MXPA00000364A - Chimeric interleukin-6 soluble receptor/ligand protein, analogs thereof and uses thereof - Google Patents

Abstract

Chimeric proteins constructed from the fusion of the naturally occurring form of the soluble IL-6 receptor and IL-6 which are useful for treatment of cancer and liver disorders, enhancement of bone marrow transplantation, and treatment of other IL-6 related conditions are provided.

Description

CHEMÉRICA PROTEIN OF RECEIVER LIGANDO OF INTERLEUCINA-6 SOLUBLE, ANALOGUES OF THE SAME AND USES OF THE SAME FIELD OF THE INVENTION The present invention generally belongs to the field of biological activities of interleukin-6 (IL-6) that depend on the agonistic action of the soluble IL-6 receptor (slL-6R). More specifically, the present invention relates to novel chimeric SLL-TR / IL-6 proteins constructed from the fusion of essentially the naturally occurring form of slL-6R and IL-6, and biologically active analogs thereof. , which are particularly useful for treating cancer by inhibiting the growth of cancer cells, to improve bone marrow transplantation, to treat liver disorders and other conditions related to IL-6.

BACKGROUND OF THE INVENTION Interleukin-6 (IL-6) is a well-known cytokine whose biological activities are mediated by a membranal receptor system comprising two different proteins called IL-6 receptor (IL-6R or gp80) and the other gp130 (reviewed by Hirano and others, 1994). The soluble forms of IL-6R (sIL-6R), which correspond to the extracellular domain of gp80, are natural products of the human body found as glycoproteins in the blood and urine (Novick et al., 1990, 1992). An exceptional property of sIL-6R molecules is that they act as potent agonists of IL-6 on many cell types, including human cells (Taga et al., 1989; Novick et al., 1992). This is due to the fact that even without the intracytoplasmic domain of gp80, slL-6R is still capable of triggering the dimerization of gp130 in response to IL-6, which in turn mediates IL-6-specific signal transduction and Subsequent biological effects (Murakami et al., 1993). The active IL-6 receptor complex is in fact a hexamerica structure formed by two gp130 chains, two IL-6R and two IL-6 ligands (Ward et al., 1994; Paonessa et al., 1995), in which slL -6R has two types of interaction with gp130, both of which are essential for IL-6-specific biological activities (Halimi et al., 1995). Treatment with slL-6R results in an increase in the biological activities of IL-6 in many cell types. One example is tumor cells whose growth is inhibited to a greater degree by IL-6 when slL-6R is added, such as murine myeloleukemic M1 cells (Taga et al., 1989), human breast carcinoma T47D cells (Novick and others, 1992) or non-small human cell lung carcinoma cells (Ganapathi et al., 1996). IL-6 has anti-metastatic activity in vivo (Katz et al., 1995), slL-6R can also increase said anti-tumor effects in vivo of IL-6 (Mackiewicz et al. 1995). Another activity of IL-6 that is increased by the addition of slL-6R is the stimulation of hematopoietic stem cells to produce colonies of multiple lineage (Sui et al., 1995). The present inventors have also observed that the survival of primary cultures of brain oligodendrocytes is supported by the combination of slL-6R and IL-6 (Oh, 1997), whereas IL-6 alone is deficiently active in those cultures (Kahn and De Vellis, 1994). This discovery indicates that IL-6, when combined with slL-6R, can mimic the activity of other neurotropic cytokines such as Ciliary Neurotropic Factor (CNTF) or Leukemia Inhibitory Factor (LIF) that also act through gp130, as is the case also for IL-11 and oncostatin M (Hirano et al., 1994). In an attempt to provide a molecule that could combine the functions of IL-6 and slL-6R mentioned above, the production in recombinant yeast cells of a fusion protein between a truncated segment of the IL-6R sequence has recently been reported. human and IL-6, bound by a glycine-rich linker (Fisher et al., 1997). This fusion protein essentially includes only the N domain of the cytokine receptor and the C domain of the cytokine receptor IL-6R, and thus essentially lacks the entire immunoglobulin (Ig) type domain of IL-6R, and the receptor pre-membrane region (the region between the C domain and the transmembrane domain). Therefore it represents a truncated form of slL-6R, this slL-6R truncated in the fusion protein being linked by means of the glycine-rich linker mentioned above essentially to the entire mature form of IL-6. Apart from lacking parts of the natural slL-6R, this fusion protein, when produced in yeast cells, does not have the glycosylation pattern of said fusion protein would have if it were produced in mammalian cells, in particular, for example, in human cells. In fact, this fusion protein produced by yeast has a molecular weight of only about 57 kDa in contrast to a fusion product which contains all the amino acid residues of natural slL-6R and IL-6 and which is completely glycosylated in cells of mammal (e.g., human), having the expected molecular weight of about 85 kDa (see Example 2 hereinafter). The common experience in the development of recombinant proteins that can be used to treat human patients has shown that it is important to stay as close as possible to the natural forms of the proteins, as they are found in the human body, to avoid the unleashing of antibodies and proteins. other side effects observed with non-natural recombinant products. For this reason, the use of recombinant mammalian cell systems to produce glycosylated proteins such as interferon-β or granulocyte colony-stimulating factor (Chernajovsky et al., 1984, Holloway, 1994) in a most similar chemical form has been advantageous. possible to the natural human product. Bacteria or microorganisms such as, for example, yeasts, which do not glycosylate properly, also cause the incorrect folding of the protein chains, leading to immunogenic reactions. This is particularly important with respect to IL-6, which is strongly modified after translation by N and O glycosylation, as well as by phosphorylation (Revel, 1989 for reference), and with respect to the natural blood and urine slL-6R human that is a glycoprotein whose N-terminal and C-terminal amino acids are constant and have been determined (Novick et al., 1990 and patents of co-ownership by the present inventors Nos. US Pat. No. 5,216,128 and corresponding EP 413909B1). Accordingly, it would appear that the aforementioned prior fusion product between part of the slL-6R and IL-6 has a number of possible disadvantages, especially as regards its use to treat humans and this, due to the fact that it lacks part of the slL-6R, as well as its production in yeast that could provide an incorrect glycosylation of the protein. To date, a fusion molecule comprising the natural slL-6R found in human body fluids and natural IL-6, and which is produced in human or other mammalian cells, has not been described. Therefore, an object of the present invention is to provide a fusion molecule comprising natural slL-6R and natural IL-6 (in any order) that is produced in mammalian cells. Another objective of the present invention is the use of a fusion protein (SIL-6R / IL-6 chimera) to inhibit the growth of highly metastatic melanoma cells at very low concentrations, these cells being resistant to IL-6 or slL- 6R separately. Another objective is the use of a fusion protein (chimera sIL-6R / IL6) for the in vivo grafting of human hematopoietic stem cells into bone marrow transplant protocols.

A further object of the present invention is the use of a fusion protein in other disorders related to IL-6, for example, liver conditions or neurological conditions. A further objective of the invention is to provide pharmaceutical compositions containing the natural slL-6R-natural-IL-6 fusion protein (SIL-6R / IL-6 chimera) for the treatment of cancer, for use in transplant procedures. bone marrow and for other disorders related to IL-6, for example, liver conditions and neurological conditions. Other objects and aspects of the present invention will be described or will originate from the following description of the present invention.

BRIEF DESCRIPTION OF THE INVENTION According to the present invention there have been produced a number of fusion proteins (chimeras) each comprising essentially all of the naturally occurring slL-6R from fluids of the human body and essentially the mature form of naturally occurring human IL-6. , and each are linked by short linker peptides that can be as short as 3 amino acid residues in length or longer, for example 13 amino acid residues in length (see below and examples 1 and 2). It should be noted, however, that in these fusion proteins the linker peptides can be omitted and that the slL-6R portion can be directly linked to the IL-6 portion. Since linkers representing non-natural amino acid sequences can be immunogenic epitopes that induce antibodies in patients, it is preferred to have a directly fused SIL-6R / IL-6 chimera having the desired biological activity, while at the same time being reduced to The risk of inducing said potentially harmful antibody formation when said chimera is administered is minimized. The conservation of the complete slL-6R sequence that includes the Ig-like domain as found in the naturally occurring molecule, as well as the adequate glycosylation and other post-translational modifications introduced by human or mammalian cells when the previous chimera occurs in said cells, they are also important to reduce the potential immunogenicity of the chimeric protein product. However, it is possible to use a very short linker of about three amino acids at the point of attachment between the slL-6R and IL-6 portions of the chimeric protein. Said short linker could not be an immunogenic epitope. It is also of course possible to use longer linkers of up to about 30 amino acids to provide separation between the two portions, but care must be taken and experiments of biological efficacy and safety must be carried out to ensure that the chimeric molecules with such linkers are not immunogenic . In fact, it has surprisingly been shown in accordance with the present invention that such longer linkers are not essential for the activity of the chimeric protein, indicating that a suitable fold of the chimera does not require a longer linker, especially when almost all the sequences that will occur aatu rally of the portions of slL-6R and IL-6 are incorporated into the chimeric molecule (see example 3 and figure 5 which also refer to a very short linker (3 amino acids) and a similar chimera having a longer linker of 30 amino acids These fusion proteins or slL-6R / IL-6 chimeras have been produced efficiently, according to the present invention, in mammalian cell expression systems to create glycosylated products that they have potent activity in tumor cells that normally do not respond to IL-6 or slL-6R separately, and which were highly effective in ensuring the success of the grafting of transplanted human bone marrow cells (see below and examples 1-4). In fact, in such bone marrow transplants, the slL-6R / IL-6 chimeras were essential for the survival and proliferation of transplanted non-compromised pluripotent hematopoietic stem cells. further, from the experimental results presented hereinafter, as well as from other analyzes, the result is that various analogs of the chimeric SIL-6R / IL-6 protein of the invention, which have essentially the same activity, can be prepared. of the SIL-6R / IL-6 chimera, these analogs being SIL-6R / IL-6 chimeras in which one or more amino acid residues have been eliminated, added or replaced by others, the only limitation in said analogs being that they retain the majority of the naturally occurring sequence of slL-6R and IL-6. For example, additions of amino acids to naturally occurring slL-6R and IL-6 sequences are preferably limited to between about 20 amino acids, and preferably these additions are the binding site between slL-6R and IL-6, i.e. , the linker molecule. Also, deletions of the slL-6R and IL-6 sequences are preferably limited to between about 20-30 amino acids; and substitutions of amino acid residues in the slL-6R and IL-6 sequences by other amino acid residues are also preferably limited to between about 20-30 amino acids. All deletions, additions and substitutions mentioned above are acceptable in accordance with the present invention when the analogs modified in this manner that are obtained retain essentially the biological activity of the slL-6R / IL-6 chimera composed essentially of naturally occurring sequences. , and retains essentially the same glycosylation pattern of the chimera composed essentially of naturally occurring sequences when expressed in mammalian cells. Accordingly, the present invention provides a chimeric interleukin-6 (IL-6) -receptor of soluble glycosylated inteleucine-6 (slL-6R) (slL-6R / IL-6) and biologically active analogs thereof, which comprises a fusion protein product between essentially all the form of slL-6R occurring naturally and essentially all naturally occurring form of IL-6, said slL-6R / IL-6 and analogues thereof being glycosylated in a similar manner to the glycosylation of naturally occurring slL-6R and IL-6.

