EP0668911A1

EP0668911A1 - Synthesis and purification of truncated and mutein forms of human ciliary neuronotrophic factor

Info

Publication number: EP0668911A1
Application number: EP92923337A
Authority: EP
Inventors: Lanfranco Callegaro; Alessandro Negro
Original assignee: Fidia SpA
Current assignee: Fidia SpA
Priority date: 1991-11-11
Filing date: 1992-11-11
Publication date: 1995-08-30
Also published as: JPH07503128A; WO1993010233A1; CA2123234A1; AU2925392A

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 MUTEIN FORMS OF HUMAN CILIARY NEURONOTROPHIC FACTOR

BACKGROUND OF THE INVENTION

Field of the Invention

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 onomeric forms of these proteins, obtained by recombinant DNA techniques. Via the present invention, it is possible to obtain human CNTF polypeptides with a smaller number of amino acids than the corresponding natural form of CNTF. These new forms of CNTF exhibit different chemical and chemical-physical characteristics from those of natural CNTF, and are useful in a variety of pharmaceutical applications.

Description of Related Art

A. THE CILIARY NEURONOTROPHIC FACTOR

The ciliary ganglion (CG) contains two populations of neurons, ciliary and choroid, both of them cholinergic. CG neurons innervate the intrinsic muscle structures of the eye in the choroid, the ciliary body and the iris. During embryonic development in chicks, about half the CG neurons die between days E8 and 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 hypothesis evolved that the innervation territories contain trophic factors for their own specific neurons. Extracts from various embryonic chick tissues at day

E8 exhibited a trophic activity of their own which differed from that of dissociated neurons from chick embryo ciliary ganglia at the same stage of development (Adler et al. (1979) Science 204:1434-1436) .

This trophic activity was subsequently associated with ciliary neuronotrophic factor (CNTF) (Barbin et al. (1984) . Neurochem. 43:1468-1478).

A third of all the CNTF trophic 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 electrophoresis

(SDS-PAGE) showed it to have a molecular weight of about

21Kd and a negative net charge. The trophic activity exhibited by the purified protein in chick CG neurons at day E8, in chick DRG neurons at day E10, and sympathetic neurons at day Ell is on the order of 10"^u 10"^I2M, the same as another neuronotrophic factor, Nerve Growth Factor (NGF) , purified from male mouse submandibular glands, with regard to its neuronal DRG and sympathetic targets.

' Like NGF, CTNF supports the growth of chromaffin 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 chick 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 survival and development 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. (1990) Soc. Neurosci. Abstr. 16:484; Magal et al. (1991) Devel. Brain Res. 63:141) and hippocampal neurons (Ip et al. (1991) J. Neurosci. 11:3124) . It has also been shown to influence _.in vitro the differentiation of the progenitor glial cells known as 0-2A which differentiate in oligodendrocytes 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, 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. RECOMBINANT DNA TECHNOLOGY

Conventional techniques for isolating biologically 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 enable study of their structures, functions and, above all, applications. Therefore, in order to obtain sufficient quantities of proteins, recombinant DNA techniques can be used to clone genes encoding these proteins in expression vectors.

It is possible, by recombinant DNA 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 proteins of interest. Various reactions are employed, such as cleaving wit restriction enzymes, ligating the fragments thus obtained with ligase, the chemical synthesis of oligonucleotides to be assembled, and other methodologies devised in various laboratories working in this field. These techniques are summarized in Sambrook et al. (1989) Molecular Cloning, 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 essential information. These elements include, for example, a replication origin, a selective marker for antibiotics, an expression promoter, transcription 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 can 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, promoters 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 DNAs of various origin (eukaryotic, bacterial, viral, etc.) in association with different gene fragments linked together to form a hybrid molecule. The transcriptional activity of a gene depends on there being suitable distances between the regulatory and coding sequences. Conventional tecnniques or obtaining genes siruated in close proximity to regulatory sequences depend on there being suitable restriction endonuclease sites which facilitate their cloning. If no compatible sites are nearby, but only different sites, it is possible to join the segments by synthesis of an oligonucleotide linker containing such a restriction site. This system is very limiting to the molecular biologist. An alternative strategy is to use the PCR technique (Saiki et al. (1988) Science 239:487-489). By this technique it is possible to amplify a gene segment up to 1 x 10⁶ 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 oligonucleotides with respect to the gene sequence determines the size of the molecule to be produced. These two oligonucleotides are so constructed that there is a restriction site within their sequence which allows subsequent cloning. This restriction site is either naturally present or is constructed ad hoc by degenerating the minimum number of nucleotides. This approach, known as site-directed mutagenesis, allows restriction sites to be constructed in positions previously decided on by the molecular biologist. The construction of sites compatible with other gene segments not only facilitates cloning, but also makes it possible to specifically join different gene segments. This technique can be defined as cloning by 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 be obtained with characteristics which are the same as or different from those of the coded protein of the wild-type gene. It is thus possible to obtain truncated forms of polypeptides with fewer amino acids than the natural protein, and deleted forms of polypeptides, lacking certain amino acid sequences which are present in wild-type protein sequences. These nex-/ polypeptide forms can possess chemical, chemical- physical, and biological characteristics that are very different from those of the natural protein. In order to obtain different 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 make it possible to block the natural protein receptor.

