CA2004261A1 - Synthetic interleukin-6 - Google Patents

Abstract

ABSTRACT
The present invention is directed to recombinant genes and their encoded proteins which are peptides with IL-6 activity. Such proteins include the cysteine-free synthetic lL-6 active peptide and the recombinant peptide with cysteines. The invention is directed to the economical commercial production of large amounts of peptides with IL-6 activity in a form which does not require the use of harsh denaturants and which does not need to be refolded after purification. The proteins of the present invention can be used to stimulate protein production by cells, including cells of the immune system and hepatocytes. The proteins of the present invention may have antiviral activity and may prevent viral infection of cells.
In a preferred embodiment, the cysteine-free synthetic IL-6 like peptide is produced by microbial cells as a soluble trihybrid fusion comprising synthetic cysteine-free IL-6 like peptide, a chemically cleavable peptide and a protein capable of expression in the host organism. After purification of the large fusion protein, the synthetic IL-6 like peptide is removed by digestion of the collagen portion of the fusion with collagenase. The synthetic cysteine free IL-6 like protein is purified by HPLC to yield a pure protein that stimulates the production of immunoglobulins by B-cells, and stimulates the production of hepatic proteins.

Description

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SYNT}IETIC INTERLEUKIN-6 This application is a oontinuat.ion-in-part of copending U.S. Application Serial No. 07/278,690, filed December 1, 198~.
_____ _ 1. INTRODUCTION
The present invention relates to genes and their encoded proteins which are recombinant mature interleukin-6 (hereinafter IL-6) and a synthetic cysteine-free protein that retains IL-6 activity. These proteins are expressed in unicellular hosts as the amino or carboxy terminal peptide portion o a tri-hybrid~fusion protein comprising either interleukin-6 or its modified synthetic form, together with a portion of a cleavage site and carrier DNA. The recombinant fusion protein is purified and digested i~ vitro with a protease that ls specific for the cleavage site to liberate the peptide with IL-6 activity, which is easily separated from the large ~-galactosidase protein expressed by the carrier DNA. The purified peptide can be used to stimulate the production of proteins, including immunoglobulins and hepatic proteins and may be used to prevent viral infections.
The protein can also be added to vaccine preparations as an adjuvant.

2. BACK5ROUND OF THE INVENTION
2.1. PEPTIDE REGULATORS
Physiological agents that regulate metabolic activity of distant cells were given the name hormone by English scientists Bayliss and Starlinger in 190~. These agents consist of amino acid derivatives, s~eroids and peptides.
More recently, a variety of peptides that activate and/or inhibit cell proliferation have been identified and termed 35 stimulatory factors or growth factors. An alternative general term for such cellular factors is cytokine although more specific ter~inology indicates the cell of origin, i.e.
lymphokines which are produced by lymphocytes. Lymphokines also belong to the interleukin class of molecules that modulate the proliferation of cells in the immune system.
Other peptides produced by the immune system result in specific antiviral activities; such peptides are termed interferons. To qualify as an interferon a factor must be a protein which exerts antiviral activiky through cellular metabolic processes involving the synthesis of both RNA and protein (Committee on Interferon Nonmenclature, 1980, Nature 86(2~:110).

2.~ INTERLEUKIN-6 Interleukin-6 (IL-6) is the term given to a peptide described alternatively as IFN~2A, BSF-2, H5F, G-CSF, CS-309, HPGF, or 26 kDa protein by a multiplicity of investigators.
These original factors now known a~ IL-6 include: I) interferon-~2 (2ilberstein et al., 1986, ~MBO J. 5:2529) or 26 kDa protein (Haegeman et al., lg86, Eur. J. ~iochem.
159:625) which was first detected in poly(rI) poly(rC) stimulated fibroblasts; 2) a potent T-cell derived lymphokine termed B-cell stimulation factor-2 (BSF-2) ~Hirano et al., 1986, Nature 324:73): 3) a fibroblast product called 8-cell hybridoma/plasmacytoma growth factor (HPGF or HGF) (Van Snick et al., 1986, Proc. Natl. Acad. Sci. 83:9679 Billiau, 1987, Immunol. Today ~:84 Van Damme et al., 1987, Eur. J. Biochem.
168:543; Tosato et al., 1988, Science 239:502): and 4) a peripheral blood monocyte protein called hepatocyte stimulating factor (HSF) (Gauldie et al., 19~7, Proc. Natl.
Acad. Sci. 84:7251).
The functions which have been ascribed to the IL-6 peptide are basic to both the inflammatory and immune response in human pathology. Those functions are diverse and 35 depend on the type of cells under examination~ IL-6 is 20~fi~
~3--expressed in leukocytes, epithelial cells, IL-I treated fibroblasts, hepatocytes, vascular endothelial cells, cardiac myxoma tissue, certain bladder carcinom~s, cert~in cervical cancer cells and glial cells. IL-6 is one of the peptides involved in the interaction of T cells with B cells to result in the prolifer~tion and diferentlation o antibody producing cells. IL-6 significantly enhances secretion of immunoglobulins in activated B-cells (Mura~uchi et al., 1988, J. EXp. Med. ~ 332; Tosato et al., 1988, Science 239:502; Hirano et al., 1985, Proc. Nat:l. Acad. Sci.
ln 82:5490).
With regard to the antiviral activity responsible for the initial identification of IL-6 as an interferon, the transformation of Chinese hamster ovary cells with a recombinant IL-6 plasmid which allowed constitutive expression of IL-6 resulted not only in the detection of the peptide in media, but also resulted in the detection of antiviral activity (Zi]berstein et al., 1986, EM~O J.
5:2529).
Whether natural IL-6 has antiviral activity or not has been subject to debate in the scientific literature.
Antiviral specific activities were first reported by Weissenbach et al., (1980, Rroc. Natl. Acad. Sci. 77:7152);
and members of this scientific group have continued to report antiviral activitie~ in their preparatlons (see Content et al., 1985, Eur. J~ Biochem. 152:253: May et al., 1988, J.
Biol. Chem. 263:7760). The values reported for antiviral activity range ~rom 5-10 x 1o6 U/mg protein to as low as 1-3 x 102 U/mg. In contrast, a large number of other 30 investigators have repoxted no significant antiviral e~fects associated with their purified recombinant IL-6 preparations.
(Poupart et al., 1987, EMBO J. 6:1219 Van Damme et al., 1988, J. Immunol. 140:1534 Reis et al., 1988, J. Immunol.
140Q5):1566; Hirano et al., I988, ImmunolO Lett. 17(1~:41).
IL-6 appears to suppress the action of TNF (Kohase et 2 ~ rZ ~

al., 1987, Mol. Cell Biol. 7:273). However, it stimulates the growth of human B-lymphoblastoid cells infected with EBV
(Tosato et Al., 19~, Science 239:502), and of human thymocytes and T-lymphocytes tLotz et al., 1988, J. Exp. Med.
1_7~ 1253). It has been identified as a growth factor for murine hybridomas (Poupart et al., 1987, EMBO J. 6:1219) and hybridoma plasmacytoma cell lines (Van Damme et al., 1987, J.
Exp. Med. 165:914). In combination with IL-3, IL-6 supports the proliferation of hematopoietic progenitor cells (Van Damme et al., 1987, J. Exp. Med. 165:914; Ikebuchi et al., 1987, Proc. Natl. Acad. Sci. 84:9035), and modulates the synthesis of a subset of hepatocyte proteins in response to injury and infection (Gauldie et al,, 1987, Proc. Natl. Acad.
5ci. 84:7251: Andus et al., 1987, FEBS Lett. 221:18). The multiplicity of its own actions, and its interactions with other peptide regulators like IL-l, TNF, IFN~l and PDGF, have led to the suggestion that IL-6 plays a pivotal role in a complex cytokine network needed for homeostatic control of cellular functions (Kohase et al., 19~7, Mol. Cell Biol.
7:273; Sehgal et al., 1987, Science 235:731; Billiau, 1987, Immunol. Today 8:84; Sporn and Roberts, 1988, Nature 332:217).

2.3. DNA SEQUENCE OF THE INTERLEUKIN-6 PROTEIN
Comparison of the primary structure of factors that were originally characterized by multiple investigators on the basis of differing biological activitie~ revealed the identity of their amino acid sequences and resulted in the renaminy of the peptides as interleukin-6 (IL-6).
3a Fibroblasts treated with either poly(rI) (rC) or cycloheximide and actinomycin D produced a 14S mRNA molecule which coded for a protein capable of inducing antiviral activity called IFN~2 (Weissenbach et al., 1g80, Proc. Natl.
Acad. Sci. 77:7152; British Patent No. 2,063,882~. Clones 35 produced from cDNA copies of this induced mRNA fraction 2~ fil.

provided a partial sequence of the IFN~2 promoter that was clearly different from IFN~l, IFN7~A, IFN7~, and IFN~D
(Chernajovsky et al., 1984, DNA 3:297; Revel et al., 1983, Interferon 5:205 1. Gresser ed., Academic Press, N.Y.). The complete lFNa2 sequence derived ~rom multiple cDNA clones defined a 212 a~ino acid long protein in addition to a probable ATG start se~uence, polyadenylation sites~ TATA
boxes and the mRNA start site which was revealed by Sl nuclease mapping (Zilberstein et al., 1986, EMB0 J. 5:2529;
European Patent Application 0220574A1, Publication Date 06-05-1987). In a different analysis of the IFN~2 sequence, May et al., 1986, Proc. Natl~ Acad. Sci. 83:8957, also defined the 212 amino a~id protein sequence using a full length cDNA
clone rather than the partial cDNA clones of Zilberstein et al., 1986, EMB0 J. 5:2529. These investigators also noted that IFN~2 was induced by TNF.
Even though their 26 kDa protein had no detectable antiviral activity, Haegemann et al., 1986, Eur. J. Biochem.
159:625 recogn.ized that the sequence of their 26 kDa protein, induced in fibroblasts by treatment with cycloheximide or interleukin-l, was identical to the IFN~2 of Zilberstein et al., 1986, EMB0 J. 5:2529. Because the 5' terminus of the protein was missing .in the cDNA clone collection~ a screen of a human gene library, testing for complementarity with an internal cDNA sequence of the 26 kDa peptide, yielded genomic clones that provided the complete 212 amino acid sequence, as well as the DNA sequence of a 162 bp intron in the 5' terminus region of the human gene. A search o~ a protein data base containing 3309 individual peptide sequences failed to reveal any significant similarities with proteins in the database.
In a separate study ~Hirano et al., 1986, Nature 324:73), BSF-2 protein was purified from a human T-cell line that constitutively produced the factor and was established using the HTLV-1 virus. The amino acid sequence data from nine peptide fragments provided the information necessary to produce synthetic oligomers which were used to probe cDNA
li~raries. The cDNA clones were se~lenced as was the amino terminus o the purified IL-6 protein. The end of the mature protein sequence was pro-val pro-pro indicating that the 212 amino acid prepeptid~ that was predicted from the nucleic acid sequence contained a 28 amino acid long signal peptide which is cleaved to produce the natural mature BSF-2 (IL-6 protein.
Continued sequence analysis of the entire BSF-2 (IL-6) genomic DNA demonstrated that the chromosomal segment contained five exons and four introns (Yasukawa et al., 1987, EMBO J. 6:2939). The organization of the BSF-2 (IL-6) gene was strikingly ~imilar ~o that of G-CSF when the two genes were compared.
After completion of the sequence of H~F (Brakenhoff et al., 1987, J. Immunol. 139:4116), the identity of HGF and lL-6 was confirmed. In addition, a study of the sequence differences in all reported analyses of the IL-6 gene (as HGF, IFN~2, the 26 kDa protein or BSF-2) revealed f~w single base changes. Neither of the two changes that occur within the peptide reading frame produce an amino acid change. Van Damme et al., 1988, J. Immunol. 140:1534, also noted that the amino terminal sequence of HGF was identical to IFN~2, the 26 kDa protein and BSF 2.
Clark et al. (International Application Number PCT/US87/01611, Publication Number W088/00206, published January 14, 19~8) also reports the cDNA sequence of IL-6.

2.4. CONSERVATION OF CYSTEINE XESIDUE5 IN IL-6 All cysteine residue positions are conserved between the two proteins, BSF-2 (IL-6) and G-CSF. Upon noting this similarity, Hirano et al., 1986, Nature 324:73, suggested that intramolecular disulphide bonds would be important in 35 the structure of BSF-2 (IL~6~ and G-C5F. Their suggestion ~O~fl2~

was based on a comparison of the ~SF-2 (IL-~) sequence with other se~lences in a personal database of multiple growth factors, interleukins and interferons which revealed that B~F-2 (IL-6) was distantly relat~d to G-CSF. However, comparison with the National Biomedical Research Foundation database or the Genetic Sequence Data ~ank revealed no significant similarity with other proteins in those databanks.
Conservation of the cysteines was also suggested by Van Snick et al., 1986, Proc. Natl. Acad. Sci. 83:9679. They sequenced murine HP-1 and found conserved homology with human IL-6. They also examined the positions of the cysteines of HP-1, IL-6 and G-CSF and noted that the cysteine positions were conserved within t~e three peptides.
As described by Yasukawa et al., ~ , the organization of exons and introns within the BSF-2 (IL-6) gene was strikingly similar to that o~ G-CSF when the two genes were compared. This finding provides further support for the shared evolutionary history-of the two proteins (see also Kishimoto, 1987, J. Clin. Immunol. 7(5):343~.
The presence of cysteines in a protein can cause problems in processing when the protein is being produced recombinantly in ~ bacterial host. Microblally produced cysteine-containing proteins may tend to form multimers which greatly complicate puri~ication of the protein product.
Several additional purification steps, such as reduction and reoxidation of the recombinant protein, may be required to obtain the protein in the proper conformation. Removal of one or more of the cysteine residues, with concurrent 30 replacement by a chemically equivalent neutral amino acid, would be desirable, in oxder to simplify the isolation and purification of the IL-6 molecule. However, the successful removal of cysteines ~rom biologically active molecules is unpredictable, in that the ~ertiary structure, in the absence 35 of the normally formed disulfide bridges, can be 2a~0~fi~.

substantially altered. It is often the case that one or more of these residues may he essential to retaining the desired activity.
Natural cysteine residues are expected to be particularly critical for cyto~ines such as IL-6, since the cysteine~ of IL-6 have been conserved (See above). Moreover, the biological activity of other cytokines has been reported to be affected if cysteines are removed. For example, only one cysteine residue out of three can be removed without af~ecting the biological activity of the IFN-~ (see U.S. Pat.
No. 4,737,462). Similarly, only one of four cysteines of IFN~l can be removed without complete loss of activity (Mark et al., 1984, Proc. Natl. Acad. Sci. 81: 5662).

