WO1996008562A1 - Biologically active molecules derived from neurotrophins - Google Patents

Abstract

Nucleic acid sequences code for naturally non-occurring (poly)peptides having the biological activity of a neurotrophic factor and a longer N-terminus than that of ripe neurotrophic factors. Also disclosed are recombinant DNA-molecules containing the disclosed nucleic acid sequences, (poly)peptides coded by these sequences, medicaments, diagnostic agents and kits containing the disclosed (poly)peptides, as well as in vitro processes to detect diseases (or their prodromes) of the nervous system.

Description

 Biologically active molecules derived from neurotrophins

The invention relates to nucleic acid sequences which encode naturally non-occurring (poly) peptides with the biological activity of a neurotrophic factor, the (poly) peptides having an N-terminus which is elongated in comparison with mature neurotrophic factors. The invention further relates to recombinant DNA molecules which contain the nucleic acid sequences according to the invention, thereby encoded (poly) peptides, medicaments, diagnostics and kits which contain the (poly) peptides according to the invention, and in vitro methods for the detection of diseases (or Precursors thereof) of the nervous system.

The development of the vertebrate nervous system is controlled by a number of factors that are both chromosomally encoded and epigenetic in nature. In the development phase of target field innervation, a relatively large proportion (around 30-60%) of postmitotic neurons die. This phenomenon is known as "naturally occurring cell death". The death of cells was already described by Ernst (Zeitschrift. Anat. U. Entw. Gesch. 79, 228-262) in 1926 as a natural component of normal embryonic development. A hypothesis based on this observation claims that a cell needs signals from other cells not only for division but also for survival (Raff, Nature 356 (1992), 397-400). The realization that neuronal cell death can be prevented by inhibiting RNA or protein synthesis (Martin et al., J. Cell. Biol. 106 (1988), 829-844; Scott and Davies, J. Neurobiol. 21 (1990) , 630-638) allows the conclusion that apoptical neuronal cell death is an active process that takes place via the expression of certain genes.

Nerve growth factor (NGF), BDNF ("brain-derived neurotrophic factor"), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and neurotrophin-6 are members of a gene family commonly known as neurotrophins (neurotrophic factors). All these factors prevent the death of a number of embryonic neurons kept in cell culture (cf., for example, Barde et al., Progress in Growth Factor Research 2 (1990), 237-248) and are therefore able to become neuronal, as described above To prevent cell death leading active process. The importance of NGF, BDNF and NT-3 for the normal development of the nervous system was underlined by a series of experiments in which the function of the neurotrophic factors NGF and NT-3 was examined with the help of specific blocking antibodies (e.g. Cohen, Proc. Natl Acad. Sci. USA 46 (1960), 302-311, Gaese et al., Development 120 (1994), 1613-1619), or in which the genes NGF, BDNF and NT-3 or the genes of the corresponding receptor tyrosine kinases trk, trk B and trk C were switched off by "gene targeting". In all of these cases, considerable neuronal loss in the peripheral nervous system has been observed (Snider, Cell 77 (1994), 627-638).

Mature, biologically active neurotrophins are obtained from precursors by cleaving an N-terminal peptide. The cleavage takes place at a 4-amino acid consensus cleavage site of the furin type (Bresnahan et al., J. Cell. Biol. 111 (1990), 2851-2859). The known neurotrophins have a high degree of homology (Barde, Progress in Growth Factor Research 2 (1990), 237-248), which is of the order of 50%. They are secretory, basic, homodimeric proteins with a relative molecular weight of 27 kD. Six conserved cysteine residues form 3 intramolecular disulfide bridges in NGF and BDNF. A similar structure is probably found in all neurotrophins. Although there are no covalent bonds between two neurotrophin monomers, they all appear to be extremely stable dimeric structures in solution. Early studies with NGF revealed that this protein exists as homodimer in very low, physiologically relevant concentrations (Bothwell and Shooter, J. Biol. Chem. 252 (1977), 8532-8536). BDNF and NT-3 also form dimers in physiologically relevant concentrations (Radziejewski et al., Biochemistry 31 (1992), 4431-4436).

The clarification of the crystalline structure of the NGF dimer showed that the two NGF monomers are held together by non-covalent, hydrophobic interactions which are distributed over the entire contact area (MacDonald et al., Nature 354 (1991), 411-414 ). A number of hydrophobic amino acid residues conserved in the neurotrophins contribute to the formation of contact between the monomers.

EP-A 0544293 describes a method for the genetic engineering production of biologically active NGF in prokaryotes. In order to increase the expression rate, NGF was extended by 3-5 amino acids of an N-terminal sequence of a protein which is expressed in the host cells used with a high expression rate and by 4-10 amino acids from the prosequence of the NGF precursor. Such an elongated NGF, like natural NGF, has an effect on neurons and has the same activity as natural human NGF.

Although the neurotrophic factors have considerable therapeutic potential due to their biological properties, their therapeutic applicability in practice is severely impaired by the fact that they are released relatively quickly in vivo, for example through the kidneys be filtered out of the bloodstream. The object of the present invention was therefore to provide new molecules with the biological activity of the neurotrophins which are characterized in the body by a longer half-life (length of stay in the body) than the naturally occurring mature neurotrophins and, consequently, can be used effectively as therapeutic agents. This object is achieved by the embodiments characterized in the claims.

The invention thus relates to a nucleic acid molecule (DNA or RNA) which encodes a naturally non-occurring (poly) peptide with the biological activity of a neurotrophic factor, the (poly) peptide having an N-terminus which is elongated in comparison with a mature neurotrophic factor with the proviso that the nucleic acid molecule does not encode NGF. The invention further relates to a nucleic acid molecule which codes for NGF and which is N-terminally extended by a peptide fragment of up to approximately 19 amino acids, preferably by 4-19 amino acids. This peptide fragment is a C-terminal fragment of the precursor part of NGF. At the C-terminus, this precursor contains a consensus cleavage site of the furin type, in which an exchange in the amino acid -1 and / or -2 has taken place. Arginine in position -1 is preferably replaced by another amino acid, particularly preferably by lysine.

The term “naturally non-occurring (poly) peptide” is understood here to mean a (poly) peptide that does not occur either as a natural precursor of a neurotrophin or as a mature neurotrophin (poly) peptide under physiological conditions. The amino acid sequences of many mammalian and fish neurotrophins and their precursors are known in the art; these are naturally occurring (poly) peptides and therefore do not fall under the term defined above. On the other hand, this expressly includes precursors of neurotrophins which have a mutation in the 4-amino acid consensus cleavage site of the furin type, it being assumed that such mutations occur very rarely or not naturally. The nucleic acid according to the invention, preferably a DNA or RNA, is preferably produced by recombinant methods (cf. Sambrook et al., "Molecular Cloning, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1989), for example by mutagenesis of Consensus cleavage site in the cleavable N-terminus of a neurotrophin precursor.

For the purposes of this invention, the term “an N-terminus elongated in comparison with a mature neurotrophic factor” relates to any elongation of the N-terminus compared to the N-terminus of a mature neurotrophic factor. The extended N-terminus, ie the additional The presence of one or more amino acids N-terminal from the N-terminus of a mature neurotrophin may be an altered sequence of the naturally occurring N-terminal, cleavable peptide of the precursor of the neurotrophin or another neurotrophin. This altered sequence can be shortened or lengthened or have the same length as the cleavable peptide, in which case it has at least one amino acid exchange with the cleavable peptide. The shortened and extended sequences can also have such amino acid exchanges, but are additionally characterized by amino acid insertions, additions or deletions or combinations thereof, which can have been carried out internally and / or terminally. In another embodiment, the extended N-terminus cannot be derived from the amino acid sequence of the cleavable N-terminal peptide of the neurotrophin precursors.