SUMMARY OF THE 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 vivo, both singly and in combination with other molecules or their derivatives, or with manufactured biomaterials, for therapeutic purposes.

Another object of the present invention is to provide pharmaceutical preparations containing as active substances one or more of the new complexes between one ^'or more isoforms of the CNTF growth factor and a natural ganglioside 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 encompasses the therapeutic 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 derivatives, with the isoforms and muteins of growth factor CNTF for the aforesaid indications. Daily dosages for humans by subcutaneous, intramuscular or intracerebral injection can vary between 0.05 mg and 5 mg of active substance per kg of body weight.

A further object of the present invention is to provide a process for producing a human CNTF mutein which possesses the advantage of facilitating the homogeneous purification of its monomers. This process includes the fusion of the mutein to at least 6 histidine residues, and the presence of an enzymatic or chemical cleavage site in the polypeptide chain. The presence of these 6 histidine 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 amino terminal end have been removed, and the only cysteine of CNTF has been replaced with serine so that the recombinant protein cannot become oxidized during purification, thus maintaining its monomeric form. This mutein maintains its biological activity after purification.

Further scope of the applicability 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 the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The acove and other objects, features, and advantages of the present invention will be better understood from the following detailed descriptions taken in conjunction with the accompanying drawings, all of which are given by way of illustration 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 1.

Figure 3 shows the nucleic acid sequence encoding a form of human CNTF lacking the first 14 amino acids at the amino terminal end of the molecule.

Figure 4 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 encoded by the nucleic acid sequence of Figure 3.

Figure 5 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 acid sequence and other data of the form of human CNTF lacking the last 26 amino acids at the carboxy terminal end of the molecule encoded by the nucleic acid sequence of Figure 5. Figure 7 shows the nucleic acid sequence encoding a form of human CNTF lacking both 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 polypeptide forms of human CNTF disclosed herein. Figure 10 shows tπe truncated forms of human CNTF reported in Examples 1-2, and the truncated forms with deleted sequences described in Example 5.

Figure 11 shows the biological activity of truncated polypeptide forms of human CNTF compared to the wild-type human CNTF protein. The data show that the specific activity, calculated as ED50, is at least 5 x 10^'πM for the various forms.

Figure 12 shows the structure of the expression vector CNTF(His)_ήΔl4Serl7CNTF.

Figure 13 shows the structure of the expression vector Δl4Serl7CNTF.

Figure 14 shows the elution profile of the bacterial starting material of Example 13, loaded in buffer A,^' washed with buffers B and C. The recombinant protein elutes in buffer D.

Figure 15 shows the elution profile of CNTF(His)_ήAsnGlyD14Serl7CNTF of Example 13, after enzymatic cleavage with hydroxylamine. Mutein GlyD14Serl7CNTF elutes in buffer C; mutein CNTF(His)_ήAsn elutes in buffer D.

Figure 16 shows the elution profile of mutein His₆D14serl7CNTF of Example 13, after enzymatic cleavage with enterokinase. The recombinant protein elutes in buffer C.

DETAILED 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 discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery. The contents of each cf the references cited in the present Specification are herein incorporated by reference in their entirety.

The present invention generally falls within the field of recombinant DNA technology. It concerns specifically mutated forms (called isoforms) of natural CNTF which differ from the corresponding natural forms due to the presence of one or more deletions in their polypeptide sequences, as well as muteins of CNTF. These isoforms and muteins can retain the biological activity of the corresponding natural protein.

General Recombinant DNA Techniques

The cleavage of DNA with restriction enzymes was effected according to manufacturer's instructions. Generally, 1 μg of plasmid was cut with 1 U of enzyme in 20 μl of reaction solution. Temperatures and incubation times depended on which enzyme was employed, but were usually 1 hr at 37°C. After incubation, the plasmids and gene fragments were purified in all cases via electrophoresis through LMP Agarose (BRL, USA) in 40 mM Tris-HCl, 20 πiM sodium acetate, 1 mM EDTA, and then eluted from the agarose using a GENECLEAN^R kit (BIO 101 Inc., La Jolla, CA, USA).