Modalities of the chimeric protein of the invention include: (i) A chimeric s? 6R / fL-6 protein and biologically active analogs thereof, wherein said slL-6R is fused to IL-6 by means of a linker molecule of peptide. (li) A chimeric slL-6R / IL-6 protein and biologically active analogs thereof, as in (i) above, wherein said linker is a very short non-immunogenic linker of about 3-4 amino acid residues. (ii) A chimeric slL-6R / IL-6 protein and biologically active analogues thereof, as in (ii) above, wherein said linker is a tripeptide of the sequence E-F-M (Glu-Phe-Met). (iv) A chimeric slL-6R / IL-6 protein and biologically active analogs thereof, as in (i) above, wherein said linker is a peptide of 13 amino acid residues of the sequence EFGAGLVLGGQFM (Glu-Phe -Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met) (SEQ ID: NO 1). (v) A chimeric SIL-6R / IL-6 protein, which is designated here as slL-6RdVal / IL-6 having a tripeptide linker of the EFM sequence between Val-356 of the C-terminus of slL-6R and Pro -29 of the N-terminus of IL-6, said chimeric protein has the sequence described in Figure 3. (vi) A chimeric slL-6R / IL-6 protein, which is designated here slL-6RdVal / L / IL-6 having a 13 amino acid peptide linker of the sequence EFGAGLVLGGQFM between Val-356 of the C-terminus of slL-6R and Pro-29 of the N-terminus of IL-6R, said chimeric protein has the sequence described in Figure 3, wherein the tf < jS¡} K.o of the E-F-M sequence between positions 357-359 of Figure 3 is replaced by said 13 amino acid peptide sequence. (vii) A chimeric slL-6R / IL-6 protein, wherein said protein is produced in mammalian cells in a fully processed manner. (viii) A chimeric SIL-6R / IL-6 protein, wherein said protein is produced in human cells. (ix) A chimeric slL-6R / IL-6 protein, wherein said protein is produced in CHO cells. (x) A chimeric slL-6R / IL-6 protein and biologically active analogues thereof, as above, wherein said chimeric protein and analogs are characterized as being capable of inhibiting the growth of highly malignant cancer cells. (xi) A chimeric SIL-6R / IL-6 protein and biologically active analogs thereof, as above, wherein said chimeric protein and analogs are characterized as being capable of inhibiting the growth of highly malignant melanoma cells. (xii) A chimeric slL-6R / IL-6 protein and biologically active analogs thereof, as above, wherein said chimeric protein and analogs are characterized by being capable of inducing the in vivo grafting of human hematopoietic cells into bone marrow transplants that is. (xiii) A chimeric SIL-6R / IL-6 protein and biologically active analogs thereof, as above, wherein said chimeric protein and analogs are characterized by being able to protect the liver against hepatotoxic agents. The present invention also provides a DNA sequence encoding a chimeric SIL-6R / IL-6 protein and biologically active analogs thereof as mentioned above, according to the invention. In addition, the present invention also provides a DNA vector comprising a DNA sequence encoding a chimeric SIL-6R / IL-6 protein and biologically active analogs thereof of the invention, as mentioned above, said vector being suitable for the expression of said chimeric protein in mammalian cells. Modalities of the DNA vector of the invention include: (i) A DNA vector wherein said vector is suitable for the expression of said chimeric protein in human cells. (ii) A DNA vector in which said vector is expressed in mammalian or human cells, the expressed chimeric protein has a sequence that allows the complete processing of the chimeric protein by the mammalian or human cell and the secretion of the protein chimeric completely processed from the cells in the culture medium in which said cells grow. (iii) A DNA vector, as above, wherein said vector is the plasmid designated here pcDNAslL-6R / IL-6 comprising a pcDNA 3 vector containing the DNA sequence encoding the slL-6R / IL protein -6 chimeric under the control of a cytomegalovirus (CMV) promoter. zsá é ** * "'^ í__S_e_fc? á * - .. jfe = fe5as = s. (iv) A DNA vector, as above, wherein said vector is the plasmid designated here pcDNA slL-6F # ßF? L-6 comprising a pcDNA3 vector containing the DNA sequence encoding the SIL-6R protein / Chimeric IL-6 under the control of a cytomegalovirus (CMV) promoter, and wherein in said DNA sequence coding for said chimeric slL-6R / IL-6 protein a linker sequence encoding a linker peptide is inserted in the EcoRI site placed between the sequence encoding the slL-6R part and the sequence coding for the IL-6 part of the protein. Also, the present invention also provides transformed mammalian cells containing a DNA vector like the one above, which is capable of expressing the sequence of the chimeric slL-6R / IL-6 protein carried by said vector, and of completely processing the protein expressed and secreted in the culture medium in which said cells grow. One embodiment of these transformed cells are the human embryonic kidney 293 cells (HEK293) described herein transfected by the pcDNA vector slL-6R / IL-6, said cells being able to express the chimeric slL-6R / IL-6 protein. , to completely process said protein and to secrete said protein in the culture medium in which said cells grow in the form of a glycoprotein of approximately 85 kDa. Another modality of transformed cells are CHO cells (Chinese Hamster Ovary) described herein transfected by the pcDNA vector slL-6R / IL-6, said cells being able to express the chimeric sIL-6R / IL-6 protein, to fully process said protein in the culture medium wherein said cells grow in the form of a glycoprotein of X approximately 85 kDa. The present invention also provides a method for producing a chimeric protein or biologically active analogs thereof, as above, which comprises culturing the aforementioned transformed cells under conditions suitable for expression, processing and secretion of said protein or analogues in the culture medium. in which said cells grow; and purifying said protein or analogs from said culture medium by immunoaffinity chromatography using monoclonal antibodies specific for slL-6R. The chimeric protein of the present invention has a number of uses including: (i) the use of a chimeric SIL-6R / IL-6 protein or the like, salts of any of them and mixtures thereof, as an inhibitor of cancer cells. (ii) the use, as in (i) above, as an inhibitor of highly malignant melanoma cells. (iii) the use of a chimeric SIL-6R / IL-6 protein or analogs, salts of any of them and mixtures thereof, as an active ingredient for inducing the grafting of human hematopoietic cells into bone marrow transplants. (iv) the use of a chimeric IL-6 slL-6R protein and the like, salts of any thereof and mixtures thereof, as an ingredient . * # * r 'ift í ^ fcfr ^ * ^^ active to increase hematopoiesis, to treat liver and neurological conditions, or for other applications in which IL-6 or slL-6R are used. Similarly, the chimeric protein of the present invention can be used to prepare drugs for a number of medical indications, namely, a chimeric SIL-6R / IL-6 protein or the like, salts of any of them and mixtures thereof , for use in the preparation of a medicament for treating cancers by way of inhibiting cancer cells, or in the preparation of a medicament for the improvement of bone marrow transplants in order to induce the grafting of human hematopoietic cells in bone marrow transplants bone, or in the preparation of a drug to increase hematopoiesis, or in the preparation of a medication to treat neurological disorders, or in the preparation of a drug for other applications in which IL-6 or slL-6R are used. further, the present invention also provides a pharmaceutical composition comprising as active ingredient a chimeric SIL-6R / IL-6 protein or analog thereof as described above, and a pharmaceutically acceptable carrier, diluent or excipient. The embodiments of this pharmaceutical composition of the invention include: (i) A pharmaceutical composition for the treatment of mammalian cells. (ii) A pharmaceutical composition for the improvement of bone marrow transplants. (iii) A pharmaceutical composition for the treatment of liver and neurological disorders, or to increase hematopoiesis or for other applications in which IL-6 or slL-6R are used. The present invention also provides a method for treating cancers in mammals, or for improving bone marrow transplants, or for treating liver and neurological disorders, or for increasing hematopoiesis, or for other applications in which IL-6 or slL-6R, which comprises administering to a patient a pharmaceutical composition as described above in a suitable dosage form and by means of an appropriate administration route. For the avoidance of doubt, the present invention relates to a chimera between IL-6 and slL-6R in any order, ie, the N-terminal and C-terminal portions can be reversed and the chimera would then be an IL-6-slL protein. 6R, although in the present it is always indicated as SIL-6R / IL-6. Other aspects and embodiments of the present invention are described or originate directly from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 (A, B) illustrates a schematic representation of the different vectors, reagents and process steps used in the construction of the chimeric DNA molecule encoding a chimeric protein in which the shell is conserved in the natural form of slL-6R ending at the Val 356 residue followed by the sequence of the mature and natural processed form of IL-6, as detailed in Example 1; Figure 2 (A, B) shows the results obtained from the analysis carried out to identify the chimera slL-6RdVal / IL-6 p86 by electrophoresis with polyacrylamide gel (A) and by bioactivity profile (B), where Figure 2A shows a reproduction of a Coomassie stained gel on which immunopurified fractions eluted from affinity chromatography columns loaded with a sample of secreted protein obtained from cultures of cells transfected with a vector encoding the chimeric protein were subjected to electrophoresis.; and Figure 2B shows a graphic representation of the biological activity (growth inhibition of melanoma cells F10.9) of each of the fractions mentioned above eluted from affinity chromatography columns, all as detailed in the examples 2 and 3; Figure 3 illustrates the amino acid sequence (one letter code) of the chimera slL-6RdVal / IL-6, in which the different domains of the molecule are shown, including the N-terminal signal peptide (line in the part of the sequence), the immunoglobulin type domain (Ig type), the cytokine receptor N domain (underlined), the cytokine C domain (line on top of the sequence) and the premembrane region of the receptor (the region between domain C and the transmembrane domain), all the slL-6R part of the chimera; ^^^^ tm ^^ i ^ t kiA as well as the mature IL-6 portion (underlined below) of the chimera, as described in examples 1 and 2; Figure 4 (A, B) shows photographs of F10.9 melanoma cells in culture without (A) and with (B) treatment with chimeric slL-6R / IL protein for 4 days, where in Figure 4B they are apparent the morphological changes induced in said metastatic melanoma cells (F10.9 cells) by treatment with the slL-6R / IL-6 chimera, as described in example 3; Figure 5 is a graphical representation of the results illustrating the inhibition of F10.9 melanoma cell growth by the chimeric slL-6R / IL-6 protein at various concentrations of the chimera ranging from about 0.12 ng / ml to about 150 ng / ml, wherein the chimera with a linker of only 3 amino acids IL-6RIL-6 as described in example 3 is purchased with a chimera with a long linker of 13 amino acids (IL-6RLIL-6); Figure 6 is a graphical representation of the results illustrating the absence of growth inhibitory effects in F10.9 melanoma cells either from isolated IL-6 alone (dotted upper curve with open squares) at concentrations ranging from 0-40 ng / ml of IL-6 and slL-6R alone (point of convergence of all curves on the vertical axis where the concentration of IL-6 is zero); as well as the inhibitory effects of growth observed when IL-6 and slL-6R are added together at various concentrations of each where the concentration of IL-6 varies from 10 ng / ml to 40 ng / ml, and slL-6R is added in three concentrations of 100 ng / ml, 200 ng / ml and 400 ng / ml for each concentration of IL-6, as illustrated in the three lower curves (two dotted curves with open triangles and circles and a complete curve with squares closed), as described in example 3; Figure 7 is a reproduction of an autoradiogram of a Southern blot showing the need for the chimeric slL-6R / IL-6 protein for successful grafting of human hematopoietic stem cells during bone marrow transplantation in SCID-NOD mice (the two right lanes representing the mice that received the chimeric SIL-6R / IL-6 protein apart from the other necessary factors, SCF, FLT-3, and this in contrast to the three left lanes representing the mice that have received only SCF and FLT-3 and SCF, FLT-3 as well as IL-6 and slL-6R isolated, ie, not fused), as described in example 4; Figure 8 is a Scatchard plot of the affinity characteristics of the slL-6R / IL-6 chimera compared to a mixture of IL-6 and sIL-6R, the values of the chimera illustrated by the filled boxes and the mix by filled diamonds, the ratio of the slopes being from 4 to 1; Figure 9 shows the highest activity of the slL-6R / IL-6 chimera in melanoma cells F10.9 compared to that of the slL-6R + IL-6 mixture, or with that of slL-6R (without IL-6); Figure 10 shows the protection of the SIL-6R / II-6 chimera against liver toxicity, the values representing an average of 4 experiments, the filled squares representing IL-6 - / - mice, the filled diamonds representing IL mice. -6 - / - who received IL-6, and the filled stars representing IL-6 - / - mice that received the chimera; Figure 11 illustrates the amino acid sequence (one letter code) of the IL-6-slL-6RdVal 3e chimera, the linker being underlined; and Figure 12 shows the biological activity in melanoma cells of figure 9 of chimera 3e (dark filled stars) compared to the SIL-6R / IL-6 chimera (filled squares) and two mutants (Mutt 39 (HD) - filled diamonds) and Mutt NHD - clear filled stars), as described in example 9.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a chimeric SIL-6R / IL-6 protein and biologically active analogs thereof having essentially all naturally occurring forms of slL-6R and essentially all forms of naturally occurring IL-6 fused between yes, the fusion site of which may be in the form of a linker peptide as short as three amino acids, and the chimeric slL-6R / IL-6 protein or analogs have a glycosylation amount and pattern similar to those of slL- 6R and IL-6 that occur naturally. It was found that said chimeric slL-6R / IL-6 protein is produced according to the present invention in mammalian cells, in particular, in human cells (see examples 1-4 below) or CHO cells (see example 6 below). expressed efficiently AtóSá * in these cells was highly glycosylated and had a potent activity in tumor cells that show no response at all to IL-6 or slL-6R alone. In a very particular way, according to the present invention it has been observed (see examples 1-3 below) that the chimeric slL-6R / IL-6 protein of the invention mentioned above causes the arrest of the growth of highly malignant mammalian cells such such as F10.9 melanoma cells at lower concentrations than necessary when using a mixture of unfL-6R and unfused IL-6. This is a particularly significant result in view of the fact that said F10.9 melanoma cells continue to grow normally when treated only with IL-6 or only with slL-6R separately, and suffer a growth arrest only when they are exposed to relatively high doses of a combination of non-fused IL-6 and slL-6R. Accordingly, the chimeric SIL-6R / IL-6 protein of the present invention is surprisingly a more potent inhibitor of these highly malignant melanoma cells than a mixture of their separate parts, i.e., a mixture of IL-6 and slL-6R not merged. The chimeric protein of the present invention is thus particularly useful as an active ingredient for treating various types of cancers. The higher activity of the chimeric slL-6R / IR-6 protein is due to its higher affinity for gp130 than that of the mixture of unfused IL-6 and slL-6R (example 7).