For NGF, the term “naturally non-occurring polypeptide” is to be understood as a (poly) peptide which is extended at the N-terminal by a peptide fragment. This peptide fragment is a fragment of a precursor portion of a neurotrophin that is up to approximately 19 amino acids long, preferably 4-19 amino acids long. Precursor content, in the sense of the invention, is to be understood as meaning a peptide which can be cleaved from the N-terminus of the mature protein and which is naturally contained in the corresponding pro-neurotrophin at the N-terminal. Such neurotrophin precursor sequences are known and are described, for example, for:

NGF: Suter et al., EMBO J. 10 (1991) 2395-2400 and

Scott et al., Nature 302 (1983) 538-540

BDNF: Leibrock et al., Nature 341 (1989) 149-152

NT-3: Hohn et al., Nature 344 (1990) 339-341

NT-4: Hallboek et al., Neuron 6, 845-858

NT-5: Ip et al., Neuron 10 (1983) 137-149

NT-6: Götz et al., Nature 372 (1994) 266-269

At the C-terminus, these precursor sequences contain a furin-type consensus cleavage site in which an exchange in the amino acid position -1 and / or -2 has taken place. Arginine in position -1 is preferably replaced by another amino acid, particularly preferably by lysine.

The DNA encoding the (poly) peptide according to the invention can be of synthetic, semisynthetic origin or can be produced recombinantly. All of the embodiments discussed above have in common that the (poly) peptide encoded by the DNA according to the invention is non-toxic when applied in pharmaceutically customary concentrations, for example in humans.

Surprisingly, it was found that the (poly) peptide encoded by the DNA according to the invention only appears in the dimeric state under physiological conditions and that these dimers have the same biological activities as mature, naturally occurring neurotrophins. This finding is particularly surprising when it was assumed in the prior art that mouse β-NGF has a tenfold higher biological activity in its mature form than its precursor; see. Edwards et al., J. Biol. Chem. 263 (1988), 6810-6815. In vitro, dimers of the (poly) peptides according to the invention have greater dimer stability than dimers of the mature neurotrophins. Since the naturally occurring neurotrophins, as already discussed above, have too short a half-life in vivo to be therapeutically effective, the (poly) peptide encoded by the DNA according to the invention offers the advantage over all neurotrophins known and available in the prior art to be used effectively for therapeutic purposes. Another significant advantage of the longer half-life in vivo is that, with the same therapeutic efficacy, a smaller amount of the (poly) peptide encoded by the DNA according to the invention can be applied compared to the amount of the corresponding wild-type neurotrophin, and thus undesirable side effects are avoided can. The increased stability of the dimers which are formed by the (poly) peptides encoded by the DNA according to the invention and the advantages associated therewith are of particular importance insofar as it has surprisingly also been found that the neurotrophin monomers have no biological activity at all under physiological conditions .

Another advantage of the (poly) peptides according to the invention is that they can be marked much better with 1251 than the mature neurotrophic factors, since the N-terminal peptide, which is usually split off when the neurotrophin precursor is converted to mature neurotrophin, in particular there are also several tyrosine residues in the BDNF precursor, which are radiolabelled when the enzyme lactoperoxidase is used. This labeling option has particularly advantageous effects when using the (poly) peptides according to the invention in diagnostics. It also has advantages for the isolation of the labeled (poly) peptide. A preferred embodiment according to the invention relates to a DNA sequence, the neurotrophic factor originating from vertebrates.

A particularly preferred embodiment of the invention relates to a DNA sequence, the neurotrophic factor originating from fish or mammals.

In a further particularly preferred embodiment of the DNA sequence according to the invention, the neurotrophic factor is a human neurotrophic factor.

Another preferred embodiment of the invention relates to a DNA sequence, the factor being NGF, NT-3, NT-4/5, NT-6 or BDNF.

In a further preferred embodiment of the invention, the DNA sequence encodes a (poly) peptide, the N-terminus, which is elongated in comparison with a mature neurotrophic factor, being a mutein of the peptide cleaved off when the neurotrophin precursor changes to the mature neurotrophin.

According to the invention, “mutein of the peptide split off during the transition from the neurotrophin precursor to the mature neurotrophin” means that part of the neurotrophin precursor that is normally split off at the 4 amino acid cleavage site of the furin type and is not converted into the mature neurotrophin and that into its amino acid sequence is preferably genetically modified compared to the naturally occurring precursor in such a way that it is not cleaved off by a corresponding protease.

The protease cleavage can either be prevented by exchanging at least one amino acid in the recognition sequence for the protease, or by changing the three-dimensional structure of the N-terminus of the precursor in such a way that binding and / or cleavage by the protease is avoided becomes.

According to the invention, muteins are also understood to mean those amino acid sequences which are extended or shortened compared to the cleavable peptide of the precursor. Such an extension or shortening can be achieved genetically by adding, inserting or deleting amino acids or by combinations thereof, within and / or terminally on the cleavable peptide. The processes required for this are standard molecular biology processes and are described, for example, in Sambrook, loc. Cit. In a particularly preferred embodiment of the DNA sequence according to the invention, the mutein has at least one amino acid exchange in the 4 amino acid consensus cleavage site of the furin type. This cleavage site has the amino acid sequence

R - X - K - R (SEQ ID NO: l),

R-X-R-R (SEQ ID NO: 2) or preferably R-V-R-R (SEQ H> NO: 3) (R: arginine, X: any amino acid, K: lysine, V: valine). This exchange in the amino acid sequence means that the N-terminal peptide can no longer be cleaved off by a protease cleaving at the cleavage site mentioned. In this embodiment, further changes already discussed above in the amino acid sequence of the N-terminal peptide can also occur. However, the change in the amino acid sequence is preferably limited to the exchange / exchanges in the consensus cleavage sequence, the exchanges preferably occurring in the amino acid position -1 and / or -2. In a further particularly preferred embodiment, the mutein has the 19 C-terminal amino acids of the N-terminal peptide which can be cleaved in the native BDNF precursor and also contains the naturally occurring potential glycosylation site.

In a further preferred embodiment of the DNA sequence according to the invention, the 4 amino acid consensus cleavage site of the arginine furin type in position -1 is replaced by lysine.

Another particularly preferred embodiment of the invention relates to a DNA sequence which encodes a (poly) peptide which has the 19 C-terminal amino acids of the N-terminal peptide which can be cleaved in the native BDNF precursor, with the exception of the arginine in position -1 which is replaced by lysine.

According to the invention, it could be shown that this amino acid exchange in the BDNF precursor prevents cleavage of the N-terminal peptide, thereby producing a molecule which is 19 amino acids longer than the mature BDNF and has a greater dimer stability in solution.

In a further preferred embodiment of the DNA sequence according to the invention, the N-terminus, which is elongated in comparison with a mature neurotrophic factor, is glycosylated. The glycosylation of the asparagine residue conserved at this or a corresponding position in all neurotrophins in the N-terminal sequence of the (poly) peptide according to the invention leads to the dimer shown here in the examples having a molecular weight which is almost twice that of the wild-type BDNF -Dimer. It seems possible that the glycosylation has an influence on the longer half-life of the (poly) peptide encoded by the DNA according to the invention, it also offers the possibility of using the sugar group for labeling, for example with biotin.

In a further preferred embodiment of the DNA sequence according to the invention, the N-terminus, which is elongated in comparison with a mature neurotrophic factor, comprises a detectable and / or biologically active amino acid sequence.

This amino acid sequence can either start N-terminally from the amino acid sequence encoding the mature neurotrophic factor. In another embodiment, it connects N-terminally to the mutein of the peptide cleaved off when the neurotrophin precursor changes to the mature neurotrophin or to the extended N-terminus, which cannot be derived from the amino acid sequence of the cleavable N-terminal peptide of the neurotrophin precursors is.