2.5. PRODUCTION OF NATURAL INTERLEUXIN-6 AS A RECOMBINANT PROTÆIN
-2.5.1. RECOMBINANT DNA TECHNOLOGY AND GENE EXPRESSION
Recombinant DNA technology involve~ insertion of specific DNA sequences into a DNA vehicle (vector) to Eorm a recombinant DNA molecule which is capable of replication in a 20 host cell. Generally, the inserted DNA sequence is foreign to the recipient DNA vehicle, i.e , the inserted DNA sequence and the DNA vector are derived from organisms which do not exchange genetic information in nature, or the inserted DNA
sequence may be wholly or partially synthetically mad~.
25 Several general methods have been developed which enable construction of recombinant DNA molecules.
Regardless of the method used for construction, the recombinant DNA molecule must be compatible with the host cell, i.e., capable of autonomous replication in the host 3~ cell or stably integra~ed into one or mor~ of the host cells chromosomes. The recombinant DNA molecule should prefexably also have a marker function which allows the selection o~ the desired recombinant DNA molecule(s;. In addition, if all o~
the proper replication, transcription, and translation signals are correctly arranged on the recombinant vector, the foreign gene will be properly expressed in, e.g., the transformed bacterial cells, in the case of bacterial expression plasmids, or in permissive cell lines or hosts infected with a recombis~ant virus or carrying a recombinant plasmid having the appropriate orig~n of replication.
Different genetic signals and processing events control levels of gene expression such as DNA transcription and messenger RNA (mRNA) translation. Transcription of DNA is dependent upon the presence of a promoter, which is a DNA
sequence that directs the binding of ~A polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promoters differ from those of procaryotic promoters. Furthermore,~ eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system and furthermore, procaryotic promoters are not recogni2ed and do not function in eucaryotic cells.
5imilarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals, which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (S/D) sequenc~e on the mRNA (Shine, J. and Dalgarno, L., 1975, Nature 254:34). This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The S/D sequences are complementary to the 3' end of the 16S rRNA (ribosomal RNA), and probably promote ~inding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome.
Although the Shine/Dalgarno sequence, consisting of the few nucleotides of complementarity between the 16S ribosomal RNA and mRNA, has been identified as an important feature o~
the ribosome binding site (Shine and Dalgarno, 1975, Nature Z63~

254: 34; Steitz, 1980, in Ribosomes: Structure, Function and Geneti-cs ed. Chambliss et al. Baltimore, Md., University Park Press pp. 479-495), computer analysis has indicated that approximately one hundred nucleotides surrounding the AUG
initiating codon are involved in ribosome/mRNA interaction as indicated by proper predlction of translation start signals (Stormer et al., 1982, Nucl. Acids Res. 10:2971; Gold et al., 1984, Proc. Natl. Acad. Sci. 81:7061). No prediction of what actually proYides the best and complete ribosome binding site for maximum translation of a specific protein can be made (see Joyce et al~, 1983, Rroc. Natl. Acad. Sci.
80:1830).
Schoner and Schoner recognized the significance of the entire ribosome/mRNA interaction region in the development of recombinant expression vectors in their charactarization of a 72 bp sequence termed the "minicistron" sequence (See Figure 1 of Schoner et al., 1986, Proc. Natl. Acad. Sci. USA 83:
8506). A one base deletion in the first cistron of the "minicistron" sequence was sufficient to increase the production of the downstream recombinant protein Met-[Ala]bGH
from 0.4~ to 24% of total cell protein (See Figure 4, pCZ143 compared to pCZ145, Schoner et al., id.).
Alternati~ely a two base insertion also resulted in significant expression of the coding peptide encoded by the second cistron. Control experiments indicated that the differences in expression were due to translational differences because mRNA levels in these constructs were essentially equi~alent (no more than 3 fold different) as compared to the expressed protein differences ~which were approximately 50 fold). The conclusion was that the position of the stop codon that termina~es translation of the first cistron of the minicistron sequence affected tha efficiency o~ translation of the second cistron, con~aining the coding sequence of the recombinant protein. Most importantly their 35 work indicated that one or two base changes in the sequence --ll--immediately preceding the coding sequence of a recombinant protein can have tremendous effects on downstream expression.
Successful expression of a cloned gene requires suficient transcripkion of DNA, translation o~ the mRNA and in some .instan~es, post-translational modification of the proteln. Expression vectors have been used to express genes under the control of an active promoter in a suitable host, and to increase protein production.

2.5.2. NONBACTERIAL PRODUCTION OF RECOMBINANT INTERLEUKIN-6 Although Weissenbach et al., 19~0, Proc~ Natl. Acad.
Sci. 77:7152 translated cDNA in oocytes to make recombinant IFN~2 in vitro, Zilberstein et al., 1986, EMBO J. 5:2529 reported the first exam~le of IFN~2A (IL-6~ cDNA expression in vivo. The restriction fragment containing the fused cDNAs o~ different primary clones was positioned downstream from the SV40 early promoter to produce the pSVCIFB2 plasmid. In this plasmid, the entire IFN~2A cDNA sequence and some adjacent nucleotides o~ the original cloning vector followed the SV40 early promoter and the first ~0 nucleotides of the T
antigen RNA. The IFN~2A sequence was followed by the T
antigen splicing region and polyadenylation site. After the steps of trans~ection into Chinese hamster ovary (CHO) tlssue culture cells and methotrexate amplification selection, CHO
cell clones labeled with [35S] were screened for radioactive IFN~2 protein produ¢tion by immunoprecipitation using nonsaturating amounts of antibodie~ In the same study, plasmid constructs, wherein the entire IFN~2A cDNA sequence was fused to the T7 RNA polymerase promoter, also were 30 transcri~ed in vitro to produce mRNA that was subseqllently translated in rabbit reticulocyte lysates. Following immunoprecipitation of the lysate, the proper sized IFN~2 (IL-6) protein could be detected on SDS-polyacrylamide gels.
BSF-2 (IL-6) was functionally expressed in COS7 cells 35 following transfection of a plasmld containing the entire .2~;~

coding region of BSF-2 adjacent to the SV40 early promoter (Hirano et al., 1986, Nature 324:73). BSF-2 activity could be detected after purification and concentration of the media from the transfected cells using immunoaffinity gel methods.
Recombinant IL~6 produced usiny this method was used to analyze the regulation of ibrinogen and albumin mRN~ in FAO
cells (Andus et al., 1987, FEBS Letts. 221: 18).
Clark et al., (International Publication Number W088/00206, published January 14, 1988) described the construction o~ pCSF30~ wherein the IL-6 CDNA sequence was ligated into the p91023B plasmid which contains the SV40 enhancer, major adenovirus late promoter, DHFR coding sequence, SV40 late message poly-A addition site and the VA I
gene. When COS cells are transfected with this plasmid, IL-6 hematopoietic stimulating activity could be recovered at a 10-4 dilution of conditioned tissue culture cell medium.
This type of recombinant preparation was used in an analysis o~ the effect af IL-6 on T-cell proliferation in the presence of either ConA or agarose beads coupled with F23.1 IgG2a anti-[mouse T cell receptor B chain variable region seqments 8.1, 8.2, and 8.3]-mouse antibodies (Garman et al., 1987, Proc. Natl. Acad. Sci. ~4:76~9). The preparation of recombinant IL-6 was also used in a study of the ef~ect of IL-6 on the differentiation of Ly-2+ cytolytic lymphocytes from murine thymocytes in the present of interleukin-2 (Takai et al~, 1988, J. Immunol. 140:508). Alternative methods o~
higher cell production of recombinant IL 6 include in vitro synthesis following injection of IL-6 mRN~ into Xenopus oocytes (Weissenbach et al., 1980, Proc. Natl. ~cad. Sci.
77:7152; Coulie et al., 1987t Eur. JO Immunol. 17:1435;
poupart et al., 1~87, EMB0 J. 6:1219), by translation of the IL-6 mRNA in xabbit reticulocytes (Poupart et al., 1987, EM80 J. 6:1219), or by cDNA expression in yeast (La Pierre et al., 1988, J. Exp. Med. 167:794~.

2.5.3. C _RIAL PRODUCTION OF RECOMBINANT_INTERLEUKIN-6 -Clark et al., (International Publication Number Woa8/oo2o6~ published January 14, l9a~) reported the expression and production o recombinant II,~6 in E. coli using pCSF~09 (deposited July 11, 1986 as accession number ATCC 67153). tlowever, the preferred emhodiment reported therein selec~ively modified the sequenca of pCSF309 a) to delete its signal peptide leader sequence and its 3' noncoding sequence: and b) to co~ple the protein coding sequence to a temperature inducible PL promoter in association with a temperature sensitive CI repressor. This strain, containing plasmid pAL309C-781, was not deposited with the ATCC. In this strain, the bacterially expressed protein was produced in an insoluble form which first had to be solubilized and then refolded ~see example V of their patent application). Alternatively, a plasmid termed pAL-Sec-IL6-181 was constructed to produce IL-6 by coupling the cDNA sequence of ~L-6 to a synthetic signal peptide leader sequence under PL promoter control. Following high temperature induction o~ the PL promoter in a temperature sensitive CI repressor strain, IL-6 was isolated from the periplasm of the transformed cells a~ a homogeneous protein from which the signal peptide had been removed. No yield o~
purified protein was reported.
Brakenhoff et al., 1987, J. Immunol. 139:~116, reported the expression of HGF, hybridoma growth factor (IL- 6), in different E. coli strains containing any one o~ seven specific IL-6 cDNA clones. Media was screened for IL-6 activity to iden~ify the clones producing IL-6. Th~ activity varied at least 5,000 fold between the clone~. The most active construct (clone 7) was missing the first 43 amino acids of the peptide, but that construct produced more than 20X as much activity as the next active clone. The constructs containing the complete amino acid coding region of HGF (IL-6) had the least activity ~for example, clone I5 fl~

starting at position -62 from the ATG start site had 1.7 kU/ml of activity compared to clone 7 which had 10,000 kU/ml). In order to detect activity, recombinant IL-6 was first separated from cytosolic proteins of clones 7 and 15.
Activity was assayed only after elutlon of indivldual SDS-PAGE slices. Even though activity was detected~ no IL-6 protein was apparent ih the gel protein profile.
Tosato et al., 1983, Science 239:502, reported the production of an antiserum after immunization with recombinant IL-6 that was produced in E. coli but the details of both the antiserum production methods and the recombinant IL-6 protein synthesis and purification were not published (see their reference 10, reported as manuscript in preparation).
Recombinant BSF-2 (IL-6) was produced in E. coli using pTBCDF-12 (Hirano et al., 1988, Immunol. Letters 17:413.
Induction of this plasmid resulted in the production of a fusion protein in which the recombinant IL-6 peptide was fused to the IL-2 peptide. Protease digestions using Kallikrein and amino peptidase-P were required to obtain mature IL-6 protein. However, the details of the procedure other than this nonenabling description were to be published elsewhere (see their manuscript in preparation citation).
May et al., 1988, J. Biol. Chem. 263:7760 reported the production of an insoluble form of IL-6 in bacterla following the fusion of the IL-6 cDNA in a REV expression vector (Repligen Corp., Cambridge, MA) to produce a product containing 34 amino acids of a prokaryotic leader peptide fused to an IL-6 peptide portion of 182 amino acids. The 30 expressed pro~ein wa~ recovered in an insoluble pellet which was solubilized in ~M urea, 5 mM DTT and 10 mM ~-mercaptoethanol in a Tris based bu~fer. The fusion product was partially purified from ~he numerous other E. coli pellet proteins by D~AE-sepharose column chromatography using a salt 35 gradient with the solubilizing buffer, followed either by ~0[)4~

immunoaffinity chromatography with a monoclonal antibody to the prokaryotic leader peptide or FPLC with a Mono Q column (Pharmacia). No yields of purified protein were reported.
The DEAE-Sepharose peak fraction exhibited antiviral activity of 0.5-1 IU/ml using a cytophatic effect reduction assay in FS-4 cell cultures and vesicular stomatitis virus. The recombinant fused IL 6 protein was injected into rabbits to produce a polyclonal antlserum. The polyclonal antiserum was used to characterize the natural IL-6 protein in fibroblastic FS4 cells following TNF and cycloheximide induction of IL-6 or in human monocytes. Incubation of the antiserum with the F54 cell medium prevented the induction of immunoglobulins in CESS cells.
Multiple investig~tors have reported a recombinant I~-6 protein made in E. coli, but no disclosure of the methodology for reco~binant IL-6 production was provided (Taga et al., 1987, J. Exp. Med. ~ 4L:967; Gauldie et al., 1987, Proc.
Natl. Acad. Sci. 84:7251: Lotz et al., 1988, J. Exp. Med.
167(3):1253; Muraguchi et al., 198~, J. Exp. Med. 167(2):332;
Reis et al., 1988, J. Immunol. ~ L:1566).

2.5.4. PRODUCTION OF HIGH YIELDS OF RECOMBINANT

Although nonenabling reports of production of natural IL-6 in bacteria exist (see Section 2.5.~), production of 25 high yields of the natural protein in bacteria has not been successful even using recombinant DNA bioengineering methods (Asagoe et al., 1988, Biotechnology 6:806) (see also Section 5.1 of this applicat~on). The Asagoe study reportC the inability to produce natural IL~6 as a detectable protein in 30 induced cell extracts using a plasmid construct very reminiscent to those reported in 5ection 2.5.3. supra. The unsuccessful Asagoe expression plasmid contained the mature processed BSF-2 (IL-6) protein coding sequence adjacent to and in frame with the PTAC promoter and ATG start sequence~

)4~

Although the plasmid was analyzed in a variety of strain backgrounds r IL-6 protein was not detected following induction. 5ignificant amounts of insoluble recombinant IL-6 activity were produced by Asagoe et al. only after induction of a plasmid containiny a multi-hybrid-fusion protein in which 1) the PTRyp promoter and ~TG start site were fused to human growth hormone coding sequence; 2) the human growth hormone se~uence was fused to an oligonucleotide sequence producing a Xa factor peptide recognition site (ile-glu-gly-arg); and 3) the Xa factor recognition site was then fused to either a glu-phe-met-BSF-2 sequence or an ala-BSF-2 coding sequence.
This recombinant IL-6 (or BSF-2) peptide was thus the carboxy terminal portio~ of the human growth hormone/Xa factor sequence/BSF-2 hybrid fusion product in this construct. The recombinant IL-6 activity, existing either as glu-phe-met-BSF-2 peptide in one plasmid construct or ala-BSF-2 peptide in the other, could be purified only after solubilization of the fusion protein in 8M urea. Cleavage of the fusion pro~ein with Xa factor required refolding the purified fusion protein hy extensive dialysis. The refolding process wa~ incomplete since the fusion protein preparation was only incompletely digested by Xa factor.
The Xa factor cleavage produced a heterogeneous mixture containing intact fusion protein, recombinant IL-6 peptides, and human growth hormona peptide, in addition to other partial cleavage products. In order to recover IL-6 activity in the purified product, the Xa factor/cleavage mixture first had to be denatured with 6M guanidinium hydrochloride. After 30 chromatographical purification of the recombinant IL-6 peptide, it was subjected to an additional round of extensive dialysis in order to recover a refolde~ active peptide. The rPsultant recombinant peptides, either glu-phe-met-BSF-2 or ala-BSF-2, were examined only for their activity in a B-cell 35 stimulatory factor assay. The yield reported for the 2~

