Synthetic peptides that inhibit IL-6 activity

Details Synthetic peptides that inhibit IL-6 activity Synthetic peptides that inhibit IL-6 activity

1998-09-02

2001-01-09

Borin, Michael (Department: 1631)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S014800, C530S326000, C530S327000

Reexamination Certificate

active

06172042

ABSTRACT:

FIELD OF THE INVENTION
The present invention is generally in the field of inhibitors of Interleukin-6 (IL-6) activity. More specifically, the present invention concerns new synthetic peptides which are capable of inhibiting the IL-6-dependent growth of myeloma/plasmacytoma cells.
BACKGROUND OF THE INVENTION AND PRIOR ART
The receptor system for Interleukin-6 (IL-6) is composed of two distinct receptor subunits designated gp80 (IL-6R) and gp130 (reviewed in Hirano et al., 1994). These two receptor proteins belong to the cytokine receptor superfamily of Bazan (1990). The 3-dimensional structure of the human Growth Hormone Receptor (hGHR), a member of this family, has been revealed by crystallography (DeVos et al., 1992), from which the residues which interact with the ligand and those which mediate the interaction between the two receptor subunits in their extracellular domains, have been determined. The alignment of part of the amino acid sequence of the extracellular domain of the IL-6R and hGHR sequences based on Bazan's model is shown schematically in FIG.
1
.
IL-6 is a pleiotropic cytokine which has a number of important biological activities (see Revel, 1992, for review). Further, IL-6 has been implicated in the growth and progression of human multiple myeloma (Klein et al., 1990).
In fact, IL-6 is a growth factor for B-lymphocyte leukemic cells of Multiple Myeloma. These leukemic cells are also called plasmacytoma or myeloma cells, as they are derived from mature B-lymphocytes or plasma cells. When fused to antibody-producing B-cells, these myeloma or plasmacytoma cells are called hybridoma cells.
In view of the fact that IL-6 is a growth factor for such plasmacytoma or myeloma cells, there has been a long-felt need to obtain specific IL-6 inhibitors which may be used to block the IL-6-mediated or IL-6-dependent growth of such plasmacytoma/myeloma cells, and thereby provide a way for treating Multiple Myeloma, a disease affecting a very large number of people worldwide. To this end, Grube and Cochrane (1994) have described a peptide derived from IL-6R, called peptide 249-264 (i.e. a peptide having the amino acid residues from residue No. 249 to residue No. 264 of the IL-6R amino acid sequence), which is capable of inhibiting the growth of murine plasmacytoma B9 cells. It is also known that IL-6 plays a role in the inhibition of other diseases, such as osteoporosis and autoimmune diseases.
Heretofore, neither the specific peptides of the present invention, nor their specific biological activities and other characteristics such as, for example, their specificity for certain monoclonal antibodies, have been described.
It is therefore an aim of the present invention to provide new peptides derived from IL-6R which are capable of inhibiting the IL-6-dependent growth of human myeloma or murine plasmacytoma cells.
It is another aim of the present invention to provide such new peptides which are further characterized by virtue of their defining linear epitopes within the IL-6R sequence, which are the binding sites of monoclonal antibodies (Mabs) which are themselves capable of blocking IL-6 activity.
Yet another aim of the invention is to provide a chemical synthesis process for the preparation of the new peptides.
A yet further aim of the invention is to provide pharmaceutical compositions containing the new peptides.
SUMMARY OF THE INVENTION
The present invention is based on the unexpected finding that short peptides within the IL-6 receptor gp80 molecule (IL-6R) could be defined by virtue of their ability to bind two different monoclonal antibodies (Mabs) which were previously known to strongly inhibit the activity of IL-6. Further, when chemically synthetized, in accordance with the present invention, these peptides when added to cultures of leukemic cells, were surprisingly shown to be capable of causing the complete inhibition of the growth of such leukemic (plasmacytoma/myeloma) cells.