Ligations were performed using T4 DNA ligase at a concentration of 1 U per 0.5 μg of DNA in a reaction volume of 20 μl at 13°C 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 μg/ml of ampicillin. The recovered cells were grown in LB containing 100 μg/ml of ampicillin, and plasmids were purified, both for the small and larger preparations, with a kit from Quiagen (DIAGEN GmbH, Dϋsseldorf, West Germany) . The cloning vectors were prepared from the bacterial cells by the method recommended by Quiagen. All ciigonucleotides -./ere synthesized in solid phase using a 3S0B DNA Synthesizer (Applied Biosystems, USA) according to the manufacturer's instructions. They were treated at 55°C for 12 hrs in NH-, and then recovered in a speed-vacuum centrifuge. They were resuspended in 2.5 M ammonium acetate and then precipitated with 3 volumes of cold ethanol (-20°C) . They v/ere rewashed with cold 80% ethanol and resuspended in water. The concentration of oligonucleotides was assessed spectrophoto etrically. Amplification was effected on a Perkin Elmer Cetus DNA Thermal Cycler, and the reagents used for amplification were those of the relative DNA™ Amplifier (Perkin Elmer- Cetus) . Briefly, a mixture containing 200 μM of each oligonucleotide was used, 0.5 μM of each of the nucleotides dATP, dTTP, dCTP, dGTP, and 0.1 μg of human DNA and reaction buffer in a total mixture of 100 μl with 0.5 U of TAQ polyroerase, the reaction mixture being then covered with liquid paraffin to prevent evaporation.

The amplification reaction was conducted by setting the instrument at 25 cycles under the following conditions: 1 min. at 95°C denaturation, 45°C alignment, 72°C extension.

Particular modifications were performed on wild-type CNTF gene sequences to obtain genes encoding proteins with partial amino acid sequences and altered sequences compared to the natural form of CNTF, possessing chemical and chemical-physical characteristics which are different from those of the natural protein.

GENES AND PROTEINS FOR

HUMAN CILIARY NEURONOTROPHIC FACTOR

Each of the nucleic acid sequences and polypeptides disclosed herein, or their biologically functional equivalents, can be used in accordance with the present invention. The term "biologically functional equivalents," as used herein, denotes nucleic acid sequences or polypeptides exhibiting the same or similar biological activity as the particular nucleic acid sequences and polypeptides described infra.

For example, the nucleic acid sequences depicted below can be altered by substitutions, additions or deletions that provide for biologically functionally equivalent molecules. Due to the degeneracy of the genetic code, other DNA sequences which encode substantially the same amino acid sequences as depicted below may be used in the 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 within 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 sequence, resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted with another amino acid of similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, 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. Also included within the scope of the present invention are CNTF fragments or derivatives thereof which are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. The examples below exemplify the expression of a biologically active recombinant CNTF isoform or mutein in E. coli . thereby 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 termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further m vitro modification. Any technique for mutagenesis known in the art can be used, including, but not limited to, .in vitro site-directed 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 therein. Preferred vectors are those whose restriction sites have been well documented and which contain the operational elements prererred :r required for transcription of the DNA sequence.

Certain embodiments of the present invention employ vectors which would contain one or more of the DNA sequences described herein. It is preferred that all of these vectors have some or all of the following characteristics: (1) possesses a minimal number 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 marker 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 one Shine-Dalgarno sequence and initiator codon, and at least one termination codon. These "operational elements" may also include one or more of the following: at least one operator, at least one leader sequence for proteins to be exported from mtracellular space, at least one gene for a regulator protein, and any other DNA 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. Regulators

Regulators serve to prevent expression of the DNA sequence in the presence of certain environmental conditions and, in the presence of other environmental conditions, will allow transcription and subsequent expression of the protein coded for by the CNTF DNA sequences. In particular, it is preferred that regulatory 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 to initiation of the expression of the CNTF isoforms or muteins. Expression of the desired protein is induced by addition of a substance to the microbial environment 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 proteins. While the lactose promoter 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 recombinant CNTF isoforms and muteins.

Transcription Terminators The transcription terminators contemplated herein serve to stabilize the vector. In particular, those sequences described by Rosenberg et al. (1979) Ann. Rev.

Genet. 13:319-353 are contemplated for use in the present invention. Non-translated Sequences

It may also be desirable to construct the 3 ' or 5' end of the coding region to allow incorporation of 3 ' or 5' non-translated sequences into the gene transcript. Included among these non-translated sequences are those which stabilize mRNA, as disclosed by Schmeissήer et al. (1984) J. Mol. Biol. 176:39-53.

Ribosome Binding Sites The microbial expression of foreign proteins requires operational elements which include ribosome binding sites. A ribosome binding 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 ribosome binding site is GAGGCGCAAAAA(ATG) .

Leader Sequences and Translational Couplers

Additionally, it is preferred that DNA coding for an appropriate secretory leader (signal) sequence be present at the 5' end of the DNA sequence, as set forth by Watson, M.E. in Nucleic Acids Res. 12:5145-5163, if the protein is to be excreted from the host cytoplasm. The DNA- for the leader sequence must be in a position that allows the production of a fusion protein in which the leader sequence is immediately adjacent to and covalently joined to CNTF, 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, Saccharo yces and strains of Bacillus and Pseudomonas, the appropriate leader sequence will allow transport of the protein through the cell membrane and into the extracellular medium. In this situation, the protein may be purified from extracellular protein. An additional DNA sequence can oe located immediately preceding the DNA sequence which codes for the CNTF isoform. The additional DNA sequence is capable of functioning as a translational coupler, i.e. , it is a DNA sequence that encodes an RNA which serves to position ribosomes immediately adjacent to the ribosome binding site of the inhibitor RNA with which it is contiguous. The translational coupler may be derived using the DNA sequence TAACGAGGCGCAAAAAATGAAAAAGACAGCTATCGCGATCTTGGAGGATGATTAAATG and methods currently known to those of ordinary skill in the art related to translational couplers.

