CA2123234A1 - Synthesis and purification of truncated and mutein forms of human ciliary neuronotrophic factor - Google Patents

Abstract

The present invention relates to truncated and mutein forms of human ciliary neuronotrophic factor, nucleic acid sequences which encode these forms, the amino acid sequences of these forms, methods of producing the same by means of recombinant DNA techniques, pharmaceutical compositions containing these truncated and mutein forms of CNTF, and methods of use thereof.

Description

SYNTHESIS AND PURIFICATION OF TRUNCATED AND M~TEIN
FORMS OF HU~ CILIARY NEURONOTROPHIC FACTOR

BAC~OU~D G.- THE ~'~VE~TION

~iela or the Inven~ion The present invention relates to processes for the synthesis and purification of biologically active truncated and mutein forms of the human ciliary neuronotrophic factor (CNTF), including monomeric forms of these proteins, obtained by recombinant DNA techniques. Via the presen~
invention, it is possible to obtain human CNTF polypeptides with a smaller num~er of amino acids than the corresponding natural form of CNTF. These new forms of CNTF exhibit different chemical and chemical-physical characteristics 15 from those of natural CNTF, and are useful in a variety of -pharmaceutical applications.

Descri~tion of Related Art A. THE CILIARY NEURONOTROPHIC FACTOR

The ciliary ganglion (CG) contains two populatl~ng of neurons, ciliary and choroid, both of them cholinergic. CG
neurons innervate the intrinsic muscl~ ctructures of the eye in the choroid, the ciliary ~ody and the iris. During embryonic development in chicks, about half the CG neurons die between days E8 an~ E15 when their axons establish connection with the intraocular innervation territory.
This neuronal death is strongly enhanced by removal of the eye, while it is significantly impeded by the implantation of a supplementary eye bud.
From this observation the hypothPsis evolved that the innervation territories contain trophic factors for their own specific neurons.

2123234 :

3 PCT/EP92/02~86 :, ~ r2c_s Lro. various e~ Dr~onic cnic~ ssues a~ dG~
Eô e~:nibi.e~ 2 Iropnic ac~ of thei~ own t:nlcn differe~
fror that of dissociated neurons from chick embryo ciliarv ganglia a. the same stage o~ developmenl (Adler e~ al - (1979) Scierlce 20~:1434-1436).
This ~rophic activity WâS subsequently associated with ciliary neuronotrophic factor (CNTF) (Barbin et al. (1984) ~;
J. Neurochem. ~3:1468-1478).
A third of all the CNTF tropAic activity in the embryo was observed in the eye, and most of this was localized in the choroid and in the intrinsic muscle structure of the eye.
Purification of this protein by ion exchange chromatography, sucrose gradient ultracentrifugation and sodium dodecyl sulfate polyacrylamide gel e~ectrophoresis (SDS-PAGE) showed it to have a molecular weight of about 2lKd and a negative net charge. The trophic activity exhibited by the purified protein in chick CG neurons at day EB, in ~hick DRG neurons at day E10, and sympathetic neurons at day E11 is on the order of lOIi lOI~M, the same as another neuronotrophic factor, Nerve Growth Factor (NGF), purified from male mouse su~mandibular glands, with regard to its neuronal DRG and sympathetic targets.
Like NGF, CTNF supports the growth of chromaffin 2S adrenal cells in culture, although the measure of their effects on the enzymes tyrosine hydroxylase and phenylethanolamine N-methyltransferase differs. CNTF
raises ChAT activity in chic~ retinal cell cultures, indicating cholinergic neuron response. CNTF has no trophic effects on the cholinergic neurons of the pontine basal region of the brain, and cannot be considered a cholinergic neuronotrophic factor.
CNTF in vitro enhances the sur~ival and developmen~ of some sympathetic neurons, spinal neurons of cranial ganglia (Barbin et al. (1984) J. Neurochem. 43:1468); motoneurons of the spinal cord (Arakawa et al. (1990) J. Neurosci.
10:3507; Wong et al. (l990) Soc. Neurosci Abstr. 16:484;

2123~

:;agai ~ c~ (1991) Devei 3~n ~es ~ 1) an~
nlppoca.~lDal neurons (Ip el 21. (199l) J. Neurosci.
'1:312~) It has also been shown to influence in vitro the differentiation of the progenilor glial cells known as 0-2A
S which differen~iate in oligodendrocy~es and astrocytes (Lillien et al. (1988) Neuron 1:48).
The characteristics of the protein are such (isoelectric point 5.8, molecular weight 24kDa) that it is relatively easy to produce up to 30% of the total proteins of a recombinant bacterium as CNTF, but the same characteristics make it necessary to employ various chromatographic steps to obtain a homogeneously pure protein. These various steps include ion exchange chromatography, inverse phase chromatography, lS polyacrylamide gel electrophoresis, ion metal interaction chromatography, and hydrophobic interaction chromatography.
As many of these steps are difficult to perform at an industrial level, thus adding considerably the costs of production of the factor, it is highly desirable to find alternative purification methods.

B. RECOM8INANT DNA ~ECHNOLOGY

Conventional techniques for isolating bioiogically active proteins include isolation of the proteins by purification from biological fluids or tissues, but the quantities of protein thus obtainable are not always sufficient to ena~le study of their structures, functions and, above all, applications.
Therefore, in order to obtain sufficient quantities of proteins, recombinant D~A techniques can be used to clone genes encoding these proteins in expression vectors.
It is possible, by recom~inant DN~ techniques, to construct a series of vectors that can express proteins of interest in large quantities~ Recombinant DNA technology allows the molecular biologist to arrange and assemble DNA
sequences to create hybrid molecules capable of producing 2 3 '~ 3 ~

~ro~e-.~C ~ eres. ~arlous reac.ions are emplo~!eà such ~s clea~lnc ;:ilh reslric~ion enzv~es ligaring the fragmen~s thus obtaine~ uith ligase, the chemical synthesis of olisonucleotides to be assembled, and other 3 me~hodologies devised in various laboratories working in this field These techniques are summarized in Sambrook et al. (1989) Molecular Cloninq A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Laboratory, NY.
In order tO obtain high levels of expression, the DNA
elements to be assembled must contain certain essen~ial information. These elements include, for example, a replication origin, a selective mar~er for antibiotics, an expression promoter, tr~nscription activators for the gene of interest, and other regulatory elements known to those of skill in the art. The combination of these elements, including a gene of interest functionally linked to - transcriptional and translational regulatory elements, forms a plasmid called an expression plasmid. Such an expression plasmid or vector facilitates the expression of the protein in host cells, which may be of eukaryotic or prokaryotic origin. The protein c~n then be obtained by subsequent purification.
Promoters which naturally control the expression of genes such as growth factors are not highly active, and are activated only under suitable natural conditions, which are often unknown. To this end, promo~ers with known activities are often employed, such as those from viruses of the Papovavirus series, or other known promoters.
The regulatory elements employed to obtain high levels of gene expression are therefore a combination of D~As of various origin (eukaryotic, bacterial, viral, etc.) in association with different gene fragmen~s linked ~ogether to form a hybrid molecule. The transcriptional activity of a gene depends on there being suitable distances between the regulatory and coding sequences.

21232~1 W093/l0233 PCT/EP92/0258 c~ c~es ~ in~nq c_~cc ~ a~e~
ciose Dro~:lmi.~ ~o reguia~or~ sequences ~eDend o. ~nere ~einc suit2ble res~ric~ion endonuclease s'tes :Jhic..
~acili~a~e their cioninc. If no compatiD'e sites axe nearDy, but only different sites, it is possible to join the segments by synthesis of an oligonucleotide linker containing such a restric~ion site. This sys~em is very limiting to the molecular biologist. An alternative strategy is to use the PCR technivue (Saiki et al. (1988) Science 239:487-489). By this technique it is possible to -amplify a gene segment up to 1 x lO~ times. The principle is based on the use of two oligonucleotides, each of which -~
can be exactly paired onto one of the DNA strands to be amplified. The distance between the two aligonucleotides with respect to the gene sequence determines t~e size of the molecule to be produced. These two aligonucleotides are so constructed that there is a restriction site within their sequence which allows su~sequent cloning. This -restriction site is either naturally present or i5 constructed ad ~hoc by degenerating the ~inimum number of nucleotides. This approach, ~nown as site-directed mutagenesis, allows restriction sites to be construct2d in positions previously decided on ~y the molecular biologist.
The construction of sites compatible with other gene segmen~s not only facilita~s cloning, but also makes it possible to specifically join different gene segments~
This technique can be defined as cloning ~y direct mutagenesis.
The gene sequence can also be suitably changed by increasing or decreasing the length of the gene obtained, after insertion of the modified gene in suitable expression vectors, as described above. Peptides can b~ obtained with characteristics which are the same as or diffe~ent from those of the coded protein of the wild-type gene. It is thus possible to obtain truncated forms of pciypeptides with fewex amino acids than the natural protein, and deleted forms of polypeptides, lac~ing certain amino acid W093/l0233 PCT/EP92/0~58h -cs~e~c~s ~ c.~ ~e ~-esen~ ln ~!1 ld-~vpe pro~in seauences.
These ne.-. pol~epliae ror~.s can D055eSS che~llcai, cnemical-physical, and bioloaic21 characteristics tha~ are very different from ~hose of the natural protein. In oràer to obtain differen~ forms of a polypeptide, mutations can be introduced in the gene therefor in order to obtain new forms of the polypeptide with the same biological activity as the natural form, possessing a smaller number of amino acids than the natural protein, or which ma~e it possible to block the natural protein receptor.