production process was approximately 5% (3 mg of purified actiYity was recovered from an initial 100 mg of fusion protein of which 58 m~J was IL-6 peptide; 3/58 = 5.17%).
A need thus still exists for a convenient, relatively inexpensive method of prod~lcing high yields of IL-6.
Recombinant methods using mammalian cel1s tend to be rather costly. Although bacterial production is a lower cost al~ernative, previous attempts to produce IL-6 in transformed bacteria have not resulted in commercially feasible yields.
The present invention provides DNA constructs and methods for producing IL-6 in bacteria which permit the production of the protein in high yields, typically about 20% of total cytosol protein. The invention also provides a cysteine-free form of IL-6 which retains the biological activity of the native IL-6 molecule.
1~
3. SUMMARY OF T~IE INVENTION
The present invention is directed to the economical production of recombinant synthetic cysteine-free IL-6 proteins produced by bacteria as well as active peptide fragments of each of the proteins. The proteins produced are either cysteine-containing or cysteine-free, and retain IL-6 biological activity. Bacterial cultures express either recombinant protein as a high percentage, generally at least 20%, of total soluble cytosol protein. The recombinant IL 6 protein does not require treatment with harsh denaturing agents like 8M urea, or ~-mercaptoethanol to solubilize the protein from a pellet. As used throughout the present specification and claims, the phrase ~Isubstantially soluble 30 in wat~r" re~ers to this property.
In the present invention, the IL-6 is produced recombinantly in a unicellular host by expression of a tripartite fusion protein. The fusion protein comprises first, second and third peptide portions. The first peptide portion has IL-6 activity; the second peptide portion is a X0~1~2Gl chemically or enzymatically cleavable sequence which links the first peptide portion to the third peptide portion; the third peptide portion is a protein or portion thereof which is capable of being expressed by the unicellular host, and which pre~erably has a detectable function. The three peptide portions ara referred to as "first", "second" or "third" only for convenience, and should not ~e read as requiring a specific order, relative to the promoter controlling expression of the protein: the positions of the first and third peptide portions are interchangeable.
The third peptide portion provides a protein or portion thereof which the unicellular host cell can make. The presence of the carrier DNA that expresses the third peptide facilitates production Qf the aucaryotic IL-6 protein. In its detectable function, (for example, enzymatic activity), the third peptide portion also provides a means by which transformed clones producing the fusion protein can be readily identified and isolated. The first peptide portion, which possesses the desired biological activity, is separated from the remainder of the fusion protein by cleavage of the second peptide portion. The recombinant IL-6 peptide is then separated and purified from the protein mixture by methods known in the art such as by higX performance liquid chromatography ~HPLC) to yield a pure protein that is active in IL-~ assays. The present invention also contemplates nucleotide sequ~nces encoding the fusion protein, recombinant vectors comprising these nucleotide sequences, as well as unicellular hosts transformed with these vectorsO
In a particular embodiment, a synthet~c peptide having IL-6 activity is produced with all four cysteines of the native IL-6 sequence replaced by serine residues. Unless otherwise specified, the term synthetic cysteine-free IL-6 or recombinant cysteine-free IL-6 is used in the specification and claims to identify a synthetic peptide having IL-6 activity with all four cysteines of the native IL-~ sequence replaced by serine residues. The peptide so produced surpr~singly retains its biological activity. For example, the cysteine-free form of IL-6 has heen shown to exhibit hepatocyte activation and B-cell stimulation. The proteins o~ the present invention may have antiviral activity and may prevent viral ~nfection of cells. The cysteine-free synthetic protein may be nonpyrogenic or at least significantly less pyrogenic than native IL-6 protein.
The proteins of the present invention also include different cysteine-free and cysteine-containing IL-6 peptides which have deletad up to 27 amino acid residues from the IL-6 amino terminus, and/or which add up to 50 amino acid residues on the amino terminus and/or up to 350 amino acids on the carboxy terminus of the~IL-6 sequence, and retain the relevant biological activity. Thus, surprisingly, and unlike many other biologically active peptides, the basic IL-6 sequence can be modified substantially without any significant loss of activity. The invention therefore encompasses the gene sequences encoding the cysteine-free peptides, as well as truncated and extended cysteine-free or cysteine-containing peptides which retain IL-6 activity.
The present method represents an improvement over known recombinant methods for producing IL 6 in that it provides means for producing IL-6 in commercial quantities in a recombinant/vector system. Previous IL-6 fusion protein constructs, such as the one described by Asagoe, supra, have not been successful in obtaining expression o~ the protein in large quantities, and also require extensive harsh purification and refolding procedures in order to obtain a 30 functional protein. This treatment with harsh denaturing agents is not required in the present method, however, nor is refolding of either the fusion protein or the cleavage protein necessary.
The peptides producad by culturing these recombinant 35 hosts retain characteristic IL-6 activity in their stimulatory effects on the immune system and on therapeutic cell àctivity. The present invention also contemplates method.s of treatment of viral disease, immunodeficiencies and hepatic disorders, as well a~ overall modulation o~ immune response, by administration of the claimed synthetic IL-G
peptide.

3.1. DEFINITIONS
ATCC : American Type Culture Collection bp : base pair ~SF-2 : B-cell Stimulation Factor cDNA : Complementary DNA
CHO : Chinese Hamster Ovary CSF : Colony Stimulating Factor DHFR : Dihydrofolate Reductase DNase : Deoxyribonucleic acid nuclease ELISA : Enzyme Linked Immunosorbent Assay G-CSF : Granulocyta Colony Stimulating Factor HGF : Hepatocyte Growth Factor HPGF : Hybridoma Plamacytoma Growth Factor HPLC : High Pexformance Liquid Chromatogxaphy HSF : Hepatocyte Stimulating Factsr IGF : Insulin-Like Growth Factor Ig~, IgM : Immunoglo~ulin G, M
IL-1, IL-3, or IL-6, : Interleukin 1, 3, or 6 IPTG : Isopropylthiogalactoside INF : Interferon kDa : kilo Dalton Kb : Kilobases LB : Luria Broth mRNA : messenger RNA
PAGE : Polyacrylamide Gel Electrophoresis fil PBS : Phosphate buffered saline PDGF : Platelet Derived Growth Factor Poly ~rI)'~rC): RNA with a strand of ribo-Inosine duplexed with a ribo-Cytosine strand PL : Promoter l.eft of Lambda PR : Promoter Right of Lambda PTAC' PTRC Synthetic Promoter of Tryptophan-Lac Combination RNase : Ribonucleic acid nuclease SDS : Sodium Dodecylsulfate S~D sequence: Shine-Dalgarno sequence SH : Sulfhydryl TNF : Tumor Necrosis factor X-gal ~Bromo-chloro-indolyl-~-D-galactopyranoside YT : Yeast-tryptone growth media 4. RIEF DESCRIPTION OF THE FIGURES
FIG. l. Diagram of the construction of p360. The plasmid p360 is a plasmid expression vector constructed to express the cysteine free synthetic IL-6 like peptide as a protein of approximately 22-23 KDa in E. coli.
Oligonucleotide 737 [AGC TGA TT~ AAT AAG GA~ GAA TAA CCA TGG
CTG CA 3'] and 73 8 [ GCC ATG GTT ATT CCT CCT TAT TT~ ATC 3'~
were fused and replaced the Hind III-Pst I fra~ment of pB~(+), a phagemid purchased from Stratagene Systems, to form p350. The sequence for the synthetic cysteine free IL-6 like peptide fro~ plasmid p337-l, which contains a peptide terminating codon, replaced the Nco I-Eco RI fragment of p350 30 to produce plasmid p360. In p360, expression ~rom the induc~ble lac promoter through the minicistron sequence and through the syn~hetic cysteine free I~-6 like sequence fails to produce a peptide of the size of IL-6 in extracts of E.
coli (see Figure 3 rox gel of expressed proteins).
FIG. 2. Diagram of the construction of p369. The fragment containing the sequence for the synthetic cysteine free IL-6 like peptide of plasmid p365, which contains no peptide terminating codon is this construction~ replaced the Nco I-Bam HI fragment of p350 tdescribed in Figure 1) to form plasmid p369 wherein the synthetic cysteine free IL-6 like peptide sequence was fused in frame to the alpha-complementing fragment of ~-galactosida~e. Induction of the lac promoter oP p369 fails to produce a detecta~le peptide which would be the fusion product between the synthetic IL-6 like peptide and the alpha-complementing portion of ~-galactosidase (see Figure 3 for gel of the expressed proteins).
FIG. 3. Expression and nonexpresslon of plasmid constructs containiny sequences for the synthetic cysteine free IL-6 peptlde. Cultures of E. coli cells carrying different plasmids were induced with IPTG. Samples of cell cultures were lysed and electrophoresed in SDS-polyacrylamide gels. The gels were stained with coomasie blue to visualize proteins. Lanes a and b are duplicate aliquots from different tubes of the same culture. Lanes la and b are from cells bearing plasmid p369. The ëxpected induction protein should be a ~usion protein o~ the alpha-complementing portion of ~-galactosidas~ and the synthetic IL-6 like peptide. No large molecular weight protein representing the fusion is present. Lanes 2a and b are from cells bearing plasmid p367, the successful expression vector ~or the fusion product of the synthetic IL-6 like peptide/collagen/~-galactosidase.
Note the large amount o~ high molecular weight fusion protein in the approximately 130 kDA position of the gel. Lanes: 3a 30 and b are from cells bearing plasmid p360 which were expected to produce a peptide of 22-23 ~Da, the size of deglycosylated IL-6. The arrowhead marXs the position of the expected small peptide.
FIG. 4. Diagram of the construction of p340-1. The 35 steps in the construction of p~40-1 are described in Section 5.2. Oligonucleotide 237 was [AAT TCT AAT ACG ACT CAC TAT
~GG GTA AGG AGG TTT AAC CAT GGA GAT CTG 3']. Oligonucleotide 238 was ~GAT CCA GAT CTC CAT GGT TAA ACC TCC TTA CCC TAT AGT
GAG TCG TAT TAG 3'] Oli~onucleotide 703 was [CAT GTA TCG ATT
F AAA TAA GGA GGA ATA ACC 3']. Oligonucleotide 704 was [CAT
GGG TTA TTC CTC CTT ATT TAA TCG AT~ 3']. PTRC is ~he hybrid promoter tryptophan/lac. "Term" represents the terminatlng codon th~t is in the same reading frame as the preceeding lnitiating methionine codon. APR represents the rightward promoter from bacterlophage lambda. Ampr represents the ampicillin resistance marker from pBR322. ori represents the plasmid origin of DNA replication from pBR322. ~-gal represents the E. coli ~-galactosidase gene region.
FIG. 5. Complete ,nucleotlde sequence (5'-3') of the synthetic recombinant IL-6 gene and its predicted amino acid sequence. The coding sequence o~ the fused gene begins at nucleotide 3 (Met). The amino acid sequence of the recombinant IL-6 protein extends to nucleotide 557 (Met) where it is fused to the collagen linker of the fusion protein via a ~erine residue. The asterisk desiqnate~ the location of the TAG stop codon normally found in the native cDNA sequence. Modified amino acid residues are shown in script below the sequence.
FIG. 6. Assembly of the gene ~or the synthetic cysteine-free IL-6 like peptide. Synthetic oligonucleotides were annealed and ligated to form the four synthetic double stranded sub-fragments of the synthetic gene. The insert contained in p333 was formed by ligating two pairs of complementary oligonucleotides~ each bearing internal ~ive-30 base 5' complementary overhangs (nucleotides 59-63 in Figure 5). Construct p334 was composed o~ three pairs of annealed oligonucleotides joined at residues 162-167 and 220-225.
Complementary overhangs for the assembly of p331 were located at nucleotides 312-315 and 358-361. Construct p332 was 35 ligated at positions 457-46i and 512-517.

2~2fi~

-~4-The synthetic fragments were ~loned into a modified pBS
M13+ Stratogene vector by insertion into the multiple cloning site of the circular vector via synthetic 4-6 base overhangs (represented by the hatched boxes). The coding sequence for the synthetic cysteine-free IL-6 like gene is shown in black.
Open boxes represent the modified pBS M13~ sequences.
Fragments were assembled by digesting with appropriate enzymes, puri~ying the desired bands by electroelution from agarose gels and religating. Removal of the termination codon in p337 was accomplished by substituting a second synthetic fragment containing a serine resldue and a compatible Eco RI overhang (RI). Ligation of this fragment, to yield construct p365, resulted in the addition of another Bgl II site and loss of.the Eco RI site. Nucleotide 564 identifies the position of the last base contained in the Bgl II site required to fuse the insert in p365 with the collagen linker of the expression vectort Restriction sites are identified as follows: B, Bam HI; 8g, Bgl II; RI, Eco RI; RV, Eco RV; H, HinD III; N, Nco I; S, Stu I; X, Xba I.
FIG. 7. Diagram showing the construction o~ plasmid p367, the success~ul expression vector fox producing the synthetic cysteine-free IL-6 peptide. The steps in the construction of p367 are described in sections 5.2-5.5 in the taxt.
FIG. 8a. Plasmid map of recombinant plasmid p367 showing the location of the IL-6 sequence (designated HSF).
The assembled synthetic cysteine free recombinant IL-6 gene contained i~ an (Nco I - Bgl II) insert was introduced between the Nco I and Bam ~ I restrîction sites of the vector. The open box (C) indicates the location of the 60 amino acid collagen linker through which recombinant ~L-6 is fused to ~- galactosidase. Expression of the complete fusion gene produces a 130 kDa hybrid protein.
FIG. 8b. Plasmid map of recombinant plasmid p367 showing the location of the sequence for the synthetic cysteine-free IL-6 like peptide (designated HSF). The vector shown is a significantly modified version of pJG200 as describin~ in the application in section 5. The assembled synth~tic IL-6 likQ gene contained in an (Nco I-BGl II) insert was introduced between the Nco I and ~am HI
r~striction sites of tha vector. The open box (C) indicates the location of the 60 amino acid collagen linker through which the synthetic IL-6 like peptide is fused to ~-galactosidase. Expression of the complete fusion gene produces a hyhrid protein of approximately 130 kDa (see Figure 3 ~or expression of the plasmid in induced E. coli).
FIG. 9. NaDodS04-PAGE analysis of synthetic cysteine-free IL-6 protein purification. Samples collected at various stages o purification ~ere electrophoresed on 10%
polyacrylamide- NaDodS04 gels under reducing conditions as shown. Protein molecular weight markers are shown in lanes M. E. coli cells grown in 10 L batch culture were lysed by addition of lysozyme followed by brief sonication (Lane 1).
The unusually hydrophobic fusion protein was purified by repeated ammonium sulfate precipitation after solubilization in PBS- Sarkosyl (Lane ~). Partially purified ~usion protein was then digested with collagenase to release the 23 kDa recombinant IL-6 moiety (Lane 3j. After removing the majority of the cleaved, soluble ~-galactosidase by precipitation in 40% ammonium sulfate, synthetic I~-6 was recovered in near homogeneous form as a pellicle upon increasing the ammonium sulfate concentration to 70%
saturation (Lane 4). Reverse phas~ ~PLC was used in the final step of the purification~ The protein was recovered in 30 a single peak fraction eluting at 54% acetonitrile (Lane 5~.
FlG. 10. Stimulation of fibrinogen sythesis by synthetic cysteine-free recombinant IL-6 as prepared according to the methods described in ~5.2 to 5.8.
Confluent FAZ~ cell monolayers were treated with varying 35 concentrations of purified recombinant cysteine-free IL-6 in '12~
- ? 6 -the presence of 10 7 M dexamethasone. After five hours of stimulation, cell medium was recovered and filtered through a nitrocellulose membrane using a dot-blot apparatus. Secreted fibrinogen level~ were cletermined by solid phase immunoassay using a polyclonal fi~rinogen antiserum as primary antibody and alkaline phosphatase conjugated anti-IqG antiserum as secondary antlbody. Bound antibody was visualized by adding chromog0nic substrate~ ~BCIP ar.d NBT) to the blots.
Concentrations of secreted protein were determined by scanning laser densitmetry and comparison of signal intensities against dilutions of a purified rat fibrinogen standard.
FIG. 11. B-cell differentiation assay. CH12.LX cells (2 x 10 cells/well) were co-cultured in the presence or absence o~ antiyen (SRBC) prior to treatment with various concentrations of recombinant synthetic cysteine-free I~-6, as prepared according to the methods described in 5.2 to 5.8. After 48 hrs, treated CH12.LX aliquots were mixed with a freshly washed suspension of SRBC containing guinea pig complement, and transferred to Cunningham chambers for incubation at 37 de~rees Centigrade. After 30 minutes, cells were returned to room temperature allowing hemolytic plaques to develop. Results of the assay are expressed as plaque forming colonies per million viable cells (shown a~ Pfc on the y axis). Bacterial lipopolysaccharide, included in the assay as a positive stimulatory control, gave a response of 10,726 pfc/million cells. The results shown represent mean values o~ duplicate assays.
FIG. 12a and b. Diagrammatic illustration of ~he 30 construction o~ pTrpE/EK/cfIL-6. The details of the construction are found in the text in Section 5~10.1.
FIG. 13. Graphic depiction of ~ime 0 colony assay for stimulation of progenitor cells by various growth factors and combinations thereof.
3~ FIG. 14. Number of nonadherent cPlls after 7 days u~

liquid culture. This assay is described in Example ll.
FIG. 15. Imclone vs. Endogen IL-6 on 7tdrl~
FIG. 16. Imclone vs. bm IL-6 on 7td.1.
FIG. 17. Imclone v. Genzyme IL-6 using 7td.1.
FIG. 18. The sequence in pTrpE/EK/cfIL-6 from the enterokinase site through the amino terminal sequence o~
cysteine-free IL-6 to the natural carboxy terminus of IL-6 followed by three stop codons.

