PT101064A - PROTEINS AND PREPRO CHEMICALS, NUCLEIC ACID MOLECULES THAT CODE THEM, AND PRODUCTION PROCESS OF A NEUROTROFIN - Google Patents

Description

74 586 6526-056-118

The present invention relates to the construction and expression in eukaryotic host cells of novel prepro proteins or novel chimeric prepro peptides expressing bioactive neurotrophic factors. The invention is based, in substantial part, on the finding that chimeric prepro proteins or prepro peptides comprising the prepro region of a first neurotrophic factor fused to the mature protein or a portion thereof of a second different neurotrophic factor, do not result in efficient post-translational processing resulting in an increased expression level of the second neurotrophic factor bioactive protein.

BACKGROUND OF THE INVENTION 2.1. NEUROTROPHIC FACTORS 1990, Science The development and maintenance of the nervous system depends on proteins known as neurotrophic factors. A neurotrophic factor is a cytokine, a protein that acts as a messenger and communicates with other cells in the coordination and regulation of biological functions. Neurotrophic factors promote the survival and / or differentiation of components of the nervous system. The generalized death of neuronal cells accompanies the normal development of the central and peripheral nervous systems and apparently plays a crucial role in regulating the number of neurons projecting to a given target field. (Berg, D. K. 1982, Neuronal Development 297-331). Studies of ablation and transplantation of peripheral target tissues during development have shown that the death of neuronal cells results from competition between neurons for limiting amounts of survival factors (" neurotrophic factors ") produced in their fields of projection. Important neurotrophic factors identified so far include nerve growth factor (NGF; Levi-Montalcini and Angeletti, 1968, Phys Rev. 48: 534); (N-3, Hohn et al., 1990, Nature 344: 339, Maisonpierre et al., 74 586 6526-056-118-2-

247: 1446), brain-derived neurotrophic factor (BDNF; Barde et al., 1982, EMBO J. 1.:549), neurotrophin-4 (NT-4; Hallbook et al., 1991, Neuron 6: 845- 858) and ciliary neurotrophic factor (CNTF; Lin et al., 1979, Science 246: 1023).

Neurotrophins are generally synthesized in vivo as " prepro " precursor proteins. The " prepro " refers to the NH 2 terminus of the precursor which is proteolytically removed during biosynthesis of the mature, biologically active form of the protein. The region " pre " refers to the signal sequence normally removed by proteolytic processing during translation through the cell membrane to give the " pro "; the " pro " is then removed by proteolytic processing to give the mature form (see e.g. Darnell et al., 1990, Molecular Cell Biology 2nd Edition, Scientific American Books, page 650-657).

2.1.1. NERVE GROWTH FACTOR Nerve growth factor (NGF) is by far the best characterized of these neurotrophic molecules and was found, in vitro and in vivo. be essential for the survival of sympathetic sensory neurons and neuronal Christian derivatives, during the early development of chicken and rat (Levi-Montalcini and Angeletti, 1963, Develop Biol., 653-659; Levi-Montalcini et al. Until recently, almost all studies on NGF had focused on their role in the peripheral nervous system, but now it seems that NGF also influences development and maintenance of specific populations of neurons in the central nervous system (Thoenen et al., 1987, Rev. Physiol Biochem Pharmacol 109: 145-178, Whittemore and Seiger 1987, Brain Res. Rev. 12: 439-464) The abundance of NGF protein in the mouse submaxillary gland allowed the determination of the primary amino acid sequence by relatively conventional protein chemistry (Angeletti and Bradshaw, 1971, Proc Natl Acad Sci 68: 2417-2442) of NGF has already been cloned from many species, i including the mouse (Scott et al., 1983, Nature, 302: 538-540, <u