SIJ?`IMAP~Y OF THF INVENTION
_ _ Based on the foregoing, an object of the present invention is the expression by host cells, and purification in quantity, of homogeneous truncated and/or mutated forms (defined as isoforms) of the human ciliary neuronotrophic factor. These new polypeptides can be employed in vitro and in vi~o, both singly and in combination with other molecules or their derivatives, or with mar.ufactured biomaterials, for therapeutic purposes.
Another object of the present invention is to provide pharmaceutical preparations containing as active su~stan~es one or more of the new complexes between one or more isoforms of the CNTF growth factor and a natural ganylioside or one of its derivatives or semisynthetic analogues, or one of its salts, or associations of the same with phospholipids, other growth factors, or natural polymers such as acidic polysaccharides, or chemically modified or transformed biomaterials.
The present invention also enco~passes the therap~utic use of all the new complexes of natural gangliosides or one of their derivatives or semisynthetic analogues, or one of their salts, or of hyaluronic acid or one of its derivati~es, with the isoforms and muteins of gr~wth factor CNTF for the aforesaid indications. Daily dosages for humans by subcutaneous, intramuscular or intracerebral WO93/10233 2 1 2 3 2 3 PCT/EP9~/02S8fi ~ c~c.- ~ v ~e~-:ee~ o o~ r~ 2nd ~ r~c c~ cCliVe suhs.~nc~ ~e- ~:- cl bodv ::eignt ~ r~r~.~er o~jec~ or the presenl invention is to provide 2 Drocess for proaucing a human CNTF mutein wnich possesses the a~vantage of facilitating the homogeneous purification of its monomers. This process includes the fusion of the ~utein to at least 6 histidine residues, and the presence of an enzyma~ic or chemical cleavage site in the polypeptide chain. The presence of these 6 h:istidine residues makes it possible for the mutein of CNTF to be separated from the complex mixture of proteins produced by the host organism in a single step via chromatography through a chromatographic column by interaction with metal ions. The mutein thus separated is chemically or enzymatically cleaved and recovered in pure form from the same column~ The mutein is a polypeptide form of CNTF, wherein the first 14 amino acids starting from the aminc terminal end have been removed, and the only cysteine of CNTF has been replaced with serine so that t~e recom~inant protein cannot become oxidized during purification, thus maintaining its monomeric form. This mutein maintains its biological activi~y after purification.
Further scope of the applicabili~y of the present invention will become apparent from the detailed description and drawings provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in tne art from this detailed descriptlon.

WO93/10233 2 1 ~ ~ 2 3 I PCT/EP92/025~

BRIEF DESCRIPTION OF THE DRAWINGS
:.
c _-c~ n~ s~ne~ c.~iec,s, ec~ures, an~ acvan~a~es of the ~resen~ invention ~ill be be~e~ unàerstood from the follo;:inc de~2iled oescri~ticns ~a};en in conjunction ~3ith the accom~anylng drawings, all of wnich are given bv way of S illus~ration only, and are not limitative of the present invention, in which:
Figure l shows the nucleic acid sequence encoding human CNTF.
Figure 2 shows the amino acid sequence encoded by the nucleic acid sequence of Figure l.
Figure 3 shows the nucleic acid se~uence encoding a form of human CNTF lac~ing the first 14 amino acids at the amino terminal end of the molecule.
Figure 4 shows the amino acid sequence and other data lS of the form of human CNTF lacking the first 14 amino acids at the amino terminal end of the molecule encoded by the nucleic acid sequence of Figure 3.
Figure S shows the nucleic acid sequence encoding a form of human CNTF lacking the last 26 amino acids at the carboxy terminal end of the molecule.
Figure 6 shows the amino acicl sequence and other ~ata of the form of human CNTF lac~ing the last 26 amino acids at the carboxy terminal end of the molecule encoded by the nucleic acid sequence of Figure S.
2S Figure 7 shows the nucleic acid sequence encoding 2 form of human CNTF lacking bo$h the first 14 amino acids at the amino terminal end of the molecule and the last 26 amino acids at the carboxy terminal end of the molecule.
Figure 8 shows the amino acid sequence and other data of the form of human CNTF lacking the first 14 amino acids at the amino terminal end of the molecule and the last 26 amino acids at the carboxy terminal end of the molecule encoded by the nucleic acid sequence of Figure 7.
Figure 9 shows the cloning scheme employed to obtain the vectors used for the expression of the new truncated polypeptiàe forms of human CNTF disclosed herein.

WO93/10233 2 1 2 3 ~ 3 ~ PCT/EP92/02586 . ' a~ J 5.~0 ,C _;:e ~-uncalec Cor~s c nul;,cn Ci~T~
re~orle~ in ~:am~iec :-_, ana ~r,~ ~runca.ec ~cr,s ; it.
deleled sequences aesc~ed in E~:am~le ~
Figure '1 shotis t.~e bioloaical ac~ivi~y c, truncatec S polypeptide forms cf human CNTF compareà to the wild-type human C~TF protein. The data show that ~he specific activity, calculatea as ED50, is at least 5 x 10llM for the various forms.
Fig~re 12 shows the slructure of the expression vector CNTF(His)~14Serl7CNTF.
Figure 13 shows the structure of the expression vector ~14Serl7CNTF.
Figure 14 shows the elution profile of the bacterial starting material of Example 13, loaded in buffer A, washed wit~ buffers B and C. The recombinant protein elutes in buffer D.
Figure 15 shows the elution profile of CNTF(His)hAsnGlyD14Serl7CNTF of Example 13, after enzymatic cleavage with hydroxylamine. Mutein GlyD14Serl7CNTF elutes in buffer C, mutein CNTF(His)~sn elutes in buffer D.
Figure 16 shows the elution profile of mutein His6Dl4serl7CNTF of Example 13, after enzymatic cleavage with enterokinase. The recombinant protein elutes in buffer C.
ETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description should not be construed to unduly limit the present invention, as modifications and variations in the embodiments herein discllssed may be made by those of ordinary skill in the art without departin~ from the spirit or scope of the present inventive discovery.

2 1 2 3 2 3 l W093/10233 PCT/EP92/OZ5g6 c ^nl - ~ C c - G _ C .- C - - .r -~ : G - e r~nc e C C ' .esen~ S~ec1Iic~lic.. â~e ne~ein i,-~or~or2~ec b~ rererence in ~heir entiretv.
The zresenl inven~ion aenerallv falls within ~he ~-~lc of recombinant DNA technoloav. It concerns specificaily mutated forms (called isoforms) of natural CNTF 7nich differ from the corresponding na~ural forms due to ~he presence of one or more deletions in their polypeptide sequences, as well as muteins of CNTF. These isoforms and muteins can retain the ~iological activity of the corresponding natural protein.

General Recombinant DNA Techniques The cleavage of DNA with restriction enzymes w~s effected according to manufacturer's instructions.
Generally, 1 ~g of plasmid was cut with 1 U of enzyme in 20 ~1 of reaction solution. Temperatures and incubation times -: depended on which enzyme was employed, but were usually 1 hr at 37C. After in~ubation, the plasmids and gene fragments were purified in all cases vi~ electrophoresis through ~MP Agarose (BRL, USA) in 40 mM ~ris-HCl, 20 mM
sodium acetate, 1 mM EDTA, and then eluted from the agar~se using a GENECLEAN~ kit (RIO 101 Inc., La 3011a, CA, USA).
Ligations were performed using T4 DNA ligase at a concentration of 1 U per 0.5 ~g of DNA in a reaction volume of 20 ~1 at 13C for 12 hrs. Analyses to confirm the correct plasmid sequence were effected by transfecting the ligation products into E. coli HB101, and selecting the transformed cells on agar plates in LB (Luria Bertani) medium with 50 ~g/ml of ampicillin. The recovered cells were grown in LB containing 100 ~gtml of ampicillin, and plasmids were purified, both for the small and larger preparations, with a ki~ from Quiagen (DIAGEN GmhH, ~usseldorf, West Germany). The cloning vectors were prepared from the bacterial cells by the method recommended by Quiagen.

WO93/10233 212 3 ~ ~ ~1 PCT/EP92/02586 1~ _ _con~cice_~ce~ c ,~nes _e~ ~- soi~d ~h~s~
us;r,_ ~ vo~, Dlir. -~ n~h~sl~ cl- (..~pllea ~los~s~e.r~.s ~ t;S~
acccrdinc ~ the mcnuf2c_u~e~'s instru~.ions. ~hev were trea~ed c. ~oc for '~ hrs ~n ~h" and then recovered in â
speed-vacuum centri uge. They were resuspended in 2.5 ammonium acetate and then precipitated with 3 volumes of cold ethanol (-20C). They ~ere rewashed .~ith cold ~0~ ;
ethanol and resuspended in water. The concentration of oligonucleotides was assessed spectrophotometrically.
10Amplification was effected on a Perkin Elmer Cetus DNA
Thermal Cycler, and the reagents used for amplification were those of the relative DNAl~' Amplifier (Perkin Elmer-Cetus). Briefly, a miY.~ure containing 200 ~M of each oligonucleotide was used, 0.5 ~M of each of the nucleotides dATP, dTTP, dCTP, dGTP, and 0.1 ~g of human DNA and reaction buffer in a total mixture of 100 ~1 with 0.5 U of TAQ polymerase, the reaction mixture being then covered with liquid paraffin to prevent evaporation.
The amplification reaction was conducted by setting the instru~ent at 25 cycles under the foll~wing conditions:
1 min. at 95C denaturation, 45C alignment, 72C
extension.
Particular modifications were performed on ~ild-type CNTF gene sequences to obtain genes encoding proteins with partial amino acid sequences and altered sequences compared to the natural form of CNTF, possessinq chemical and chemical-physical characteristics which a e different from those of the natural protein.