The amino acid sequence can be any sequence that is detectable and or biologically active. Examples of such sequences are antibody chains or parts thereof, or sequences which are recognized by antibodies available in the prior art, e.g. c-myc or HA, furthermore biotin, enzymes or catalytically active parts thereof or enzyme substrates, or labels, such as radioactive, fluorescent, chemiluminescent or bioluminescent labels.

In a particularly preferred embodiment of the DNA sequence according to the invention, the amino acid sequence is detected by binding to an antibody or an enzyme. The detecting antibody is preferably labeled. Labeling methods for antibodies are known in the prior art; see. e.g. Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor 1988. If an enzyme is used for the detection, the reaction catalyzed thereby is preferably a color reaction.

In a further particularly preferred embodiment of the DNA sequence according to the invention, the biologically active amino acid sequence comprises biotin. Biotin has long been known in the prior art as a component of enzymatic detection systems. Another object of the invention is an RNA sequence which is complementary to the DNA sequence according to the invention or the DNA sequence complementary thereto.

The RNA sequence according to the invention can be of synthetic, semi-synthetic or recombinant origin. It can be used, for example, for in vitro translation or as anti-sense RNA or as a ribozyme.

Another object of the invention is a pair of primers that hybridize to the DNA or RNA sequence according to the invention.

The Primeφaar according to the invention preferably hybridizes under suitable conditions at the two ends of complementary strands of the DNA sequence according to the invention and can thus be used for the specific amplification of the DNA sequence according to the invention by PCR (cf. e.g. Sambrook et al., Loc. Cit.).

The invention further relates to a recombinant DNA molecule which contains at least one DNA sequence according to the invention.

The recombinant DNA molecule according to the invention is preferably a recombinant vector which is suitable for expressing the DNA sequence according to the invention. One or more DNA sequences according to the invention can be cloned into the recombinant DNA molecule according to the invention. If several DNA sequences according to the invention are cloned into the recombinant DNA molecule, these can originate from genes which code for the same or different neurotrophins. The recombinant DNA molecules according to the invention can furthermore contain genes which encode other (poly) peptides, e.g. Wild type precursors of neurotrophins.

In addition, the recombinant DNA molecules according to the invention can code fusion proteins of the (poly) peptide according to the invention, for example with β-galactosidase.

In addition to the expression of the DNA sequence according to the invention, the recombinant DNA molecule can also only be used to multiply it.

The invention further relates to a host that is transformed with at least one recombinant DNA molecule according to the invention. This recombinant DNA molecule / these recombinant DNA molecules can have the same factor or different Encode factors or, if it codes the same factor, have different amino acid sequences.

In a preferred embodiment, the transformed host is a bacterium, a fungal, yeast or mammalian cell or a transgenic animal.

If the host according to the invention is a bacterium, E. coli is preferred. In contrast, if the host is, for example, a transgenic animal, mice are preferred. The production of transgenic animals is now one of the standard methods in modern biology (see e.g. Hanahan, Science 246 (1989), 1265-1275) and is therefore not described in detail here. The expression of the (poly) peptides according to the invention can be examined in a tissue-specific manner in transgenic animals (cf. e.g. Barinaga, Science 265 (1994), 26-28).

The invention further relates to a (poly) peptide which is encoded by the DNA sequence according to the invention, the RNA sequence according to the invention or the recombinant DNA molecule according to the invention.

The (poly) peptide according to the invention can be produced, for example, by translation of an mRNA which has been transcribed by the DNA molecule according to the invention. In another embodiment, the (poly) peptide according to the invention can be produced by chemical processes known in the art or else by a combination of chemical and biological (e.g. translation) processes.

Also included in the invention are (poly) peptides that have been post-translationally modified. As is known in the prior art, the respective post-translational modification of the (poly) peptides depends on the organism used for expression or on the cell used for expression.

Another object of the invention is a homodimer which is formed by two (poly) peptides according to the invention which have the biological activity of the same neurotrophic factor. The amino acid sequence of the (poly) peptides according to the invention can differ within the homodimer as long as they are still capable of dimerization and the biological activity does not change in their nature. For example, the two (poly) peptides forming the homodimer may have been transcribed or translated by different DNA-RNA sequences that are in the same vector or in different vectors. ren can be located and in the latter case produced by different hosts / host cells and thus can also be modified differently post-translationally.

As was determined according to the invention, the neurotrophin dimers have a significantly higher biological activity than the corresponding monomers, and the dimers according to the invention also have a greater stability than the wild-type neurotrophin dimers, which predestines them for therapeutic use.

It has been shown according to the invention that improved stability of the dimers according to the invention is essentially achieved by N-terminal extension of the protein and modification of the consensus cleavage site of the furin type in such a way that the N-terminal peptide can no longer be split off. The dimers according to the invention show improved stability both in the N-terminal peptide glycosylated and in the non-glycosylated form. The neurotrophin is preferably extended by up to about 19 amino acids, preferably by about 4 to 19 amino acids, N-terminally. An improvement in stability is obtained if the N-terminal peptide does not contain a furin-type cleavage site. A non-glycosylated protein according to the invention can be obtained recombinantly in eukaryotes if there is no potential glycosylation site, for example asparagine, in the sequence of the N-terminal extension. If the neurotrophin is produced in prokaryotes, there is no glycosylation, even if the sequence contains asparagine.

However, the N-terminal peptide preferably corresponds to up to approximately 19 amino acids of the C-terminal end of the neurotrophin prosequence, which is naturally cleaved off when the mature neurotrophin is formed. Preference is also given to sequences which differ from this sequence by up to 20%, preferably by up to 10%.

The invention further relates to heterodimers which are formed by two (poly) peptides according to the invention which have the biological activity of different neurotrophic factors. The statements made for the homodimers according to the invention apply analogously to the heterodimers according to the invention. It is also important here that the (poly) peptides are still capable of dimerization. Because of the relatively large homologies between the individual neurotrophic factors, it is assumed according to the invention that heterodimers can be formed between the (poly) peptides according to the invention derived from all neurotrophins. The formation of heterodimers of various neurotrophic factors has already been shown by Radziejewski et al., Biochemistry 32 (1993), 13350-13356. Another object of the invention is a method for producing a (poly) peptide, homodimer or heterodimer according to the invention, wherein

(a) cultivating at least one host according to the invention in a suitable culture medium under conditions which allow the expression of the (poly) peptide / the (poly) peptides and, if appropriate, the formation of dimers; and

(b) purification of the (poly) peptide, homodimer or heterodimer thus obtained from the culture / from the culture medium.

The culture medium used for culturing the host according to the invention can be any medium which allows the expression of the (poly) peptides according to the invention. As is known in the prior art, the composition of the medium depends on the host used and, if appropriate, on the regulatory sequences present in the vector. For example, when E.coli is used as the host, Luria Broth (Sambrook et al., Op. Cit.) Can be used, but when using mammalian cells, the modified Eagle medium, which is also known in the art, can be used.

If the (poly) peptides, homodimers or heterodimers according to the invention are secreted into the medium, depending on the properties of their N-terminus, they are isolated from the culture supernatant and purified by standard methods in protein chemistry. If, on the other hand, they are not secreted, they are isolated from the hosts themselves after disruption, also using standard methods.

The invention relates to an antibody which is specifically weighted against the (poly) peptide, homodimer or heterodimer according to the invention. In one embodiment, the antibody according to the invention does not cross-react with mature neurotrophic factors. In this embodiment the antibody can e.g. be used in diagnostic methods that require a distinction between the mature neurotrophic factor and the (poly) peptide according to the invention.

In a further embodiment, the antibody according to the invention cross-reacts with mature neurotrophic factors and can therefore be used therapeutically and diagnostically wherever antibodies against mature neurotrophic factors can also be used; see. eg WO 91/03568. In a preferred embodiment, the antibody according to the invention is a polyclonal antibody.