In addition, it has also been found in accordance with the present invention (see example 4) that a chimeric slL-6R / IL-6 molecule of the present invention is particularly useful for improving bone marrow transplantation. In fact, the use of a known protocol for the grafting of human bone marrow cells in severe combined immunodeficient mice (SCID), in which the stem cell factor (SCF) and the ligand Flt3 are used to make survival possible and proliferation of more primitive pluripotent hematopoietic stem cells capable of long-term grafting in recipient bone marrow, it was found that these two factors, SCF and Flt3 ligand, were not sufficient to promote the grafting of human cells into the bone marrow of the recipient mouse , and that only when the chimeric slL-6R / IL-6 protein was also added this graft was successful. This discovery indicates that the chimeric protein may be essential in said graft protocols. In the same experiments, IL-6 and slL-6R not fused when added separately, were insufficient to promote a successful bone marrow transplantation, and when added together they were much less active than the sIL-6R / IL-6 protein. chimeric, ie, at an effective concentration of 100 ng / ml the chimeric SIL-6R / IL-6 protein promoted a successful bone marrow transplantation, whereas the two slL-6R and IL-6 not separately fused, when added together at even higher concentrations. (slL-6R of 125-1250 ng / ml, IL-6 of 50-200 ng / ml), were much less active to promote said transplantation. The chimeric slL-6R / IL-6 protein of the invention described above is preferably a recombinant glycosylated SIL-6R / IL-6 chimera produced in human cells or in any other suitable mammalian cell expression system, such as hamster CHO cells that are able to glycosylate proteins as human cells do and that introduce the same post-translational modifications as human cells. An important feature is that the chimeric glycoprotein produced in this way is processed and modified as the molecules of natural slL-6R and IL-6 origin found in the human body, without truncation and without the addition of foreign non-natural polypeptide sequences, with the exception of of the very short tripeptide or when a longer linker peptide is incorporated between the slL-6R and IL-6 portions of the chimeric protein. To prepare the preferred chimeric protein of the invention mentioned above, the following characteristics of the naturally occurring slL-6R and IL-6 portions were considered: it is known that IL-6R present in membranes of human cells is produced by a cDNA that codes for 468 amino acids comprising a signal peptide, an immunoglobulin-like domain (Ig), a cytokine binding domain, a transmembrane region and a cytoplasmic domain (Yamasaki et al., 1988). A soluble form of slL-6R is found in body fluids which, like the mature IL-6R of the membranes, have an N-terminus corresponding to Leu-20 (Novick et al., 1990) and a corresponding C-terminus. to Val-356 just before the transmembrane region of IL-6R (see co-owned US Patent No. 5,216,128 and EP 413,908 B1). To merge this sequence from slL-6R to IL-6, an EcoRI restriction site was introduced after Val-356. The sequence of mature IL-6 that starts in Pro-29 of the AD fcde IL-6 and that concludes in Met-212 (Zilberstein et al., 1986; Hirano and otf Pwd?) Was introduced after this EcoRI site. At this EcoRI site one could also, but need not, introduce a linker peptide of desired length to distance the slL-6R and IL-6 portions from one another in the chimeric protein. As described in the examples below, two different chimeric proteins were produced as examples of said possible chimeric proteins, one having a tripeptide linker and the other having a 13 amino acid residue linker at its EcoRI site, both being essential also biologically active. The present invention also relates to protein analogues Chimeric SIL-6R / IL-6 of the invention described above, which retain essentially the same biological activity of the chimeric protein having essentially only the naturally occurring sequences of slL-6R and IL-6. Said analogs can be ones in which up to about 30 amino acid residues can be eliminated, added or substituted by others in the slL-6R and / or IL-6 portions of the chimeric protein, so that modifications of this type do not substantially change the biological activity of the chimeric protein analogue to the chimeric protein itself and in which the slL-6R portion of said analogs essentially retains the naturally occurring structure (prior to processing - see Figure 3) of a peptide of signal, Ig type domain, cytokine receptor N domain, cytokine receptor domain C and premembrane receptor domain. Likewise, said analogs of the chimeric protein must essentially retain the naturally occurring mature form of the IL-6 portion. The different analogs can differ greatly from each other and from the basic chimeric protein molecule (that essentially only with the naturally occurring sIL-R and IL-6 sequences) at the site of the linker peptide that binds the sIL-6R portions and IL-6 in the chimeric protein. Said linker may have a length of up to about 30 amino acids, and serves to separate the sIL-6R and IL-6 portions from each other in the chimeric protein. With respect to said linker, care must be taken to choose its sequence (and therefore also to biologically test in suitable standard tests each of said analogues) in such a way that, for example, it does not result in an incorrect folding of the protein chimeric that could make it inactive, or does not result in making the analog of the chimeric protein an immunogenic protein that induces antibodies against it in a patient who will be treated with it, with the result that said analog will be ineffective at least as a medication in the medium or long term. As for the above analogs of the chimeric protein of the invention, these analogs are those in which one or more and up to about 30 of the amino acid residues of the basic chimeric protein of the invention are replaced by different amino acid residues, or are deleted, or one or more amino acid residues are added to the original sequence of the chimeric protein of the invention (which essentially has only the naturally occurring slL-6R and IL-6 sequences) without substantially changing the activity of the resulting products compared to v-i_ «.-_ & fe__í. -, the basic chimeric protein of the invention. These analogs are separated by known synthesis and / or by known site-directed mutagenesis techniques, or any other suitable technique for the same. Any of these analogs preferably has an amino acid sequence sufficiently duplicative of that of the basic SIL-6R / IL-6 chimera, to have substantially similar activity thereto. Thus, it can be determined whether any given analog has substantially the same activity as the basic chimeric protein of the invention by means of routine experimentation comprising subjecting said analog to the biological activity tests described in Examples 2-4 below. Analogs of the chimeric protein that can be used according to the present invention, or the nucleic acids encoding them, include a finite set of substantially corresponding sequences such as substitution peptides or polynucleotides that can be routinely obtained by one skilled in the art. , without undue experimentation, based on the teachings and guides presented herein. For a detailed description of the chemistry and structure of proteins, see Schulz, G.E. and others., Principies of Protein Structure, Springer-Verlag, New York, 1978; and Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are incorporated herein by reference. For a presentation of substitutions of nucleotide sequences, such as codon preferences, see Ausubel et al., Supra, at §§ A.1.1-A.1.24, and Sambrook et al., Current Protocols in Molecular Bioloqy. Interscience N.Y. § § 6.3 and 6.4 (1987, 1992), in Appendices C and D. The preferred changes for analogs according to the present invention are those known as "conservative" substitutions. Conservative amino acid substitutions of those in the chimeric protein that essentially have the naturally occurring slL-6R and IL-6 sequences may include the same amino acids within a group that have sufficiently similar physicochemical properties so that substitution between the members of the group retain the biological function of the molecule; Grantham, Science. Vol, 185, pp. 862-864 (1 +74). It is clear that the insertions and deletions of amino acids can also be made in the sequences defined above without altering their function, particularly if the insertions or deletions involve only a few amino acids, for example, less than thirty, and preferably less than ten, and not removing or displace amino acids that are critical to a functional conformation, eg, cysteine residues, Anfinsen, "Principies That Govern The Folding of Protein Chains", Science, Vol. 181, pp. 223-230 (1973). Analogs produced by said deletions and / or insertions are within the scope of the present invention. Preferably, the same amino acid groups are those defined in Table I. Most preferably, the same amino acid groups are those defined in Table II and more preferably the same amino acid groups are those defined in Table III.