HUMAN CILIARY NEURONOTPcOPHIC ~.CTOR

Each of the nucleic acid sequences and polypeptides disclosed herein, or their bioloqically functional equivalents, can be used in accordance with the presen~
invention. The term "~iologically functional equivalents,"
as used r.erein, cenotes nucleic acid sequences or ~l2~23l;l WO93/10233 PCT/EP92/02~86 polypep-~ ~s e~:r.l~i~ln~ =se s3m~ cr ~ OlC_iC2i ac~ivi~ cis _ne par~lcul~ nucleic ~ici~ sequences 2na polypep~ides aescribed inrr~.
For e~ar..Dle, the nucleic acic seauences àeDic,ed below can be altered by substi utions, additions or deletions that provide for biologically func~ionally equivalent molecules. Due to the degeneracy or the gene~ic code, other DNA sequences which Pncode substantially the same amino acid sequences as depicted below may be used in the 10 practice of the present invention. These include, but are ~:
not limited to, nucleotide sequences comprising all or portions of the CNTF genes depicted below which are altered by the substitution of different codons that encode a physiologically functionally equivalent amino acid residue 15 ~ithin the sequence, thus producing a silent change. -~
Similarly, the CNTF proteins, or derivatives thereof, of the present invention include, but are not limited to, :. those containing all of the amino acid sequences substantially as depicted below, including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the se~uence, resulting in a silent change. For example, one or more amino acid residues within ~he sequence can be substituted with another amino acid of similax polarity which acts as a functional equivalent, resulting in a ~ilent alteration.
Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid ~elonqs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neut~al amino acids include glycine~ serine, t~reonine, cysteine, tyrosine, asparagine, and glutamine.
The positively charged (basic) amino acids include :
arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

21232~ 1 W093/l0233 PCT/EP92/02586 -lcc :~cluàec '.~ n ~i~e àco~e c- ~~e presen.
in~e~.~cr cre CliTF ~raamenls c~ ce~iva~iv~c .hereor ~hicn are differenlially modified durina or a.ter ,ransla~ on, e g , by glycos~lation, proteolytic cleavage, linKage to an antibody ~olecule or olher cellular ligand, etc The examples below exemplify the expression of a biologically active recombinant CNTF isoform or mutein in r, coli, there~y indicating that non-glycosylated forms of CNTF are biologically active In addition, the recombinant CNTF encoding nucleic acid sequences of the present invention may be engineered so as to modify processing or expression of CNTF For example, and not by way of limitation, a signal sequence may be inserted upstream of CNTF encoding sequences to permit secretion of CNTF, and thereby facilitate harvesting or bioavailability.
Additionally, a given CNTF isoform or mutein can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or te~mination sequences, or to create variations in coding regions andlor form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification Any technique for mutagenesis known in the art can be used, including, but not limi~ed to, in vi~ro site-direct d mutagenesis (Hutchinson et al. (1978) J. Biol Chem. 253:6551~, use of TAB~ linkers (Pharmacia), etc EXPRESSION VECTORS FOR HUMAN CNTF

The vectors contemplated for use in the present invention include those into which a DNA sequence as discussed below can be inserted, along with any necessary operational elements Such vectors can then be subsequently transferred into a host cell and replicated 3~ therein ~referred vectors are those whose restriction sites have been well documented and which contain the 2123~

~~cnsc~ cr, or ,ne D:;. seau~nce Ce~ ir e~bo~i~.en~s o- ~ne presenL :~venlion e~pio~
vec.crs ~.~hic.~ wouid oont2ir one o~ ~,ore o. the DNA
seauences àescribeà herein. It is preferred that all of these vectors have some or all of the following characteristics: (1) possesses a minimal num~er of host-organism sequences; (2) be stably maintained and propagated in the desired host; (3) be capable of being present in high copy number in the desired host; (4) possess a regulatable promoter positioned so as to promote transcription of the gene of interest; (5) have at least one ~arker DNA sequence coding for a selectable trait present on a portion of the plasmid separate from that where the DNA sequence will be inserted; and ~6) contain a DNA sequence capable of terminating transcription.
The cloning vectors capable of expressing the DNA
sequences of the present invention contain various operational elements. These "operational elements" can include at least one promoter, at least o~e Shine-Dalgarno sequence and initiator codon, and at least one termination codon. These "operational elements" may also includelone or more of the following: at least one operator, at least one leader sequence for proteins to be exported from intracellular space, at least one gene for a regulator protein, and any other ~NA sequences necessary or preferred for appropriate transcription and subsequent translation of the cloned CNTF DNA.
Certain of these operational elements may be present in each of the preferred vectors of the present invention.
It is contemplated that any additional operational elements which may be required may be identified and added to these vectors using methods known to those of ordinary skill in the art, such as those described by Sambrook et al., supra.
-2123~23,1 .
WO 93/10233 PCltEP92tO2586 _ C ~ _ _ C
~~eculci.crs _er~e ~ pre~e~ rescii~.r ~ l~.e ~;;r. .-seaue.~ce ~n ~ e ~rese.~ce cr i~er~2ln envlron~e~lai conài~ions ana, in the ~resence c~ cthe~ envlronmen~al concitions, ;~ill 2Ilo~ transcr~ tion ana subsequen expression o~ ~he ~ro~ein codeà for by Ihe CNTF ~NA
seauences. In particular, it is preferred that regulator~
segments be inserted into the vector such that expression of the DNA sequence will not occur, or will occur to a greatly reduced extent, in the absence of, for example, isopropylthio-beta-D-galactoside (IPTG). In this situation, the transformed microorganisms containing the DNA sequence may be grown to a desired density prior ~o initia~ion of the expression of the CNTF isoforms or muteins. xpression of the desired protein is induced by addition of a substance to the microbial enviro~ment capable of causing expression of the DNA sequence after the -desired cell density has been achieved.

Promoters The expression vectors must contain promoters which can be used by the host organism for expression of its own pro~eins. While the lactose pro~noter system is commonly used, other microbial promoters have been isolated and characterized, enabling one skilled in the art to use them for expression of the recom~inant CNTF isoforms and muteins.

Transcri~tion Terminators The ~ranscription termina~ors contemplated herein serve to stabilize the~ vector. In particular, those sequences described by Rosenberg e~ al. (1979~ ~nn. Rev.
Genet. 13:319-353 are contemplated for use in the present invention.

2 1 ~ 3 ~ ~ 1 ::on---crsi~ec ~cu~,c~-T ~ ~12`~ Gisc ~e ~es yc~ ~ e -c cc.-slruc. ;~.e 3' cr ~ c or the coding region ~c allo:i incorpora~ion o~ 3' or _' non-transl2~eà seauences into .he gene transcri~ .
Included among these non-~ranslated sequences are those ~hich stabilize mRNA, 25 disclosed by Schmeissner et 21.
(1984) J. Mol. Biol 176:3-S3.

Ribosome Bindina Sites The microbial expression of foreign proteins requires operational elements which include ribosome binding sites.
A ri~osome hinding site is a sequence which a ribosome recognizes and binds to in the initiation of protein synthesis as set forth in Gold et al. Ann. Rev. Microbiol.
35:557-580 and Marquis et al. (1986) Gene 42:175-183. A
preferred ri~osome binding site is GAGGCGCAAAAA(ATG).

Leader Seauences and ranslationa~ Cou~lers Additionally, it i5 preferred that DNA coding for an appropriate secretory leader ~si~nal) sequence be present at the S' end of the DNA sequence, as set forth by Watson, M.E. in Nucleic Acids Res. 12:5145-~163, if the protein is to be excreted from the host cytoplasm. The DNA for the leader sequenc~ must be in a position that allows the production of a fusion protein in which ~he leader sequence is immediately adjacent to and covalently joined to CNTE, i.e., there must be no transcription or translation signals between the two DNA coding sequences.
In some species of host microorganisms, the presence of an appropriate leader sequence will allow transport of the completed protein into the periplasmic space, as in the case of some E. coli. In the case of certain E. coli, Saccharomvces and strains of Bacillus and Pseudomonas, the appropriate leader sequence will allow transport of the 35 protein through the cell membrane and into the --extracellular medium. In this situation, the protein mav be purified from e~lracellular protein.

WO 93/10233 2 1 2 3 ~ ~ 1 PCl tE~P9~/0258fi .-.n G~ _^r.z ~ ~u~nce oc~ ~e lcca,ea ~- ei~e~
pre~ecir,c ~~,e 3i~r. -_a~)e c~ :nlcl coaes c. .~e C.TF
isoforr ~he cda~_~on2l DNA seauence is cap2Dle Or functionina 25 a ~-2nsia~ionai cou~ler, i.e., _. is a Dl~'~
sequence tha~ encodes an ~NA .~hich serves ~o Dosition ribosomes im~ediatel~ adjacent to the ribo~ome ~inding site of the inhibitor RhA s.~ith which it is contiguous. The translational coupler may be derived using the DNA sequence TAACGAGGCGCAAAAAATGAAAAAGACAGCTATCGCGATCTTGGAGGATGATTAAATG
and methods currently known to those of ordinary s~ill in the art related to translational couplers.