Accordingly, the present invention provides a peptide or biologically active analogs thereof capable of inhibiting the activity of IL-6, wherein said peptide is characterized by being derived from the gp80 (IL-6R) subunit of the IL-6 receptor system and by being a linear epitope recognized by one or more monoclonal antibodies (Mab) specific to IL-6R, with the proviso that said peptide is other than the group of peptides consisting of: (i) the 16 amino acid peptide having the amino acid sequence of residues 249-264 of the IL-6R molecule; (ii) the 14 amino acid peptide having the amino acid sequence of residues 255-268 of the IL-6R molecule; (iii) the 6 amino acid peptide having the amino acid sequence of residues 249-254 of the IL-6R molecule; (iv) the 10 amino acid peptide having the amino acid sequence of residues 259-268 of the IL-6R molecule; and (v) the 10 amino acid peptide having the amino acid sequence of residues 249-258 of the IL-6R molecule.
An embodiment of the above peptide or analogs thereof of the invention is a peptide selected from the group of peptides having between about 4 and about 25 amino acid residues derived from the portion of the IL-6R molecule extending between residue 223 and residue 272 as depicted in
FIG. 2
, said group of peptides comprising:
(a) a peptide having at least the 10 amino acid sequence LRYRAERSKT from position 255 to position 264 of the IL-6R molecule (residues 143-152 of SEQ ID NO:1);
(b) a peptide having at least the 10 amino acid sequence YRAERSKTFT from position 257 to position 266 of the IL-6R molecule (residues 145-154 of SEQ ID NO:1);
(c) a peptide having at least the 10 amino acid sequence AERSKTFTTW from position 259 to position 268 of the IL-6R molecule (residues 147-156 of SEQ ID NO:1);
(d) a peptide having at least the 10 amino acid sequence RSKTFTTWMV from position 261 to position 270 of the IL-6R molecule (residues 149-158 of SEQ ID NO:1);
(e) a peptide having at least the 10 amino acid sequence KTFTTWMVKD from position 263 to position 272 of the IL-6R molecule (residues 151-160 of SEQ ID NO:1);
(f) a peptide having at least the 10 amino acid sequence SFYRLRFELR from position 247 to position 256 of the IL-6R molecule (residues 135-144 of SEQ ID NO1);
(g) a peptide having at least the 10 amino acid sequence YRLRFELRYR from position 249 to position 258 of the IL-6R molecule (residues 137-146 of SEQ ID NO:1);
(h) a peptide having at least the 10 amino acid sequence LRFELRYRAE from position 251 to position 260 of the IL-6R molecule (residues 139-148 of SEQ ID NO:1); and
(i) an analog of any one of the peptides of (a)-(h) in which one or more amino acid residues have been added, deleted or substituted by another amino acid residue, and wherein said peptides and analogs of (a)-(i) are characterized by defining all or part of a linear epitope recognized by anti-IL-6R monoclonal antibodies and by being capable of inhibiting IL-6 activity.
Another embodiment of the above peptides or analogs of the invention is a peptide or analog wherein said peptide or analog defines a linear epitope recognized by one or both of the Mabs, herein designated Mab 34.4 and Mab 50.6.
A yet further embodiment of the above peptides of the invention is a peptide selected from any one of the herein designated peptides: (i) 1062 having the sequence from position 247 to position 260 of the IL-6R molecule as depicted in
FIG. 2
; (ii) 1063 having the sequence from position 255 to position 270 of the IL-6R molecule as depicted in
FIG. 2
; (iii) 1086 having the sequence from position 226 to position 245 of the IL-6R molecule as depicted in
FIG. 2
; (iv) 1085 having the sequence from position 234 to position 245 of the IL-6R molecule as depicted in
FIG. 2
; and (v) 1122 having the sequence from position 260 to position 269 of the IL-6R molecule as depicted in FIG.
2
.
The present invention also provides in another aspect, a peptide or biologically active analogs thereof capable of inhibiting IL-6 activity, wherein said peptide is characterized by being derived from the gp80 (IL-6R) subunit of the IL-6 receptor system and by being part of all of the region of IL-

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