CUAPj j Anesthetics groups that are preferred TABLE II Same amino acid groups that are preferred TABLE Groups of equal amino acids that are most preferred Examples of the production of amino acid substitutions in proteins that can be used to obtain analogs of the chimeric protein for use in the present invention include any known method steps, such as those presented in the U.S. Patents. Nos. RE 33,653, 4,959,314, 4,588,585 and 4,737,462, to Mark et al .; 5,116,943 to Koths et al., 4,965,195 to Ñamen and others; 4,879,111 to Chong et al .; and 5,017,691 to Lee and others; and the lysine substituted proteins presented in the US patent. No. 4,904,584 (Shaw et al.). In another preferred embodiment of the present invention, any analog of the chimeric protein for use in the present invention has an amino acid sequence that essentially corresponds to that of the basic chimeric protein of the invention mentioned above. The term "essentially corresponds to" is designed to comprise analogs with minor changes to the basic chimeric protein sequence that do not affect the basic characteristics of the same, particularly as regards its ability to inhibit the proliferation of cancer cells or to promote transplants. of bone marrow, for example, is concerned. The type of changes that are generally considered within the language "essentially corresponds to" are those that could result from conventional mutagenesis techniques of the DNA encoding the chimeric protein of the invention, resulting in few minor modifications, and the analysis of the desired activity in the manner described above. Analogs according to the present invention include those encoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA under stringent conditions and which codes for a chimeric protein according to the present invention, comprising essentially all the natural sequences that code for slL-6R and IL-6. For example, said DNA or hybridizing RNA may be one that codes for the same protein of the invention having, for example, the sequence described in Figure 3, but which is different in its nucleotide sequence from the derived nucleotide sequence. naturally by virtue of the degeneracy of the genetic code, that is, a slightly different nucleic acid sequence can still code for the same amino acid sequence, thanks to its degeneracy. In addition, as also mentioned above, the amount of amino acid changes (deletions, additions and substitutions) is limited to about 30 amino acids, such that even with the maximum amount of changes, the analogs according to the present invention are those that essentially retain the leader sequence (before processing), Ig-like domain, N and C domains of the cytokine receptor and premembrane region of the receptor (the region between the C domain and the transmembrane domain) in the slL-6R portion and essentially the entire IL-6 portion. Said nucleic acid would be an ideal candidate to determine if it codes for a polypeptide that preserves the functional activity of the chimeric protein of the present invention. The term "astringent conditions" refers to the hybridization and subsequent washing conditions that those skilled in the art conventionally know as "astringent". See Ausubel et al., Current Protocols in Molecular Biology. supra. Interscience. N. Y., for. 6.3 and 6.4 (1987, 1992) and Sambrook et al., Supra. Without limitation, examples of astringent conditions include wash conditions 12-20 ° C below the calculated Tm of the hybrid under study in, for example, 2 x SSC and 0.5% SDS for 5 minutes, 2 x SSC and 0.1% SDS for 15 minutes. minutes; 0.1 x SSC and 0.5% SDS at 37 ° C for 30-60 minutes and then 0.1 x SSC and 0.5% SDS at 68 ° C for 30-60 minutes.

Those skilled in the art will understand that stringency conditions also depend on the length of DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferred to use tetramethylammonium chloride (TMAC) instead of SSC. See Ausubel. supra. The term "salts" here refers both to salts of carboxyl groups and to acid addition salts of amino groups of the chimeric protein of the invention or analogs thereof. The salts of a carboxyl group can be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids, such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid. Of course, all of these salts must have activity substantially similar to that of the chimeric protein of the invention or its analogues. The present invention also relates to DNA sequences that encode the chimeric protein of the invention and its analogs, as well as DNA vectors carrying said DNA sequences for expression in suitable mammalian cells, preferably human. One embodiment of a vector of the invention is a pcDNA slL-6R IL-6 plasmid comprising the "^ ^ ^ l ^, ^^, ^! - ^^ - ^^^^^ .iA- ^ vector pcDNA3 (Invitrogen) containing the merged sequences of slL-6R / IL-6 under the control of a promoter of Cytomegalovirus (CMV) The present invention also relates to transformed mammalian cells, preferably of human, capable of expressing the above proteins of the present invention, One embodiment of said transformed cells are human embryonic kidney cells 293 (HEK 293, ATCC CRL 1573) transfected by pcDNA SIL-6R / IL-6 that secrete chimeric SIL-6R / IL-6 fused as an 85 kDa glycoprotein.An additional modality is a pcDNA SIL-6R / L / IL-6 plasmid that differs from the previous pcLNA slL-6R / IL-6 by the insertion in the EcoRI site of short linkers that code for 10 additional amino acids A number of different sequences, of various lengths, can be introduced to optimize the distance between slL-6R and IL The invention also includes a chimeric protein in which A portion of IL-6 precedes SLL-6R (as in Figure 11). The present invention further relates to a method for producing and purifying the chimeric protein of the invention or its analogs, which comprises culturing the above transformed cells under conditions suitable for the expression and secretion of the chimeric protein product in the culture medium and then purify the secreted protein by immunoaffinity chromatography using anti-slL-6R monoclonal antibodies 34.4 as mentioned in examples 2 and 5 below.

The invention also relates to a pharmaceutical composition comprising as an active ingredient a SIL-6R / IL-6 chimera or analogs thereof or mixtures thereof or salts thereof and a pharmaceutically acceptable carrier, diluent or excipient. One embodiment of the pharmaceutical composition of the invention includes a pharmaceutical composition for an increased IL-6 type action, for the treatment of cancers, for bone marrow transplantation, for the increase of hematopoiesis, in particular thrombopoiesis, for the treatment of conditions neurological, for the treatment of liver disorders and other applications of IL-6 or related cytokines. The pharmaceutical compositions of the invention are prepared for administration by mixing the chimeric protein, or its analogs, with pharmaceutically acceptable carriers., and / or stabilizers and / or excipients, and is prepared in a dosage form, for example, by lyophilization in dosage containers. The method of administration can be by any of the modes of administration accepted for similar agents and will depend on the condition to be treated, for example, intravenously, intramuscularly, subcutaneously, by local injection or topical application, or continuously by infusion , etc. The amount of active compound to be administered will depend on the route of administration, the disease to be treated and the condition of the patient. For example, local injection will require a lower amount of the protein on a body weight basis than an intravenous infusion.

The present invention also relates to uses of the chimeric protein of the invention or its analogs or mixtures thereof for the treatment of cancers, for bone marrow transplants, for increasing hematopoiesis, especially thrombopoiesis, for the treatment of neurological conditions, for protection of liver tissues in patients with necrotic diseases due to chemicals (eg, carbon tetrachloride, alcohol, paracetamol) or other causes (eg, viral, surgical) and for use in other IL-6 or related cytokine applications . Also, the present invention also relates to the chimeric protein or analogs thereof or mixtures thereof for use in the preparation of medicaments for treating the conditions mentioned above or for use in the above mentioned indications. In addition to the treatment methods mentioned above, ex vivo procedures and gene therapy with the qimera or DNA encoding it are also contemplated. The present invention will now be described in more detail in the following non-limiting examples and the accompanying drawings.

EXAMPLE 1 Construction of the expression vector of the slL-6RdVal / IL-6 chimera Figure 1 shows a schematic flow chart of the steps taken to construct the expression vector carrying the sequence encoding the chimeric protein slL-6RdVal / IL-6, inclusive of all the different starters and intermediates, different reagents and reaction steps. This construction procedure was essentially done using techniques well known in the art to construct expression vectors of choice (see, for example, Sambrook et al., 1989). The procedure was, briefly, as follows: A library of cDNA molecules from human breast carcinoma T47D cells was cloned into the bacteriophage lamda (?) Gt11 and analyzed with oligonucleotide probes derived from the IL-6R sequence of Yamasaki and others (1988). A clone of? Gtl 1 cDNA having the complete human IL-6R coding sequence was isolated. The insert was cut from? Gtl 1 by EcoRI and cloned into the Multiple Cloning Site (MCS) of the phagemid of E. coli Blue script pBS / SK (Stratagene Cloning Systems, LaJolla, California). This plasmid pBS / SK-IL-6R (figure 1) was cut by EcoRI and then shaved at its ends and re-cut with EcoRV to isolate the 5'-fragment of 959 base pairs (bp) from IL-6R. concludes at the EcoRV site of IL-6R (coordinate 1203). This fragment extracted from an agarose gel electrophoresis was cloned into a new open pBS / SK vector in the EcoRV of the SCM (pBS / SK-slL-6R-RV in Figure 1). The pBS / SK-IL-6R DNA mentioned above and obtained above was subjected to Polymerase Chain Reaction (PCR) to amplify a 368 bp fragment between the forward primer 1137-1156 and the reverse primer 1505-1488. The reverse primer was synthesized with an EcoRI site _____ i ____ fc ____-__ »~, ^. immediately after the codon for Valine-356 of IL-6R (see figure 1), since this Valine residue previously determined as a carboxy-terrminal amino acid of the natural form of soluble slL-6R excreted in human urine (Novick and others, 1990; OH? _ others, 1996; US patent in co-ownership No. 5,216,128 and EP patent No. EP 413908 B1). The PCR product was cut by EcoRV and by EcoRI and ligated into pBS / SK-slL-6R-RV between the EcoRV site of IL-6R and the EcoRI site of the MCS (Figure 1). The resulting pBS-sIL-6R-dVal-RI plasmid was then shortened to remove the 5 'untranslated sequences by ligation of the HindIII site of the MCS with the Ncol site in base pair 410 of IL-6R (both sites being first shaved at their ends), to produce pBS-slL-6R-dVal-RI-Ncol (figure 1). The sequence of IL-6 was derived from the plasmid pKKß2-7 which, as described above (Chen et al., 1988), was constructed by inserting the cDNA of IFN-β2 / BstNI-cut IL-6 (Zilberstein et al., 1986) at the EcoRI site of the E. coli expression vector pKK223-3 (Pharmacy, Uppsala, Sweden) using a synthetic oligonucleotide with an EcoRI site followed by a methionine codon and the codon for proline-29 of IL-6 and concluding in a BstNI site (EcoRII). The IL-6 cDNA insert of pKKß2-7 concludes 7 base pairs after the stop codon at a N1alV site and is then followed 11 bp by the HindIII site of vector pKK223-3 (Figure 1). The pKKß2-7 DNA was cut with Hindlll, its ends shaved and re-cut with EcoRI and the IL-6 cDNA was inserted into pBS-slL-6R-dVal-RI-Ncol to fuse the mature sequence of IL -6 (starting in proline 29) immediately after valine 356 of IL-6R and separated only by 3 codons (Glu-Phe-Met). The resulting plasmid pBS / SK-slL-6R / IL-6 (FIG. 1) was then cut again at the SalI and NotI sites of its MCS and the insert was cloned into the EcoRV site of pcDNA3 (Invitrogen Corporation, San Diego, California). The resulting pCDNA3-slL-6R / IL-6 plasmid (Figure 1) contains the insert towards the 3 'end of the strong cytomegalovirus (CMV) promoter and is followed by a polyadenylation site ensuring efficient transcription of the slL-6RdVal chimera / IL-6. The conservation of the 5 'end of the slL-6R in the chimera ensures that after expression in mammalian cells the function of the signal peptide and the processing of the N-terminus of the chimeric protein will be the same as in natural slL-6R. As indicated above, an advantageous feature of the slL-6RdVal / IL-6 construct is that it is essentially the fusion of the natural form of slL-6R and the natural form of IL-6 as they exist in the human body, and without sequences of foreign polypeptides. However, the conservation of the EcoRI site in the slL-6RdVal / IL-6 construct (Figure 1) allows easy insertion of linker polypeptide segments between the slL-6R and IL-6 moieties. A construction was also made (slL-6RdVal / L / IL-6) with the 13-amino acid linker sequence Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met introduced between Val-356 of slL-6R and Pro-29 of IL-6.