(Meier, et al., 1986, EMBO J. 5: 1489-1493), and the presence of a protein in the cell. rat (Whittemore, et al., 1988, J. Neurosci. Res., 20: 402-410) using standard molecular biology techniques based on the availability of the mouse NGF protein sequence to design suitable oligonucleotide probes. The mouse NGF gene encompasses approximately 45 kb, containing several small 5 'exons, resulting in alternating (&quot; splicing &quot;) splitting in four distinct mRNA species (Serby, et al., 1987, Mol. Cell Biol. .: 3057-3064). The major transcripts result in prepro peptides of NGF &quot; long &quot; 1 &quot; short &quot; (Edwards, et al., 1986, Nature, 319: 784-787: Serby, et al., 1987, Mol. Cell Biol., 7: 3057-3064). The precursor &quot; short &quot; contains a conventional signal sequence (pre-region) at the NH2 terminus flanking the pro region. The long precursor contains a &quot; pro region &quot; additional at its NH 2 terminus (see, e.g., Suter et al., 1991, EMBO J. 10: 2395-2400, Figure 1). To date no functional distinction has been elucidated between the prepro precursor of NGF &quot; long &quot; and &quot; short &quot;. However, the shorter mRNA transcript is more abundant in most tissues (Edwards et al., 1986 J. Biol. Chem. 263: 6810-6815). The biologically active form of the mouse NGF is a 7S complex, comprising a fully processed mature form dimer of β-NGF together with two members of the kallikrein family of the serine proteases, subunit a and subunit) of NGF (Varon et al., Biochemistry 7: 1296-1303). Mason et al., 1983, Nature, 303: 300-307). Translation, processing and secretion of the NGF precursor to form a biologically active form of NGF is well documented. Darling, et al. (1983, Cold Spring Harbor Symp., Quot; Biol., 48: 427-433). based on the strength of the reported sequence of cDNA encoding mouse NGF (scott, et al., 1983, Nature 302: 538-540). used an in vitro cell free translation system to identify key intermediates in the biosynthesis of the NGF 7S complex. The signal sequence of the NGF prepro precursor is removed by proteolytic processing to give a NGF precursor signal sequence.

pro-NGF species of approximately 31 kD. The pro-NGF intermediate pro region contains a pair of arginine residues known to be endoproteolytic processing sites. Proteolytic processing in any of these residues results in a major intermediate (21 kD) and secondary (18/5 kD) intermediate species. The mature form of NGF can be derived proteolytically from any of the aforementioned intermediary species. At any point in the biosynthesis of the mature form of NGF, a COOH-terminal dipeptide (arg-gly) is proteolytically released.

It has been shown in vivo that the subunit proteolytically cleaves the pro-NGF precursor in the mature form of NGF (Edwards, et al., 1988, J. Biol. Chem. 263: 6810-6815). Attempts to mimic the in vitro process were unsuccessful, resulting in unprocessed processing of the pro-NGF precursor, presumably due to the aberrant folding of the in vitro translation product. Silen and Agard (1989, Nature 341: 462-464) have demonstrated that the pro region may facilitate adequate folding of the lytic protease precursor. Accordingly, the NGF precursor pro region may also be required for proper folding prior to endoproteolytic processing in the mature form and association in the 7S biologically active NGF complex. Evidence for this hypothesis is documented in Suter et al. (1991, EMBO J. 10: 2395-2400), which assigned functions to two of the partially conserved domains within the NGF pro region. Domain 1 was shown to be essential for the expression of NGF in COS cells. In addition, Domain II, located in the pro region of NGF near the mature coding region, was found to be involved in proteolytic processing.

Recently, proteolytic processing of pro-NGF has been shown in vivo. is controlled by the human fur gene product, a membrane-associated endoprotease that shares structural homology with the KEX2 gene, which encodes a yeast endoprotease (Bresnahan, et al., 1990, J. Cell Biol., 111: 2851-2859).

Accordingly, the initiation of the biosynthesis of the active form

Mouse NGF involves transcription of the NGF gene and possible alternative junction of the transcription product to generate mRNAs capable of translation of the long or short prepro precursor of NGF. The prepro precursor of the long or short NGF is subsequently subjected to a series of endoproteolytic processing events, possibly induced by proper folding of the precursor through the structural characteristics of the pro region, resulting in the mature form of NGF.