Translation Terminators The translation terminators contemplated herein serve to stop the translation o~ mRNA. They may be either natural, as descri~ed by Kohli, J., Mol._Gen Genet.
182:430-439, or synthetic, as described by Pettersson, R.F.
(1983~ Gene 24:15-27.

Selectable Markers Additionally, it is preferred that the cloning vector contain a selectable marker, such as a drug resistance marker or other marker which causes expression of a selectable trait by the ho~t microorganism. In one embodiment of the present invention, the gene for ampicillin resistance is include~ in ~he vector. In other plasmids, the qene for tetracycline resistance or the gene for chloramphenicol resistance can be includied.
Such a drug resistance or other selectable marker facilitates the selec~ion of transforman~s. Additionally, the presence of such a selectable marker in the cloning vector may be of use in keeping contaminating microorganisms from multiplying in the culturg medium. A
pure culture of the transformed host microorganisms would be obtained by culturing the microorganisms under conditions ~!hich reuuire the induced pheno~ype ,or survival.

2:1~32~1 ~8 .qe o~era~lonc ~le~e.~.C _~scusse~ :~e~eln are ~ou~lnei~ sel~c~eà b~. ~hose o, c~airar~ in .ne ar. in ligh. OL prior litera~ure and ~;~e ~eachings con~ainea herein. Generai e~amples of these opera~ional elements are set forth in B. Lewin (1903) Genes, l~iley & Sons, New York.
various examples of suitable opera~ional elements may be found in the vec~ors discussed above, and may be gleaned via review of the pu~lications discussing the basic characteristics of the aforementioned vectors.
Upon synthesis and isolation of all the necessary and desired component parts, the vector can be assem~led by methods generally known to those of ordinary skill in the art. Assem~ly of such vec~ors is within the ordinary skill in the art, and, as such, is capable of being performed without undue experimentation.
Multiple copies of the DNA sequences of the present invention and their accompanying operational elements may r be inserted into each vector. In such case, the host organism would produce greater amounts per vector of the desired CNTF isoform. The number of multiple copies of the DNA sequence which may be inserted into the vector is limited only by the ability of the resultant vector t due to its size, to be transferred into and replica.ted and transrribed in an appropriate host cell.
Other Host Microorqanisms Vectors suitable for use in microorganisms other than E. coli are also contemplated for use in the present invention. Such microorganism include, for example, Bacillus, Pseudomonas, and yeast. Also contemplated within the scope of the present invention is the expression of the truncated and mutein forms of human CNTF disclosed herein in mammalian cells, including, for example, Chinese Hamster Ovary cells.

WO93~10233 PCT/EP92/0258~

~ rO~ C~pression lr ~ C c~-~e~ CI~rs S~.OUl~
inciuae c reaula~ea ~ro o~e- S~C.I as ~ e aipha a~ias~
promoler, the subtilisin p~omc~er, the ~-3 promoter, or the spac-126 promoler. ~serui transcription terminalors include rrn and rrn BT.T. Transcriptional start sites and leader peptides can be chosen from among those from B.amyloliauefaciens neutral protease, B.amvloliauefaciens alpha-amylase, and B.subtilis subtilisin. Useful antibiotic markers are Kan'and Cam'. ~ibosome binding sites can be obtained from the B.amvloliquefaciens neutral protease and B.amvloliquefaciens alpha-amylase genes. A
preferred expression sys~em in hosts of the genus Bacillus involves the use of plasmid pUB110 as the cloning vehicle~
For expression in Pseudomonas, promoters can be lS selected from Trp, Lac, and Tac. Useful transcriptional start sites and leader peptides can be obtained from the phospholipase C and exotoxin A qenes. Useful antibiotic markers are those for sulfonamides and streptomycins. A
useful ri~osome binding site can be obtained from ~Le Trp promoter of E. coli. Particularly preferred vectGrs would employ the plasmid RSF1010, and derivatives thereof.
In the case of yeast, useful promoters include Gal 1 and 10, Adh 1 and 11, and Pho S. Transcription terminators can be chosen from among Cyc, ~na, Alpha Factor, and Sac 2.
Transcriptional start sites and leader p~ptides can be obtained from the inverta~e, acid phosphatase, and Alpha ~actor genes. Useful selection markers are Ura 3, Leu 2, His 3, and Tap 1.
Finally, in the case of expression in mammalian cells, the DNA encoding the present truncated or mutei~ CNTFs should have a sequence e~ficient at binding ribosomes.
Such a sequence is described by Kozak in Nucleic Acids Research (1987) 15:8125-8132. The CN~F-encoding fragment can De inserted into an expression vector containin~ a transcriptional promoter and a transcriptional enhancer as descri~ed by Guaren~e i~ Cell (1988) 52:303-305 and Kadonaga et al. (1987) Cell ,1:1079-1090. A regulata~le ~093/10233 2 ~ 2 ~ 2 ~ I PCTtEP92/02586 he .~i-cr~.a~ cS.~ c ~seG, _ ~cCcs2r~ or aeslrec ~he ~ec~o- s,~ou 'G ciso ~ossess G
co~?le.e polvaaenylation s~gn2i 2S desc~ibed b~ Ausube~ e~
cl. (1907) i~ Curren~ Dro~ocols i~ olecular ~ioloa~, l~ile~, so that mRNA transcribed f rom ~he veclor is properly processed. Finall~, the vector may 21so contain the replica~ion origin and at leas~ one antibio~ic resistance marker from a plasmid such as pBR322, to allow replication and selection in E. coli.
In order to select a stable cell line that produces CNTF as descrihed herein, the expression vector can carry the gene for a selectable marker such as a drug resistance ~-marker or a complementary gene for a deficient cell line, such as a dihydrofolate reductase (dhfr) .gene for transforming a dhfr cell line, as æescribed by Ausubel et al., su~ra. Alterna~ively, a separate plasmid carrying the selectable marker can be cotransformed along with the expression vector. -`~
Vectors for mammalian cells can be introduced therein by several techniques, including calcium phosphate:DNA
coprecipitation, electroporation, or protoplast fusion.
Coprecipitation with calcium phosphate as descri~ed by Ausubel et al., s~ra, is the preferred method.
Preferred vectors include, for example, a PSVT7 2S eukaryotic expression plasmid.

To o~tain a form of CNTF lacking the first 14 amino acids, two oligonusleotides were synthesized. The first (A) had the sequence 5'AACATATGGACCTGTAGCCGCTCTATC 3', and starts mapping from the 42nd oligonucleotide after the initial oligonucleotide of the human CNTF sequence. An NdeI
restriction site was added to facilitate subsequent cloning, and a methionine codon was added in order to initiate transcription of the gene.

21232~

~ `v ~:slr~ c~ a '~ ... C.leC c~ c~r.ucico~ ~ c_ _c:z~ne~
;: ,h an oliqonucl~o~ ~e (B) :!l.rl _ne seauence S~TTGTCGACTC.~CATTTTC~TGTT~-TTrGCAATr.T ~~ icr. inve~sel~
maps at .he end or the sequence encodinc C`ITF, it is possible, using the piasmid pT7.7hC~'TF (Negro e~ al. (1991) J. Neurosci. Res. ~9:270) ~o amplify the sequence shown in Figure 3 This sequence was cut with NdeI, and SalI, anà cloned using T4 DNA ligase in plasmid pT7.7 (Figure 9) in order to obtain transcription of the amino acid sequence shown in Figure 4, together with molecular weight data, number of residues, and amino acid composition This polypeptide lacks the firsl 14 amino acids, starting from the amino terminal end of the natural polypeptide. The methionine was introduced at the start of the amino acid sequence. The isoelectric point (pI) of this new polypeptide form is 6.05, compared to 6.38 of the natural protein.

To obtain a form of CNTF lacking the car~oxy terminal 26 amino acids, two oligonucleotides were synthesized. The first (C) had the sequence ~'TTCATAGGCTTTTACTGAAGCATTCA 3', and starts mapping from the ~eginning of the sequence encoding the CNTF gene. The second oligonucleotide (D) had the sequence S'TTGTCGACTCAATGGATGGACCTTACTGTCCAC 3', and maps inversely at th~ end of the CNTF gene. Moreover, a stop codon w~s added at the end of the gene at position 523 with respect to the start of the gene, so as to eliminate the last 17 amino acids. A SalI restriction site was also added to facilitate subsequent cloning. By using oligonucleotides (C) and (D) together with the plasmid pT7.7hCNTF, the nucleotide sequence shown in Figure 5 can be~ amplified.
This sequence was cut with NdeI and SalI, and cloned using T4 DNA ligase in the plasmid pT7.7 so as to o~tain the transcrip~io~ c_ the amino acid sequence shown in r igure 6.