EXAMPLE 2 Expression of slL-6RdVal / IL-6 chimera in human cells Using essentially standard techniques of mammalian cell culture, transfection of cells and analysis of transfected cells for the expression of the newly introduced DNA sequence to be expressed (for procedures, see, eg, Sambrook et al., 1989) , the above plasmid construct (Example 1) was used to transfect human cells, and their expression was determined therein. Briefly, the following methods were employed: Human HEK 23 cells (ATCC, CRL 1573, transformed primary cells of human embryonic kidney) were transfected with plasmid construction pCDNA3-slL-6R / IL-6 DNA (described in example 1 above) . The logarithmic phase cultures of HEK 293 cells were treated with trypsin and seeded on 9 cm Nunc plates (2.5x10 6 cells / plate). One day later, transfection was carried out with 10 μg of pCDNA3-slL-6R / IL-6 DNA by the CaP04 precipitation method (Sambrook et al., 1989), and one hour later the medium was changed to DMEM- FCS at 10%, and the culture was continued for another 16 hours. After changing the medium to DMEM-FCS at 2%, the secreted proteins were collected for 2 consecutive periods of 48 hours. The remains were removed by centrifugation at 1,000 rpm for 10 minutes, and the supernatant was tested by ELISA test for slL-6R using rabbit polyclonal anti-slL-6R and mouse McAB 17.6 (Novick et al., 1991 ). A concentration of 1.2 μg / ml equivalents of slL-6R was found, indicative of highly efficient expression of the chimeric slL-6R / IL-6 protein in transfected human cells. The immunopurification of the secreted chimeric protein (slL-6R / IL-6) was carried out with monoclonal antibody 34.4 specific for an epitope in the extracellular domain of human slL-6R (Novick et al., 1991; Halimi et al., 1995; ). Hybridoma 34.4 cells were cultured in the peritoneal cavity of mice, and the immunoglobulin (Ig) fraction of the ascites fluid was obtained by precipitation with ammonium sulfate. Affigel-10 (Bio-Rad Labs, Richmond, California) was used to immobilize McAB 34.4 (15 mg of Ig coupled to 1 ml of Affigel-10). Supernatants containing the secreted proteins of HEK 293 cells transfected by pCDNA3-slL-6R / IL-6 were adsorbed onto McAB 34.4 columns (0.3 ml column for 15 ml supernatant). After washing with PBS, the bound proteins were eluted by citric acid at 25 mM, pH 2.5, then immediately neutralized by pH buffer Hepes at 1 M, pH 8.5, and dialysed overnight (approximately 8 to 12 hours) against PBS . The analysis of the immunopurified protein by polyacrylamide gel electrophoresis in SDS showed a single protein band stained by Coomassie blue (figure 2). The molecular weight of the protein was 85 kilodaltons, as expected from the fusion of the glycosylated forms of slL-6RdVal (60 kDa, as shown in Oh et al., 1996) and glycosylated IL-6 (23- 26 kDa, as shown in Zilberstein et al., 1986). The Y __*_*___- ' ? * .- * "_. < * J _____ §_El = _ ^ Í_í _ @ _ §llíí _'_-_? ___ J ^ '- ^ amino acid sequence of slL-6R / IL-6 is 543 amino acids, which after processing the signal peptides would test a protein of 524 amino acids or approximately 58 kDa (figure 3). The much larger size of the slL-6R IL-6 chimera produced from recombinant DNA in human cells indicates that glycosylation constitutes a measurable portion of the molecule.

EXAMPLE 3 Chimera slL-6R IL-6 stops growth and induces differentiation of metastatic melanoma cells Clone F10.9 derived from B16 melanoma cells forms highly metastatic tumors in C57Black / 6 mice, which cause their death by pulmonary metastasis after 2 to 3 months (Katz et al., nineteen ninety five). The addition of the chimeric protein slL-6R / IL-6 to the culture of F10.9 cells produces a profound morphological change in the cells, and arrest of their growth (figure 4). The F10.9 cells treated by the chimera become elongated, with protruding dendritic extensions, resembling the husoid differentiation of embryonic melanocytes or glial cells. Cell growth was quantified 4 days after seeding 3x103 cells in wells of a 96-well microplate in 0.2 ml of RPMI 1640 medium with 10% FCS. Cells were fixed in glutaraldehyde at 12.5% for 30 minutes, washed in water and stained with 0.1% violet crystal for 30 minutes. After washing and drying completely, the dye was extracted with 10% acetic acid, and the optical density was determined at 540 nm. The chimera produced a dose-dependent growth inhibition with complete inhibition of growth at concentrations as low as 10 ng / ml of the chimeric protein (p85) (FIG. 5). The chimeric proteins slL-6RdVal / IL-6 and slL-6RdVal / L / IL-6 (chimera with the longest linker between the portions of slL-6R and IL-6, see Example 1), were active in the same way. This result also serves to show that the peptide linker between the portions of sIL-6R and IL-6 in the chimera is not essential for the activity of the chimera, since the chimera slL-6RdVal / IL-6 above has only one very short linker of 3 amino acids, whereas the chimera slL-6RdVal / L / IL-6 above has a longer linker peptide of 13 amino acids, but both show essentially the same activity to inhibit the growth of metastatic cells. In contrast, neither IL-6 alone, nor slL-6RdVal alone, inhibits the growth of these melanoma cells (Figure 6), demonstrating the unique activity of the chimeric protein slL-6R / IL-6 (p85). To obtain a similar effect, a mixture of 200-400 ng / ml of IL-6 and 125 ng / ml of slL-6RdVal is required (figure 6). When calculated in molar concentrations, maximal inhibition of F10.9 cells requires IL-6 at 7.5 nM and slL-6RdVal at 2 nM, against only 0.12 nM of the SIL-6R / IL-6 chimera. The growth inhibitory activity of the chimeric protein SIL-6R / IL-6 p85 was followed during the immunopurification on McAB 34.4 columns (see Example 2). The activity pattern corresponded to the intensity of the p85 band observed in the different fractions of SDS polyacrylamide gel electrophoresis in Figure 2.

EXAMPLE 4 Chimera slL-6R / IL-6 is essential for grafting transplanted human bone marrow cells The grafting of human bone marrow hematopoietic stem cells can be studied after transplantation in combined severe immunodeficient mice (SCID) (Vormoor et al., 1994). SCID-NOD mice were subjected to sublethal irradiation, and injected into the tail vein with 3x105 human bone marrow CD34 + cells. Prior to injection, purified CD34 + cells were maintained for 3 days in liquid cultures with different combinations of cytokines. After one month, the mice were sacrificed and bones were removed to collect bone marrow cells. The grafting of human cells into the SCID-NOD ptor mice was evaluated by means of Southern blot hybridization for human repetitive DNA. It has been found that stem cell factor (SCF, factor steel or ligand ckit) and ligand Flt3 (ptor ligand flt3 / tyrosine kinase flk2) are important for the survival and proliferation of the most primitive pluripotent hematopoietic stem cells capable of being grafted long-term into pient bone marrow (McKenna et al., 1995). As seen in Figure 7, these two factors by themselves were insufficient for bone marrow of the SCID-NOD ptor mice. The adra f of the chimeric protein slL-6R / IL-6 was required for the graft to be defecated at significant levels. At 100 ng / ml, SIL-6R / IL-6 chimera was much more active than isolated IL-6 (50-200 ng / ml) and SIL-6R (125-1250 ng / ml) (figure 7). and termination of chimera slL-6R / IL-6 indicates that this protein is essential for the survival and proliferation of non-compromised pluripotent hematopoietic stem cells, which can be housed in, and repopulated, the environment of the bone marrow, indicating that this protein may be useful in the clinical protocols of bone marrow transplantation. This is the first demonstration that the slL-6R / IL-6 chimera has at least the following two newly found activities: (i) when it is added together with the SCF and ligand Flt3 factors to primitive human hematopoietic progenitor cells, promotes its survival and proliferation; and (ii) it is active (and apparently essential) in an in vivo model of a human bone marrow transplant in immunodeficient mice. highly purified primitives Human umbilical blood mononuclear cells were subjected to fractionation of low density mononuclear cells (NMC) on Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) followed by the use of a MACS mini-kit (Miltney Biotec, Bergisch Gladbach, Germany), to prepare an 80% pure population of CD34 + cells. These cells were then passed on immobilized anti-CD38 monoclonal antibody or clustered by fluorescence-activated cell selection, and the CD34 + CD38 cell population was vered, which corresponds to approximately 0.1% of the original cells. (20,000 cells) were placed in suspension cultures in 0.5 ml of RPMI medium, 10% fetal calf serum (FCS), 1% bovine serum albumin containing 50 ng / ml of stem cell factor (SCF) and 100 ng / ml ligand flt3 (FL) (both from R &D Systems, Minneapolis, MN) Half of the cultures were supplemented with 100 ng / ml chimera slL-6R / IL-6, and the others were The incubation was carried out at 37 ° C in C0 at 5%, for 6 days The number of cells that returned to colonize the bone marrow was evaluated by intravenous injection of all the cells from these cultures in vitro in NO mice D-SCID subletically irradiated. The mice were kept in germ-free conditions. After 6 weeks, the mice were sacrificed, and the bone marrow vered from their long bones. These bone marrow (BM) cells were deposited on 0.9% semisolid methyl cellulose plates with 30% FCS, 50 μM β-mercaptoethanol, 50 ng / ml SCF, 5 ng / ml IL-3, 5 ng / ml of GM-CSF and 6 u / ml of erythropoietin (all from R & amp; amp; amp;; D Systems). The cultures also contained human serum, conditions that prevent the growth of mouse cell colonies. The results (Table IV) indicated that the addition of the slL-6R / IL-6 chimera to the suspension cultures produced an increase of 30 to 50 times the number of units of human colony-forming cells (CFU) recovered from the mice transplanted, comparatively with SCF and FL alone. This represents a large increase in the number of stem cells from recolonizing SCID mice present in the suspension cultures on day 6, compared to day 0. In the absence of the SIL-6R / IL-6 chimera, SCF and FL did not produce any increase in the number of stem cells during the 6 days of culture in suspension. The DNA of the BM cells recovered from the transplanted NOD / SCID mice was analyzed by Southern blot as in example 4. The amount of human DNA recovered was 10 times higher when the mice received the cells cultured with chimera, compared to chimera. The CFU progenitors of the bone marrow of NOD / SCID mice as in Table IV, gave rise to hematopoietic cells of different myeloid lineages (macrophages and granulocytes), as well as erythroid and lymphoid lineages (eg, CD19 +, CD56 +) only when human blood cells had been cultured with the slL-6R / IL-6 chimera before transplantation.