5. DETAILRD DESCRIPTION OF THE INVENTION
5.1. INITIAL EXPRESSION CONSTRUCTS
Initial attempts to express high levels of interleukin-6 in bacterial cells resulted in the production of no detectable or commercially useful amounts of the 15 protein. One attempt to produce commercially useful amounts of protein resulted in the synthesis of plasmid p360. In this expression vector, the synthetic cysteine-free IL-6 sequences from p337-1 were inserted immediately downstream from the minicistron sequence produced by the fusion of oligo 737 ~AGCTGATTA~ATAAGGAGGAATAACCATGGCTGCA-3'] and oligo 738 ~GCCATGGTTATTCCTCCTTATTTAATC-3']. In this construct the synthetic IL-6 peptide had a termination codon and was not fused to the ~-galactosidase protein. Induction was expected to yield a non-glycosylated synthetic IL-6 peptide slightly larger than 22 KDa. The construction of plasmid p360 is graphically diagrammed, but not to scale, in Figure 1. The inductiQn of expresslon of the synthetic IL-6 peptide by the addition of IPTG to cells carrying p360 resulted in no detectable peptide (see Figure 3, lanes 3a and 3b for absence 30 of protei~ at the arrow).
Similarly induct~on of a strain carrying plasmid p369, constructed as diagrammed in Figure 2, wherein the synthetic IL-6 like p~ptide was fused in frame with the alpha-complementing fragment of ~-galaotosidase resulted in no 35 detectable protein (see lanes la and b, Figure 3).

These unsuccessful attempts to produce synthetic IL-6 c~n ~e compared in Figure 3 with the successful production, as described i _ a, of the synthetic cysteine-free IL-6/collagen/~-galactosidase fusion protein (see the large amount o~ the 130 KDa protein in lanes 2a and b o~ Figure 3).

5.2. CONSTRUCTION OF AN IL-6 EXPRESSION VECTOR
5.2.1. THE INITIAL VECTOR pJG200 Plasmid pJG200 was the starting material that was modified to produce a successful IL-6 expression vector. The initial plasmid, pJG200, contained target cistrons that were fused in ~he correct reading frame to a marker peptide with a detectable activity via a piece of DNA that codes for a protease sensitive linker pepkide (Germino and Bastia, 1984, Proc. Natl. Acad. Sci. USA 81:~692; Germino et al~, 1983, Proc. Natl. Acad. Sci. USA 80:6848). The promoter in the ori~inal vector pJG200 was the PR promoter of phage lambda.
Adjacent to the promoter is the CI857 thermolabile repressor, followed by the ribosome-binding site and the AUG initiator triplet of the Cro gene of phage lambda. Germino and Bastia inserted a fragment containing the triple helical region of the chicken pro-2 collagen gene into the Bam HI restriction site next to the ATG initiator, to produce a vector in which the collagen sequence was ~used to ths lacZ ~-galactosidase gene sequence in the correct translational phase. A single Bam HI restriction sites was regenerated and used to insert the plasmid R6K replication initiator protein coding sequence.
The plasmid pJG200 expressed the R6K replicator initiator protein as a hybrid fusion product following a temperature shift which inactivated the CI~57 repressor and allowed transcription initiation ~rom the PR promoter. Both the parent vector construct with the ~TG initiator adjacent to and in frame with the collagen/~-galactosidase fusion (non~nsert vector), and p~G200 containing tbe R6K r~plicator initiator prot~in joined in frame to the ATG initiator codon (5') and the collagen/~-galactosidase fusion t3') (insert vector), produced ~-yalactosidase activity in bacterial cells transformed witll the plasmids. As a result, strains containing plasmids with inserts are not distinguishable from strains containing the parent vector with no insert.

5~2.2. REMOVAL OF THE P CI857 REPRESSOR
AND AMINO TE~MINBS OF CRO
The first alteration to pJG200 in this invention was 10 the removal and replacement of the Eco RI~Bam HI fragment that contained the PR promoter, CI857 repressor and amino terminus of the cro protein which provided the ATG start site for the fusion proteins. An oligonucleotide linker was inserted to produce the p258 plasmid, which maintained the 15 Eco RI site and also encoded the additional DNA sequences recognized by Nco I, Bgl II and Bam HI restriction endonucleases. This modificatlon provided a new ATG staxt codon that was out of frame with the collagen/~-galactosidase fusion. As a result, there is no ~-galactosidase activity in 20 cells transformed with the p258 plasmid. In addition this modification removed the cro protein amino terminus so that any resultant recombinant fusion products inserted adjacent to the ATG start codon will not have cro encoded amino acids at their amino terminus. In contrast, recombinant proteins 25 expressed from the original pJG200 vector all have cro encoded amino acids at their amino terminus.

5.2.3. AD~ITION OF THE P PROMOTER, SHINE
DA~GARNO SEQUENCET~D ATG CODON
In the second step oP construction of the IL-6 expression VeGtOr~ a restriction fragment, the Eco RI-Nco I
fragment of pKK233-2 (Pharmacia Biochemicals, MilwauXee, WI~, was inserted into the Eco RI-Nco I restriction sltes o~
plasmid p25~ to produce plasmid p277. As a result, the p277 ZO~

plasmid contained the PTAC ~also known as PTRC) promoter of pXK233-2, the lacZ ribosom~ binding site and an ATG
initiation codon. In the p277 plasmid, the insertion of a target protein sequence allows its transcription from an IPTG
inducible promoter in an appropriate strain background. The appropriate strain background provides sufficient lac repressor protein to inhibit transcription from the uninduced Pq,AC promoter. B~ca~se cells can be induced by the simple addition of small amounts of the chemical IPTG, the p277 plasmid provides a significant commercial advantage over promoters that require temperature shifts for induction such as the PR promoter of pJG200. Induction of commercial quantities of cell cultures containing temperature inducible promoters would otherwise require heating large volumes of cells and medium to produce the temperature shift necessary for induction. For example, induction by the PR promoter requires a temperature shift to inactivate the CI857 repressor inhibiting pJG200's promoter. One additional benefit of the promoter change is that cells are not subjected to high temperatures or temperatuxe shifts. High temperatures and temperature shi~ts result in a heat shock response and the induction of heat shock response proteases capable of degrading recombinant proteins as well as host proteins (See Grossman et al., 1984, Cell 38:383; Baker et al., 1984, Proc. Natl. Acad. Sci. 81:6779).

5.2.4. IMPROVEMENT OF T~E RIBOSOME BINDING_SITE
The p277 expression vector was further modified by insertion o~ twenty-nine base pairs, namely 5'CATGTATCGATTAAATAAGGAG~AAT~AC3' into the Nco I site of p277 to produce plasmid p340. This sequence i5 related to, but different than, one portion of the Schoner "minicistron"
sequence (described in section 2.5.1). The inclusion of these 29 base pairs provides an optimum Shine/Dalgarno site for ribosomal/mRNA interaction. The final p340 vector significantly differs from pJG200 because it contains a highly inducible promoter suitable ~or the high yields needed for commercial preparations, and improved synthetic ribosome binding site region to improve translation, and means to provide a visual indicator of fragment insertion. The steps in the construction of vector p340-1 are diagrammed in Figure .

5.3. M FICATIONS OF THE INTERLEUKIN-6 SEQUENCE
The coding sequence used for the exprassion of synthetic cysteine-free recombinant IL-6 was based on the cDNA sequence of human IL-6. The coding sequence was cons~ructed using 11 complementary synthetic oligonucleotide pairs. Several mutatio~s were introduced into the recombinant IL-6 coding sequence during oligonucleotide synthesis to enable proper assembly of the gene sub-fragments on the on2 hand, and to ensure efficient expression of the assembled gene on the other. Nucleotide sequence modifications, designed to introduce novel restriction sites for use in joining the gene sub-fragments, were incorporated within the coding sequence in such a manner as to avoid altering the amino acid composition of tha synthetic gene with respect to the native IL-6 protein sequence. The modifications included: 1) replacement of the 5' terminal 118 nucleotides, which encode th2 28 amino acid signal sequence normally found in the native IL-6 gene, with a methionine codon (nucleotides 3-5); ii) replacement o~ the [Pro] residue in the native IL-6 protein sequence with tGly]
(nucleotides 6-8) in the syn hetic IL-6 sequence; iii) replacement of the normal TAG stop codon with a serine codon (nucleotides 558-560) to effect fusion of the synthetic lL-6 protein with the collagen linker. and iv) replacement of the four internal cysteine residues with serines (nucleotides 135-137, 153-155, 222-224, 252-25~) to produce a synthetic 35 lL-6 protein that is unable to form disul~ide bonds. ~he ~0~26~

eequence of the modified synthetic cysteine-free protein is included in Figure 5. Those skilled in the art will recognize that other modifications in the sequence of the synthetic peptide are useful in the present invention. The invention as contemplated includes the modi~ication of other amino acids of the peptide sequence or other nucleotides of the DNA sequence.

5.4. OLIGONUCLEOTIDE SYNTHESIS AND AS5EMBLY
Assembly o~ the synthetic oligomers was carried out in three ~teps. Initially, oligonucleotides bearing complementary overhangs were annealed and ligated to produce four separate double stranded fragments, one composed of four oligonucleotides (2 per strand) and three composed of six oligonucleotides each (3 per strand). Before assembly, synthetic oligomers were kinased with 10 units of T4 polynucleotide kinase. To prevent concatenation during ligation, the 5' terminal oligomers on either strand were not phosphorylated. Subsets of these double stranded oliqonucleotides were assembled in separate annealing and 2 ligation reactions to produce four sub ~ra~ments, each repre~entin~ approximately one fourth of the recombinant synthetic cysteine-free IL-6 coding sequence.
A modified plasmid was constructed to allow for DNA
amplification and ease in sequencing each oligomer. A pBS
M13+ cloning vector (Stratagene) was modified by insertion of a 28 base oligonucleotide adapter (5'AGC~TCCATGGTCGCGACTCGAGCTGCA-3') between the Hind III and Pst I site~ of its multiple cloning region. As a result, the modified plasmid, designated p2~7, no longer contains its original Sph I restriction site bu~ encodes additional sites for Nco I, Nru I and Xho I.
The synthatic oligomers were separately cloned into the modified pBS M13+ vector p287 to allow DNA amplification and 35 sequence verification by dideoxy-nucleotide sequencing.

~o~

Insertion of the assembled fragments into th~ modified vector produced recombinant plasmids p333, p334, p331, and p332, each containing a portion of the synthetic cysteine-free IL-6 protein coding region proceeding ~rom amino to carboxy terminus respectively. Following ligation, each plasmid DNA
was transformed separately into competent E. coli JMlol.
The second step of the assembly involvQd the construction of two vectors that encoded the amino and carboxy halve~ ~f synthetic cysteine-free IL-6. These were obtained by ligating the inserts of p333 and p334 to produce the N- te~minal coding vector p336 and by joining the inserts of p332 and p331, to form the C-terminal coding vector p335.
In the third and final step of the construction, inserts were subsequently combined to yield the entire coding sequence of the recombinant synthetic cysteine-free IL-6 gene. The insert released from plasmid p335 was ligated into p336. The resulting plasmid p365 contained the complete coding sequence o~ synthetic IL 6 inserted between the Nco I
and Eco RI sites o the modified pBS M13~ vector p2~7. Th~
constructions are shown diagrammatically in Figure 6.
2n 5.5. CONSTRUCTION OF THE p367 VECTO~ FOR
EXPRES5ING THE SYNTHETIC IL-6/COLLAGEN/~
GALACTOSIDASE FUSION PRODUCT
The assembled cysteine-free recombinant IL-6 gene was 25 excised from p365, and inserted into plas~id p340 between the Nco I site, which encompasses the initiating methionine, and the BamH I site adjacent to the collagen linker as depicted in Figurs 7. The resulting vec~or p367, diagrammed in Figure 8 but not to scale, was used to transform E. coli JM101.
30 Recombinant colonies were selected on the basis of antibiotic resistance and by appearance o~ blue coloration in the presence o~ X-Gal. The size of ~he insert DNA was con~irmed by mini-lysate extraçtion ~ollowed by polyacrylamide gel electrophoresis.

~4~fi~1.

A tripartite fusion protein composed of synthetic cysteine-free IL-6, a sixty amino acid collagen linker and fl-galactosidase was produced in tran~formed bacteria ~See Figure 3 and Figure 9). As predicted from the gene sequence, ampicillin resistant transformants carrying the modified IL-6 expression plas~id produced blue colonies upon addition of the inducing agent IPTG and the chromogenic ~-galactosidase substrate X-Gal. Synthesis of the fusion protein by IPTG-induced transformants was independently confirmed by western blot analysis of a total E.coli lysate using a monoclonal anti-~-galactosidase antibody obtained from Promega BiotecO
The monoclonal anti-~-galactosidase antibody used in the Western blot recognized a band with an apparent molecular weight of 130,000 kDa.

5.6. INDUCTION OF LARGE AMOUNTS OF THE MODIFIED

Transformed JM101 containing plasmid p367 were grown in 10 L batch cultures using a Magnaferm fermentor tNew Brunswick Scienkific). Cells were grown in 2x YT containing 20 100 ug/ml ampicillin and induced at A550 = 1.5 by addition of 5 mM isopropylthio-~D-galactopyranoside (IPTG, Sigma). At to 12 hours post-inoculation, when tlle c 11 numbers and ~-galactosidase activity had reached maximal levels, cells were pelleted by centrifugation at 5000 x g and stored frozen at 25 -20~C until needed.

5.7. PURIFICATION OF THE FUSION PROTEIN
Frozen E. coli cell pellets were processed in aliquots of 100 g (wet wei~ht) by washing with TNS buffer ~30 mM
30 Tris.Cl, pH 7.4; 30 mM NaCl, 0.05% sodium lauroyl sarcosine~.
Washed cells were lysed in 450 ml TNS containing 1.5 mM EDTA
and 0O5 mg/ml lysozyme. After incubating the suspension on ice for 30 minutes, complete lysis was ensured by subjecting cells to three cycles of freeze-~hawing and brief sonication.