2l23231 ~ h~c -~uenc~ e 2sr c~ c~s ~ T~
so ~ha~ e ~oi~pe~ia~ nas l,~ ar,lr,c 2Cl~S. ~ne ~esul~inc molecul2~ :.elch. is 20,007 KDa. ~he ~I ot this ne: fcrm is ~-5.67 .
EXAMPL~ 3 To obtain a polypeptide form of human CNTF lacking the first 14 amino acids at the amino terminal end and the last 26 amino acids at the carboxy terminal end, two oligonucleotides, i.e., (A) in Example l and (D) in Example 2, and the plasmid pT7.7hCNTF, were used to amplify the ;
sequence shown in Figure 7.
This sequence was CUI with NdeI and SalI, and cloned using T4 DNA ligase in the plasmid pT7.7 so as to obtain `
the transcription of the amino acid sequence shown in Figure 8.
This sequence lacks the first 14 amino acids and the last 26 amino acids of CNTF. Therefore, the polypeptide contains 161 amino acids, taking into account the methionine added initially.
The resulting polypeptide has a molecular weigh~ of 18,476 KDa, and an isoelectric point of 5.14.

Figure 9 shows the cloning scheme used to obtain the vectors employed for the expression of the new polypeptide forms of human CNTF disclosed herein.

EXA~PLE 5 From the plasmid pT7.7hCNTF it is possi~le to amplify the whole sequence using two oligonucleotides, suitably chosen according to the desired deletion.
Described in detail below is a method for obtaining a ` `~
polypeptide sequence with the se~ment 137-150 deleted with respect to the wild-type human CNTF polypeptide sequence.

W093/10233 212 3 ~ 3 ~-~ PCT/EP92/02586 T~ iiO;ilna cilgonUcieollaec bC..~ 2~r, ~ r.
res=~ . si~e; s~cn slles cre nc~ ~reser,.
plcs.la:

S AGCATGCGGATCTTGTATTCCAGGAG

TTGCATGCTCTTTGAGAAGAA~CTGT

These two oligonucleotides are employed in the PCR
technique as descri~ed previously. The purified DNA is CUt with SphI, and then ligated with T~ DNA ligase.
By this method it is possible to obtain linear segments linked via a ligase. The distance in nucleotides between the two selected oligonucleotidos determines the extent of deletion in the mutant.
Figure 10 illustrates all the truncated forms of human CNTF reported in ~xamples 1 to 3, and the forms with deleted sequences produced as described in Example 5.
EXAMP~E 6 ~ACTERIAL GROWTH AND PRQPAGATION
A colony of E. coli BL~l(D3) containing the mutated plas~id pT77RCNTF ~egro et al. (19gl) J. Neurosci. Res.
29:2513 is inoculated into 5 ml of LB medium containing 200 ~g/~l of ampicillin. As pre~iously described, each cell line expresses the polypep~ide sequence encoded by the gene seauence inserted in the expression vector.
All constructions were performed in E. coli HB101.
~o obtain protein expression, the plasmids containing the truncated or deleted forms o~ CNTF were transformed into different E. coli lines, including B~21(D3).
Cells were grown overnight at 30C. An aliquot was diluted 1/100 in M9 medium containing 200 ~g/ml of ampicillin. The cells were grown to an absorDancy of 0.O OD
a_ ,co nr. ..t this point, isoprop~l thiogaiactoside (IPTC-) W093/10~33 2 12 3 ~ ~ ~ PCT/EP92/02586 ..cs c~e~, -. m.~ ci .once.rlLra,lon. T!le cells; ~re ~ a~
-G~ c~O~ hours, cen~ri~uged, 2na r-esus~enàea ;?. ~-iS-~C', p~ G . O, 10 m~ DT~, after tinich they were read~ -or sUDseqUen~ e~:lrac~iorls.
:
EXA~PLE 7 PURIFICATION PROCEDURE
The pellet obtained after centrifugation was washeà at least 3 times with 50 mM Tris, pH 7.4, and 50 mM NaCl (C).
After each washing, the pellet was recovered by cen~rifuging at 600 rpm for 10 minutes.
The final pellet was resuspended in buffer and passed at least twice through a Montain Gaulin at 300 psi~
The resulting material was centrifuged again at 500 rpm for 10 minutes and resuspended in 8 M urea, 50 mM Tris, pH 7.4, 1 mM PMSF, 2 mM EDTA, at room temperature.
After centrifugation for 10 minutes at 600 rpm, the soluble material was dialyzed against 1 M urea.
The supernatant obtained after further centrifugation for lS minutes at 600 rpm was loaded onto an ion exchange column such as a Mono Q column, suitably equilibrated, at a pH based on the pI of the mutated pxoteins.
Under these conditions, the recombinant proteins were Z5 eluted at NaCl concentrations of 10 mM to 1.0 M. The material obtained was dialyzed against SO mM ammonium acetate and loaded onto an inverse-phase column.
The column employed was a Vydac C18 7 ~m (0.46 x 26 cm); the solvents used were (A) 0.05% TFA in water, and ~B) 0.05% TFA in acetonitryl.
The column was equilibrated with A and 10% B, and the protein was loaded.
A gradient was thus produced wherein the percentage of buffer ~B) reached 60% in 60 minutes, after which the column was washed with 100% (B) for 10 minutes.
The recom~inan~ pro~ein eluted from the column 2S 2 single ~eak.

WO 93tlO233 212 3 2 3 ~ PCI/EP92~02586 ' !`i P T _ , D~TE~MIN.~TIOI! C~F '~`IOLCC-C.`.L ~.CTI~ITV
The ac~ivi~v OI the poiypeD~ides obtaine~ cs ae~c.-lbec in Examples l, 2 and , was assessed by ~hei~ capaci~ O
maintain chic}: e~Dryo dorsal root neuronal ceils a, à2v El0 in culture. These cells were removed from chic~ emDryCs at day E10, dissociated with trypsin, and enriched by subsequent preplating steps.
The neurons were then seeded in 96-well tissue-~ulture dishes, treated with polyornithine (100 llg/ml in 15 mM
borate buffer, pH 8.4) and laminin (10 ~g/ml). The neurons were seeded at a concen~ration of 4,000 neurons per well, the culture medium was Dulbecco's minimal essential medium (DM~M) with 100 ~g/ml of penicillin, 2 mM L-glutamine and 10% inactivated fetal calf serum. The surviving neurons were counted after 24 hours in culture (Skaper et al.
(1985~ Dev. ~rain Res. 24:39-46)~
Figure ll shows exa~ples of the ~iological ac~ivity of truncated polypeptide forms compared to wild-type human CNTF protein. The results of this experiment show that the specific activity, calculated as ED50, was at least 5 x 10~lM for the various forms.

EXPRESSION VECTQR FOR THE CNTF POLYPEPTIDE Delta 14 Ser 17 CNTF
On the basis of the human CNTF sequence ~Negro et al.
(1991) J. Neurosci. Res. 29:251), the following oligonucleo~ide was constructèd:

BqlII
5'CCAGATCTGGCACCATCATCATCACCATAACGGCGACCTCAGTAGCCGCTCTA~' IleTrpHisHisHisHisHisHisAsnGlyAspLeuSerSerArgSerIle 212~23~
WO 93/10233 PCr/EP92/02~86 his C'~Cc~nuciea~l'e car.~ .s c E~c1lI Cl~
-egion. si~ s coaons cre Gresen., ~ollo~Jea b~ G2~ 0_ .he C~TF sequence s~ar~inS fro~ l_th codon. !~oreove~ .he 17th codon (TC-T) encoding cvsteine was replaced with (TCT), encoding serine. This modification was necessarv Decaus~
in the presence of cysteine, two molecules of CNTF can combine to form a dimer, thus lowering the bioloaical activity. This oligonucleotide, together with the oligonucleotide SalI of the following sequence:
5'AGGTCGACTACATTTTCTGTTGTTAG 3', was used for PCR. The amplified product was cut with BglII and SalI, and cloned into the plasmid pT7.7hCNTF (Negro et al. (1991) J.
Neurosci. Res. 29:270) between the BamHI and SalI sites.
BamHI and BglII are compatible restriction sites.
Figure 12 represents the expression vector. It includes resistance to ampicillin ~AMP'~, the T7 promoter, the restriction sites N (NdeI), Nh (NheI), B (BamHI), Bg (BglII) and S (SalI). The position of the histidines (~) - and cleavage site for hydroxylamine (H) are also indicated.

Fusion Proteins The fusion protein containing the chemical cleavage site H is shown below:

hydroxylamine CNTF(l-l86)l3is-His-His-His-l;~s-Hi~ sn-Gly-Asp-Leu-ser-cNTF(lS-200) The fusion protein is a CNTF molecule fused to another CNTF molecule by 6 histidines and the amino acids asparagine and glycine. These two amino acids represent the cleavage site for hydroxylamine.