2.1.2. NEUROTROPHIC FACTOR DERIVED FROM THE BRAIN

Using pig brain as a starting material, Barde et al. (1982, EMBO J., 1: 495-553) reported a factor, now called brain derived neurotrophic factor (BDNF), which appeared to promote the survival of dorsal root ganglion neurons of chick E10 / E11 embryos. The neurotrophic activity was found to reside in a highly basic protein (isoelectric point, 10.1) migrating during sodium dodecylsulfate (SDS) gel electrophoresis as a single band of 12.3 kD. It was reported that the highly basic nature and molecular size of BDNF were very similar to those of the NGF monomer. Cloning of the BDNF gene was first performed as described in co-pending U.S. Patent Application Serial No. 07 / 400,591, filed Aug. 30, 1989, which is hereby incorporated by reference in its entirety (see also PCT International Publication No. WO 91/03568, published March 21, 1991). Genomic BDNF cDNAs and / or genes were cloned complete from a variety of species, including man, pig, rat and mouse, and the sequences of these genes were determined. Recombinant BDNF expression was achieved in COS cells. The first demonstration of neuronal specificity of BDNF different from that of NGF consisted of the in vitro demonstration that purified BDNF supports the 40-50% survival of cleaved nodes of the nodal ganglion derived from the chick embryo neural placent at E6, E9 or E12 (Lindsay et al .. 1985 74 586

/ RTI &gt; &lt; / RTI &gt; 6526-056-118 -6- J. Cell Sci. Supp. 3: 115-129). NGF had no apparent effect on these neurons by itself or in conjunction with BDNF. It has later been shown in explant culture studies that BDNF appeared to support the survival and outward growth of neurite (&quot; neuritis outgrowth &quot;) from other sensory ganglia derived from neural placodes, including petrosal ganglia , geniculate and trigeminal venterolateral (Davies et al., 1986, J. Neurosci. 6: 1897-1904), none of which were considered to be sensitive to NGF. In addition to their effects on cultured peripheral ganglia neurons, BDNF was found to stimulate the survival and neuronal differentiation of cultured cells of quail neuronal Christian (Kalcheim and Gendreau, 1988, Develop.Research 41: 79-86 ).

Two recent BDNF studies (Kalcheim et al., 1987, EMBO J. 6: 2871-2873; Hofer and Barde, 1988, Nature 331: 261-262) have, however, indicated a physiological role of BDNF in the development of SNP in birds In addition to its effect on peripheral sensory neurons originating from neuronal Christian and neuronal placode, BDNF has been found to support the survival of developing CNS neurons: Johnson et al (1986, J. Neurosci. 3031-3938) showed data indicating that BDNF supports the survival of retinal ganglion cells cultured from E17 rat embryos.

In addition to its effects on the survival of developing neurons in culture, BDNF has been shown to have effects on cultured neurons of the peripheral and central nervous system of adults. Analysis of the predicted primary structure of mature BDNF revealed a striking similarity to NGF; with only three gaps introduced into the NGF sequences to optimize pairing, are common to the various NGF 51 identities (from Snake to Man) and BDNF. It is important to note that these identities include six cysteine residues.

74 586 6526-056-118 -7- 2.1.3. NEUROTROFIN-3

Another member of the neurotrophin family, designated neurotrophin-3, was found, and the NT-3 gene was cloned from the mouse, rat and human (see U.S. Application Serial No. 07 / 490,004, filed 7 March 1990, incorporated herein by reference in its entirety, see also International PCT Publication WO 91/03569, published March 21, 1991). The overall structure of mature mouse NT-3 protein, consisting of 119 amino acids with an estimated pi of about 9.5, was found to be similar to that established for NGF and BDNF; a putative 18 amino acid signal sequence (showing 5 and 9 amino acid identities with BDNF and NGF, respectively) appears to be followed by a pro sequence of 121 amino acids (as compared to a pro sequence of 103 amino acids in the mouse and a sequence pro ratio of 112 amino acids in mouse BDNF). A comparison of NGF, BDNF and mature mouse NT-3 revealed 54 amino acid identities. All 6 cysteine residues, which are known in NGF and BDNF are involved in the formation of disulfide bridges (Leibrock et al., 1989, Nature 341: 149-152: Angeletti, 1973, Biochem. 1: 100-115). are among the conserved residues. Similarly, rat mature NT-3 appears to share a 57% amino acid homology with rat NGF and 58% amino acid homology such as rat BDNF; 57 of the 120 amino acid residues (48%) appear to be shared by all three proteins. Again, the six cysteine residues of rat NGF and BDNF were found to be absolutely conserved in rat NT-3 and regions of higher homology among the three proteins appear to cluster around these cysteine residues.