2~,~

Soluble prot2ins, devoid of ~-galactosidase activity, were removed by three repeated washings in TNS followed by centrifugation at 10,000 x g for 20 minutes. The ~inal pellet, weighing approximately 40 g, was resuspended in 60 ml of 10% sarkosyl and diluted to 2~4 liters with phosphate buff~red saline ~PBS). Insoluble material was removed by centrifugation and the supernatant made 40% with respect to a saturated solution of ammonium sulfate. After incubating the extract on ice for 30 min. precipitated protein was again recovered by centrifugation and sub~ected to two additional rounds of ammonium sulfate precipitation. The ~inal extract was resuspended in 250 mls of 20 mM Tris.Cl, pH 7.4 and 150 mM NaCl, divided into 4 ml aliquots and stored at -20-C until ready for further proce~sing.
5.8. CLEAVAGE OF THE FUSION PROTEIN AND PURIFICATION

The recombinant synthetic cysteine-free IL-6 protein was purified to homogeneity using r~verse phase HPLC. Thawed extract (4 mls) was sonicated briefly to disperse aggregates, 20 added to pre- treated collagenase, and incubated for 45 minutes at room temperature. The majority of the cleaved ~-qalactosidase was removed by adding 0.5 volumes of saturated ammonium sulfate, incubating on ice for 30 minute~ and pelleting the insoluble material. The cleaved recombinant 25 cysteine-frea IL-6 was concentrated by bringing the total ~olum~ of the supernatant to 13 mls with saturated ammonium sulfate, incubating on ice for 30 minutes, and centrifuging to compact the insoluble protein into a floating pellicle.
Liquid was drained by puncturing the tube, and the remaining 30 pellicle was resuspended in 0.5 mls of Tris buffered saline.
The resuspended recombinant cysteine-free IL-6 was prepared for reverse phase HP~C by adding an equal volume 9f 60% acetonitrile, 0.1% trifluroacetic acid. Insoluble material was pelleted and the clarified supernatant was ~ 3~

loaded onto a 250 mm, 4.6 mm ID reverse phase column (Vydac, 218Tp, Cl8, 10 ~Im- Alltech Associates) in an injection volume of 0.5 to 1 ml. The mobile phase consisted of varying concentrations of solvent B (60% acetonitrile and 0~1%
triflurooacetic acid) relative to solvent ~ (0.1 trifluoracetic acid). The flow rate was 0.5 ml 1 min , and the system programmed to deliver two consecutive linear gradients, from 25~ to 80% B in five minutes, and 80% to 100%
B over 54 minutes. Protein eluted from the column at 54%
acetonitrile and collected in a single peak fraction was the purified synthetic cysteine-free interleukin-6 peptide which migrated at 2~ kDa following SDS-PAGE. The steps of the purification are indicated in Figure g and the yields at each step are provided in Table 1.

x~

Yield Purification step _ (m~lOOg wet cell weiqh~) Total Fusion protein protPin Galactosidase rIL~6 Whole cells12000 1080 --- 156a 10 ~ashed Lysateb1420 570 ~~~ 81a Collagenased lysate 1220 --- 491 75 Digest supernatantd 80 --- 22 27 HPLC fractione16 16 ~5 Relative Purificatio Purification step _(~raction of total_protein~
Fusion Protein Galactosidase rIL-6 Whole cells 0.09 --- 0.013a Washed Lysateb0.40 --- 0.057a 20 Collagenased lysateC --- 0.40 0.061 Diges~ supernatantd --- 0.27 0.34 HPLC fractione ~ - 1.0 a Hypothetical value based on a calculated molecular weigh~
ratio of about 1:7 for rIL-6:fusion protein.
25 b Insoluble cellular material after washing with TNS buîfer, detergent solubilization, and repeated ammonium sulfate c precipitation.
Washed lysate fraction after digesting 1 hour with d collagenase.
Supernatan~ fraction after precipi~a~ion of collagenase~
lysate with 3~% satura~ed ammonium sulfate.
30 e Fraction containing rIL-6 after running the digest supernatant on reverse phase HPLC column.

,, To confirm the identity of the 23 kDa protein, HPLC-35 purified material was subjected to direct N-terminal automated protein sequencing. The amino acid residues tMGVPPGED) identified after seven cycles of sequential degradation coincided with the predicted N-terminal amino acid composition of the recombinant protein as deduced from the synthetic recombinant cysteine free IL-6 gene.
Confirmation o~ the protein sequence revealed that the preparation contained a small fraction of recombinant cysteine-free IL-~ protein with a terminal methionine residue.
In a particular embodiment the isolated active fraction consisted of a mixture of amino terminal methionine-containing and amino terminal methionine-free synthetic cysteine-free IL-6 proteins. ~mino acid sequence analysis indicates that in one preparation of the mixture 90% of the mixture was methionine-free at the amino terminus and 10%
d contained an amino terminal methionine. In this embodiment, the active preparation of the cysteine-free protein varies from natural IL-6 in that 1) no intramolecular disulfide bonds occur; 2) a methionine amino acid at position one in the bioengineered protein replaces the signal sequence amino 20 acids 1-28 of the natural unprocessed protein; 3) amino acid two in the bioengineered protein, glycine, replaces the proline amino acid present ~n the natural IL-6 protein: 4) the carboxy terminus contains additional amino acids.
Collagenasa generally cleaves after Y in the sequence P-Y-G-P wherein Y represents a neutral amino acid. See Keil et al. FEBS Letters 56: 29~-296 (1975). In the present example of the fusion protein with II,-6 sequences the neutral amino represented by Y i5 valine. Accordingly, the carboxy terminus of the protein produced by induction of the fusion protein coded by the p367 vector after digestion of the fusion protein with collagenase is expected to be mainly . .
. P-G-P-V-G-P-V and/or . . . P-G-P-Vo If suf~icient collagenase is present under sufficiently rigorous ~6 conditions, the carboxy terminus is exclusively . . .

P-G-P-V. I the amino acid sequence starting with the third amino acid (i.e. V) in Figure 5 and ending with the fourteenth amino acid from the end (i.e~ M) is called pep, a mixture of the following peptides may be prepared by the present example:

G-pep-SDPGPVGPV ~Protein I) G-pep-SDPGPV (Protein II) MG-pep-SDPGPVGPV (Protein III) MG-pep-SDPGPV (Protein IV) Such a mixture was used in the asYays described in Sections 5.11l 7, R, 9, 10 and 11. The termini of the peptides of this mixture differ from that of native IL-6 by the presence of G or MG instead of P at the amino terminus and by the pre~ence of SDPGPV and SDPGPVGPV at the carboxy terminus. ~hese diffarences in the termini of cysteine-free IL-6 do not affect the activity of the protein.

5.9. ALTERNATE METHODS OF PREPARATION

The foregoing description is but one specific example of a useful method by which the Rynthetic cysteine-free IL-6 peptide of the present invention may be prepared. However, ~5 those ~killed in the art will readily recognize that other vector constructs, as well as other unicellular host, are also useful in the method of the present invention. In very general term~, for example, the skilled artisan will recognize that to eventually achieve transcription and tran~lation of the inserted gene, the gene must be placed under the control of a promoter compatible with the chosen host cell. A Promoter is a region of DNA at which RNA
polymerace attaches and initiates transcription. The promoter selected may be any one which ha~ been i olated 6~
-39a-from the host cell organism. For example, E coli, a commonly used host system, has numerous promoters such as the lac or rec~ promoter associated with it, its __ bacteriophages or its plasm.ids. Also, synthetic or recombinantly produced promoters, such as the ~ phage PL and PR promoters may be used to direct hiqh leval production of the segments of DNA
adjacent to it. Similar promoters have also been identified for other bacteria, and eukaryotic cells.
Signals are also necessary in order to attain efficient transcription and translation of the gene. For example, in E. coli m*NA, a ribosome binding site includes the translational start codon (AUG or ~UG) and other sequence complementary to the bases of the 3' end of 16S ribosomal RNA. Several of these latter sequences (Shine-Dalgarno or S-D) have been identified in E. coli and other suitable host cell types. Any SD-ATG sequence which is compatible with the host cell system, can be employed. These SD-ATG sequences include, but are not limited to, the SD-ATG sequences o~ the cro gena or N g~ne of coliphage lambda, or the E. coli tryptophane E, D, C, B or A genes.
A number of methods exist for the insertion of DNA
fra~ments into cloning vectors ln vitro. DNA ligase is an enzyme which seals single-stranded nicks between adjacent nucleotides in a duplex DNA chain; this enzyme may therefore be used to covantly join the annealed cohesive ends produced by certain restriction enzymes. Alternatively, DNA ligase can be used to catalyze the formation of phosphodiester bonds between blunt-ended fragments. Finally, the enzyme terminal deoxynucleotidyl transferase may be employed to form homopolymeric 3'-single-stranded tails at the ends of fragments; by addition of oligo ~dA) sequences to the 3' end of one popula~ion, and oligo (dT) blocks to 3' ends of a second population, the two types of molecules can anneal to form dimeric circles. Any of these methods may be used to ligate the control ~lements into specific sites in the vector. Thus, the sequence coding for the cysteine-free or cysteine-containing IL 6 fusion protein is ligated in~o the chosen vector in a specific relationship to the vector promoter and control elements, so that th~ sequence is in the 2~

correct reading frame with respect to the vector ATG
sequence. The vector employed will typically have a marker function, such as ampicillin resistance or tetracycline resistance, so that transformed cells can be identified. The method employed may be any of the known expression vectors or their derivatives; among the most frequently used are plasmid vectors such as pB~ 3~2, pAC 105, pVA 5, pACYC 177, pKH 47, pACYC 184, pUB 110, pmB9, pBR325, col El, pSClOl, pBR313, pML21, RSF212~, pCRl or Rp4; bacteriophage vectors such as lambda gtll, lambda gt-WES-lambda B, chain 28, chain 4, lambda gt-I-lambda BC, lambda-gt-l lambda ~, M13mp7, M13mp8, M13mp9; SV40 and adenovirus vectors; and yeast vectors. ThP
vector is selected for its compatibility with the chosen host cell system. Although bacteria, particularly E. coli, have proven very useful in high yield production of the synthetic IL~6 peptide, and are the preferred host, the invention is not so limited. The present method contemplates the use of any cultuxable unicellular organism as host; for example, eukaryotic hosts such as yeast, insect, and mammalian cells, are also potential hosts for IL-6 production. The selection of an appropriate expression system, based on the choice o~
host cell, is well within the ability of the s}cilled artisan.
One skilled in the art will readily recognize that variations on the described fusion protein are also possible.
For example, the order of the first and third peptide portions can be reversed, so that the third peptide segment is positioned at the amino terminus and the sequence coding fox peptides with IL-6 activity is a~ the carboxy terminu~, with thQ second, cleavable peptide portion remaining as a 30 link between the two segments.
The identity of each of these segments may also be varied. For example, substantial variation is possible within and around the basic IL-6 peptide sequence. A
particularly interesting observation is that a substantial 35 portion of the amino terminus can be deleted, not only without loss of activity, but with a resultant 2-3 fold increase in activity in both cysteine-containing and cysteine free IL 6 sequences. However, removal o~ the last 20 residues in the sequence results in a complete loss of activity, in both cysteine-containing as well as cysteine-free forms. Also, as noted above, the presence o~ additional amino acid residues on the carboxy terminus of the IL-6 peptide doe~ not affect the biological activity of the molecule.
It will also be understood by those ski:Lled in the art that any amino acid in the known sequence of IL-6 may be substituted with a chemically equivalent amino acid. In other words, "silent changes" may be made in the amino acid sequence without affecting the activity of the molecule as a whole. For example, as has be~n shown, substitution of all cysteine residues with serine residues allows the modified IL-6 molecule to retain its biological activity. Alternative choices as substitutes for cysteine are other ne-ltral amino acids such as valine, proline, isoleucine and glycine, serine, threonine or tyrosine. Negatively charged residues, such as aspartic acid and glutamic acid may be interchanged, as may be positively charged residues such as lysine or arginine. Hydrophobic r~sidues including tryptophan, phenylalanine, leucine, isoleucine, valine and alanine may al50 be exchanged. Alteration of the sequence by amino acid substitution, deletion, or addition and subsequent testing of the resultant molacule to determins if biological activity is retained is well within the ability of one skilled in the art, without neces~ity for undue expPrimentation.
The identity of the cleavable linker peptide sequence is also a matter of choice and may be accomplished using chemical or enzymatic means. The sequence employed may be any one which can be chemically cleaved, so tha~ t~e peptide with the biological activity of IL-6 can be released from the 35 remainder of the fusion protein. In a preferred embodiment, -the cleavable sequence is one wllich is enzymatically degradable. A collagenase-susceptible sequence is but one example. Other useful sites include enteroklnase- or Factor Xa-cleavable site. For example, enterokinase cleaves after the lysine in the sequence Asp-Asp Asp-~,ys. Factor Xa i~
specific to a site having the sequence Ile~Glu-Gly-Arg, and cleaves after th~ arginine. Another useful cleavage site is that of thrombin which recognizes the sequence Leu-Val-Pro-Ary-Gly-Ser-Pro. Thrombin cleaves between the Arg and Gly residues. Other enz~me-cleavable sites will also be recognized by those skilled in the art. Alternately, the sequence may be selected so as to contain a site cleavable by cyanogen bromide; cyanogen bromide attac~s methionine residues in a peptide sequence.
It is preferabla, although not essential, to select a linker sequence which, when cleaved, leaves a minimal number of residues attached to the IL-6 sequence, so that the terminus of the released IL-6 active peptide is as near to the native sequence as possible. In a particular embodiment the IL-6 active portion is at the carboxy terminal end of the 2 fusion protein, and the cleavage site i5 specific for a protease that is capable of leaving the natural pro-val-pro amino terminal peptide sequence. Examples of such cleavage sites axe those that are cleaved by enterokinase or Factor Xa.
The identity of the third peptide seguence may also be varied. This portion of the tripartite structure potentially serves two purposes: (1) the use of a correctly selected protein, capable of being expressed in the chosen host, can 30 place the produotion of peptides having activity Ih-6 under the control of a qtrong promoter, and thus ~acilitate the production of those peptides; and (2) it can provide a convenient means for identifying transformed clones producing the fusion protein. For example, in the discussion provided 35 above, the full sequence encoding ~-galactosidase was used;

this protein provides a visual means of detection by the addition of the proper substrate.
Alternatively, the third peptide portion can ~e a fraction of such a protein, provided that the portion remaininq i~ still readily expressed by the host cell. This portion can also be a peptide which is not necessarily visually detectable, but the presence of which may be detectable by other means, such as by calculation of the expected molecular weight of the fusion protein or insertion into a vector with a detectable marker. Another useful alternative sequence for use in a prokaryotic host is the ~E gene product, or a portion thereof (Kleid et al., Science 214:1125-1129, 1981). Additional choices include sequences coding for the cro gene of ~ phage or othex portions of the lac genes than the lac~ sequence coding for ~-galactosidase. Those skilled in the art will recognize additional choices which may provide the basis for the third peptide portion of the claimed fusion protein.

5.10. ALTERNATE DNA CONSTRUCTS
The followins Examples illustrate the preparation of DNA constructs in which the position of the first and third peptide portions of the tripartite ~usion proteins is reversed. Also illustrated is the use of alternative linkers and third peptide portions. The quantity of production of IL-6 protein using this construct is also high, ranging ~rom about 1-~0% of total soluble cellular protein.

5.10.1. TrpE - ENTEROKINASE CLEAVAGE SITE -IL-6 MUTEIN FUSION PROTEIN _ _ ~a) A fusinn protein that encodes beta alacto~idase, followed by an enzymatic cleavage site, followed immediately by a synthetic IL-6 peptide sequence having C-terminus and N-terminus ends of the natural IL-6 peptide and all four cysteines replaced by serinss, is expressed by a new recombinant plasmid p-beta-Gal/cfIL-6. To prepare p-beta-GalJcfII.-6, th~ plasmid p365 - which has been described in Section 5.5 and Figure ~ of this specification - is digested with the enzymes EcoRII (to cut the EcoRII site that is located 14 bases from the 5' NcoI site) and ~glII (to cut the ~glII site ~hat is shown in Figure 13 aæ BglII'). The plasmid is digested with 5 units of each enzyme per 10 ~g plasmid at 37C :~or 2 hours. The 0.492 Kb EcoRII/BglII
fragmen-t is isolated by standard procedures such as electro-el~tion. This 0.492 Kb fragment is called sequence A. This sequence A and the subsequent sequences noted in the following text of this section refer to the illustrations in Figure 12.
(b) An ~dditional aliquot of plasmid p365 15 (approximately 10 ~g) is digested with HindIII and ~glII as described above at pH 7.5 in a bu~fer of 25 mM Tris HCl, 100 mM MgC12, 10 mg~ml BSA and 2 mM BME. The large (3.0 Kb) fragment that results from cutting the unique HindIII site and the BglII site referred to in Figure 13 as BglII' is called sequence B
~ .
(c) A synthetic, double-stranded oligonucleotide (sequencP C) is prepared and ligated to sequence A and sequence B. The oligonucleotide starts with overlapping HindIII and BclI sites, encodes a sequence of amino acids con~aining an enterokinase cleavage site followed immediately by the first three amino acids of natural IL-6, Pro-Val-Pro (PVP), and ends with an EcoRII site. The sequence o~ the oligonucleotide is:

BclI EK sit~
5'AG CTT GAT CAG GCG GAT CCG GAA GGT GGT AGC GAC GAC GAC GAC AAA
3' A CTA GTC CGC CTA GGC CTT CCA CCA TCG CTG CTG CTG CTG TTT
P V P 3' CCG GTT CCG
GCC CAA GGC GGT CC 5"

61.