2 1 2 ~

F~ ,~ l`i P L ~ - -_~'p~rSC~OJi ~iFCTO~ FO~ THE POL~PEP~TDF DETTrl~Serl?C,~TF.
On the basis of the sequence ~~ human CNTF (Nearo e~
5 âl. (19gl) J Neurosci Res 29:251), the rollo~inc oligonucleotide was constructed:

5 I TCTAGATCTCTCTAGCCGCTCTAT 3' AspLeuSerSerArgSerIle In this oligonucleotide, â BglII restriction site is present in the 5' region. Moreover, the 17th codon (TGT) encoding the amino acid cysteine is substituted with (TCT), encoding the amino acid serine. This oligonucleotide, together with the oligonucleotide SalI, previously described, was used for PCR. The amplified product was cut with BglII and SalI and cloned in the expression plasmid p~'SETB (Invitrogen) between the restricti~n sites BamHI and Xhol, which are compati~le with BglII and SalI, respectively.
Figure 13 shows the expression ~ector. It illustrates the resistance to ampicillin (AMP'), the T7 promoter; the restriction sites N (NdeI), B (BamHI~, Bg (BglII), S
(SalI), and X (XhoI). Moreover, t~e position of the histidines (M) and the cleavage site for enterokinase are illustrated.
The initial amino acids of this fusion protein are:

Met-Arg-Gly-Ser-His-His-His-His-His-His-Gly-Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Asp-Leu-Tyr-Asp-Asp--Asp-Asp-Lys-Aspl5-Leul6-Ser~

As can be seen, the transcri~ed protein is a fusion protein containing six histidines in the amino terminal region, followed bv a cleavage site for enterokinase W093/l0233 21 2 3 2 ~ 4 PCT/EP92/02586 ~el;-een ;~s an~ ~.sp~ hicn fG~li,c.~s ~ c e2vage o .~.e sequence ~s~-~s~-~sp-~s~-Lys-~sp!~ c ~e~ e i~.~sine.

EXA~PT~ 12 s 3ACTERIAL_G OWTH
Bac~erial growth was as described in Example 6 PURIFICATION PROCEDURE
The pellet obtained after centrifugation was washed at leas~ 3 times with buffer (50mM Tris, pH 7.4, 50 mM NaCl) and each time the pellet was recovered by centrifugation.
The final pellet was resuspended in the above b~lffer and passed at least twice through a Montain Gaulin at 800psi.
The resulting material was ag~in centrifuged at 600 rpm for 10 minutes and resuspended in buffer A ~6M guanidine, 0.lM
NaH7PO" lOmM Tris/ pH 8). It was centrifuged again as before, and the supernatant was loaded on~o an IMA~-NTA
chromatographic column (~uiagen) to interact with metal ions. 8 ml of NTA resin was loaded with nickel and washed -with 70 ml of buffer A. The supernatant was loadéd in buffer A at a rate of 30-50 ml/hour using a peristaltic pump. The column was then washed with buffer B ~8M urea, 0.lM NaH.PO~, 10mM Tris, p~ 8), then washed again with 4~ml of buffer C (8M urea, 0.1M NaH,PO4, lQmM Tris, pH 6.3). The recombinant muteins were eluted with buffer D ~8M urea, 0.lM NaH,PO~, lOmM Tris, pH 4.5).
The mutein CNTF~His)6AsnGlyD14Serl7CNTF was dialyzed against water, and the precipitate resuspended in 6M
guanidine and 2M hydroxylamine. The p~lypeptide was incubated for 12 hrs at 37C, during which time the hydroxylamine cleaves the polypeptide into two portions, CNTF(His)6Asn and GlyD14Serl7CNTF, respectively.

WO93/10233 2 1 2 3 2 ~ ~ PCT/EP9V02586 The ..,u~ein ~is6Dl;S~',C.'TF _-. the c~ne~ .,2r., :.a5 dialvzea agains~ ,OmM CcCl~, ~O~: ~r~s-HCl, p~ na diges~ed with entero~inase (Boehringer) usin~ an enzy~.e substrate ratio of l:500 a~ 37C for 12 hrs. Durinc .r.is time, the polypeptide divides into two por~ions: His6 and Dl4Serl7CNTF. The muteins thus digested were dialyzeà
against buffer A and loaaed onto the column as described previously.
The final product of the polypeptide CNTF(His~AsnGlyDl4Serl7CNTF is represented by the following muteins: GlyDl4Serl7CN~F, which elutes in buffer C, and CNTF(His)~Asn, which elutes in buffer D.
~he final product of the polypeptide His6Dl4Serl7CNTF `;
is the mutein Dl4Serl7CNTF, which elutes in buffer C.
The two recombinant muteins o~tained by this method are purified as indicated in the following flow schemes:

Mutein: CNTF~His?6AsnCl~Dl4Serl7CNTF
Bacterial pellet Solubilization in buffer A
Loading of IMAC-NTA column in buffer A
Washing with buffer B (histidine-free proteins) Washing with buffer C (Bacterial proteins with few histidines) Washing with ~uffer D (Mutein CNTF(His)6AsnGlyDl4Serl7CNTF) Dialysis Centrifugation Pellet Solubilization with ~M guanidine, 2M
hydroxylamine, 37C, 12 hrs Dialysis with buffer A
Loading of IMAC-NTA column in buffer A
Washing with buffer B
Washing with buffer C (GlyDl4Serl7CNTF) Washing ~ith buffer D (CNTF~His)6Asn) 21~32~
WO93/10233 PCT/EP92~02586 !~utein~ s)~Dl~Ser'7C'~TF
Baclerial peile~
Solubilization in buffe~ .-Loading or II~C-NTA colu~n Washing with buffer B (his~idine-free pro~eins) Washing with buffer C (Bacterial pro~eins with f ew histidines) Washing with buffer D (His)hD14Serl7CNTF
Dialysis Digestion with enterokinase, 1:500, at 37C for 12hrs `~
Dialysis with buffer A ~.
Loading of I~AC-NTA column Washing with buffer B ~-i5 Washing with buffer C (D14Serl7CNTF) -' Washing with buffer D ((His)~

Figure 14 shows the elution profile of the bacterial starting material, loaded in buffer ~, washed with buffers B and C. The recombinant protein elutes in buffer D.
Figure 15 shows the elution profile of CNTF(His)~snGlyD14Serl7CNTF, after enzymatic cleavage ~ith hydroxylamine. Mutein GlyD14Serl7CNTF elutes in buffer C, - mutein CNTF(His)6Asn elutes in buffer D.
2S Figure 16 shows the elution profile of mutein His~D14Serl7CNTF, after enzymatio cleavage with enterokinase. The recom~inant protein elutes in buffer C.
It is also possible to purify the muteins under non-denaturating conditions. In this case, IMAC columns (Pharmacia) can be used. The various steps include the growth of bacteria at 30C, solubilization of the muteins in a phosphate buffer, pH 7.4, and loadiny onto the column under non-denaturating conditions. The protein elutes at the same pH values as indicated for denaturating conditions.

WO93/10233 2 ~ 2 3 2 3 -1 PCT~EP92/02~86 L ~; .2, 1,~, p L - 1 -DETEPMIN~TION OF ~MINO ACID SEOUENCFS
The amino ~erminal sequences o~ the recom~inant muteins were determined by Edman degradation using an Applied Biosystem automatic protein sequencer, Model t77A.
The phenylthiohydantoins derived from the amino acids were instrumentally determined. The seauence of the first 10 amino acids starting from the N-terminal amino acid for GlyD145erl7CNTF was: Gly-Asp-Leu-Ser-Ser-Arg-Ser-Ile-Trp-Leu-Ala-Arg, and for D14Serl7CNTF was: Asp-Leu-Ser-Ser-Arg-Ser-Ile-~rp-~eu-Ala-Arg-Lys. This sequence correlates with that of CNTF (Figure 2) when the first 14 amino terminal amino acids in ~lyD14Serl7CNTF are absent, a glycine amino terminal amino acid is present, and the cysteine in position 17 is substituted with serine. In the case of Dl4Serl7, the first 14 amino acids are deleted and cysteine .; in position 17 is substituted with serine.

~ oeL~

DETERMINATION OF BIOLOGICAL ACTIVITY
The biological activity of the recombinant muteili was assessed on the ~asis of the mutein's efficacy in maintaining in culture embryonic neuron cells at day E10, extracted from chick doxsal ganylia. The cells were removed from chick embryos at day El0, and dissociated and enriched via a series of pre-plating steps. The neurons were then seeded in g6-well dishes for tissue culture, treated with polyornithine (100 ~g/ml in borate buffer, pH
8.43 and laminin 10 ~g/ml). The neurons were seeded a~ a concentration of 4,000 neurons per well. The culture medium was Dulbecco minimal essential medium (DMEM~ with 100 ~g/ml of penicillin, 2mM L-glutamine, and 10%
inactivated fetal calf serum. Surviving neurons were counted after 24 hrs in culture (Skaper et al. (1985) Dev.
Brain Res. 24:39-46).

2 1 2 3 ~ ? ~ .