In addition to the homology between NT-3, NGF and BDNF within a species, a high degree of conservation in the nucleic acid sequence between rat and human NT-3 was observed within the region encoding the mature polypeptide (119 amino acids ). The deduced amino acid sequences of mature NT-3 from rat and human (as well as from mouse NT-3)

be identical, which mirrors the high degree of BDNF conservation, which shows complete identity to the amino acid sequence of the mature polypeptide between rat, mouse, human and pig. In contrast, the amino acid sequences of mature human and rodent NGF (mouse or rat) differ by approximately 10%.

Studies of the neurotrophic activity of NT-3 have indicated that NT-3 is capable of promoting the survival and outward growth of neurites of dissociated dorsal root ganglion neurons in culture. Furthermore, it was observed that NT-3 promoted neurite outgrowth growth from nodal ganglion and sympathetic ganglion explants, whereas BDNF promoted outward growth from the nodose but not from the ganglion ganglion and the NGF promoted growth abroad from explants of the sympathetic ganglion but not from explants of the ganglia nodosa. Accordingly, it appears that NT-3 has a broader action specificity than either BDNF or NGF. 2.1.4. Neurotrophin-4 is a new member of the NGF family that has recently been cloned and isolated (Hallbook et al., 1991, Neuron 6: 845-858). PCR fragments corresponding to the Xenopus NT-4 and viper gene were obtained and a Xenopus genomic clone was subsequently isolated. Analysis of the nucleotide sequence of this clone revealed an open reading frame for a 236 amino acid protein, with various structural characteristics similar to those of NGF, BDNF and NT-3. These features include a putative, amino terminal signal sequence, and a potential N-glycosation site near a proteolytic cleavage site. As is true for NGF, BDNF and NT-3, the entire Xenopus prepro-NT-4 protein is encoded by a single exon.

2.2. PRODUCTION OF NEUROTHROPHINS

Various expression vectors and hosts were used

ai and

Attempts to produce recombinant neurotrophins.

All who used animal cell expression systems (mammalian kidney cells), Leibrock et al. [Nature 341; 149 (1989)] reported the expression of active porcine BDNF, and Rosenthal et al. [Neuron 4: 767 (1990)], Maisonpierre et al. [Science 247: 1446 (1990)] and Hohn et al. [Nature 344: 339 (1990)] reported separately the biologically active NT-3 expression of several species. In addition, Chan et al. [EP Publication No. 370171, published May 1990] reported the expression of mature, biologically active human BDNF from insect cells through a baculovirus expression system.

In relation to microbial neurotrophin production, Iwai et al. [Chem. Pharm. Bull. 34: 4727 (1986)] reported the expression &quot; genes &quot; synthetic methods for human NH2 and their fusion in E. coli. The product was only characterized by the molecular mass after treatment with a reducing agent and there was no information regarding the presence of biological activity.

Dicou et al. Neuroscience Res. 22:13 (1989)] reported the separate expression of fusions of mouse NGF and hNGF in E. coli. Dicou et al. (1989, J. Neurosci. Res. 22: 13-19) fused the entire mouse prepro nerve growth factor DNA sequence to the carboxyl terminus of the beta-galactosidase gene of Escherichia coli and also fused a DNA fragment genomic DNA corresponding to codons 11 to 106 of the human nerve growth factor gene to the fifth amino terminal codon of beta-galactosides. Both bacterial vectors were associated with expression of large amounts of chimeric proteins. Although after lysis of the bacterial cells, most of the mouse chimeric prepro nerve growth factor appeared to be insoluble, most of the human chimeric beta nerve growth factor appeared to exist in the supernatant. Neurotrophic activity was not reported.