EcoRL I

Each strand of the oligonucleotide ls prepared separately, treated with polynucleotide kinase in the pre~ence of 1 mM rATP in a ~uita~le reaction buffer at 37-C
for 30 minutes, ~nd annealed by heating to 85-C for 5 minutes, followed by slow cooling to 25-C.
To ligate sequence C to sequences ~ and B, approximately 1 ~g of synthetic cysteine-free IL-6 EcoRII/BglII fragment tsequence A) is coprecipitated with 200 ng of the synthetic oligonucleotlde (sequence C) and ligated to the HindIII/BglII vector component ~sequence B) of p36S.
Li~ation is accomplished in a 20 ~1 reaction volume containing 20 mM Tris HCl, pH 7.6, 0.5 mM rATP, 10 mM MgC12, 5 mM DTT at 16- overnight. The new plasmid is pABC. pABC is cloned by adding a 5 ~1 aliquot of the reaction mixture to competent HB101 bacteria. ~mpicillin-resistant colonies are selected after overnight incubation at 37C.
(d) The 3' end of the recombinant synthetic cysteine-free IL-6 gene expressed in p365 is reconstructed to encode the natural I~-6 carboxy terminus, which ends with methionine.
To accomplish this, the following oligonucleotide is synthesized as above~

S' GA TCT TTC AAA GAA TTC CTG CAG TCC TCC CTG CGT GCT CTG CGT
3' A AAG TTT CTT AAG GAC GTC AGG AGG GAC GTA CGA GAC GCA

CAG ATG TAA TGA TAG GTA C 3' GTC TAC ATT ACT ATC 5' This oligonucleotide, reading from left to right, starts with a BglII site, encodes the natural amino acid sequence of IL-6 that follows the BglII' site of p365, and 35 concludes with a methionine residue that is followed Z~ 2~.

immediately by three stop codons an~ a KpnI site (sequence D).
(e) p~BC (step C) is di~ested with HindIII and BglII
(sequence E).
(f) PATH 23 (available from A. Tzajaloff, Columbia University, New YorJc City) is an ampicillin-resistance plasmid containing a gene that encodes the amino-terminal 3~7 a~ino acids of TrpE (anthranilate synthetase component I) adjacent, and in reading frame at its 3' end with, a polylinker containing a HindIII site. A general description of the TrpE operon may be found in ~iller and Reznikaff, eds., The Operon, Cold Spring Harbor Laboratory, pp. 263-302 (1978). Other sources of DN~ that encode all or part of trpE
and lacZ are readily available. Such other sources may be found, for example, in Pouwels et al., Cloning Vectors, A
Laboratory Manual, Elsevi~r, 1985. For example, trpE
sequences may be isolated from plasmids haviny the following identifying codes in the Pouwels et al. manual:
I-A-ii-3 (pDF41 and 42), I-A-iv-23 (pRK353), I-B-ii-4 (pMBL24), I-B~ii-1 (ptrpED5~ D-i-3 (pEP70-pEP75~, and I-D-i-4 (pEP165 and pEP168).
10 ~g PATH 23 is digested with 5 units each of HindIII
and XpnI at 37 C for 2 hours. The large fragment is isolated by gel chromatography, followed by electro-elution and ethanol precipitation (sequence F).
(g) Approximately ~00 ng of sequenc~ D are mixed with approximately 1 ~g of sequence E. The resulting mixture is coprecipitated with ethanol in tha presence o~ sequence F and ligated as described above. The resulting fragment i5 called PTrpE/EK/cfIL-6.
(h) Competent E. coli host cells are trans~ormed with pTrpE/EK/cfIL-6. For example, the E. coli HB101 strain is used as host cell for transformation in one embodiment.
Ampicillin-resistant colonies are gathered. These colonies express a fusion protein comprising a TrpE segment, an amino 6~.

acid segment recogni2ed and cleaved by enterokinase, and the synthetic cysteine-free IL-6 amino acid sequence that has the termini at both carboxy and amino ends of the natural IL-6 peptide, starting with PVP and ending with M.
The structure of the protein following cleavage is shown in Figure 18.

5.10.2. ~ETA-~ALACTOSIDASE-ENTEROKINASE
CLEAVAGE SITE - SYNTHETIC CYSTEINE-FREE
IL-6 MUTEIN FUSION PRO?EIN
The protocol described in Section 5.10.1 for producing the TrpE enterokinase - cleavage site synthetic cysteine-free IL~6 fusion protein is ollowed, except the pEx-l vetor is substituted for PAT~ 23 in step f. pEx~
digested with BamHI and XpnI. ~amHI and BclI have compatible restriction sites. rrhe resulting construct contains a thermoinducible ~-galactosidase gene followed by a cloning polylinker. The truncated gene produces a peptide that i3 approximately 48 Rd of the beta-galactosidase protein (Stanley, K.K. and Luzio, J.P., 1984, EMBO J., Vol.
3, pp. 1429-1434). The resulting construct is called ~ gal/
EK/cfIL-6. Another source of beta-galactosidase DNA
includes pHg~000 described in 5.2.1. The fu~ion protein is produced after transformation of E.coli N4830 cells with p~gal/EK/cfIL-6 and thermoinduction. The plasmid is replicated in E.coli strain N99. N99 and N4830 are available from Pharmacia. The fusion protein is cleaved by enterokinase using methods known and used in the art.
It is routine to cleave proteins having an enterokinase recognition site with enterokinase. See, for example, Hopp et al., ~iotechnology 6: 1204-1210 (1988~.

-48a-5.10.3. FUSION PROTEINS WITH FACTOR Xa CLEAVAGE SITE
A factor Xa site is substituted for an enterokinase site by modifying step C of Section 5.10.1 and 2. The synthetic oligonucleotide (sequence C) shown in step (C) of Section 5.10.1 comprises a DNA sequence that encodes Asp.Asp.Asp.Asp.Lys. This DN~ sequence is modified so as.to X~

encode the factor Xa cleavage recognition site, Ile.Glu.Gly.Arg. The resulting construct is called p~gal/Xa/cfIL-6. The plasmids pTrpE/EK/cfIL-6 and p~gal/Xa/cfIL-6 are expressed as described in ~5.10.1 and 5.10.2. The resulting fusion proteins are cleaved with factor Xa, which cleaves after the arg in its recognition site by methods known in the art. See, for example, Nagal &
Thogerson, Nature 309: 810-812 (1984).

5.11. ASSAYS OF ACTIVITIES OF THE
SYNTHETIC CYSTEINE-FREE PROTEIN

5.11.1. HEPATOCYTE STIMULATION ASSAY
FAZA 967 rat hepatoma cells were grown in DMEM/F12 supplemented with 10% NuSerum (Collaborative Research), 15 penicillin and streptomycin. Assays were performed on one day old confluent monolayers s~eded in 48 well plates (Costar). Prior to treatment with cysteine-free recombinant IL-6, as prepared according to the methods described in 5.2 to 5.8, and other conditioned media, cells were washed with 20 serum-free medium con~aining 107 M dexamethasone. Treated cells were subsequently maintained in serum-free/dexamethasone medium for the duration of the assay.
Cells were incubated in the pressnce o~ cysteine free recombinant IL-6 and other conditioned media for five hours 25 at 37-C in a tissue culture incubator. After treatment, cell supernatants were removed and ~tored at -20C or assayed directly for fibrinogen as follows. Two-fold serial dilutions of cell supernatants were prepared in a ~eparate 96-well microtiter plate and spotted onto a 0.45 7m 30 nitrooellulose filter using dot~blot apparatus (Bio-Rad).
~ibrinogen levels were datermined by solid phase enzyme-linked immunoassayO Fibrinogen was detected using a 1:1000 dilution of rabbit anti-rat fibrinogen polyclonal anti~erum (obtained from Dr. Gerald R. Crabtree, Stanford University).

2~

Secondary antibody was affinity purified alkaline phosphatase-colljugated goat anti-rabbit antisera (Promega , Biotec). Bound antibody was visualized by addition of substrates nitro-blue tetrazolium (NBT) and 5-bro~o-4-chloro-3-indovl-phosphate, p-toluidine salt (BCIP; Sigma).
Positive controls consisted of cells treated with supernatant obtained from PMA stimulated MRC-5 fibroblasts. Quantitation of the assay was carried out by scanning laser densitometry.

5.11.2. _-CELL DIFFERENTIATION ASSAY
Murine B-cell clone CH12.LX (N , d+ LY-l+) was grown and maintained in ~PMI 1640 containing 5~ heat-inactivated fetal bovine serum, 300 Ng/ml glutamine, 0.04 mM 2-mercaptoethanol and anti~iotics CH12.LX cells bear surf~ce IgM spècific for the phosphatidyl choline moiety of sheep erythrocytes (SRBC).
The differentiation assay was performed by culturing 2 x ~05 B-cells in the presence or absence of various concentrations of synthetic cysteine-free IL-6, as prepared according to the methods described in ~5.2 to 5.B, in 2 ml B-cell medium in Costar 24-well plates. SRBC (ASA Biological Products, Einston-Salem, NC) were washed three times in RPMI
1640 prior to use; 1 x 10~ erythrocytes were included in each test culture. Positive controls consisted of mitogen-stimulated B-cells using 50 Nq/ml lipopolysaccharide (Difco).
Cultures were incubated at 37C in an atmosphere of 5% CO2.
Direct hemoly~ic plaque forming colonies (pfc) in CH12.LX
cultures were determined.

5.11~3. IN VITRO ~ONE MA~ROW ASSAYS
Synthetic cysteine-free IL-S prepared in accordance with the methods described in Section 5.2 - 5.8, has also been shown to have therapeutic utility in art-recognized ln vitro testing. Delta (~) assays of the e~fect u~ IL-6, on ~luorouracil treated bone marrow cells, indicates that 2~

synthetic cysteine--free IL-6 has good stimulatory activity on progenitor cells. The protocol for these assays is found in Figure 14. Particularly effective stimulation is observed when synthetic cysteine-free IL-6 is combined with IL-l, and also when these two cytokines are comhined with either M-CSF
or IL-3. Tabular presentation of ~ values are found in Table 2, graphic depiction of Time 0 colony assays are shown in Figura 13.

5.11.4. 7TDl ASSAY OF DELETION MUTATIONS
... .
In a 7TDl assay (Van Snick et al~, Proc. Nat'l~ Acad.
Sci. USA 83:9679-9683, lg86), which utilizes the proliferation of an IL-6 dependent murine hybridoma cell line to quantify biological activity, various deletion~ of amino acid residues from IL-6 sequence were tested. Table 3 shows these results, expressed as percent activity compared with equimolar amounts of recombinant cysteine-containing IL-6 (Amgen). The IL-6 sequences tested are all cysteine-containing. Plasmid p478 i~ a plasmid construct identical to plasmid p367 except that in p478, the cysteines in th2 IL-6 sequence have not been replaced. The IL-6 active protein fraction is the IL~6 pep~ide cut from the fusion protein ("p478 cut" in Table 2) with collagenase. This peptide fraction for each deletion mutation tested is, therefore~ a mixture of cysteine-containing IL-6 peptide~ having discrete amino acids added to the carboxy terminal end of the IL-6 peptide sequence. In addition, the proteins tested are the fusion proteins themselves ('7p478 uncut'l). The wild typet cysteine-containing, natural IL-~ sequence fusion protein produced by p478 ("478 uncut") retains the biological activity of IL-6 (3% activity) as compared to the cleaned cysteine-containing IL-6 peptide ("478 cu~ hat i-~ produced from it . The ~ 478 cut ep~ide is the s~andard against which the nine deletion mutations are tested and as such, is 10û%
active in the test. Thus in Table 3 478 cut refers to the ~O~

wild type plasmid expressing cysteine-containing IL-6 fusion protein that has had tlle IL-6 cleaved from the fusion protein with collagenase and 478 uncut in Table 3 refers to the uncut fusion protein. Mutant lAE3 is a deletion mutation produced by deletion of amino acids 4 through 23 in the cysteine-containing IL-6 peptide where met is amino acid number one of the peptide sequence for the analogous IL-6 cysteine-free peptide found in Figure 8b. Mutant 2AB, 3AB
etc. all refer to the corresponding twenty amino acid deletions at the appropriate position in the sequence as identified in Table 3 in the second column labeled amino acid residues deleted~

Colony Synergizing ~ctivity in Liquid Culture Stimulating Actlvity in Liquid 5 Culture Mediu~ IL-l IL-6 IL-l ~ IL-6 Readout Value Readout Value Readout Value Readout Value CSA CSA CSA CSA

10 Medium M 0 M 7 M 2 M 27 (G) GM 0 ' GM 25 GM 7 GM 58 1~i CSF-l M 0 M 29 M 2 M 97 (M) GM 0 GM 43 GM 9 GM 99 (GM) GM 0 GM 54 GM 53 GM 157 G 14 G 509 G 866 G 1,781 IL-3 24 IL 3 102 IL-3 62 IL~3 313 Z5 _ _ _ . _ -54~

Amino Acids ~utantResldues AcClvity in Na~e Deleted 7TDl Assay ~i _ lAB 4-23 280%
2AB 24-43 0.001 3AB 44-63 0.0003 4AB 64-83 0.0003 5AB 84-103 0.0003 6AB 104-123 0.006 7AB 124-143 (0.0000) 8AB 144-163 (0.0000) 9AB 164-183 ~(0.0000) 478 Cut 100 478 Unc~t 3 6. _XAMPLE: MATERIALS AND METHODS
6.1. CONDITIONS FOR RESTRICTION ENZYME DIGESTION
Enzymes were obtained from commercial sources (New England Biolabs) and digestion were carried out as recommended ~y the manufacturer.

6.2. BACTERIAL STRAINS AND PLASMIDS
E. coli JM101 (P-L Pharmacia~ was transformed as de~cribed in Hanahan, 1~83, J. Mol. Biol. 166:557. Plasmid pKK233-2 was obtained from P-L Pharmacia; plasmid pBSt was from Stratogene. Other plasmid constructs are as described in this application.

6. 3 . OLIGONUCLEOTIDE ASSEMBLY
Oligonucleotides were synthesized from CED
phosphoramidites and tetrazole from American Bionetics.
Oligonucleotides were kinased with T4 polynucleotide kinase according to manufacturers suggestions (New England Biolabs).
The kinase was inactivated by heating at 65C.
Oligonucleotide mixtures were annealed by heating at 85C for 15 minutes and cooled slowly to room temperature. The annealed oligonucleotides were ligated with 10 U T4 ligase, ligated products were separated on a 6% polyacrylamide gel, and the fragments were recovered by elertroelution.
6.4. DNA SEQUENCING
The DNA sequence of inserted fragments and oligonucleotides were determined by the chain termination method of Sanger et al., 1977, Proc. Natl. Acad. Sci.
3~ 74:5463, in~orporating the modifications o~ Biggen et al., 1983, Proc. Natl. Acad. Sci. 80 3963/ ~attori and Sakakai, 1986, Anal. Biochem. 152:232, and Bankier et al., 1988, Methods Enz~mology, in press.

