cytokines of the invention also contain four &agr;-helical regions, at least two of which are derived from the corresponding &agr;-helical regions of different members of this group. The linking amino acid sequences preferably are also derived from the four factors described herein.
Thus, in one aspect, the invention is directed to a hybrid cytokine comprising a first, second, third and fourth &agr;-helical region. Each of these regions is derived from the corresponding &agr;-helical region of LIF, G-CSF, IL-6, or OSM. At least one of these regions is derived from a different factor of this group than at least one other.
In additional aspects, the invention is directed to DNA sequences encoding the hybrid cytokines, to expression systems capable of expressing these DNAs, to host cells transformed with these expression systems and to methods
Rose Timothy M.
Todaro George J.
Carlson Karen Cochrane
Foley & Lardner
Fred Hutchinson Research Center
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