- 3~ -The ~inale ~.u~eins had the ~ol'cwirl~ s~ec~ ~
activi-ies, calculated as ED50:

CNTF(His)6AsnGlyDl~Serl7CNTF 20ng/ml GlyDl~Serl7CNTF 0.25ng/~
CNTF(His)6Asn O.Sng/r.l (His)6D14Serl7CNTF 0.4ng/mi D14Serl7CNTF 0.12ng/ml PHARMACEUTICAL COMPOSITIONS

The formulation of pharmaceutical compositions containing solutions of the present human CNTF molecules with and withou~ gangliosides, phospholipids, hyaluronic acid or their derivatives or semisynthetic analogues or one of their salts, includes well known methods for the preparation of pharmaceutically acceptable compositions, to -. be administered to patients, wherein an effective quantity of the CNTF molecule is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles and their formulations~ including other proteins, are described, for example, in Reminaton's Ph~rmaceuti~cal Sciences ~1985) Mack Publishing Company, Easton, Pa., USA.
Such vehicles include injectable "depot formulations." On this basis~ the pharmaceutical formulation includ~s, albeit not exclusively, solutions of CNTF growth factor or its free2e-dried powders in association with one or more pharmaceutically acceptable vehicles or diluents, contained in buffer at a suitable pH, and isosmotic with physiological fluids. In the case of freeze-driea preparations, support excipients can be used, such as, but not exclusively, mannitol or glycine, and suitable buffered solutions of the desired volume will be supplied in order to obtain adequate isotonic buffered solutions with the desired pH. Similar solutions can be used for pharmaceutical compositions or the present CNTF ~olecules in isotonic solutions of the desired volume and incluae, `:

212~234 W093/10~33 PCT/~P92/02586 bul not ~::c sl~e~ he use or Dhyslologic2' ~lffe~ed solutions ~ n nospna~e or ci~rale a~ sui~able concentra~io~s i~ order to consistently obtain iso~onic pharmaceulic21 prepara~ions ~ith the desireG ~, for example neulral pH.
Pharmaceutical preparations can ~e employed for oral, topical, reclal, parenteral, local, inhalant, intracerebral or nasal use. They can therefore be in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, non-woven products, tubelets, threads, microspheres, and sponges. ~or parenteral and intracerebral uses, those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral in~ection can be used and can therefore be prepared as solutions of the active compounds or as freeze-dried powders of the active compounds to be mixed with one or more pharmaceutically acceptable excipients or diluents, suitable for the aforesaid uses and with an osmolarity which is compatible with physiological fluids. For local use, those preparations in the form of creams or ointments for topical use or in the form of sprays can be used; for inhalant purposes, preparations in the form of sprays, for example nose sprays, can ~e used; for use in association with polymers, those preparations in the form of membranes, sponges, non-woven products, ~ubelets, threads, microspheres, nanospheres, and nanocapsules can be used.
The preparations of the present inven~ion can be used for administration to humans or animals~ They preferably contain between 0.01% and 10% of active component in the case of solutions, sprays, ointments and creams, and between 1% and 100%, and preferably between 5% and 50% of active compound, in the case of solid form preparations.
Dosages to be administered depend on individual needs, on the desired effect, and on the chosen rou~e of administration.

WO93/10233 212 3 ~ PCT/EP92J02586 The ~nar,a_eu~ re~ara~lons also lncl~a~, ~u. are no~ limilec ~_, su~osi.ories for rectal adminis~ralicn .~ith lipopniiic e~:ci~ien~s, for eY.ample, ;.ater soluble excipients, self-emulsifying excipients such as glycogelatin, or others. In these preparations, the C~'TF
can be present in quantities varying between 0.01~ and 1%
by weight of the entire excipient. The suppositories can contain, but without being limited to these, suitable quantities of acetylsalicylate. The pharmaceutical formulations also include microspheres, nanospheres, nanocapsules for nasal, inhalatory, and intramuscular administration. Moreover, such formulations may also include other particular forms, according to their intended use, such as membranes, sponges, tubes, guide channels, etc.

,-.
A) EXAMPLES OF INJECTABLE SOLUTIONS
PREPARATION No. l - a 2-ml vial contains:
active substance ~g 0.5 to 1 (2,500 BU) sodium chloride mg 16 citrate buffer pH = 7 ml 2 in water for injection q.s.
PREPARATION No. 2 - a 2-ml vial contains:
active substance ~g 5 to 10 (25,000 BU) sodium chloride mg 16 citrate ~uffer pH = 7 ml 2 in water for injection q.s.

PR~PARATION No. 3 - a 2-ml_vial contains:
active su~stance ~g 0.5 to 1 (2,500 BU) sodium chloride mg 16 35 ganglioside sodium salt mg 100 citrate buffer p~ = 7 ml 2 in water for injection q.s.

212323~

PREP~.~.r.-IC:: t:o. ~ - ^. ^-ri vlal con~zins:
ac~ive substance ~g , to l0 (25,000 ~U) sodiu~ chloride m~ 16 ganglioside sodium sal~ mg 50 5 citrate buff2r pH = / ml 2 in water for injection q.s.

PREPARA~ION No. 5 - a 2-ml vial contains:
active substance ~g 0.5 to l (2,500 BU) l0 sodium chloride mg 16 monosialotetrahexosylganglioside (GMl) sodium salts mg l00 citrate buffer pH = / ml 2 in water for injection q.s. ~:
~-' PREPARATION No. 6 - a 2-ml vial contains:
active substance ~g 5 to l0 (25,000 BU) sodium chloride mg l6 monosialotetrahexosylganglioside 20 (GMl) sodium salts mg l00 , .:
citrate buffer pH = 7 ml 2 ~;
in water for injection q.s.

PREPARATION No. 7 25 a) a 2-ml amPule contains:
freeze dried active substance ~g 2 to 4 (l0,000 BU) glycine mg 30 b) a 2-ml ampule of solvent contains:
__ 30 sodium chloride mg 16 citrate buffer in water for injection q~.s. ad ml 2 PREPARATION No. 8 35 a) a 2-ml vial contains:
freeze-dried active substance ~g 2 to 4 (l0,000 BU) :-mannitol ma ~o `-2~ 2~2~ ~
W093/10233 PCT/EP~2/0258S

5j ~ 2-.. ` 2~.DUle 0-- _oi~en~ CC~2' ns:
soaiu~l c.^ioride ~g '~
_i.rate buffe~ in !a~e~
~or inieclion a.s. ad r.l -P~EP~RATION No. 5 a) a 3-ml vial contains:
-freeze-dried active subs~ance ~g 5 to 10 (25,000 BU) glycine mg 45 b) a 3-ml amPule of solvent contains:
sodium chloride mg 24 ~:
citrate buffer in water for injection q.s. ad ml 3 PREPARATION No. 10 a) a 3-ml vial contains:
freeze-dried active substance ~g 5 to 10 ~25,000 BU~
ganglioside sodium salts mg 100 20 glycine mg 4S

b) a 3-ml ampule of solvent contains:
sodium chloride mg 24 citrate buffer in water 25 for in~ection q.s. ad ml 3 PREPARATION No. 11 a) a 3-ml vial contains:
freeze-dried active substance ~g 5 to 10 (25,000 BU) 30 ganglioside sodium salts mg 50 glycine mg 45 b) a 3-ml amDule of solvent contains:
sodium chloride mg 24 35 citrate buffer in water for injection a.s. ad ml 3 WO93/10233 2 1 2 3 2 3 !~ PCT/EPg2/02586 '-~F~`R~TOt~ `'^ -, a) 2 ~ 'iZl con~2~n.C:
freeze-dried ac~ive substance ~g 0.5 to l ( G, 500 BU) monosialotetrahexos~lganglioside 5 (GM1) sodium szlts mg 100 glycine mg ~5 ~:

b) a 3-ml am~ule of solvent contains:
sodium chloride mg 24 10 citrate buffer in water for injection q.s. ad ml 3 PREPARATION No. 13 a~ a 3-ml vial contains:
freeze-dried active substance ~g 5 to 10 (25,000 BU) ~:
monosialotetrahexosylganglioside (GMl) sodium salts mg 100 glycine mg 45 ~::
b) a 3-ml ampule of solvent contains: :
20 sodium chloride mg 24 citrate buffer in water :~
for injection q.s~ ad ml 3 , ~
PREPARATION No. 14 25 a) a 3-ml vial contains:
freeze-dried active substance ~g S to 10 (25,000 BU)3-sn-phosphatidylserine mg 50 lecithin mg 15 mannitol mg 100 b) a 4 ml am~ule of solve.nt contains:
mannitol mg 60 in water for injection q.s. ad ml 4 WO93/10233 2 12 3 2 3 4 PCT/EPg2/02586 - 3~ - :
?REPAP..~.-IO~ c. 1-2j a 3-~i via' conlains:
freeze-ariea aclive substance ~g 5 to l0 (25,000 BU) mannitol mg 60 b) a 3-ml am~ule of solvent contains:
sodium chloride mg 2 citrate buffer in water for injection q.s. ad ml 3 B) EXAMPLES FOR SUBCUTANEOUS INJECTION

P~EPARATION No. 16 a) a 2-ml vial contains:
freeze-dried active substance ~g 2.5 to 5 ~12,500 BU) mannitol mg 30 - b) a 2-ml am~ule of solvent contains:
sodium chloride mg 16 20 citrate buffer in water for injection q.s. ad ml 2 C) EXAMPLES OF SUPPOSITORIES FOR RECTAL A~MINISTRATION

PREPARATION No~ 17 , active subs~ance ~g 5 to l0 (25,000 BU) cocoa butter mg 2.5 PREPARATION No. lo active substance ~g 5 to l0 (25,000 BU) carbowax lS40 g 1.75 carbowax 6000 g 0.75 PREPARATION No. l9 active su~stance ~g 5 to l0 t25,000 BU) Tween 61 g 2.125 lanolin g 0.25 WO 93/10233 2 12 3 2 3 4 PCI`/EP92/02586 ~ 39 --r ~ EP.`.~AT I O ~ . ^ 0 2CIi~/e suDs~ance ~g 5 to 10 (~5,000 B~) alycerin g 1.5 ~iater 9 0.25 5 gelatin g 0.25 PR~PARATION No. 21 Cream containing a partial ester of hyaluronic acid with benzyl alcohol, wherein 100 gr contain:

active substance ~g 10 to 20 (50,000 BU) ~:
- partial ester of hyaluronic acid with benzyl alcohol gr 0,2 polyethylene glycol monostearate 400 gr 10.000 cetiol V gr 5.000 lanette SX gr 2. oao methyl paraoxybenzoate gr 0.075 propyl paraoxybenzoate gr 0.050 20 sodium dehydroacetate gr 0.100 glycerin F.U. gr 1.500 sorbitol 70 gr 1.500 test cream gr 0~050 wa~er for injection q.s. ad gr 100.000 PREPARATIO~J No. ~2 Cream containing a total ester of hyaluronic acid with benzyl alcohol, wherein 100 gr contain:
active substance ~g10 to 20 (50,000 BU) 30 total ester o~ hyaluronic acid with benzyl alcohol gr 0.
polyethylene qlycol - monostearate 400 gr10.000 cetiol V gr 5.000 35 lanette SX gr 2.000 methyl paraoxybenzoate gr 0.07~
propyl paraoxybenzoate gr 0.050 ~1~323 i WO93t10233 PCT~EP92/02586 u.n _eh~ oace_c~.- 5~ 3.100 ce~ . gr '.500 sc~bilcl ,o gr i.500 ~es~ c-eam gr 0.050 _ -.. ater for injection q.s. ad gr lOo.000 The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims..