Translation Terminators

The translation terminators contemplated herein serve to stop the translation of mRNA. They may be either natural, as described 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 host microorganism. In one embodiment of the present invention, the gene for ampicillin resistance is included in the vector. In other plasmids, the gene for tetracycline resistance or the gene for chloramphenicol resistance can be included.

Such a drug resistance or other selectable marker facilitates the selection of transformants. Additionally, the presence of such a selectable marker in the cloning vector may be of use in keeping contaminating microorganisms from multiplying in the culture medium. A pure culture of the transformed host microorganisms would be obtained by culturing the microorganisms under conditions which require the induced phenotype for survival. The operational elements discussed herein are routinely selected by those of ordinary skill in the art in light of prior literature and the teachings contained herein. General examples of these operational elements are set forth in B. Lewin (1983) Genes, Wiley & Sons, New York. Various examples of suitable operational elements may be found in the vectors discussed above, and may be gleaned via review of the publications discussing the basic characteristics of the aforementioned vectors. Upon synthesis and isolation of all the necessary and desired component parts, the vector can be assembled by methods generally known to those of ordinary skill in the art. Assembly of such vectors 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 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, due to its size, to be transferred into and replicated and transcribed in an appropriate host cell.

Other Host Microorganisms

Vectors suitable for use in microorganisms other than ______ 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. For expression in Bacillus , prererred vectors snouid include a regulated promoter εucn as the alpha amyiase promoter, the subtilisin promoter, the P-43 promoter, or the spac-126 promoter. Useful transcription terminators include rrn and rrn BT.T. Transcriptional start sites and leader peptides can be chosen from among those from B.amyloliguefaciens neutral protease, B.amyloliquefaciens alpha-amylase, and B. subtilis subtilisin. Useful antibiotic markers are Kan^r and Cam^r. Ribosome binding sites can be obtained from the B.amyloliquefaciens neutral protease and B.a yloliguefaciens alpha-amylase genes. A preferred expression system in hosts of the genus Bacillus involves the use of plasmid pUBHO as the cloning vehicle.

For expression in Pseudomonas, promoters can be selected from Trp, Lac, and Tac. Useful transcriptional start sites and leader peptides can be obtained from the phoεpholipase C and exotoxin A genes. Useful antibiotic markers are those for sulfonamides and streptomycins. A useful ribosome binding site can be obtained from the Trp promoter of E_j_ coli. Particularly preferred vectors 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 5. Transcription terminators can be chosen from among Cyc, Una, Alpha Factor, and Sac 2. Transcriptional start sites and leader peptides can be obtained from the invertase, acid phosphatase, and Alpha factor 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 mutein CNTFs should have a sequence efficient at binding ribosomes. Such a sequence is described by Kozak in Nucleic Acids Research (1987) 15:8125-8132. The CNTF-encoding fragment can be inserted into an expression vector containing a transcriptional promoter and a transcriptional enhancer as described by Guarente in Cell (1988) 52:303-305 and Kadonaga et al. (1987) Cell 51:1079-1090. A regulatable promoter as in the Pharmacia plasmid pMSG can be used, if necessary or desired. The vector should also possess a complete polyadenylation signal as described by Ausubel et al. (1987) in Current Protocols in Molecular Biology, Wiley, so that mRNA transcribed from the vector is properly processed. Finally, the vector may also contain the replication origin and at least one antibiotic resistance marker from a plasmid such as pBR322, to allow replication and selection in |J_ coli. In order to select a stable cell line that produces CNTF as described 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 described by Ausubel et al. , supra. Alternatively, 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 described by Ausubel et al. , supra, is the preferred method.

Preferred vectors include, for example, a PSVT7 eukaryotic expression plasmid.

EXAMPLE 1

To obtain a form of CNTF lacking the first 14 amino acids, two oligonucleotides 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 Ndel restriction site was added to facilitate subsequent cloning, and a methionine codon was added in order to initiate transcription of the gene. By using the aforementioned oligonucieotiάG together with ah oligonucleotide (B) with the sequence 5 'TTGTCGACTCACATTTTCTTGTTGTTAGCAATAT 3' , hich inversely maps at the end of the sequence encoding CNTF, it is possible, using the plasmid pT7.7hCNTF (Negro et al. (1991) J. Neurosci. Res. 29:270) , to amplify the sequence shown in Figure 3.

This sequence was cut with Ndel, and Sail, and 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 first 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.