In a further preferred embodiment, the antibody according to the invention is a monoclonal antibody.

The production of both polyclonal antibodies (antisera) and monoclonal antibodies is known in the prior art and is described, for example, by Harlow and Lane, cited above.

The invention relates to a medicament which contains the (poly) peptide, homodimer and / or heterodimer according to the invention, optionally in combination with a pharmaceutically acceptable carrier. The respective indication determines whether the medicament contains a combination of (poly) peptide, homodimer and heterodimer. In addition to the active substance and one or more further active substances such as a native wild-type neurotrophin, the medicament according to the invention can be used in any biocompatible pharmaceutical carrier, e.g. physiological saline, dextrose or water. If necessary, the medicinal product can also be used to prevent the diseases listed below.

The amount of drug or active ingredient to be administered depends on the type of disease and the condition of the patient to be treated. If possible, dose-response curves should be drawn up for the active substances before the actual administration in vitro. This can be done, for example, with suitable biological test systems. Such test systems can easily be established by a person skilled in the art with neurons that originate from the central or peripheral nervous system of chickens or rats. The data obtained i_ vitro can then be confirmed in the animal model. The treating doctor can determine the optimal dose to be administered for the treatment of the patient from the data obtained in this way, if necessary after carrying out preclinical tests.

Methods of administration include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intrathecal, intraventricular and intranasal administrations. Intrathecal and subcutaneous administration are preferred for the purposes of this invention. It may be necessary to introduce the drug of the invention directly into the CNS using any suitable route of administration, including intraventricular and intrathecal. The intraventricular injection can be facilitated by an intrathecal catheter, which is connected, for example, to a reservoir, such as an Ommaya reservoir.

Furthermore, it may be necessary to apply the medicament according to the invention locally. This is achieved, for example, by local infusion during the operation, by injection, with the aid of a catheter or by an implant. The implant can consist of porous, non-porous or gelatinous material, for example of membranes such as sialastic membranes ("sialastic membranes") or of fibrous materials.

The medicaments according to the invention can also be administered via liposomes, microparticles or microcapsules. For individual embodiments according to the invention it may be useful or necessary to achieve a delayed release of the (poly) peptides according to the invention.

According to the invention it is also provided that for therapeutic purposes cells are administered which contain the (poly) peptides according to the invention, antagonists thereto or antibodies against them.

In a preferred embodiment of the invention, the medicament serves to increase the survival rate of neurons. In particular, the increase in the survival rate of sensory neurons, e.g. the petrous, knee, or ventrolateral trigeminal ganglia.

In a further preferred embodiment of the invention, the medicament serves to improve neuronal growth. The medicament according to the invention can be used, for example, to increase neurite growth, e.g. of sensory neurons.

In another preferred embodiment, the medicament according to the invention is used to support differentiated cell functions. In this embodiment, the medicament according to the invention is used, for example, to treat disorders which relate to the differentiation of the neural crest. In a further preferred embodiment of the invention, the medicament is used for the treatment of diseases of the nervous system.

In a particularly preferred embodiment, the medicament according to the invention is used for the treatment of degenerative diseases of the nervous system. For example, the medicament according to the invention can be used to treat hereditary spastic paraplegia with degeneration of the retina (Kjellin and Bamard-Scholz syndromes), retinis pigmentosa, Stargardt's disease, Usher syndrome (Retinis pigmentosa with congenital hearing loss) and Refsum syndrome (retinis pigmentosa, hereditary hearing loss and polyneuropathy) can be used. A defect in the synthesis of one or more neurotrophic factors can be the etiology underlying a syndrome, which is characterized by a combination of retinal degeneration and sensory dysfunction.

In a further particularly preferred embodiment, the medicament according to the invention is used for the treatment of diseases of the nervous system which include injury or damage to the nervous system.

In a further particularly preferred embodiment, the medicament according to the invention is used to treat injuries of this type which are attributable to trauma, surgery, an infarction, an infection or a malignant disease.

In addition to the diseases listed above, the medicament according to the invention is also used to treat ischemia, nutritional deficiencies or metabolic diseases. In addition, the medicament according to the invention can be used for the treatment of neuropathic side effects which are caused by the use of anti-static active substances in cancer patients.

The medicament according to the invention is furthermore preferably used in cases where the above-mentioned disease, injury or damage to the nervous system is due to the action of a toxic active substance. In particular, the medicament according to the invention can be administered locally to injured or damaged sensory neurons which occur, for example, in dorsal root ganglia, temporal bone, knee joint or gangrion nodosum or in the other tissues mentioned below: the vestibuloacoustic complex of VIII. Cranial nerve, the ventrolateral pole of the maxillomandibular lobe of the trigeminal ganglion and the mesencephalic trigeminal nucleus). there it can be advantageous if the (poly) peptide contained in the medicament is applied to a membrane, for example a sialastic membrane, which can be implanted in the vicinity of the injured nerve. The medicament according to the invention can also serve to accelerate the regeneration of patients suffering from diabetic neuropathies, for example mononeuropathy multiplex.

In a further preferred embodiment, the medicament according to the invention is used for the treatment of diseases of the nervous system which comprise an innate disease of the retina.

In another preferred embodiment, the medicament according to the invention is used to treat a degenerative disease of the nervous system, which is Alzheimer's disease, Huntington's chorea, a retinal disease, Parkinson's disease or a Parkinson's plus syndrome.

According to the invention, it is also assumed that the medicinal product is used in combination with surgical implantation of tissue in the treatment of Alzheimer's disease or Parkinson's disease.

In a particularly preferred embodiment, the medicament is used to treat a Parkinson's plus syndrome, which is the progressive supranuclear pulsy (Steele-Richardson-Olszweski) syndrome, olivopontocerebral atrophy, the Shy-Drager syndrome or the Guam-Parkinson's dementia complex is.

A further preferred embodiment of the invention relates to a medicament with the (poly) peptide according to the invention, which is used to treat the disease of sensory neurons or a tumor.

In a particularly preferred embodiment, the medicament according to the invention is used to treat a tumor that is a neuroblastoma.

In a further preferred embodiment, the medicament according to the invention is used to increase the survival rate of dopaminergic neurons. The pharmaceuticals according to the invention can be used, for example, to promote the survival rate of dopaminergic (substantia nigra) neurons in a dose-dependent manner. In addition, the medicament according to the invention can be used in the treatment of irregularities in the radio tion of dopaminergic neurons of the CNS, which occur, for example, in Parkinson's disease.

In a further preferred embodiment, the medicament according to the invention is used to increase the survival rate of cholinergic neurons. The medicament according to the invention can be used, for example, to support the survival of cholinergic neurons of the CNS, in particular cholinergic basal neurons of the forebrain, and thus for the treatment of Alzheimer's disease. Indeed, it has been shown in the past that about 35% of patients with Parkinson's disease also suffer from Alzheimer's-like dementia. The medicament containing the (poly) peptide according to the invention may prove to be useful in the single-agent therapy of this complex of diseases. In addition, the medicament according to the invention can be used for the treatment of Alzheimer's disease in connection with Down syndrome. The medicament according to the invention can also be used in the treatment of a number of dementias and congenital learning difficulties. In addition, the medicament according to the invention should prevent the multiplication of astroglial cells and thus contribute to the prevention of scarring in the CNS (which occurs, for example, following trauma, surgery or an infarction). The medicament according to the invention can also be used for the treatment of tumors of the CNS which have arisen from degenerate astroglial cells. In addition, the medicament according to the invention can be used to increase the expression of receptors in neurotrophic factors and can thus be administered, for example, before or together with NGF. The medicament according to the invention is thus preferably also used to increase the survival rate of cholinergic neurons which are cholinergic neurons of the basal forehead or cholinergic neurons of the septum.