35~.5. PROTEIN BLOT ANALYSIS

2~

Samples equivalent to 50 ~L cell culture were run on 8 NaDodSO4-polyacrylamide gels under reducing conditions according to L~emlli, 1970, Nature 227:680~ Gels were either stained with Coomasie blue or electroblotted onto two layers of nitrocellulose in order to have duplicate blots of the same gel. Prestained molecular weight markers (BRL) were used to monitor transfer. After being blocked with 0.25%
gelatin, the blots were incubated with a commercial antibody to ~-galactosidase (Promega siotech). The cross reacting bands were visualized with a phosphatase-linked, affinity-purified, goa~ anti-mouse IgG antisera (1:7,500 dilution, Promega Biotech) using bromo-chloro-iodoyl phosphate and nitro-blue tetrazolium as recommended by Promega Biotech.

6.6. CELL LYSIS AND TRIHYBRID ASSAY

Cell lysis was performed according to Germino et al., Proc. Natl. Acad. Sci. 1983, 80:6848. E. coli were harvested by centrifugation, and the cell pellets were suspended in on~-fifth volume of 0.05 mol/L Tris-HCl p~8, 0.05 mol/L EDTA, 15~ sucrose with freshly dissolved lysozyme at l mg/ml.
After 15 minutes at room temperature, the lysates were ~rozen at -70C, thawed rapidly at 37C, and sonicated briefly to shear DNA. ~rihybrid fusion protein was quantitated by colorimetric assay for ~-galactosidase activity using 0-nitrophenyl-~-D galactopyranoside as substrate.

6.7. COLLAGENASE DIGESTION OF FUSION PROTEIN
_.
Prior to digestion of the fusion protein, non-speci~ic proteolytic activities in the collagenas~ preparation were reduced by treatment with ~-hydroxy-mercuribenzoate according to Lecroisey et al., 1975, FEBS Le~. 59:167. One unit of collagenase o~ Achromobacter iophagus ~EC 3~4-24.8;
Boehringer Mannheim~ was dissolved in 100 ~l of buffer containing 100 mM Tris-HCl, pH 7.4; 250 mM NaCl; 1 mM CaC12:
and 40 ~g/ml ~-hydroxy-mercuribenzoate. The dissolved collagenase was transferred to a 15 ml siliconized polyp~opylene tube and incubated at room temperature for 30 minutes. Thawed cell extract (4 mls) was sonicated briefly to disperse aggregates, added to the pretreated callagenase, and incubated for ~5 minutes at room temperature. The majority of the cleaved ~-galactosidase was removed by adding o.~ volumes of saturated ammonium sulfate, incubating on ice for 30 minutes and pelleting the insoluble material. The cleaved recombinant synthetic cysteine-free IL-6 was concentrated by bringing the total volume of the supernatant to 13 mls with saturated ammonium sulfate, incubating on ice for 30 minutes and centrifuging to compact the insoluble protein into a floating pellicle. Liquid was drained by puncturing the tube and the remaining pellicle was resuspended in 0.5 ml of Tris buffered saline.

7. EXAMPLE: HEPATOCYTE STIMULATION
Synthetic cysteine-free IL-6, as prepared according to the methods described in 5.2 to 5.8, stimulates hepatocytes as shown in Figure 11. The hepatocyte stimulating activity 2 was detected according to the assay described in 5O9.2 supra. The hepatocyte stimulation exhibited ~y synthetic cysteine-free IL-6 occurs at conc~ntrations of 10 8M, indicating a specific activity o~ 104 U/mg protein. This activity is 100 fold different than observed by May et al., 1988, J. Biol. Chem. 263:7760~ The level of fibrinogen synthesis observed in our FAZ~ cells is similar to the response obtained using crude conditioned medium obtained from PMA induced fibxoblasts. At concentrations greater than 30 1 ~M, the peptide causes a decrease in the number of hepatocytes resulting in a decrease in the detectable fibrinogen.

8. EXAMPLE: B-CELL DIFFERENTIATION
_ ~5 The most sensitive method ~o evaluat~ the functionality of the synthetic cysteine-free IL-6 protein, as prepared according to the methods described in 5.2 to 5.8 was the B-cell differentiation assay. A hemolytic plaque assay assessed the ability of the synthetic IL-6 to induce Ig secretion in resting ~-cells. The hemolytic assay is superior for studies of differentiation and proliferation at the cellular level (Gronowicz et al., 1976, Eur. J. Immunol.
6:5~8). Our protein had a maximal stimulatory concentration of about 0.1 ng/ml (43 p) (see Figure 12). This value is similar to values reported elsewhere for natural IL-6 (Van Snick et al., 1986, Proc. Matl. Acad. Sci. 83:9679;
Brakenhoff et al., 1987, J. Immunol. 139:4116; Poupart et al., 1987, EMBO 3. 6:1219; Kishimoto, 1985, Ann. Rev.
Immunol. 3:133). At concentrations above 43 pM, synthetic cysteine-free IL 6 can cause a decreas~ in the number of differentiated B-cells.

9. EXAMPLE: CYSTEINE-FREE IL-6 RETAINS BIOLOGICAL ACTIVITY
Threa assays o different activities of the natural IL-6 protein have shown that cysteine-free IL-6 retains biological activity. Our invention has shown that it i5 the primary sequence of the peptide that is necessary to fold the peptide chain into an active conformation.
Vaccines are often formulated and inoculated in comb1nation with various adjuvants. The adjuvants aid in attaining a more durable and higher level of immunity using smaller amounts of antigen or fewer doses than if the immunogen were administered alone. The mechanism of adjuvant action is romplex. It may involve the stimulation of 30 production of cellular cytokines (such as the cytokine IL-6), phagocytosis and other activities of the reticuloendothelial system as well as a delayed release and degradation of the antigen. Examples o~ adjuvants include Freund's adjuvant (complete or incomplete), Adjuvant 65 (containing peanut oil, 35 mannide monooleate and aluminum monostearate), surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and mineral gels such as aluminum hydroxide or aluminum phosphate. Freund's adjuvant is no longer used in vaccine formulation for hum~ns because it contains nonmetabolizable mineral oil and is a potential carcinogen.
Purified synthetic IL~6 like peptides of the present invention can be added to vaccine preparations to modulate an immune response, i.e., to act as an adjuvant; this includes vaccine preparations used to immunize animals such as mice, guinea pigs, rabbits, chickens, horses, goats, sheep, cows, chimpanzees as well as other primates, and humans. Methods of introduction of the vaccine with peptides like IL-5 as adjuvant include oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or any other route of immunization. The purified IL-6 active peptides can be used as an antigen for host immunization and ultimate production of IL-6 specific monoclonal antibodies.
Such antibodies in turn may be used as Ih-6 inhibitors and as such may be useful in the treatment of certain conditions in which dysfunction in immunoglobulin production has been implicated. This includes treatment of multiple myeloma, and autoimmune disease. For example, intraarticular injection of these monoclonal antibodies could be employed in treating rheumatoid arthritis The purified synthetic IL~6 like peptide of either the cystelne-frae form or the form with cysteines i~ adjusted to an appropriate concentration, ~ormulated with any suitable additions such a~ o~her cytokine peptides or vaccines and packaged for use. The peptide with IL--6 activity can also be incorporated into liposomes for use as an adjuvant in vaccine formulations or as a pharmaceutical product by itself. The synthetic peptide with IL-6 activity can also be added to preformed antibodies that are provided for passive immunotherapy.

In an alternative embodiment, the purified peptides with IL-6 activity can be used as an immunostimulant pharmaceutical product; for example, IL-6 peptldes are useful generally for stimulation of hemopoietic stem cells, and specifically for the stimulation of antibody production in disease-caused and drug or radiation-induced immunodeficiencies. As such, the peptides are useful in the treatment of immunosuppressed AIDS patients. They are also useful for the stimulation of production of hepatic proteins in hepatic dysfunction. The peptide can also be used as a reagent for inducing antibodies in vitro, or to modify the expression of other growth factors in culture. In another embodiment, the purified peptides with IL-6 activity, prefarably in combination with other antiviral compounds, can be used for the prevention and/or the treatment of viral dis~ases, including ~IV and HBV. In an alternative embodiment, the purified pepti~es with IL-6 activity can be used, alone or in combination with other purified cytokines, to invoke the terminal differentiation of B-cells in leukemias and other disease states. In another embodiment, 20 the synthetia IL-6 like peptide of either the cysteine-free form or the form containing cysteines can be used to induce antibodi~s that recognize any portion of the peptide.
.

10. EXAMPLE: EFFECT OF CYSTEINE-FREE IL-6 AND OTHER GROWTH FACTORS ON PROLIFERATION
AND DIFFERENTIATION OF BONE M~RROW CELL5 IN VIVO (DELTA ASSAY3 Delta assay is designed to determine whether there is renewal of a highly proliferatiYe population (HPP) in bone marrow (BM) stem cells tha~ are stimulated with variou~
growth factors~ Mice are treated with 5-fluorouracil (5FU) to remove cells that are of low proliferative potential (LPP).
The HPP cells are incubated in a first semi solid 35 agarose culture ~or 12 days in the presence of growth factors. The growth factors used include IL-l, IL-6, and a 1:1 mixture of IL-l and IL-6. BM stem cells are grown in the presence of each of these growth factors in the absence of other growth factors, and in the presence of granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte macrophaye colony stimulating factors (GM-CSF), and IL-3. The number of colonies stimulated by the growth factors is determined by a double-layer agarose clonal assay and exhibited in Figure 16 labelled "Time O Colony Assay". This figure shows the total colony forming units per 2.3 x 105 mouse bone marrow cells 2 hours after mice are treated with 5-FU. The first four bars show the effect of IL-l, IL-6 (cysteine free), and a 1:1 mixture of IL-l and IL 6 (cysteine-free) on colony stimulating activity in the absence of other growth factors.
The next set of four bars repxesents the effect of G-CSF
alone and in combination with IL-l, IL-6 ~cysteine-free), and a 1:1 mixture of IL-1 and IL-6 (cysteine-free) on colony stimulating activity. The next three sets of four bars each represent the colony stimulating activity of M-CSF, GM-CSF, and IL-3 alone and with IL-l, IL-6 ~cyst~ine-freP), and a 1:1 mixture of IL-1 and IL-6 (cysteine-free). The results show that IL-1, IL-S (cysteine-free), and a combination of IL-l and IL-6 (cysteine-free) stimulate colony formation when used in combination with M-CSF more than in combination with IL-3, GM-CSF and G-CSF.
In a separate assay, the HPP cells are incubated with the same growth factors and combination of growth factors in liquid culture. The number of non-adherent cells are counted 30 after 7 days, and the results are illustrated in Figure 15 labelled "Number o~ Non-Adherent Cells after 7 Dayq Liquid Culture." This figure shows the to~al number of non-adherent cells per ml. Each of ~he five sets of four bars has the same significance as the corresponding set in the figurP
35 labelled "Time O Colony Assay." The largest number of non-adherent cells is observed when a mixture of IL-1 and IL-6 (cysteine-free) are used in combination with IL-3, GM-CSF and M-CSF.
The cells from the liquid culture ar~ washed and again grown in the pr~sence of the growth factors in semi-solid agarose, and subjected to a second double-layer agarose clonal assay. This second agarose clonal assay is referred to as the "readout" assay. A Delta value is calculated by dividing the number o bone marrow colonies in the readout assay by the number of bone marrow colonies in the Time o Clonal Assay. The results are shown in ~able 2.
The data from the Delta assay describes which growth factors have synergizing activity in liquid culture when various factors are used in the readout assay. When medium alone is used in the liquid culture, the ability of the added cytokines to facilitate colony formation is IL-1 + IL-6 IL-l or IL-6. This pre~erence of IL-l ~ IL-~ over the ability of IL-l or IL-6 to synergize with other growth factors is evident when ~-CSF, GN-CSF, M-CSF and IL-3 are used in the liquid culture. The synergy is most evident when G-CSF is used in the readout assay. When GM-CSF and IL-3 are used in the liquid culture in conjunction with IL-6 or IL-l, IL-6 causes a greater Delta value than IL-l. However, when CSF-1 is used in conjunction with IL-1 or IL-6, IL~l causes a greater 5ynergistic value. The best results in the entire assay wer~ seen when IL-l ~ IL~6 were used in conjunction with I~-3 in the liquid assay and G-CSF wa used in the readout.
These data suggest that cysteine-free IL-6, when used 30 in conjunction with other growth factors, may be an important tool in bone marrow transplantation and cancer treatment.
high Delta value indicates that a certain combination of growth factors yields both growth and renewal of stem cells.
This particular charac~eristic would be required ln a growth 35 factor that would be used to stimulate bone marrow growth and differentiation in vivo.

Protocol for Delta Assay 1) aDFl mice treated with 150 mg/kg 5-fluorouracil ~5FU).
2) Kill mice, harvest bone marrow (BM) 24 house after 5FU
treatment.
3) Wash BM cells in IMDM medium with 20% fetal bovine serum (FBS3 and antibiotic (gentamicin).
4) Grow cells in a double layer agarose clonal assay:
Growth factors in IMDM with 20% FBS are plated in 35mm petri dishes in 0.5% agarose. BM cells are added to an overlayer at a concentration of 2 5 x 104 up to 2 x 10 BM cells in .5 ml per plate. This is called the Time O Clonal Assay. Grow at 37C under approximately 7% 2 for 12 days.
5) BM cells are grown in 1 ml liquid culture (IMDM 20% FBS
+ antibiotic) in 24-well cluster plates. Cells are incubated for 7 days starting at 2.5 x 105 cells/ml.
6) After 7 days liquid culture, the non-adherent BM cells are collected. The cell numbers are counted, cytospin preparations are made, and the remaining cell6 are washed over 5ml of cold FBS to remove any growth factors.
7) The cells are hen diluted 20-100-fold and plated into the clonal agarose assay. These cultures are grown for 7-12 days at 37C! 7% 2 in a fully hu~idified atmosphere. This is called the Readout Assay.
8) The Delta value is calculated by dividing the number of BM colonies in th~ Readout Assay by the number of BM colonies in the Time O Clonal Assay 11. EXAMPLE: COMP~RISON WITH OTHER COMMERCIAL
SOURCES OF IL-6 IN 7td 1 ASSAYS
.
The biological activity o~ the cysteine-free IL-6 peptide purified ~rom the f~sion product induced by cells harboring p367 was compared with cysteine-containing IL-6 from commercial sources using a 7td.1 assay (Van Snick et al., Proc. Nat'l. Acad. Sci. USA 83:9679-9683, 1986). Those commercial sources were Boehringer Mannheim (bm) which produced an E. coli derived IL-6; Endogen which was a non-recombinant product; and Genzyme which produced a yeast-derived IL-6. When the cysteine-free synthetic peptide in phosphate buffer~d saline purified from induced p367 containing cells was compared to Boehringer Mannheim's IL-6, Boehringer's showed higher activity at low concentrations and lower activity at high concentrations as shown in Figure 17.
The highest maximal activity was reached using the cysteine-free synthetic IL-6.
Comparison of the cysteine-free synthetic IL-6 peptide with Endogen IL-~ revealed that the cysteine-free IL 6 peptide had superior activity over all concentrations analyzed (0.1 ng to So ng1 as shown in Figure 16.
Cysteine-free synthetic IL-6 also gave higher biological activity than Genzyme's IL-6 when concentrations between 0~1 ng and 50 ng were tested as shown in Figure 18.
Description of_the 7td.1 Growkh Assay This 3.5 day long assay is performed in 96 well culture plates. Peptides with IL-6 activity are measured in HG~
units where 100 HGF units equal approximately 1 PCT-GF unit.
Typically 2000 cells of the 7td.1 strain are incubated in highly humidified chambers of 5% C0, in 200 ~1 of medium containing a dilution of the peptide exhibiting IL-6 activity. After 8~ hours, cells are pulsed for ~ hours with the tetrazolium salt 3-(4,5 dimethylthiazol-2-y~-2,5-diphenylformazan bromide ~M~T) and the supernatants are then removed following centrifugation at lOOOx G Por 5 minutes.
The formazan crystals ar dissolved with 100 1~ of DMSO and the plates are read at 570 nm wavelength. The optical density is proportional to the number o~ live cells~

~ 0 0~

Theamount of IL-6 in HGF units is defined as the reciprocal of the dilution required to give 50% of the maximum optical density.