EXAMPLE 2 To obtain a form of CNTF lacking the carboxy terminal

26 amino acids, two oligonucleotides were synthesized. The first (C) had the sequence 5 'TTCATAGGCTTTTACTGAAGCATTCA 3', and starts mapping from the beginning of the sequence encoding the CNTF gene. The second oligonucleotide (D) had the sequence 5 'TTGTCGACTCAATGGATGGACCTTACTGTCCAC 3', and maps inversely at the end of the CNTF gene. Moreover, a stop codon was 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 Sail 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 Ndel and Sail, 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 6. This sequence lacks the last 25 amino acids cr CNTF, so that the polypeptide has 174 amino acids. The resulting molecular weight is 20,007 KDa. The pi of this new form is 5.67.

EXAMPLE 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 1 and (D) in Example 2, and the plasmid pT7.7hCNTF, were used to amplify the sequence shown in Figure 7.

This sequence was cut with Ndel and Sail, 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 weight of 18,476 KDa, and an isoelectric point of 5.14.

EXAMPLE 4 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.

EXAMPLE 5 From the plasmid pT7.7hCNTF it is possible 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 segment 137-150 deleted with respect to the wild-type human CNTF polypeptide sequence. The following ciigonucleoti es both contain an SphI restriction site; such sites are not present in the plasmid:

AGCATGCGGATCTTGTATTCCAGGAG

TTGCATGCTCTTTGAGAAGAAGCTGT

These two oligonucleotides are employed in the PCR technique as described previously. The purified DNA is cut with SphI, and then ligated with T4 DNA ligase.

By this method it is possible to obtain linear segments linked via a ligase. The distance in nucleotides between the two selected oligonucleotides determines the extent of deletion in the mutant.

Figure 10 illustrates all the truncated forms of human CNTF reported in Examples 1 to 3 , and the forms with deleted sequences produced as described in Example 5.

EXAMPLE 6

BACTERIAL GROWTH AND PROPAGATION

A colony of E\_ coli BL21(D3) containing the mutated plasmid pT77RCNTF (Negro et al. (1991) J. Neurosci. Res. 29:251) is inoculated into 5 ml of LB medium containing 200 μg/ml of ampicillin. As previously described, each cell line expresses the polypeptide sequence encoded by the gene sequence inserted in the expression vector. All constructions were performed in J≡J. coli HB101.

To obtain protein expression, the plasmids containing the truncated or deleted forms of CNTF were transformed into different E_j_ coli lines, including BL21(D3) .

Cells were grown overnight at 30°C. An aliquot was diluted 1/100 in M9 medium containing 200 μg/ml of ampicillin. The cells were grown to an absorbancy of 0.8 OD at 590 nm. At this point, isopropyl thiogalactoside (IPTG) was added, 4 M final concentration. The cells were grown for another 3 hours, centrifuged, and resuspended in Tris- HC1, pH 8.0, 10 mM EDTA, after which they were ready for subsequent extractions.

EXAMPLE 7

PURIFICATION PROCEDURE

The pellet obtained after centrifugation was washed at least 3 times with 50 mM Tris, pH 7.4, and 50 mM NaCl (C) . After each washing, the pellet was recovered by centrifuging at 600 rpm for 10 minutes.

The final pellet was resuspended in buffer and passed at least twice through a Montain Gaulin at 800 psi. The resulting material was centrifuged again at 600 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 15 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 proteins.

Under these conditions, the recombinant proteins were eluted at NaCl concentrations of 10 mM to 1.0 M. The material obtained was dialyzed against 50 mM ammonium acetate and loaded onto an inverse-phase column.

The column employed was a Vydac C18 7 μm (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 recombinant protein eluted from the column as a single peak. EXAMPLE 3

DETERMINATION OF BIOLOGICAL ACTIVITY

The activity of the polypeptides obtained as described in Examples 1, 2 and 3 was assessed by their capacity to maintain chick embryo dorsal root neuronal cells at day E10 in culture. These cells were removed from chick embryos at day E10, dissociated with trypsin, and enriched by subsequent preplating steps. The neurons were then seeded in 96-well tissue-culture dishes, treated with polyornithine (100 μg/ml in 15 mM borate buffer, pH 8.4) and laminin (10 μg/ml) . The neurons were seeded at a concentration of 4,000 neurons per well, the culture medium was Dulbecco's minimal essential medium (DMEM) with 100 μg/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. Brain Res. 24:39-46) .

Figure 11 shows examples of the biological activity 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^"UM for the various forms.