TABLE IV Human stem cells capable of re-colonizing the bone marrow of NOD / SCID mice Additions during Days of culture Number of colonies culture in suspension of hematopoietic cells CD34 + CD38"human formed to human blood from BM of umbilical mice NOD / SCID transplanted 0 4 SCF + FL 6 2-3 SCF + FL * SIL- 6R / IL-6 6 50-100 In additional experiments the effect of slL-6R IL-6 on the population of CD34 + CD38 + cells in umbilical blood was compared with the effect on the highly purified CD34 + CD38 stem cells.The in vitro expansion of highly purified cells was much more markedly increased by SIL-6R / IL-6 than that of less purified cells (Table V) .This indicates that the most primitive stem cells are the preferential target of the effect of sIL-6R / IL-6 on the expansion of the cells.

CUADR® V In vitro expansion of hematopoietic stem cells Cell population Number of cells in number of cells in sown (20,000 on day 6 with SC + FL on day 6 with SC + FL + cells) SIL-6R / IL-6 Experiment 1 CD34 + CD38 + 780,000 675,000 (x 0.86) CD34 + CD38"42,000 153,000 (x 3.6) Experiment 2 CD34 + CD38 + 330,000 507,000 (x 1.5) CD34 + CD38"3,000 18,000 (x 6.0) In vitro maintenance of the bone marrow recolonizing activity was measured by increasing the duration of the suspension of cultures of CD34 + CD38"highly purified stem cells before being injected into the NOD / SCID mice.The graft was evaluated by the proportion of DNA in the bone marrow of recipient mice 6 weeks after iv injection of cultured cells When SIL-6R / IL-6 was added to SCF and FL during cultures, a high graft potential was 1% of human DNA) after two weeks of culture, and the graft was higher than in the uncultivated cells.In contrast to this, experiments with cultures containing SCF, FL, GM-CSF and IL-3 have shown that no cells of recolonizing SCID mice remain after one week of culture (Bhata, M. et al., J: Exp. Med. 186, 619-624, 1997.) These results show that SIL-6R / IL-6 allows to expand and maintain human primitive stem cells ca be grafted from being grafted into the recipient bone marrow. The stem cells remain active in an undifferentiated state, while multiplying. The SIL-6R / IL-6 chimera provides new means to grow hematopoietic cells that can be grafted. This may also allow the use of retroviral vectors to introduce genes into stem cells that can be grafted, into gene therapy protocols. So far, this has not been possible with human stem cells because these primitive cells could not be maintained in vitro in the circulating state, as required for the integration of retroviral DNA. Chimera slL-6R / IL-6 solves this problem.

EXAMPLE 6 Production of the IL-6R / IL-6 chimera in CHO cells Plasmid DNA SIL-6R / IL-6 pcDNA3 as in the figure, was co-transfected into Chinese hamster ovary (CHO) cells, together with plasmid DNA pDHFR, as described in Mory et al. (DNA 5, 181-193 , 1986). Among the transfectants growing in methotrexate at 50 nM, clone L12- was isolated fi & r.- [IL-6R / IL-6]. It was found that this clone is stable for many passages, and semi-effluent cultures usually secrete 2.5 μg / ml of the IL-6R / IL-6 chimera into the culture medium. For the purification of IL-6R / IL-6 chimera, 3.25 liters of culture medium of clone L12 in 2% bovine serum were concentrated to 200 ml. This was adsorbed onto an 18 ml column of anti-human sIL-6R monoclonal antibody 34.4 coupled to Affigel 10 spheres, and eluted as described (Novick et al., Hybridoma, 10, 137-146, 1991). A product eluted from citric acid at 25 mM was immediately neutralized with pH regulator Hepes, pH 8.6. The proteins were concentrated on an Amicon membrane for 10 kDa separation at a final concentration of 1 mg / ml. After SDS-PAGE, an individual band of 85 kDa was observed corresponding to the IL-6R / IL-6 chimera. Glycosylation was demonstrated by reduction in size after glucosidase treatment (Boehringer, Mannheim). It was found that the biological activity of the IL-6R IL-6 chimera produced by CHO cells is stable for at least 5 months at 4 ° C. Usually, storage is at -70 ° C.finity of IL-6R chimera IL-6 by qp130 The IL-6R / IL-6 chimera produced by CHO cells and a mixture of IL-6 and human slL-6R were compared in terms of their binding to the soluble form of gp130 (sgp 130), which is the second chain of the receptor system for IL-6 (see background). A 96-well microtitre plate (Nunc) was coated with monoclonal antibody gp130 ant? human, and 50 ng / ml of sgp130 (both from R &D Systems, Minneapolis) were added. After washing in phosphate-buffered saline, the IL-6R / IL-6 chimera was added in different wells at different concentrations ranging from 0.1 to 50 ng / ml. In separate wells, rhulL-6 (Ares-Serono, Geneva) was added at 500 ng / ml together with human slL-6RdVal at concentrations of 2 to 500 ng / ml. After incubation overnight at 4 ° C, rabbit polyclonal anti-IL-6R (Oh et al., Cytokine, 8, 401-409, 1996) was added, followed by goat anti-rabbit Ig conjugated with horseradish peroxidase. , which was detected by colored reaction (Sigma, St. Louis). Figure 8 shows a Scatchard plot of the results. It was found that the affinity of the IL-6R / IL-6 chimera for gp130 is four times higher than that of the two parts of the molecule added separately (6.3 x 10"1M versus 2.6 x 10" 10M). This result is online and explains the greater activity of the chimera, in comparison with the IL6 + slL-6R combination on melanoma cells and on hematopoietic cells (figure 9 and example 4).

EXAMPLE 8 The IL-6R / IL-6 chimera provides protection against hepatotoxicity Injection of carbon tetrachloride (CCI4) in mice produces severe necrosis of the liver, which leads to the death of the animals (Slater TF et al., Philos. Trans. R. Soc. Biol. Sci. 311, 633-645, 1985). When mice which are genetically deficient in IL-6 (IL-6'A) are given relatively low doses of CCI4 (2-3 ml / kg body weight) by intraperitoneal injection, lethality rates at 24 hours are of around 70% (Fig. 10). The injection of the IL-6R / IL-6 chimera produced by CHO cells one hour before the administration of CCI4 and again 4 hours after the administration thereof, protects the animals and none of them dies at 24 hours. In contrast, free rhulL-6 injected in the same way had no effect (Fig. 10). The chimera IL-6R IL-6 was effective at doses of 2 to 3 μg per injection, which in molar ratio are 10 times lower than the dose of IL-6, which was not effective. At higher doses of CCI4 (for example, 3.5 ml / kg in Figure 10), the chimera also provided protection, with mortality being lower than with IL-6 or without cytokine. The difference in mortality between mice treated with the chimera and mice not treated with it, both receiving the same dose of CCI, was significant at p < 0.01. Histological observation of liver sections stained with hematoxylin-eosin confirmed that CCI4 produces liver tissue necrosis, and that the IL-6R / IL-6 chimera protects hepatocytes from this toxic chemical effect (not shown). ___ £ & _ * fc? * _r. • 3ü-3 * __ &- An application of the IL-6R / IL-6 chimera may be for the protection of liver tissue in patients with necrotic diseases due to chemical compounds (eg alcohol, paracetamol) or other causes (for example, viral hepatitis).

EXAMPLE 9 Construction and activity of IL-6 / slL-6RdVal chimaera A chimeric molecule was constructed in which the IL-6 portion is at the N-terminus, while the slL-6R portion is at the C-terminus. The plasmid pBS-slL-6RdVal was cut in Sau3a (bp 1086), and in Hindlll after the stop codon after Val-356 (see example 1). A linker containing three restriction sites was synthesized in the following manner: Spel, Smal and BamHl: Spel Smal BamHl 5 'CT AGT GGG CCC GGG GTG GCG GG A CCC GGG CCC CAC CGC CCC TAG_5_' (SEQ ID NO: 2) This Sau3a site of slL-6R was ligated to the BamH1 of the linker and cloned into the multiple cloning site of a Bluescript pBS SK plasmid. The IL-6 sequence was amplified by PCR from pKKß2-7 DNA using the primers (underlined initiation codon): Spel Forward 5 'GA CTA GTA GCT ATG AAC TCC TC TC (SEQ ID NO: 3) Haelll Back 5 'AG GGC CAT TTG CCG AAG AGC C (SEQ ID NO: 4) The PCR product cut with Spel and Haelll was introduced between the Spel and Smal sites of the previous linker. Another linker BamHl -Ncol with an internal Smal was synthesized as follows: Smal 5 'GAT CCG GGC GGC GGG GGA GGG GGG CCC GGG C [Ncol] [BamHl] GC CCG CCG CCC CCT CCT CCC GGG CCC GGT AC 5' (SEQ ID NO: 5) This was cloned between BamHI of the previous linker and Ncol 1464 of the IL-6R sequence. A fragment of IL-6R from Smal 867 for Ncol 1464 was then introduced between Smal of the second linker and Ncol of IL-6R. The resulting chimeric DNA was sequenced and recloned in pCDNA3 for expression in human HEK 293 cells. The amino acid sequence of this chimera IL-6-IL-6R 3e is shown in Figure 11 (underlined linker). Chimera 3e was purified by affinity chromatography on an anti-IL-6 monoclonal antibody (as in Novick et al., Hybridoma 8, 561-567, 1989).

On SDS-PAGE, a band of 75 kDa was observed. The biological activity of IL-6-IL-6R 3e chimera to inhibit the growth of F10.9 melanoma cells is shown in Figure 12. It is clearly active comparatively with IL-6R / IL-6 chimera (preparation 1 -3) of the same experiment, although a greater amount of it is required to achieve 50% inhibition of growth. Two IL-6R / IL-6 mutants were obtained in which the His-280 and Asp-281 amino acids of the IL-6R portion of IL-6R / IL-6 (Figure 3) were respectively changed by Ser and Val by mutagenesis induced by PCR (mutant 39 or HD), or where Asn-230 was also changed by Asp (mutant NHD). As can be seen from figure 12, these two mutants had almost no activity, in comparison with the chimeras IL-6R / IL-6 and IL-6-IL-6R. Since in IL-6R these amino acids interact with gp 130, as shown by the formation of molecular models (Halimi et al., 1995), this shows that the SIL-6R / IL-6 chimera retains this essential interaction site. Chimera IL-6-IL-6R 3e is lacking in the immunoglobulin-like domain of IL-6R, which is present in IL-6R / IL-6. However, removing only this Ig domain of IL-6R / IL-6 did not reduce its biological activity on F10.9 cells. The binding of IL-6-IL-6R 3e chimera to gp130 was approximately 30% that of another IL-6R / IL-6 chimera (not shown).

This minor binding is in line with the minor activity on the growth of melanoma cells.

These results demonstrate that blocking the carboxy terminus of IL-6 by fusion through a linker with slL-6R, retains a good biological activity in said novel chimeras.