As already mentioned above, a further preferred embodiment of the invention relates to the use of the medicament in the suppression of the proliferation of astroglial cells.

In a further preferred embodiment of the invention, the medicament has at least one further, mature neurotrophic factor.

In a particularly preferred embodiment, this further factor is NGF, NT-3, BDNF or NT-4/5. It is intended to use the (poly) peptide or medicament according to the invention in conjunction with other cytokines in order to achieve the desired neurotrophic effects. For example, the medicament according to the invention can be used with NGF or skeletal muscle extract in order to exert a synergistic stimulating effect on the growth of sensory neurons.

In a further embodiment, the invention comprises the use of a (poly) peptide, homodimer or heterodimer according to the invention in the manufacture of a medicament for the treatment of the abovementioned. Diseases in a patient for whom a half-life of the neurotrophin in plasma or the place of application is advantageous compared to the half-life of the naturally occurring neurotrophin, the (poly) peptide, homodimer or heterodimer according to the invention forming a (poly) peptide, homodimer or heterodimer, whose plasma half-life is longer than that of the corresponding natural neurotrophin.

Half-life is the time in which the effective concentration of the therapeutic agent (activity) in the plasma is reduced by half. The measurement is usually carried out after administration of a therapeutically effective dose. Compared to the naturally occurring neutrotrophins, the neurotrophins according to the invention have an at least two, preferably a two to five times longer half-life.

Another object of the invention is a method for improving the stability of a solution or a lyophilizate of a neurotrophin, which is characterized in that the neurotrophin is extended N-terminally by 1-19 amino acids and this extended neurotrophin is dissolved or lyophilized.

The neurotrophin is preferably extended by an N-terminal peptide which corresponds 80%, preferably at least 90%, to the C-terminal peptide of the same length which can be split off from the native neurotrophin precursor form.

The invention further relates to an in-ro method for diagnosing one of the diseases listed above, wherein

a) brings a tissue sample into contact with a labeled DNA or RNA according to the invention under conditions which allow hybridization; and b) the hybridization is established.

The tissue sample for hybridization is prepared according to customary conditions. The hybridization takes place under stringent conditions.

These hybridization tests can be used to detect and diagnose, predict or monitor the above-mentioned diseases or precursors, provided that these diseases are accompanied by a change in the expression of neurotrophic factors. Such diseases generally affect e.g. the violation of neurons and the degeneration of neurons of the retina. They also include trauma to the CNS, an infarction, an infection, degenerative nerve diseases, malignant diseases and postoperative changes, Alzheimer's disease, Parkinson's disease, Huntington's chorea and degenerative diseases of the retina.

In the method according to the invention, for example, the total RNA in a patient's tissue sample can be examined for the presence or absence of the RNA of a neurotrophic factor, the decrease in the amount of BDNF mRNA, for example, indicating neuronal degeneration.

The invention further relates to an in vitro method for diagnosing one of the diseases listed above, wherein

a) bringing a tissue sample or a cell, which normally expresses a neurotrophin receptor, into contact with a labeled (poly) peptide according to the invention or a labeled homodimer according to the invention under conditions which normally allow the receptor to bind to the corresponding neurotrophin ; and

b) Detects abnormalities in neurotrophin receptor expression.

Such abnormalities can e.g. inherited changes or changes in neurons caused by toxic influences.

The desired conditions for the in vitro method according to the invention are carried out and set using customary methods known in the art. In a preferred embodiment of the method according to the invention, the neurotrophin receptor is the receptor for NGF, NT-3, BDNF, NT-4/5 or NT-6.

The invention also relates to a medicament which has an antibody according to the invention in combination with a pharmaceutically acceptable carrier. This medicament can be used, for example, to neutralize the action of the (poly) peptides according to the invention after a certain point in time, if this is desired.

If the antibodies in the medicament according to the invention cross-react with mature neurotrophic factors, they can also be used to combat diseases in which these factors are overexpressed.

The invention further relates to a kit for the diagnosis of one of the diseases listed above, which comprises at least

a) a DNA according to the invention and / or an RNA according to the invention; and or

b) a Primeφaar invention;

c) a (poly) peptide according to the invention; and or

d) an antibody according to the invention.

The kit according to the invention is usually used to carry out the methods according to the invention described above.

In the kit according to the invention, the DNA or RNA molecules or sequences complementary thereto can themselves be provided with a marker, such as an enzyme, or radioactive marker. If the DNA or RNA molecule according to the invention is used to detect a desired nucleic acid sequence, a second nucleic acid can also be used to detect the nucleic acid complex. This second nucleic acid sequence is marked with a detectable marker and hybridizes to a different region of the DNA or RNA molecule according to the invention than the region which hybridizes to the nucleic acid sequence to be detected. - 21 -

Applications for the use of the prime pair according to the invention have already been discussed above. It can be used, for example, to multiply the nucleic acid sequence according to the invention, which in turn can be used below for diagnostic purposes.

Applications for the use of the antibodies according to the invention have already been discussed above. The antibodies can be labeled for diagnostic purposes, for example using conventional methods with 1251.

In addition to the aforementioned methods, the kits according to the invention can be used in other processes, the design of which belongs to the skill of the average person skilled in the art. Such methods can be, for example, ELISAs or RIAs. The person skilled in the art is also able to supplement the kit according to the invention with the respective necessary buffers or to carry out any dilutions of the reagents obtained in the kit according to the invention.

Finally, the invention relates to a diagnostic composition which comprises at least

a) a DNA according to the invention and / or an RNA according to the invention; and or

b) a Primeφaar invention;

c) a (poly) peptide according to the invention; and or

d) an antibody according to the invention.

The areas of application for the diagnostic composition according to the invention essentially correspond to those of the kits according to the invention discussed above.

The following examples, figures and the sequence listing further explain the invention, the scope of which is evident from the patent claims. The described methods are to be understood as examples which describe the subject matter of the invention even after modifications.

Figure 1:

Elution profiles of the neurotrophins using reverse phase chromatography as the final step in the purification process. Neurotrophin peaks caused by SDS Gel electrophoresis and Western blot analysis were identified, indicated by their retention times. Unlabeled peaks do not indicate neurotrophins. Recombinant BDNF and recombinant NT-3 as well as naturally occurring BDNF elute in two different peaks, while the (R-1- K) mutant (an incompletely processed BDNF form) elutes as a single peak.

Figure 2:

FIG. 2A shows a partial amino acid sequence of mouse wild-type NT-3 and mouse wild-type BDNF and of (R-1- ^* > K) BDNF. The sequences determined by amino acid sequencing are underlined, the remaining sequences were derived from the corresponding DNA sequence. The consensus sequence for cleavage by furin-like enzymes is printed in bold. The open arrow indicates the glycosylated asparagine residue which is part of an N-glycosylation consensus sequence in all neurotrophins.

The asterisks serve to optimally stack the sequences together.

Figure 2B shows that (R-1-> K) -BDNF is glycosylated, whereas wild-type BDNF is not. The sugar residues were detected with anti-digoxigenin antibodies after the proteins were electrophoretically separated on SDS gels and transferred to membranes by vest blot. The molecular weight markers are given on the right in kDa. Transferin (T) was used as a positive control for a glycosylated protein. It is worth considering that the broad (R-1-- K) -BDNF band is glycosylated, but wild-type BDNF is not.

Figure 3:

Molecular weights of neurotrophins determined by gel filtration chromatography. The retention time was plotted against the molecular weight of standard proteins in a semi-logarithmic diagram and the straight line thus obtained was used to determine the molecular weights of the various forms of recombinant neurotrophins. The neurotrophins injected as early-eluting peaks from the urine phase chromatography elute with a molecular weight which corresponds to that of dimers, while the injection of the same peaks after incubation with guanidine hydrochloride showed that these were quantitatively converted into neurotrophin monomers.