EXAMPLE 9

EXPRESSION VECTOR 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 oligonucleotide was constructed:

BolII 5 ^■CCAGATCTGGCACCATCATCATCACCATAACGGCGACCTCAGTAGCCGCTCTAT3 ' IleTrpHisHisHisHiεHisHisAsnGlyAspLeuSerSerArgSerlle This oligonuc_leotide contains a Bglll site m tr.e Ξ' region. Six His codons are present, followed by part of t_he CNTF sequence starting from 15th codon. Moreover, the ₁₇th codon (TGT) encoding cysteine was replaced with (TCT) , encoding serine. This modification was necessary because in the presence of cysteine, two molecules of CNTF can combine to form a dimer, thus lowering the biological activity. This oligonucleotide, together with the oligonucleotide Sail of the following sequence: 5-AGGTCGACTACATTTTCTGTTGTTAG 3', was used for PCR. The amplified product was cut with Bglll and Sail, and cloned into the plasmid pT7.7hCNTF (Negro et al. (1991) J_^ Neurosci. Res. 29:270) between the BamHI and Sail sites. BamHI and Bglll are compatible restriction sites. Figure 12 represents the expression vector. It includes resistance to ampicillin (AMP^r) , the T7 promoter, the restriction sites N (Ndel) , Nh (Nhel) , B (BamHI) , Bg (Bglll) and S (Sail) . The position of the histidines (M) and cleavage site for hydroxylamine (H) are also indicated.

EXAMPLE 10

Fusion Proteins

The fusion protein containing the chemical .cleavage site H is shown below: hydroxylamine

CNTF(l-186)His-His-His-His-Kis-His-Asn 4--Gly-Asp- eu-Ser-CNTF(18-200)

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. EXAMPLE 11

EXPRESSION VECTOR FOR THE POLYPEPTIDE DELTA14Serl7CNTF.

On the basis of the sequence of human CNTF (Negro et al. (1991) J. Neurosci. Res. 29:251) , the following oligonucleotide was constructed:

5 'TCTAGATCTCTCTAGCCGCTCTAT 3 •

AspLeuSerSerArgSerlle

In this oligonucleotide, a Bglll 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 Sail. previously described, was used for PCR. The amplified product was cut with Bglll and Sail and cloned in the expression plasmid pRSETB (Invitrogen) between the restriction sites BamHI and Xhol, which are compatible with Bglll and Sail, respectively.

Figure 13 shows the expression vector. It illustrates the resistance to ampicillin (AMP^r) , the T7 promoter, the restriction sites N (Ndel) , B (BamHI) , Bg (Bglll) , S (Sail) , and X (Xhol) . Moreover, the 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-Asp₁₅-LeUι₆-Ser₁₇-

As can be seen, the transcribed protein is a fusion protein containing six histidines in the amino terminal region, followed by a cleavage site for enterokinase between Lys and Asp_[ , which facilitates the cleavage of the sequence Asp-Asp-Asp-Asp-Lys-Asp₁ after the lysine.

EXAMPLE 12

BACTERIAL GROWTH

Bacterial growth was as described in Example 6,

EXAMPLE 13

PURIFICATION PROCEDURE

The pellet obtained after centrifugation was washed at least 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 buffer and passed at least twice through a Montain Gaulin at 800psi. The resulting material was again centrifuged at 600 rpm for 10 minutes and resuspended in buffer A (6M guanidine, 0.1M NaH₂P0₄, lOmM Tris, pH 8) . It was centrifuged again as before, and the supernatant was loaded onto an IMAC-NTA chro atographic column (Quiagen) 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 loaded 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, 0.1M NaH₂P0₄, lOmM Tris, pH 8) , then washed again with 40ml of buffer C (8M urea, 0.1M NaH,P0₄ lOmM Tris, pH 6.3) . The recombinant muteins were eluted with buffer D (8M urea, 0.1M NaH₂P0₄, 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 polypeptide was incubated for 12 hrs at 37°C, during which time the hydroxylamine cleaves the polypeptide into two portions, CNTF(His)6Asn and GlyD14Serl7CNTF, respectively. The mutein His6Dl4Serl7CNTF, on the other hand, was dialyzed against 50mM CaCl , 50mM Tris-HCl, pH S, and digested with enterokinase (Boehringer) using an enzyme substrate ratio of 1:500 at 37°C for 12 hrs. During this time, the polypeptide divides into two portions: His6 and D14Serl7CNTF. The muteins thus digested were dialyzed against buffer A and loaded onto the column as described previously.

The final product of the polypeptide CNTF(His)₆AsnGlyD14Serl7CNTF is represented by the following muteins: GlyD14Serl7CNTF, which elutes in buffer C, and CNTF(His)_fiAsn, which elutes in buffer D.

The final product of the polypeptide His6D14Serl7CNTF is the mutein D14Serl7CNTF, which elutes in buffer C. The two recombinant muteins obtained by this method are purified as indicated in the following flow schemes:

Mutein: CNTFfHis) 6AsnGlvD14Serl7CNTF

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 buffer D (Mutein

CNTF(His)₆AsnGlyD14Serl7CNTF)

Dialysis

Centrifugation

Pellet Solubilization with 6M guanidine, 2M hydroxylamine, 37°C, 12 hrs

Dialysis with buffer A

Loading of IMAC-NTA column in buffer A

Washing with buffer B Washing with buffer C (GlyD14Serl7CNTF)

Washing with buffer D (CNTF(His)₆Asn) Mutein: fHis)<D14Serl7CNTF Bacterial pellet Solubilization in buffer A Loading of IMAC-NTA column Washing with buffer B (histidine-free proteins)

Washing with buffer C (Bacterial proteins with few histidines) Washing with buffer D (His)₆D14Serl7CNTF Dialysis Digestion with enterokinase, 1:500, at 37°C for

12hrs Dialysis with buffer A Loading of IMAC-NTA column Washing with buffer B Washing with buffer C (D14Serl7CNTF)

Washing with buffer D ((His)₆)

Figure 14 shows the elution profile of the bacterial starting material, loaded in buffer A, washed with buffers B and C. The recombinant protein elutes in buffer D.