REFERENCES Chen L, Mory Y, Zilberstein A and Revel, M. Growth inhibition of human breast carcinoma and leukemia / lymphoma cell lines by recombinant interferon-beta 2 / IL-6. Proc. Natl. Acad. Sci. USA, 85: 8037-8041, 1988. Chernajovsky Y, Mory Y, Chen L, Marks Z, Novick D, Rubinstein M and Revel M. Efficient constitutive production of human fibroblast interferon by hamster cells transformed with IFN-ß1 gene fused to an SV40 early promoter. DNA, 3: 297-308, 1984. Fischer M, Goldschmitt J, Peschel C, Brakenhoff JPG, Kallen K-J, Wollmer A, Grotzinger J and Rose-John S. A bioactive designer cytokine for human hematopoietic progenitor cell expansion. Nature Biotechnology 15: 142-145, 1997. Ganapathi MK, Weizer AK, Borsellino S, Bukowski RM, Ganapathi S, Rice T, Casey G and Kawamura K. Resistance to lnterleukin-6- in human Non-small cell lung carcinoma cell lines : Role of receptor components. Cell Growth and Differentiation, 7: 923-929, 1996. Halimi H, Eisenstein M, Oh J, Revel M and Chebath J. Epitope peptides from interleukin-6 receptor which inhibits the growth of human myeloma cells. Eur. Cytokine Netw., 6: 135-143, 1995.

£ ¡| ^ | «Gj- Hirano T, Yasukawa K, Harada H, Taga T, Watanabe Y, Matsuda T, Kashimura S. Nakajima K, Koyama K, Iwamatsu K, Tsunasawa S, Sakiyama F, Matsuí H, Takahara Y, Taniguchi T and Kishimoto T. Complementary DNA for a novel interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulins. Nature, 234: 73-76, 1986. Hirano T, Matsuda T and Nakajima K. Signal transduction through gp 130 that is shared among the receptors for the interleukin 6 related cytokine sufamily. Stem cells, 12: 262-227, 1994. Holloway CJ. Applications of recombinant DNA technology in the production of glycosylated recombinant human granulocyte colony stimulation factor. Eur. J. Cancer, 30: S2-6, 1994. Kahn MA and De Vellis J. Regulation of an oligodendrocyte progenitor cell line by the interleukin-6 family of cytokines. Glia, 12: 87-98, 1994. Katz A, Shulman LM, Pergador A, Revel M, Feldman M and Eisenbach L. Abrogation of B16 melanoma metastases by long-term low-dose lnterleukin-6 therapy. J. Immunother. 13: 98-109, 1993. Mackiewicz A, Winznerowicz M, Roeb E, Nowak J, Pawlowski T, Baumann H, Heinrich P and Rose-John S. lnterleukin-6-type cytokines and their receptors for gene therapy of melanoma. Ann. New York Acad. Sci., 762: 361-374, 1995. McKenna HJ, de Vries P, Brasel K, Lyman SD and Williams DE. Efect of flt3 ligand on the ex vivo expansion of human CD34 + hematopoietic progenitor cells. Blood 86: 3413-3420, 1995.

Murakami M, Hibi M, Nakagawa N, Nagakawa T, Yasukawa K, Yamanishi K, Taga T and Kishimoto T. IL-6 induced homodimerization of gp130 and associated activation of a tyrosine kinase. Science, 260: 1808-1810, 1993. Novick D, Englemann H, Wallach D, Leitner O, Revel M and Rubinstein M. Purification of soluble cytokine receptors from normal urine by lingad-affinity and immunoaffinity chromatography. J. Chromatogr., 510: 331-337, 1990. Novick D, Engelmann H. Revel M, Leitner O and Rubinstein M. Monoclonal antibodies to the soluble IL-6 receptor: affinity purification, ELISA and inhibition of ligand binding. Hybridoma, 10: 137-146, 1991. Novick D, Shulman LM, Chen L and Revel M. Enchancement of interleukin-6 cytostatic effect on human breast carcinoma cells by soluble IL-6 receptor from urine and reversion by monoclonal antibodies. Cytokine, 4: 6-11, 1992. Oh J-W, Revel M and Chebath J. A soluble nterleukin-6 receptor isolated from conditioned medium of human breast cancer cells is encoded by a differentially spliced mRNA. Cytokine, 8: 401-409, 1996. Oh J-W. Expression of recombinant soluble human interleukin-6 receptors and analysis of their functions. Doctoral thesis, Weizmann Institute of Science (Revel M, supervisor), 1997. Paonessa G, Graziani R, DeSerio A, Savino R, Ciapponi L, Lahmm A, Salvati AL, Toniatti C and Ciliberto G. Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling. EMBO J., 14: 1942-1951, 1995.

Revel M. Host defense against infections and inflammations: Role of the multifunctional IL-6 / IFN-β2 cytokine. Experientia 45: 549-557, 1989. Sambrook J, Fritsch EF and Maniatis T. Molecular cloning: A laboratory manual. Cold Spring Harbor Press, 1989. Sui X, Tsuji K, Tanaka R, Tajima S, Muraoka K, Ebihara Y, Ikebuchi K, Yasukawa K, Taga T, Kishimoto T and Nakahata T. Gp130 and c-kit signalings synergize for ex vivo expansion of human primitive hemopoietic cells. Proc. Natl. Acad. Sci. USA 92: 2859-2863, 1995. Taga T, Hibi M, Hirata Y, Yamasaki K, Yasukawa K, Matsuda T, Hirano T and Kishimoto T. lnterleukin-6 triggers the association of its receptor with a possible signal transducer gp 130. Cell, 58: 573-581, 1989. Vormoor J. Lapidot T, Pflumio F, Risdon G, Patterson B, Broxmeyer HE and Dick JE. SCID mice as an in vivo model of human cord blood hematopoiesis. Blood cells 20: 316-320, 1994. Ward LD, Howlett GJ, Disciple G, Yasukawa K, Hammacher A, Moritz RL and Simpson RJ. High affinity interleukin-6 receptor is a hexameric complex consisting of two molecules each of interleukin-6, interleukin-6 receptor and gp130. J. Biol. Chem., 269: 23286-23289, 1994. Yamasaki K, Taga T, Hirata Y, Yawata H, Kawanishi Y, Seed B, Tanguchi T, Hirano T and Kishimoto T. Cloning and expression of the human interleukin- 6 (BSF-2 / Interferon beta-2) receptor. Science, 241: 825-828, 1988. A, Ruggieri R, Korn HJ and Revel M, Structure and expression of cDNA and genes for human interferon-beta-2, a distinct species inducible by growth-stimulatory cytokines. EMBO J "5: 2529-2537, 1986.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Yeda Research and Development Co. Ltd.

(B) STREET: Weizmann Institute of Science, P.O.B. 95 (C) CITY: Rehovot (E) COUNTRY: Israel (F) ZIP CODE (ZIP): 76100 (G) TELEPHONE: 972-8-9344093 (H) TELEFAX: 972-8-9470739 A) NAME: REVEL, Michel (B) STREET: Beit Brazil 5, Weizmann Institute of Science C) CITY: Rehovot E) COUNTRY: Israel F) ZIP CODE: 76100 (A) NAME: CHEBATH, Judith B) STREET: Rehov Miller 13 C) CITY: Rehovot E) COUNTRY: Israel (F) ZIP CODE: 76284 (A) NAME: LAPIDOT, Tsvee (B) STREET: Rehov Boxer 6 (C) CITY: Ness-Ziona (E) COUNTRY: Israel (F) ZIP CODE: 74046 (A) NAME: KOLLET, ORIT (B) STREET: Rehov Ramat Chen 14 (C) CITY: Ramat Gan (E) COUNTRY: Israel (F) ZIP CODE (ZIP): 52232 (ii) TITLE OF THE INVENTION: Chimeric receptor / ligand of soluble heteroferin-6, analogous thereof and uses thereof (iii) SEQUENCE NUMBER: 8 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: FLEXIBLE DISC (B) COMPUTER: COMPATIBLE WITH IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAMS: Patentln Relay # 1.0, Version # 1.30 (EPO) (vi) PREVIOUS INFORMATION OF THE APPLICATION: (A) APPLICATION NUMBER: IL 121284 (B) DATE OF SUBMISSION: JULY 10, 1997 (vi) PREVIOUS INFORMATION OF THE APPLICATION: (A) APPLICATION NUMBER: IL 122818 (B) DATE OF SUBMISSION: 30-DEC-1997 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Glu Phe Gly Wing Gly Leu Val Leu Gly Gly Gln Phe Met 1 5 10 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: CTAGTGGGCC CGGGGTGGCG GGACCCGGGC CCCACCGCCC CTAG_44_(2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple "Tfrlftfi 'thiiliil. I (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: GACTAGTAGC TATGAACTCC TTCTC 25 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: AGGGCCATTT GCCGAAGAGC C 21 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: GATCCGGGCG GCGGGGGAGG GGGGCCCGGG CGCCCGCCGC CCCCTCCTCC CGGGCCCGGT 60 AC 62 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 6: < *. % Gly Gly Gly Gly Asp Pro Gly Gly Gly Gly Gly Gly Pro Gly 1 5 10 5 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 543 amino acids 10 (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide 15 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: Met Leu Ala Val Gly Cys Ala Leu Leu Ala Ala Leu Leu Ala Ala Pro 20 1 5 10 15 Gly Ala Ala Ala Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala Ala 20 25 30 < - * i iy &x * £ »,» i & li Gly Val Leu Thr Ser Leu Pro Gly Asp P to Thr Leu Thr Cys Pro 35 4f- 45 Gly Val Glu Pro Glu Asp Asn Wing Thr \ J # £ His Trp Val Leu Arg Lys 50 55 60 Pro Ala Ala Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg 65 70 75 80 Leu Leu Leu Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys 85 90 95 Tyr Arg Wing Gly Arg Pro Wing Gly Thr Val His Leu Leu Val Asp Val 100 105 110 Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro Leu Ser 115 120 125 Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr Thr 130 135 140 Lys Ala Val Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp 145 150 155 160 «* - ** - * - - * Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys 165 170 175 Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe Tyr lie Val Ser Met 180 185 190 Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe 195 200 205 Gln Gly Cys Gly lie Leu Gln Pro Asp Pro Pro Wing Asn lie Thr Val 210 215 220 Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp 225 230 235 240 Pro His Ser Trp Asn Ser Being Phe Tyr Arg Leu Arg Phe Glu Leu Arg 245 250 255 Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp 260 265 270 Leu Gln His His Cys Val lie His Asp Ala Trp Ser Gly Leu Arg His 275 280 285 Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser 290 295 300 Glu Trp Ser Pro Glu Wing Met Gly Thr Pro Trp Thr Glu Ser Arg Ser 305 310 315 320 Pro Pro Wing Glu Asn Glu Val Being Thr Pro Met Gln Wing Leu Thr Thr 325 330 335 Asn Lys Asp Asp Asp Asn He Leu Phe Arg Asp Ser Wing Asn Wing Thr 340 345 350 Ser Leu Pro Val Glu Phe Met Pro Val Pro Pro Gly Glu Asp Ser Lys 355 360 365 Asp Val Ala Ala Pro His Arg Gln Pro Leu Thr Ser Ser Glu Arg He 370 375 380 Asp Lys Gln He Arg Tyr He Leu Asp Gly He Ser Wing Leu Arg Lys 385 390 395 400 Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Ser Ser Lys Glu Ala Leu 405 410 415 Ala Glu Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu Val Lys He lie Thr 435 440 445 Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe 450 455 460 Glu Be Ser Glu Glu Gln Ala Arg Ala Val Gln Met Ser Thr Lys Val 465 470 475 480 Leu He Gln Phe Leu Gln Lys Lys Wing Lys Asn Leu Asp Wing He Thr 485 490 495 Thr Pro Asp Pro Thr Thr Asn Wing Ser Leu Leu Thr Lys Leu Gln Wing 500 505 510 Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His Leu He Leu Arg Ser 515 520 525 Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala Leu Arg Gln Met 530 535 540 (2) INFORMATION FOR SEQ NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 471 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala Phe Ser Leu 1 5 10 15 Gly Leu Leu Leu Val Leu Pro Ala Wing Phe Pro Pro Pro Pro Wing 20 25 30 Gly Glu Asp Ser Lys Asp Val Ala Pro Wing His Arg Gln Pro Leu Thr 40 45 Ser Ser Glu Arg He Asp Lys Gln lie Arg Tyr lie Leu Asp Gly He 50 55 60 Be Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Ser 5 65 70 75 80 Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala 85 90 95 Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu 100 105 110 Val Lys He lie Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr 115 120 125 15 Leu Gln Asn Arg Phe Glu Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135 140 Met Ser Thr Lys Val Leu lie Gln Phe Leu Gln Lys Lys Ala Lys Asn 20 145 150 155 160 Leu Asp Ala He Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170 175 ftMMM_if > M_ír * f, '-,' - ff? Thr Lys Leu Gln Wing Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His 180 185 190 Leu He Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala 195 200 205 Leu Arg Gln Met Gly Gly Gly Gly Asp Pro Gly Gly Gly Gly Gly Gly 210 215 220 Pro Gly Val Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser 225 230 235 240 Pro Leu Ser Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser 245 250 255 Leu Thr Thr Lys Wing Val Leu Leu Val Arg Lys Phe Gln Asn Ser Pro 260 265 270 Wing Glu Asp Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys 275 280 285 Phe Ser Cys Gln Leu Wing Val Pro Glu Gly Asp Ser Ser Phe Tyr He 290 295 300 í _, "_ í_» ííÉ ._ # __ l__ .. ._ &.3? E _ £ ___ B ___ SSíÉ_¡ _.-.