Figure 4:

Time-dependent conversion of neurotrophin dimers into monomers after incubation with

Guanidine hydrochloride. (A) Aliquots (about 5 μg) of each neurotrophin were resuspended in 20 μl 6M guanidine hydrochloride at room temperature and the time-dependent dissociation of the dimers was measured by gel filtration chromatography. It was found for each neurotrophin that the amount of later eluting peak (monomers) increased with time. After incubation for one hour, both NT-3 and wild-type BDNF were predominantly converted to the monomeric form, but not (R-1-K) -BDNF.

Figure 4B:

Kinetics of Dimer Dissociation: To determine the rate of dissociation of NT-3, penta-NT-3, BDNF and (R-1-> K) BDNF dimers, the areas of the dimer and monomer peaks for each Gel filtration run calculated using a computer program (Maxima 820, Millipore) and the relative amount of monomer compared to the total amount of neurotrophin per run.

Figure 5:

Restoration of NT-3 dimers from monomers monitored by gel filtration. NT-3 monomers were incubated at a concentration of 150 ng / ml PBS and applied to a gel filtration column after the respective incubation time. The absorption was measured at 280 nm. In each field, the peak with the shorter retention time represents NT-3 dimers (~ 11.4 minutes) and the one with the longer retention time represents NT-3 dimers (~ 13.17 minutes). An almost quantitative conversion of the monomers into dimers took place after about 24 hours.

Figure 6:

Biological activity of recombinant neurotrophins measured by a neuron survival test. The values obtained are expressed as a percentage of surviving neurons after a 24-hour incubation period. The neurons were isolated from the ganglia nodosa of E8 chicken embryos.

(A) Biological activity of NT-3 dimers and monomers after isolation by reverse phase chromatography. The specific biological activity of the monomers is 40x lower than that of the dimers. The values are mean values from 2 measurements from 3 different experiments and the error bars indicate the lower and upper range.

(B) After restoration of the NT-3 dimers after incubation in PBS for 24 hours (see FIG. 5), the NT-3 monomers achieve a specific activity that that of the NT-3 dimers - 24 -

comes close that have not been dissociated. The experimental conditions of the biological test correspond to those in (A).

Figure 7:

Biological activity of (R-1-> K BDNF) measured based on survival on E8 neurons of the ganglion nodosum. The effectiveness of (R-1-> K) BDNF is indistinguishable from that of wild-type BDNF. For this comparison, the molecular weight for (R-1- K)

BDNF is set at 50 kDa and for wild-type BDNF at 27 kDa. The values are mean values from 3 experiments ± the standard deviation from 2 independently carried out

Experiments.

Figure 8:

Phosphorylization of trkC receptors by NT-3 dimers and monomers. TrkC-expressing fibroblasts were incubated with NT-3 dimers and monomers for 3 minutes at various concentrations. After immunoprecipitation, SDS gel electrophoresis and a Western blot, the tyrosine phosphorylation was determined with an anti-phosphotyrosine antibody and a fluorescent signal.

(A) After isolation by reverse phase chromatography, the NT-3 dimers and monomers were resuspended and immediately then added in different concentrations to cultured fibroblasts which expressed trkC.

(B) After the recovery of NT-3 dimers from monomers, these were incubated with the trkC-expressing fibroblasts. C represents the negative control without the addition of a factor.

example 1

Production of a BDNF precursor derivative

Site-specific mutagenesis

A conservative amino acid exchange (asparagine for lysine, (R-1-> K)) in the basic processing cleavage site of BDNF was introduced using an oligonucleotide-directed mutagenesis kit (Amersham, version 2; see FIG. 2A). For this purpose, the oligonucleotide 5-ATGAGGGTTCGGAAACACTCCGACCCT-3 '(SEQ ID NO: 4) coding for the site to be mutated was attached to single-stranded DNA and the second strand was produced using the Klenow fragment in the presence of phosphorothioate deoxynucleotides. The template strand was cleaved with a restriction enzyme and removed by digestion with exonuclease m. The mutated second strand was synthesized using DNA polymerase I and the plasmid 11KATA18 (Götz et al., Eur. J. Biochem. 204 (1992), 745-749) was amplified in bacteria. The mutation in the sequence was confirmed by DNA sequencing and the sequence encoding the above-described BDNF precursor derivative was cloned in a suitable vector for recombination with the vaccinia virus genome, as described by Götz et al., Supra.

Example 2

Biochemical characterization of mature neurotrophins and mutants of

Neurotrophin precursors

Recombinant neurotrophins were produced in a vaccinia virus expression system and purified by chromatography on glass beads with a controlled pore size. This was followed by a reverse phase high performance liquid chromatography (HPLC) step as described by Götz, supra. described.

Pig brain BDNF was purified as described by Hofer and Barde, Nature 331 (1988) 261-262.

Recombinant neurotrophins were applied to a reverse phase C8 chromatography column (Aquapore RP-300, 4.6 mm x 220 mm, Applied Biosystems), which had been equilibrated with 0.1% trifluoroacetic acid (TFA; Spectro-grade, Pierce) and with a linear acetonitrile gradient (21% - 56% acetonitrile in 50 minutes, flow rate 1.0 ml / min) eluted. BDNF from porcine brain was purified from reverse phase HPLC using the same buffer system, with the exception that a C8 column with a smaller diameter (Aquapore RP-300; 250 mm x 1.0 mm, Applied Biosystems) and a flow rate of 0.2 ml / min. were used. The protein peaks were detected at an absorption of 214 nm or 280 nm and collected by hand. The fractions were dried under vacuum in a rotary vacuum dryer and the protein thus obtained was stored at 4 ° C.

Neurotrophin monomers were generated by incubating the dimers in 6M guanidine hydrochloride (pH 5.9, Sigma) in a concentration of 100 ng / μl at room temperature for 24 hours. Guanidine hydrochloride was removed by reverse phase chromatography. Wild-type BDNF was derived from the (R-1 »K) BDNF mutant by gel filtration using a TSK G3000SW column (7.5 mm × 600 mm) containing 0.1% TFA and 30% acetonitrile had been equilibrated, separated. In another embodiment, gel filtration was performed using a Pharmacia Superdex ™ 75 HR 10/30 column equilibrated with 0.1 M sodium phosphate buffer, pH 7.0 containing 0.5 M NaCl and 10% acetonitrile. Proteins used as molecular weight markers and the neurotrophins were applied in aliquots of 20-40 μl in concentrations between 100 and 200 ng / μl. Dextran blue and tyrosine were used in standard runs as markers for the empty volume and the enclosed volume. The samples were run at a flow rate of 1.0 ml min. eluted and detected at 280 nm. The molecular weights of the various neurotrophins were determined by comparing the retention times with those of the proteins used as molecular weight markers. The conversion of dimers to monomers and vice versa was followed using a peak integration program (Maxima 820 software, Millipore).

After purification from conditioned medium using reverse phase chromatography as the last step in the purification process, both recombinant NT-3 and recombinant BDNF eluted as two individual peaks (see FIG. 1). By SDS-PAGE and Westem blot analysis with sequence-specific antibodies (a polyclonal antiserum, which is prepared according to conventional methods as described by Jungbluth et al., Eur. J. Biochem. 221 (1994), 677-685 was confirmed that the two peaks correspond to NT-3 and BDNF. In some preparations in which the cells were used in very high density, the later eluting peak from NT-3 up to over 90% contained a form of NT-3 which the 5 N-terminal amino acids of mature NT-3 lacked. This form was able to form dimers, had the same biological activity as mature NT-3 and was called des-Penta-NT-3. The BDNF isolated from porcine brain also eluted in two individual peaks; see. Fig. 1. Both peaks contained fully processed, mature BDNF, as could be shown by amino acid analysis and sequencing of the N-terminus. A second chromatography of the first peak of purified NT-3 or BDNF over the same column consistently revealed a second, smaller peak that had the same retention time as the later eluting peaks observed during purification; see. Fig. 1. However, not all neurotrophins in two peaks elute from the reverse phase chromatography column: BDNF with the mutated cleavage site elutes only as a single peak, the retention time being comparable to that of the first NT-3 and BDNF peaks; see. Figure 1. Sequencing of the N-terminus of the (R-1-> K) BDNF mutant revealed that approximately 80% of the material had the N-terminal sequence LEEYKNYLDA (SEQ ID NO: 5) while the remaining 20% started one or three amino acids further downstream; see. Figure 2A. Analysis of the (R-1-> K) BDNF mutant on 15% polyacrylamide gels in the presence of SDS showed a difius band with a relative molecular weight of about 25 kDa (FIG. 2B).