Figure 15 shows the elution profile of CNTF(His)₆AsnGlyD14Ser17CNTF, after enzymatic cleavage with hydroxylamine. Mutein GlyD14Serl7CNTF elutes in buffer C, mutein CNTF(His)₆Asn elutes in buffer D. Figure 16 shows the elution profile of mutein

His₆D14Serl7CNTF, after enzymatic cleavage with enterokinase. The recombinant 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 30°C, solubilization of the muteins in a phosphate buffer, pH 7.4, and loading onto the column under non-denaturating conditions. The protein elutes at the same pH values as indicated for denaturating conditions. EXAMPLE 14

DETERMINATION OF AMINO ACID SEQUENCES

The amino terminal sequences of the recombinant muteins were determined by Edman degradation using an Applied Biosystem automatic protein sequencer, Model 477A. The phenylthiohydantoins derived from the amino acids were instru entally determined. The sequence of the first 10 amino acids starting from the N-terminal amino acid for GlyD14Serl7CNTF was: Gly-Asp-Leu-Ser-Ser-Arg-Ser-Ile-Trp- Leu-Ala-Arg, and for D14Serl7CNTF was: Asp-Leu-Ser-Ser-Arg- Ser-Ile-Trp-Leu-Ala-Arg-Lys. This sequence correlates with that of CNTF (Figure 2) when the first 14 amino terminal amino acids in -GlyD14Serl7CNTF are absent, a glycine amino terminal amino acid is present, and the cysteine in position 17 is substituted with serine. In the case of D14Serl7, the first 14 amino acids are deleted and cysteine in position 17 is substituted with serine.

EXAMPLE 15

DETERMINATION OF BIOLOGICAL ACTIVITY

The biological activity of the recombinant mutein was assessed on the basis of the mutein's efficacy in maintaining in culture embryonic neuron cells at day ElO, extracted from chick dorsal ganglia. The cells were removed from chick embryos at day ElO, and dissociated and enriched via a series of pre-plating steps. The neurons were then seeded in 96-well dishes for tissue culture, treated with polyornithine (100 μg/ml in borate buffer, pH 8.4) and laminin 10 μg/ml) . The neurons were seeded at a concentration of 4,000 neurons per well. The culture medium was Dulbecco minimal essential medium (DMEM) with 100 μg/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) . The single muteins had the following specific activities, calculated as ED50:

CNTF(His) 6AsnGlyD14Serl7CNTF 20ng/ml GlyD14Serl7CNTF 0.25ng/ml

CNTF(His) 6Asn 0.5ng/ml

(His) 6D14Serl7CNTF 0.4ng/ml

D14Serl7CNTF 0.12ng/ml

PHARMACEUTICAL COMPOSITIONS

The formulation of pharmaceutical compositions containing solutions of the present human CNTF molecules with and without 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 Remington's Pharmaceutical Sciences (1985) Mack Publishing Company, Easton, Pa. , USA. Such vehicles include injectable "depot formulations." On this basis, the pharmaceutical formulation includes, albeit not exclusively, solutions of CNTF growth factor or its freeze-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-dried preparations, support excipients can be used, such as, but not exclusively, mannitol or gl cine, 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 of the present CNTF molecules in isotonic solutions of the desired volume and include. but not exclusively, the use of physiological buffered solutions with phosphate or citrate at suitable concentrations in order to consistently obtain isotonic pharmaceutical preparations with the desired pH, for example neutral pH.

Pharmaceutical preparations can be employed for oral, topical, rectal, 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, icrospheres, and sponges. For parenteral and intracerebral uses, those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral injection 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 be used; for use in association with polymers, those preparations in the form of membranes, sponges, non-woven products, tubelets, threads, microspheres, nanospheres, and nanocapsules can be used.

The preparations of the present invention 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 route of administration. The pharmaceutical preparations also include, but are not limited to, suppositories for rectal administration with lipophilic excipients, for example, water soluble excipients, self-emulsifying excipients such as glycogelatin, or others. In these preparations, the CNTF 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.

EXAMPLE 16

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

PREPARATION No. 3 - a 2-ml vial contains: active substance μg 0.5 to 1 (2,500 BU) sodium chloride mg 16 ganglioside sodium salt mg 100 citrate buffer pH = 7 ml 2 in water for injection q.s. PREPARATION No. 4 - a 2-ml vial contains: active substance μg 5 to 10 (25,000 BU) sodium chloride mg 16 ganglioside sodium salt mg 50 citrate buffer pH = 7 ml 2 in water for injection q.s.