~eaqents of the 7td.1 Assay Diluent medium is RPMI 1640 with 10% fetal calf serum.
Supplemented RPMI contains RPMI 1640 with 10~ fetal calf serum, 0.1 mM 2-mercaptoethanol and 2x antibiotic solution.
The labeling reagent is 5 mg/mL MTT in PBS.

12. DEPOSIT OF MICROORGANISMS
The following plasmids have been deposited with the American Type Culture Collection (ATCC), ~ockville, MD on November 29, 1988, and have been assigned the indicated accession numbers:

Plasmid A C
p340 ATCC 40516 p367 ATCC 40S17 pTrpE/EK/cfIL-6 ATCC _ _ p~gal/EK/cfIL-6 ATCC

The present invention is not to be limited in scope by the plasmids deposited since the deposited embodiments are intended as single illustrations o~ one aspect of the invention and any which are functionally eguivalent are within the scope of this invention. Indeed, various 3~ modifications o~ the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing descrip~ion and accompanying drawings~
Such modification are intended to fall within the scope of the appended claims.

2~

It is also to be understood that all base pair and amino acid residue numbers and sizes given for nusleotides and peptides are approximate and are used for purposes of description.

3a

Claims (130)

1. A recombinant gene comprising a nucleotide sequence which encodes a synthetic trihybrid protein, which trihybrid protein has a first peptide portion, a second peptide portion and a third protein portion, which first peptide portion has IL-6 activity, which second peptide portion comprises a chemically or enzymatically cleavable peptide link between the first peptide portion and the third protein portion, which third protein or portion thereof portion is capable of being expressed by the host cell.

2. The recombinant gene of claim 1 in which the first peptide portion with IL-6 activity is incapable of forming sulfhydryl bonds.

3. The recombinant gene of claim 1 or 2 in which the second peptide portion comprises a peptide link which is reactive to a proteolytic enzyme.

4. The recombinant gene of claim 3 in which the second peptide portion comprises a collagenase sensitive site.

5. The recombinant gene of claim 3 in which the second peptide portion comprises an enterokinase sensitive site.

6. The recombinant gene of claim 3 in which the second peptide portion comprises a Factor Xa sensitive site.

7. The recombinant gene of claim 1 or 2 in which the third protein portion comprises a .beta.-galactosidase sequence or portion thereof.

8. The recombinant gene of claim 1 or 2 in which the third protein portion comprises a TrpE gene product.

9. The recombinant gene of claim 1 in which the first peptide portion is incapable of forming sulfhydryl bonds; the second peptide portion comprises a peptide which has a collagenase sensitive site, and the third protein portion comprises a protein with .beta.-galactosidase enzymatic activity.

10. The recombinant gene of claim 1 in which the first peptide portion is incapable of forming sulfhydryl bonds; the second peptide portion comprises a peptide which has an enterokinase sensitive site, and the third protein portion comprises a protein which is a TrpE gene product or portion thereof.

11. The recombinant gene of claim 1 in which the first peptide portion is incapable of forming sulfhydryl bonds; the second peptide portion comprises a peptide which has a Factor Xa sensitive site, and the third protein portion comprises a protein which is a TrpE gene product or portion thereof.

12. The recombinant gene of claim 1 in which the first peptide portion is incapable of forming sulfhydryl bonds; the second peptide portion comprises a peptide which has an enterokinase sensitive site, and the third protein portion comprises a .beta.-galactosidase sequence or portion thereof.

13. The recombinant gene of claim 1 in which the first peptide portion is incapable of forming sulfhydryl bonds; the second peptide portion comprises a peptide which has a Factor Xa sensitive site; and the third protein portion comprises a .beta.-galactosidase sequence or portion thereof.

14. The recombinant gene of any one of claims 9-13 in which the first peptide portion, after cleavage from the second peptide portion, does not retain residual amino acids from the second peptide portion.

15. The recombinant gene of claim 2 which comprises a DNA sequence encoding the IL-6 amino acid sequence depicted in Figure 5.

16. The recombinant gene of claim 3 which comprises a DNA sequence encoding the IL-6 amino acid sequence depicted in Figure 5.

17. The recombinant gene of claim 4 which comprises a DNA sequence encoding the IL-6 amino acid sequence depicted in Figure 5.

18. The recombinant gene of claim 5 which comprises a DNA sequence encoding the IL-6 amino acid sequence depicted in Figure 5.

19. A recombinant vector comprising the gene of claim 1.

20. A recombinant vector comprising the gene of claim 2.

21. A recombinant vector comprising the gene of claim 3.

22. A recombinant vector comprising the gene of claim 4.

23. A recombinant vector comprising the gene of claim 5.

24. A recombinant vector comprising the gene of claim 6.

25. A recombinant vector comprising the gene of claim 7.

26. A recombinant vector comprising the gene of claim 8.

27. A recombinant vector comprising the gene of claim 9.

28. A recombinant vector comprising the gene of claim 10.

29. A recombinant vector comprising the gene of claim 11.

30. A recombinant vector comprising the gene of claim 12.

31. A recombinant vector comprising the gene of claim 13.

32. A recombinant vector comprising the gene of claim 14.

33. The recombinant vector of any one of claims 19 to 32 which is a plasmid.

34. A plasmid from the group consisting of pTrpE/EK/cfIL-6; p.beta.Gal/EK/cfIL-6; pTrpE/Xa/cfIL-6; or p.beta.Gal/Xa/cfIL-6.

35. A unicellular host containing the vector of claim 19.

36. A unicellular host containing the vector of claim 20.

37. A unicellular host containing the vector of claim 21.

38. A unicellular host containing the vector of claim 22.

39. A unicellular host containing the vector of claim 23.

40. A unicellular host containing the vector of claim 24.

41. A unicellular host containing the vector of claim 25.

42. A unicellular host containing the vector of claim 26.

43. A unicellular host containing the vector of claim 27.

44. A unicellular host containing the vector of claim 28.

45. A unicellular host containing the vector of claim 29.

46. A unicellular host containing the vector of claim 30.

47. A unicellular host containing the vector of claim 31.

48. A unicellular host containing the vector of claim 32.

49. A unicellular host containing the vector of claim 33.

50. The unicellular host of any one of claims 35-49 which is a prokaryote.

51. The unicellular host of claim 50 which is a bacterium.

52. A method for producing a peptide having IL-6 activity which comprises:
(a) culturing a unicellular host microorganism containing a recombinant gene comprising a nucleotide sequence encoding a synthetic trihybrid protein which has a first peptide portion having IL-6 activity, a second peptide portion which comprises a chemically cleavable peptide link between the first peptide portion and a third protein portion capable of being expressed by the unicellular host, said gene being capable of being replicated, transcribed, and translated in the host;
(b) identifying tripartite proteins produced by the host;
(c) chemically or enzymatically cleaving, the second peptide portion; and (d) recovering the first peptide portion having IL-6 activity.

53. The method of claim 52 in which the second peptide portion comprises a peptide link which is reactive to a proteolytic enzyme.

54. The method of claim 53 in which the second peptide portion comprises a collagenase sensitive site.

55. The method of claim 53 in which the second peptide portion comprises an enterokinase sensitive site.

56. The method of claim 53 in which the second peptide portion comprises a Factor Xa sensitive site.

57. The method of claim 53 in which the third protein portion comprises a .beta.-galactosidase sequence or portion thereof.

58. The method of claim 53 in which the third protein portion comprises a TrpE gene product.

59. The method of claim 53 in which the first peptide portion is incapable of forming sulfhydryl bonds; the second peptide portion comprises a peptide which has a collagenase sensitive site, and the third protein portion comprises a protein with .beta.-galactosidase enzymatic activity.

60. The method of claim 59 in which the first peptide portion is incapable of forming sulfhydryl bonds; the second peptide portion comprises a peptide which has an enterokinase sensitive site, and the third protein portion comprises a protein which is a TrpE gene product or portion thereof.

61. The method of claim 59 in which the first peptide portion is incapable of forming sulfhydryl bonds; the second peptide portion comprises a peptide which has a Factor Xa sensitive site, and the third protein portion comprises a protein which is a TrpE gene product or portin thereof.

62. The method of claim 59 in which the first peptide portion is incapable of forming sulfhydryl bonds; the second peptide portion comprises a peptide which has an enterokinase sensitive site, and the third protein portion comprises a .beta.-galactosidase sequence or portion thereof.

63. The method of claim 53 in which the first peptide portion is incapable of forming sulfhydryl bonds; the second peptide portion comprises a peptide which has a Factor Xa sensitive site; and the third protein portion comprises a .beta.-galactosidase sequence or portion thereof.

64. The method of claim 59 in which the first peptide portion, after cleavage from the second peptide portion, does not retain residual amino acids from the second peptide portion.

65. The method of any of claims 52-64 in which the trihybrid protein comprises at least about 20% of total protein produced by the host organism.

66. The method of any one of claims 52-64 in which the host organism is a prokaryote.

67. The method of claim 66 in which the host organism is a bacterium.

68. The method of claim 65 wherein the host is a bacterium.

69. The method of any one of claims 52-64 in which the first peptide portion has no sulfhydryl bonds.

70. The method of any one of claims 52-64 wherein the first peptide portion comprises the IL-6 amino acid sequence depicted in Figure 5.

71. The method of claim 70 in which the trihybrid protein comprises at least about 20% of total protein produced by the host organism.

72. A recombinant plasmid having accession number ATCC
40516 (p340).

73. A recombinant plasmid having accession number ATCC
40517 (p367).

74. A substantially pure recombinant protein comprising a first peptide portion having IL-6 activity, a second peptide portion comprising a cleavable peptide, and a third protein portion capable of being expressed by a chosen host organism.

75. A recombinant protein encoded by the gene of claim 1.

76. A recombinant protein encoded by the gene of claim 2.

77. A recombinant protein encoded by the gene of claim 3.

78. A recombinant protein encoded by the gene of claim 4.

79. A recombinant protein encoded by the gene of claim 5.

80. A recombinant protein encoded by the gene of claim 6.

81. A recombinant protein encoded by the gene of claim 7.

82. A recombinant protein encoded by the gene of claim 8.

83. A recombinant protein encoded by the gene of claim 9.

84. A recombinant protein encoded by the gene of claim 10.

85. A recombinant protein encoded by the gene of claim 11.

86. A recombinant protein encoded by the gene of claim 12.

87. A recombinant protein encoded by the gene of claim 13.

88. A recombinant protein encoded by the gene of claim 14.

89. A recombinant protein having the IL-6 and collagen amino acid sequence depicted in Figure 8b.

90. A recombinant protein having IL-6 activity, which is the product of chemical or enzymatic cleavage of the protein of any one of claims 75-88.

91. A recombinant protein having IL-6 activity, which contains no cysteines, which is the product of chemical or enzymatic cleavage of the protein of any one of claims 75-78, and which retains no residual amino acids derived from the second peptide portion after cleavage.

92. A synthetic peptide having IL-6 activity, and which is substantially soluble in water.

93. The peptide of claim 59 which is incapable of forming a sulfhydryl bond.

94. A synthetic peptide comprising the IL-6 amino acid sequence of Figure 5, which sequence contains no cysteines;
or any homologue, analogue or active portion thereof.

95. A synthetic peptide having the IL-6 aminoacid sequence of Figure 5, which sequence contains no cysteine, wherein the amino acids of the collagen linker are partially or entirely deleted.

96. A synthetic peptide having the IL-6 amino acid sequence of Figure 5, which sequence contains no cysteine, wherein all the amino acids of the collagen linker and the serine residue bridging the collagen to the IL-6 protein are deleted.

97. A synthetic peptide according to any of claims 94 to 96 containing proline instead of glycine as the second amino acid of the N-terminal sequence.

98. A synthetic peptide according to any one of claims 94 to 97 lacking the initial methionine at the N-terminal sequence.

99. A synthetic peptide which is a derivative of a peptide according to any one of claims 94 to 98 by way of amino acid deletion, substitution, insertion, inversion, addition or replacement.

100. A synthetic peptide comprising the portion of the amino acid sequence of native IL-6 from amino acid 29 to amino acid 212, which sequence does contain cysteins, wherein, however, the amino acid 29 is glycine instead of proline.

101. A synthetic peptide according to claim 100 having an initial methionine residue.

102. A recombinant gene comprising a nucleotide sequence which encodes the synthetic peptide of claim 92 to 101.

103. A vector comprising the recombinant gene of claim 102.

104. A unicellular host comprising the recombinant gene of claim 102.

105. A unicellular host comprising the vector of claim 103.

106. A method of stimulating antibody production in a host which comprises administering to said host an effective amount of the peptide of any one of claims 90 to 101 in combination with the effective antigen.

107. A method of preventing or treating viral infection in a host which comprises administering to said host an effective amount of the peptide of any one of claims 90 to 101.

108. A method for stimulating production of hepatic proteins in a host which comprises administering to the host an effective amount of the peptide of any one of claims 90 to 101.

109. A method for stimulating terminal B cell differentiation in a host comprising administering to the host an effective amount of the peptide of any one of claims 90 to 101.

110. A method for stimulating hematopoietic stem cells in a host which comprises administering to the host an effective amount of the peptide of any one of claims 90 to 101.

111. A method of treating an immunosuppressed host which comprises administering to the host an effective amount of the peptide of any one of claims 90 to 101.

112. A method of treting a disease condition involving a dysfunction in immunoglobulin production which comprises administering to the host an effective amount of a monoclonal antibody reactive with the peptide of any one of claims 90 to 101.

113. A pharmaceutical formulation comprising an effective amount of the peptide of any one of claims 90 to 101 in combination with a pharmaceutically acceptable carrier.

114. The formulation of claim 113 wherein the peptide is combined with at least one other cytokine or hematopoietic growth factor.

115. A formulation according to claim 114 wherein the cytokine or hemopoietic growth factor is chosen from erythropoietin, an interleukin and a colony stimulating factor.

116. A formulation according to claim 114 wherein the interleukin is IL-1, IL-2 or IL-3 and the colony stimulating factor is G-CSF, GM-CSF or M-CSF.

117. A synthetic peptide according to any of claims 90 to 101 for therapeutic use in simultaneous or serial co-administration with a cytokine or homopoietic growth factor as defined in claim 114 to 116.

118. The formulation of claim 113 wherein the peptide is present as an adjuvant, in combination with an effective amount of a protective antigen.

119. A recombinant nucleic acid vector comprising a nucleic acid sequence encoding a first portion which is an inducible promoter, a second portion which is a minicistron sequence, a third portion which is a cloning site for insertion of a DNA fragment comprising a protein that lacks a termination codon, a fourth portion which is a nucleotide sequence for a cleavable peptide, and a fifth portion which is a nucleotide sequence for .beta.-galactosidase which is in frame with the cleavable peptide of the fourth portion; and in which the nucleic acid sequence the third portion cloning site is out of a translational reading phase with the .beta.-galactosidase fifth portion.

120. The vector of claim 119 which comprises the NDA
sequence depicted in Figure 8b.

121. A recombinant gene according to any one of claims 1 to 14 which comprises a DNA sequence encloding a peptide according to any of claims 92 to 101.

122. A recombinant vector comprising a gene according to claim 121.

123. A unicellular host containing the vector of claim 122.

124. A recombinant vector comprising the p340 plasmid.

125. A recombinant vector comprising the mutant 1AB
DNA sequence.

126. A peptide produced by the vector of claim 125.

127. A DNA sequence encoding a peptide having IL-6 activity, the peptide having the sequence defined by mutant 1AB.

128. The peptide encoded by the DNA sequence of claim 127.

129. A peptide having IL-6 activity, the peptide having the sequence of native IL-6, with amino acid residues 4-23 inclusive deleted.

130. A DNA sequence encoding the peptide of claim 129.