In all neurotrophins described so far, an N-glycosylation consensus site has been found N-terminal from the furin-type cleavage site; see. Figure 2A. By oxidation of the sugar residues and subsequent derivatization with digoxigenin in combination with a subsequent western blot using anti-digoxigenin antibodies, indications could be found that (R-1-> K) BDNF is actually glycosylated (see FIG. 2B) . This result was confirmed by the incubation of the mutant prior to performing SDS-PAGE with N-glycanase F, which reduced the relative molecular weight from 25 kDa to 15 kDa.

Since the early and late eluting peaks observed with the native neurotrophins cannot be explained by differences in their primary structure, it was examined whether the two peaks were caused by the buffer conditions (0.1% TFA and acetonitrile) used during the cleaning process, represent structurally different conformations. For this purpose, the early-eluting neurotrophins were incubated for one day at 37 ° C in 6M guanidine hydrochloride, which has been shown to influence the conformation of NGF, BDNF and NT-3 (e.g. Greene et al., Neurobiology 1 (1971), 37-48). Subsequent reverse phase HPLC showed that wild-type BDNF and wild-type NT-3, as well as the R-1-> K) mutant, had been converted quantitatively into individual peaks. The retention times of these peaks corresponded exactly to those of the late-eluting peaks described above for wild-type BDNF and wild-type NT-3.

NT-3 as well as wild-type BDNF and the BDNF mutant were further analyzed by gel filtration before and after the incubation with guanidine hydrochloride. While wild-type BDNF and wild-type NT-3, which eluted from the reverse phase HPLC with shorter retention times, were detectable as a single peak with a calculated molecular weight of 27 kDa, the forms with longer retention times showed a calculated molecular weight of 13.7 kDa, which corresponds to that of ribonuclease A; see. Fig. 3. The relative molecular weight of the BDNF mutant was also determined in this experiment. Before the incubation with guanidine hydrochloride, (R-1- ^* > K) -BDNF eluted with a calculated molecular weight of 50 kDa, whereas after the incubation a relative molecular weight of 25 kDa was determined; see. Fig. 3. In order to be able to compare the stability of the wild-type NT-3, wild-type BDNF and (R-1- ^* > K) BDNF dimers and of des-Penta-NT-3 (cf. Example 6) in solution, the dissociation rate of the dimers was measured after incubation with guanidine hydrochloride. For this purpose, the neurotrophin dimers were aliquoted, dried, resuspended in 6 M guanidine hydrochloride and incubated therein for different times before application to a gel filtration column; see. Figure 4A. The areas of the two peaks were calculated for each gel filtration run using the Maxima 820 (Millipore) software program and the extent of dissociation of the dimers was shown as a relative amount of monomers .; see. Figure 4B. These results confirm the results obtained with reverse phase chromatography (Fig. 1): they show that the (R-1-K) BDNF mutant forms dimers which are measurably more stable than the wild-type neurotrophins; see. 4B at the time 1 hour. Mature NT-3, which lacks 5 amino acids at the N-terminal (des-penta-NT-3), on the other hand has a much lower dimer stability than mature NT-3 and BDNF (cf. Example 6 and FIG. 4B).

After dissociation into monomers, the neurotrophins can subsequently form dimers again. After an hour's incubation of NT-3 monomers (150 ng / ml) in PBS at 4 ° C, it was found that after gel filtration about half of the monomers were already present as dimers. After 24 hours, essentially all of the monomers had been aggregated back into dimers (FIG. 5). These newly formed dimers cannot be distinguished by reverse-phase HPLC from the early-eluting peaks described in FIG. 1. Interestingly, the total amounts of NT-3 monomers and dimers did not change during the entire incubation period, as was determined by the integration of the monomer and dimer peaks after each run. This result leads to the conclusion that the monomers and dimers are freely convertible.

Example 3

Biological activities of the recombinant neurotrophins

In example 2 it was shown that neurotrophins can exist as monomers and as dimers in solution. It was therefore of interest to find out whether the neurotrophin monomers have measurable biological activity. Since it is known that neurotrophins prevent the death of neurons in cell culture, this property was used to determine the biological activity of the monomers. First, 30 to 40 ganglia nodosa from 8-day-old chicken embryos (E8) were prepared, incubated for 0.5 hours in a 0.1% trypsin solution (Worthington), twice with medium containing serum (F 14/10% Horse serum) and carefully dissociated in this medium. The biological activity of NT-3 and BDNF monomers and dimers was also investigated on neurons of the dorsal root ganglia (E9). The biological activity of (R-1-> K) BDNF was compared with that of mature BDNF on sensory neurons and sympathetic neurons (express). The cell suspension was first plated out to remove non-neuronal cells. 3 hours later, the neurons were carefully resuspended, counted, and at a density of 2000 neurons per well on plates (48 wells) that had previously been coated with polyornithine and either laminin or conditioned medium from the Schwannona cell line RN22, with or without neurotrophic Factors sown. For this purpose, the NT-3 monomers and dimers were freshly dissolved in PBS and added to the culture medium immediately before the neurons were plated.

The surviving neurons were counted after 24 hours in culture. While both monomers and dimers in the saturation range had the same maximum effect on the survival of the neurons (the dorsal root ganglia and the ganglion nodosum), their specific biological activities differed significantly: the half-maximum effect was found for the NT-3 monomers survival of the neurons at a concentration of 1.2 ng / ml was observed. It is therefore 40 times lower than that observed for the NT-3 dimers (30 pg / ml); see. Figures 6A and B. Very similar results were obtained when BDNF monomers and dimers were compared. Furthermore, it was found that the NT-3 dimers formed again after dissociation had a biological activity which did not differ from that of homodimers which had not previously been subjected to a disassociation step; see. Figure 6B.

The same test system (dorsal root ganglia and nodosum ganglion neurons) was then used to test the effectiveness of the (R-1-K) BDNF mutant on the survivability of neurons. Despite its high molecular weight, this partially processed BDNF dimer proved to be indistinguishable in terms of its biological activity from fully processed !, mature BDNF. The use of both forms in saturating concentrations led to the survival of equal numbers of neurons, whereas the half-maximal effect of the mutant and the wild type was achieved at concentrations of 1.2 × 1012 M (FIG. 7). (Rl- ^ K) -BDNF, like mature BDNF, had no survival activity on El 1 -sympathetic neurons. Example 4 TrkC autophosphorylation