PREPARATION No. 5 - a 2-ml vial contains: active substance μg 0.5 to 1 (2,500 BU) sodium chloride mg 16 monosialotetrahexosylganglioside

(GM1) sodium salts mg 100 citrate buffer pH = 7 ml 2 in water for injection q.s.

PREPARATION No. 6 - a 2-ml vial contains: active substance μg 5 to 10 (25,000 BU) sodium chloride mg 16 monosialotetrahexosylganglioside (GM1) sodium salts mg 100 citrate buffer pH = 7 ml 2 in water for injection q.s.

PREPARATION No. 7 a) a 2-ml ampule contains: freeze-dried active substance μg 2 to 4 (10,000 BU) glycine mg 30

b) a 2-ml ampule of solvent contains: sodium chloride mg 16 citrate buffer in water for injection q.s. ad ml 2

PREPARATION No. 8 ^a) a 2-ml vial contains: freeze-dried active substance μg 2 to 4 (10,000 BU) mannitol mg 40 b) a 2-ml ampule of solvent contains: sodium chloride mg 16 citrate buffer in water for injection q.s. ad ml 2

PREPARATION No. 9 a) a 3-ml vial contains: freeze-dried active substance μg 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 μg 5 to 10 (25,000 BU) ganglioside sodium salts mg 100 glycine mg 45

PREPARATION No. 11 a) a 3-ml vial contains: freeze-dried active substance μg 5 to 10 (25,000 BU) ganglioside sodium salts mg 50 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. 12 a) a 3-ml vial contains: freeze-dried active substance μg 0.5 to 1 (2,500 BU) monosialotetrahexosylganglioside (GM1) sodium salts mg 100 glycine mg 45

PREPARATION No. 13 a) a 3-ml vial contains: freeze-dried active substance μg 5 to 10 (25,000 BU) monosialotetrahexosylganglioside (GM1) sodium salts mg 100 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. 14 a) a 3-ml vial contains: freeze-dried active substance μg 5 to 10 (25,000 BU)

3-sn-phosphatidylserine mg 50 lecithin mg 15 mannitol mg 100

b) a 4-ml ampule of solvent contains: mannitol mg 60 in water for injection q.s. ad ml 4 PREPARATION No. 15 a) a 3-mJ vial contains: freeze-dried active substance μg 5 to 10 (25,000 BU) mannitol mg 60

B) EXAMPLES FOR SUBCUTANEOUS INJECTION

PREPARATION No. 16 a) a 2-ml vial contains: freeze-dried active substance μg 2.5 to 5 (12,500 BU) mannitol mg 30

C) EXAMPLES OF SUPPOSITORIES FOR RECTAL ADMINISTRATION

PREPARATION No. 17 active substance μg 5 to 10 (25,000 BU) cocoa butter mg 2.5

PREPARATION No. 18 active substance μg 5 to 10 (25,000 BU) carbowax 1540 g 1.75 carbowax 6000 g 0.75

PREPARATION No. 19 active substance μg 5 to 10 (25,000 BU) Tween 61 g 2.125 lanolin g 0.25 PREPARATION No. 20 active substance uq 5 to 10 (25,000 BU) glycerin g 1.5 water g 0.25 gelatin g 0.25

PREPARATION No. 21

Cream containing a partial ester of hyaluronic acid with benzyl alcohol, wherein 100 gr contain:

active substance μg 10 to 20 (50,000 BU) partial ester of hyaluronic acid with benzyl alcohol gr 0.2 polyethylene glycol monostearate 400 cetiol V lanette SX methyl paraoxybenzoate propyl paraoxybenzoate sodium dehydroacetate glycerin F.U. sorbitol 70 test cream water for injection q.s. ad

PREPARATION No. 22

Cream containing a total ester of hyaluronic acid with benzyl alcohol, wherein 100 gr contain: active substance μg 10 to 20 (50,000 BU) total ester of hyaluronic acid with benzyl alcohol gr 0.2 polyethylene glycol monostearate 400 cetiol V lanette SX methyl paraoxybenzoate propyl paraoxybenzoate

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

CLAIMS :

1. A nucleic acid sequence, comprising a 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 D14Serl7CNTF.

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 of the genetic code.

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

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 D14Serl7CNTF, 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 D14Serl7CNTF, 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 factor in a transformed prokaryotic or eukaryotic ceil.

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, 2, 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

D14Serl7CNTF.

20. The recombinant expression vector according to claim 18, wherein said vector is CNTF(His)₆Δl4Serl7CNTF.

21. The recombinant expression vector according to claim 18, wherein said vector is Δl4Serl7CNTF.

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_j. coli. a member of the genus Bacillus. a member of the genus Pseudomonas, a yeast, or a 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 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 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 methcd for the treatment cf 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 neuronotrophic 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 neuronotrophic 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 neuronotrophic 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 chromatography, IMAC; and

(g) recovering said mutein form of CNTF.

40. The method according to claim 39, wherein said mutein is D14Serl7CNTF.