Val Ser Met Cys Val Wing Ser Ser Val Gly Ser Lys Phe Ser Lys Thr 305 310 315 320 Gln Thr Phe Gln Gly Cys Gly He Leu Gln Pro Asp Pro Pro Wing Asn 325 330 335 He Thr Val Thr Wing Val Wing Arg Asn Pro Arg Trp Leu Ser Val Thr 340 345 350 Trp Gln Asp Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe 355 360 365 Glu Leu Arg Tyr Arg Wing Glu Arg Ser Lys Thr Phe Thr Thr Trp Met 370 375 380 Val Lys Asp Leu Gln His His Cys Val He His Asp Wing Trp Ser Gly 385 390 395 400 Leu Arg His Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly 405 410 415 Glu Trp Ser Glu Trp Ser Pro Glu Wing Met Gly Thr Pro Trp Thr Glu 420 425 430 Have you been Arg Ser Pro Pro Wing Glu Asn Glu Val Being Thr Pro Met Gln Wing 435 440 445 Leu Thr Thr Asn Lys Asp Asp Asp Asn He Leu Phe Arg Asp Ser Wing 450 455 460 Asn Ala Thr Ser Leu Pro Val 465 470

Claims (31)

NOVELTY OF THE INVENTION CLAIMS

1. - A soluble interleukin-6 receptor chimeric protein (slL-6R) -interleucine-6 (IL-6) (slL-6R / IL-6) glycosylated, and biologically active analogues thereof, characterized in that it comprises a fusion protein product between essentially all the slL-form 6R that occurs naturally and essentially all naturally occurring form of IL-6, said slL-6R / IL-6 and analogs thereof being glycosylated in a manner similar to the glycosylation of naturally occurring slL-6R and IL-6.

2. The chimeric slL-6R / IL-6 protein and biologically active analogs thereof according to claim 1, further characterized in that said slL-6R is fused to IL-6 by a peptide linker molecule.

3. The chimeric slL-6R / IL-6 protein and biologically active analogs thereof according to claim 2, further characterized in that said linker is a very short non-immunogen linker of about 3 amino acid residues.

4. The chimeric slL-6R / IL-6 protein and biologically active analogs thereof according to claim 3, further characterized in that said linker is a tripeptide of sequence E-F-M (Glu-Phe-Met).

5. - The chimeric slL-6R / IL-6 protein and biologically active analogs thereof according to claim 2, further characterized in that said linker is a peptide of 13 amino acid residues of sequence EFGAGLVLGGQFM (Glu-Phe-Gly-Ala- Gly-Leu-Val-Leu-Gly-Gly-GIn-Phe-Met).

6. The chimeric SIL-6R / IL-6 protein according to any of claims 1 to 4, being designated herein as sIL-6RdVal / IL-6 and having a tripeptide linker of EFM sequence between Val-356 C-terminal of slL-6R and the Pro-29 N-terminus of IL-6R, said chimeric protein having the sequence described in figure 3.

7.- The chimeric SIL-6R / IL-6 protein in accordance with any of claims 1, 2 and 5, being designated herein as sIL-6RdVal / L / IL-6 and having a 3 amino acid peptide linker of sequence EFGAGLVLGGQFM between the C-terminal Val-356 of slL-6R and the Pro -29 N-terminal of IL-6R, said chimeric protein having the sequence described in Figure 3, wherein the tripeptide of the EFM sequence between positions 357-359 of Figure 3 is replaced by said sequence of 13 amino acid peptides .

8. The chimeric SIL-6R / IL-6 protein according to claim 1, being designated herein as IL-6 / slL-6R and having the complete sequence of IL-6 that precedes the sequence of slL- 6 with a 14 amino acid peptide linker of sequence GGGGDPGGGG-GGPG (SEQ ID NO: 6) between the C-terminal Met-212 of IL-6 and Val-112 of sIL-6R, said chimeric protein having the sequence described in Figure 11.

9. The chimeric slL-6R / IL-6 protein according to any of claims 1 to 8, further characterized in that said protein is produced in mammalian cells in fully processed form.

10. The chimeric slL-6R / IL-6 protein according to claim 9, further characterized in that said protein is produced in human cells.

11. The chimeric SIL-6R / IL-6 protein according to claim 9, further characterized in that said protein is produced in CHO cells.

12. The chimeric SIL-6R / IL-6 protein and biologically active analogs thereof according to any of claims 1 to 11, further characterized in that said chimeric protein and analogs thereof are characterized as being capable of inhibiting the growth of highly malignant cancer cells.

13. The chimeric SIL-6R / IL-6 protein and biologically active analogs thereof according to claim 12, further characterized in that said chimeric protein and analogs thereof are characterized as being capable of inhibiting the growth of highly malignant melanoma.

14. The chimeric slL-6R / IL-6 protein and biologically active analogs thereof according to any of claims 1 to 11, further characterized in that said chimeric protein and analogs thereof are characterized by being capable of inducing the in vivo grafting of human hematopoietic cells into bone marrow transplants.

15. The chimeric slL-6R / IL-6 protein and biologically active analogs thereof according to any of claims 1 to 11, further characterized in that said chimeric protein and analogs thereof are characterized by being able to protect the liver from hepatotoxic agents.

16. A DNA sequence encoding a chimeric sIL-6R / IL-6 protein and biologically active analogs thereof according to any one of claims 1 to 11.

17. A DNA vector comprising a sequence of DNA encoding a chimeric SIL-6R / IL-6 protein and biologically active analogs thereof according to any of claims 1 to 11, characterized in that said vector is suitable for the expression of said chimeric protein in mammalian cells.

18. The DNA vector according to claim 17, further characterized in that said vector is suitable for the expression of said chimeric protein in human cells.

19. The DNA vector according to claim 17, further characterized in that said vector is suitable for the expression of said chimeric protein in CHO cells.

20. - The DNA vector according to claims 17 to 19, further characterized in that said vector is expressed in human or mammalian cells, the chimeric protein expressed having a sequence that allows the complete processing of the chimeric protein by human or mammal, and the secretion of the fully processed chimeric protein from the cells in the culture medium in which said cells are cultured.

21. The DNA vector according to any of claims 17 to 20, further characterized in that said vector is designated herein as plasmid pcDNAslL-6R / IL-6 comprising a vector pcDNA3 containing the DNA sequence encoding for the chimeric slL-6R / IL-6 protein under the control of a cytomegalovirus (CMV) promoter.

22. The DNA vector according to any of claims 17 to 20, further characterized in that said vector is designated herein as plasmid pcDNAslL-6R / L / IL-6 comprising a vector pcDNA3 containing the DNA sequence encoding the chimeric SIL-6R / IL-6 protein under the control of a cytomegalovirus (CMV) promoter, and wherein in said DNA sequence encoding said chimeric slL-6R / IL-6 protein is inserted a sequence of linker encoding a peptide linker at the EcoRI site located between the sequence encoding the slL-6R part and the sequence encoding the IL-6 part of the protein.

23. - Transformed mammalian cells containing a DNA vector according to any of claims 17 to 22 that are capable of expressing the chimeric slL-dR / IL-6 protein sequence carried by said vector, and of fully processing the protein expressed and secreting it into the culture medium in which said cells are cultured.

24. The transformed cells according to claim 23, further characterized in that said cells are described herein as human embryonic kidney 293 cells (HEK293) transfected by the vector pcDNAslL-6R / IL-6, said cells being capable of expressing the chimeric SIL-6R / IL-6 protein, completely processing said protein and secreting it in the culture medium in which said cells are cultured in the form of a glycoprotein of approximately 85 kDa.

25. A method for producing a chimeric protein or biologically active analogs thereof according to any of claims 1 to 14, characterized in that it comprises developing transformed cells according to claims 23 or 24 under conditions suitable for expression, processing and secretion of said protein or analogs thereof into the culture medium in which said cells are cultured; and purifying said protein or analogs thereof from said culture medium.

26. The method according to claim 25, further characterized in that the purification is carried out by immunoaffinity chromatography using monoclonal antibodies specific for slL-6R.

27. The use of the chimeric slL-6R / IL-6 protein or analogs thereof according to any of claims 1 to 11, salts of any of them and mixtures thereof, for the preparation of a medicament. for treating mammalian cancers by inhibiting mammalian cancer cells and highly malignant melanoma cells, or in the preparation of a medicament for improving bone marrow transplantation by inducing the grafting of human hematopoietic cells in bone marrow transplantation, or in the preparation of a medicament for increasing hematopoiesis, or in the preparation of a medicament for treating neurological or liver disorders, or in the preparation of a medicament for other applications in which IL-6 or slL-6R is used.

28. A pharmaceutical composition comprising as active ingredient a chimeric slL-6R / IL-6 protein or analogs thereof according to any of claims 1 to 11, and a pharmaceutically acceptable carrier, diluent or excipient.

29. The pharmaceutical composition according to claim 28, for the treatment of cancers.

30. The pharmaceutical composition according to claim 28, for the improvement of bone marrow transplantation.

31. The pharmaceutical composition according to claim 28, for the treatment of neurological or liver disorders, or s¬ to increase hematopoiesis Oj for other applications in which IL-6 or SIL-6R is used.