The ability of NT-3 monomers and dimers to induce receptor autophosphorylation was tested with NTH 3T3 cells that express rat trkC more fully (Tsoulfas et al., Neuron 10 (1993), 975-990) . For this, cell culture plates with about 5 x 106 NIH 3T3 cells were incubated for 3 minutes at 37 ° C. with NT-3 monomers and dimers of various concentrations. After washing twice with 5 ml ice-cold TBS, pH 7.5, the cells were washed for 15 to 20 minutes in 600 μl lysis buffer (TBS, pH 7.5, containing 1% Triton X100, 10% glycerol, 0.5 mM phenylmethanesulfonyl fluoride (PMSF ), 0.5 mM ortho vanadate, each 5 mg / ml pepstatin and trypsin inhibitor) incubated. The nuclei were pelleted by centrifugation at 12000 rpm (Sigma 3K12, rotor 12153). Then 10 .mu.l specific rabbit antiserum specific for the 14 C-terminal amino acids of human trkA full length were added to the supernatants and these were incubated for 2 hours at 4 ° C. After adding 50 .mu.l Protein A-Sepharose and subsequent incubation for 60 min., The suspension was washed twice with PBS containing 1% Triton XI 00 and 1% deoxycholate. 30 ml of SDS-PAGE sample buffer were added and an SDS-PAGE (Laemmli, Nature 227 (1970), 677-685) on a 7.5% polyacrylamide gel and a Western blot were carried out in succession. In the latter, the proteins were transferred to Immobiion-P membranes (Millipore) using a discontinuous buffer system. Buffer 1 was 300 mM Tris-HCl, pH 10.4; Buffer 2 was 25 mM Tris-HCl, pH 10.4; and buffer 3 was 40mM norleucine, 25mM Tris-HCl, pH 9.4. Sodium borate was added to each buffer at a final concentration of 10 mM before use. The membrane used for the blot was washed in methanol and water and then incubated in buffer 2. The gels were incubated in buffer 3 for 5 min. The proteins were transferred to the membranes using a semi-dry blotting chamber using a current of 0.9 mA / cm ² for one hour. The free binding sites on the membranes were blocked by incubating the membranes for ten minutes in a PBS solution containing 2.2% polyvinylpyrrolidone (a 1: 5: 5 mixture (% by weight) of PVP 360, PVP 40 and PVP 10) . After washing with 0.17% PVP and 0.1% Tween, 20 containing PBS, the membranes were incubated with a monoclonal anti-phosphotyrosine mouse antibody (UBI), which was used in a final dilution of 1: 1000 in washing buffer has been. After a further washing step as described above, the membranes were incubated with anti-mouse IgG (1: 10,000 dilution in washing buffer) which was coupled to horseradish peroxidase. The signals were detected via the ECL Westem Detection System (Amersham) according to the manufacturer's information on chemiluminescence. After three minutes of incubation with various concentrations of the NT-3 dimers, a tyrosine phosphorylation signal could already be observed at a concentration of 10 ng ml. Maximum signal strength was reached at 100 ng / ml; see. 8. In contrast, only an extremely weak phosphorylation signal was achieved even at the highest concentrations of NT-3 monomers (1 μg / ml); see. Figure 8A. However, when the NT-3 monomer solution was incubated long enough to allow dimer formation, complete tyrosine phosphorylation activity was achieved; see. Figure 8B. These results indicate that the NT-3 monomers are 100 to 1000 times less active than the NT-3 dimers in inducing trkC phosphorylation.

Example 5 Protein Quantifications

The amounts of NT-3 and BDNF wild-type proteins were routinely determined by SDS-PAGE, subsequent Coomassie blue staining and densitometric quantification (Ultroscan XL Enhanced Laser Densitometer, LKB). In this case, known amounts of egg protein lysozyme (Sigma) were used as set standards.

The (R-1-> K) -BDNF mutant was quantified by analysis of the amino acid composition using the ninhydrin method known in the art.

Example 6

Relative stability of the neurotrophin dimers

In order to further investigate the importance of the N-terminus for dimer stability, identical dried aliquots of NT-3, des-Penta NT-3, BDNF and (R-1- ^* > K) BDNF were incubated at RT in 6 M GnHCl and the time-dependent dimer dissociation is determined by gel filtration. The areas of the monomer and dimer peaks were determined per run using the "Maxima 820" computer software (Millipore) and the relative amount of monomer was plotted against the total amount of monomer + dimer. The total amount of monomer and dimer remained unchanged per run. (R-1- ^ K) Despite its glycosylated, extended N-terminus, BDNF forms more stable, des-Penta NT-3, however, less stable dimers than mature NT-3 and BDNF (Fig. 4B). This result confirms that the N-terminus influences the relative stability of the individual neurotrophin dimers. SEQUENCE LOG

(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: MAX PLANCK SOCIETY TO PROMOTE THE

SCIENCES e. V. Berlin

(B) STREET: Hofgartenstr. 2

(C) LOCATION: Munich

(E) COUNTRY: Germany

(F) POSTAL NUMBER: D-80539

(ii) DESCRIPTION OF THE INVENTION: Biologically active molecules derived from neurotrophins

(iii) NUMBER OF SEQUENCES: 5

(iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30B (EPA)

(vi) DATA OF THE URN REGISTRATION:

(A) REGISTRATION NUMBER: DE P 44 32 463.4

(B) REGISTRATION DAY: SEP 12, 1994

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LONG: 4 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Arg Xaa Lys Arg

1

(2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE LABEL:

(A) LONG: 4 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Arg Xaa Arg Arg

1

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE LABEL:

(A) LONG: 4 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Arg Val Arg Arg

1

(2) INFORMATION ON SEQ ID NO: 4:

(i) SEQUENCE LABEL:

(A) LONG: 27 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ATGAGGGTTC GGAAACACTC CGACCCT 27

(2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE LABEL:

(A) LONG: 10 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Leu Glu Glu Tyr Lys Asn Tyr Leu Asp Ala 1 5 10

Claims

claims

1. DNA molecule encoding a naturally non-occurring (poly) peptide with the biological activity of a neurotrophic factor, the (poly) peptide having an elongated N-terminus in comparison with a mature neurotrophic factor, with the proviso that the sequence of the DNA -Molecule not encoded NGF.

2. DNA molecule encoding a naturally non-occurring NGF which is N-terminally extended by a peptide fragment of up to about 19 amino acids, this peptide fragment being a C-terminal fragment of the precursor portion of NGF which is a consensus cleavage site of the Contains furin types in which an amino acid exchange in the amino acid position -1 and / or -2 has taken place, as a result of which the precursor portion modified in this way can no longer be split off by a protease cleaving at the consensus cleavage site.

3. DNA molecule according to claim 1, wherein the factor is NT-3, NT-4/5, NT-6 or BDNF.

4. DNA molecule according to claims 1 or 3, characterized in that the peptide fragment, which represents the extended N-terminus, contains a 4 amino acid consensus cleavage site of the furin type in the C-terminal, in which an amino acid exchange in the amino acid position of -1 and / or -2 has occurred, as a result of which the peptide fragment modified in this way can no longer be split off by a protease cleaving at the consensus cleavage site.

5. DNA molecule according to one of claims 1 to 4, wherein the N-terminus, which is elongated in comparison with a mature neurotrophic factor, is glycosylated.

6. RNA molecule which is complementary to the DNA sequence according to one of claims 1 to 5 or to the complementary DNA sequence.

7. A host cell transformed with at least one recombinant DNA molecule according to claim 16 and which is a bacterium, a fungal, yeast or mammalian cell or a transgenic animal.

8. (poly) peptide which is encoded by the DNA sequence according to any one of claims 1 to 5 or an RNA sequence according to claim 6.

9. homodimer or heterodimer, which is formed by two (poly) peptides according to claim 8, which have the biological activity of the same or different neurotrophic factors.

10. A process for the preparation of the (poly) peptide according to Anspmch 8 or 9, wherein

(a) cultivating at least one host cell according to claim 7 in a suitable culture medium under conditions which allow the expression of the (poly) peptide / the (poly) peptides and, if appropriate, the formation of dimers; and

(b) purification of the (poly) peptide, homodimer or heterodimer thus obtained from the culture / from the culture medium.

11. Antibody which is specifically directed against the (poly) peptide according to Claim 8 or 9.

12. Medicament containing the (poly) peptide according to Claim 8 or 9, optionally in combination with a pharmaceutically acceptable carrier.