Claims (40)

CLAIMS:

1. A nucleic acid sequence, comprising sequence encoding a truncated form of human ciliary neuronotrophic factor, CNTF, lacking at least one amino acid at the amino terminal end of wild-type human CNTF, lacking at least one amino acid at the carboxy terminal end of wild-type human CNTF, or lacking at least one amino acid at both the amino terminal and carboxy terminal ends of wild-type human CNTF.

2. The nucleic acid sequence according to claim 1, comprising a sequence encoding a polypeptide having biological activity functionally equivalent to the amino acid sequence shown in Figure 4.

3. The nucleic acid sequence according to claim 1, comprising a sequence encoding a polypeptide having biological activity functionally equivalent to the amino acid sequence shown in Figure 6.

4. The nucleic acid sequence according to claim 1, comprising a sequence encoding a polypeptide having biological activity functionally equivalent to the amino acid sequence shown in Figure 8.

5. A nucleic acid sequence, comprising a sequence encoding a truncated, mutein form of human ciliary neuronotrophic factor, CNTF, lacking at least one amino acid at the amino terminal end of human CNTF, and containing at least one amino acid substitution within said CNTF.

6. A nucleic acid sequence according to claim 5, comprising a sequence encoding a polypeptide having biological activity functionally equivalent to the amino acid sequence of D14Ser17CNTF.

7. The nucleic acid sequence according to claim 2, comprising the nucleic acid sequence as shown in Figure 3, or variants thereof in accordance with the degeneracy or the genetic code.

8. The nucleic acid sequence according to claim 3, comprising the nucleic acid sequence shown in Figure 5, or variants thereof in accordance with the degeneracy of the genetic code.

9. The nucleic acid sequence according to claim 4, comprising the nucleic acid sequence shown in Figure 7, or variants thereof in accordance with the degeneracy of the genetic code.

10. The nucleic acid sequence according to claim 6, comprising the nucleic acid sequence encoding D14Ser17CNTF, or variants thereof in accordance with the degeneracy of the genetic code.

11. A truncated form of human ciliary neuronotrophic factor, comprising the amino acid sequence shown in Figure 4, or an amino acid sequence having functionally equivalent biological activity.

12. A truncated form of human ciliary neuronotrophic factor, comprising the amino acid sequence shown in Figure 6, or an amino acid sequence having functionally equivalent biological activity.

13. A truncated form of human ciliary neuronotrophic factor, comprising the amino acid sequence shown in Figure 8, or an amino acid sequence having functionally equivalent biological activity.

14. A truncated, mutein form of human ciliary neuronotrophic factor, comprising the amino acid sequence of D14Ser17CNTF, or an amino acid sequence having functionally equivalent biological activity.

15. A recombinant expression vector, comprising a nucleic acid sequence encoding a truncated form of human ciliary neuronotrophic factor, CNTF, lacking at least one amino acid at the amino terminal end of human CNTF, lacking at least one amino acid at the carboxy terminal end of human CNTF, or lacking at least one amino acid at both the amino terminal and carboxy terminal ends of human CNTF, wherein said vector is capable of expressing said truncated form of human ciliary neuronotrophic facter in a transformed prokaryotic or eukaryotic cell.

16. The recombinant expression vector according to claim 15, wherein said nucleic acid sequence has a nucleotide sequence as defined in any one of claims 2, 3, or 4.

17. The recombinant expression vector according to claim 15, wherein said nucleic acid sequence has the nucleotide sequence as shown in any one of Figures 3, 5, or 7.

18. A recombinant expression vector, comprising a nucleic acid sequence encoding a truncated, mutein form of human ciliary neuronotrophic factor, CNTF, lacking at least one amino acid at the amino terminal end of human CNTF, and containing at least one amino acid substitution within said CNTF, wherein said vector is capable of expressing said truncated, mutein form of human ciliary neuronotrophic factor in a transformed prokaryotic or eukaryotic cell.

19. The recombinant expression vector according to claim 18, wherein said nucleic acid sequence encodes D14Ser17CNTF.

20. The recombinant expression vector according to claim 18, wherein said vector is CNTF(His)6.DELTA.14Ser17CNTF.

21. The recombinant expression vector according to claim 18, wherein said vector is .DELTA.14Ser17CNTF.

22. A prokaryotic or eukaryotic cell transformed with the vector according to claim 15.

23. A prokaryotic or eukaryotic cell transformed with the vector according to claim 18.

24. The cell according to claim 22, wherein said cell is E. coli, a member of the genus Bacillus, a member of the genus Pseudomonas, a yeast, or a mammalian cell.

25. The cell according to claim 23, wherein said cell is E. coli, a member of the genus Bacillus, a member of the genus Pseudomonas, a yeast, or 2 mammalian cell.

26. A pharmaceutical composition, comprising an effective nerve treatment amount of human ciliary neuronotrophic factor according to any one of claims 11, 12, or 13, and a pharmaceutically acceptable carrier or diluent.

27. A pharmaceutical composition, comprising an effective nerve treatment amount of human ciliary neuronotrophic factor according to claim 14, and a pharmaceutically acceptable carrier or diluent.

28. A pharmaceutical composition according to claim 26 or 27, further comprising a natural ganglioside, or a derivative, or a semisynthetic analogue, or a salt of such ganglioside.

29. A pharmaceutical composition according to claim 26 or 27, further comprising a natural polysaccharide, or a derivative, or a semisynthetic analogue of such polysaccharide.

30. The pharmaceutical composition according to claim 29, wherein said polysaccharide is hyaluronic acid.

31. A method f or the treatment of nervous disorders by maintaining, preventing loss or recovering nervous function, comprising administering to a patient an effective amount of human ciliary neuronotrophic factor according to any one of claims 11, 12, or 13.

32. A method for the treatment of nervous disorders by maintaining, preventing loss or recovering nervous function, comprising administering to a patient an effective amount of human ciliary neuronotrophic factor according to claim 14.

33. A method for the treatment of nervous disorders by maintaining, preventing loss or recovering nervous function, comprising administering to a patient an effective amount of a pharmaceutical composition according to claim 28.

34. A method for the treatment of nervous disorders by maintaining, preventing loss or recovering nervous function, comprising administering to a patient an effective amount of a pharmaceutical composition according to claim 29.

35. A method for the treatment of neuropathological conditions caused by aging of the nervous system or diseases affecting the immune system, comprising administering to a patient an effective amount of human ciliary neuronotropic factor according to any one of claims 11, 12, or 13.

36. A method for the treatment of neuropathological conditions caused by aging of the nervous system or diseases affecting the immune system, comprising administering to a patient an effective amount of human ciliary neuronotropic factor according to claim 14.

37. A method for the treatment of neuropathological conditions caused by aging of the nervous system or diseases affecting the immune system, comprising administering to a patient an effective amount of a pharmaceutical composition according to claim 28.

38. A method for the treatment of neuropathological conditions caused by aging of the nervous system or diseases affecting the immune system, comprising administering to a patient an effective amount of a pharmaceutical composition according to claim 29.

39. A method for producing a mutein form of human ciliary neuronotropic factor, CNTF, wherein said mutein comprises a form of CNTF lacking at least one amino acid in the amino terminal region of human CNTF and containing at least one amino acid substitution within the remainder of said CNTF, comprising the steps of:
(a) expressing in a host cell a nucleic acid sequence comprising a first nucleic acid sequence encoding a form of human CNTF lacking at least one codon encoding an amino acid present in the amino terminal region of wild-type human CNTF and containing at least one codon modified to encode an amino acid not present in wild-type human CNTF in the remainder of said CNTF, wherein said first nucleic acid sequence is fused to a second nucleic acid sequence comprising a sequence encoding at least six histidine residues and a chemical or enzymatic cleavage site;
(b) producing an extract of said host cell;
(c) purifying said extract by immobilized metal affinity chromatography, IMAC;
(d) recovering the recombinant mutein CNTF;
(e) incubating said CNTF of step (d) with a chemical or enzymatic cleaving agent;
(f) purifying the reaction products produced in step (e) by immobilized metal affinity chromotography, IMAC; and (g) recovering said mutein form of CNTF.

40. The method according to claim 39, wherein said mutein is D14Ser17CNTF.