Finally, Hu et al. [Gene 70: 57 (1988); and Abstract 343.16 of 20th Ann. Meeting of the Soc. For Neuroscience (1990) reported the expression of mouse NGF in E. coli. 74 586 6526-056-118 -10-

SUMMARY OF THE INVENTION

The present invention relates to novel chimeric prepro proteins or prepro peptides comprising bioactive neurotrophic factors and the use of these precursors and their nucleic acid sequences to produce proteins or peptides having one or more biological activities of a neurotrophin. The chimeric prepro molecules provided by the present invention contain a heterologous prepro region fused to a mature neurotrophin sequence or a biologically active moiety or derivative thereof. The mature neurotrophin sequences that may be used in accordance with the present invention are those of the NGF / BDNF family of homologous molecules including but not limited to NGF, BDNF, NT-3 and NT-4. Similarly, the prepro regions may be derived from those neurotrophin molecules of the NGF / BDNF family including but not limited to NGF, BDNF, NT-3 and NT-4. The invention is based in substantial part upon the finding that chimeric prepro proteins or prepro peptides, comprising the prepro region of nerve growth factor, fused to the mature portion of brain derived neurotrophic factor (NGF / BDNF prepro) are processed more efficiently by a eukaryotic host cell than the prepro-homologous brain-derived neurotrophic factor (BDNF prepro). It is further based on the finding that stably transfected, eukaryotic and stably amplified host cells expressing chimeric prepro NGF / BDNF secrete only the form The invention also is based on the finding that the prepro proteins or peptides of the present invention may be used in the manufacture of the NGF and / or short regions of the NGF in the construction of chimeric neurotrophic genes. prepro, chimeric, comprising the prepro region of NT-3 fused to the mature coding region the brain-derived neurotrophic factor (NT-3 / prepro BDNF) are more efficiently processed than the derived neurotrophic factor prepro brain counterpart (prepro BDNF). It is further based on the finding that stably transfected and amplified eukaryotic host cells expressing chimeric NT-3 / BDNF express only the mature form of BDNF for the media The present invention provides nucleic acids encoding chimeric neurotrophic prepro proteins or prepro peptides and methods for the expression of these chimeric neurotrophic proteins and peptides through the use of such nucleic acids.

4. DESCRIPTION OF THE FIGURES

Figure 1. Polyacrylamide gel electrophoresis of BDNF, recombinant NGF and chimeric precursor forms. Supernatants of metabolically labeled CHO-DG44 cells stably transfected with various constructs were resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Lane 1: wild type, control CHO-DG44 cells. The following constructs were used: pCDM8 expression vector containing the human NGF gene (lane 2); short prepro BDNF construction (lane 3); chimeric construct of long prepro NGF / BDNF (lane 4). Range 5: molecular mass markers.

Figure 2. Bioactivity of recombinant BDNF. Crude supernatants of CHO cell lines transfected with embryonic dorsal root ganglia (E8) ganglia were tested and growth out of neurites was recorded. Filled diamonds: DGC-N / B-2.5- # 23 cell line (containing the long chimeric NGF / BDNF prepro construct). Square to dotted: cell line DGZ1000-B-3-2.5 (containing the short construction of BDNF prepro).

Figure 3. Human BDNF (SEQ ID Nal) cDNA and deduced amino acid sequence (SEQ ID N02) and comparison of pig DNA sequences (SEQ ID N23 and SEQ ID NO4), rat (SEQ ID NO: 5 and SEQ ID NO: Na6) and chicken (SEQ ID NO: 7 and SEQ ID NO: 8). The figure shown is from PCT International Publication No. WO 91/03568, published March 21, 1991. 74 586 6526-056-118 -12-

Figure 4. Nucleotide sequence (SEQ ID Na9) and deduced from amino acids (SEQ ID N210) of human NGF. The numbers -187 to -1 indicate the long prepro region. The information for the sequence is from EP 121,338, issued October 10, 1984 to Gray and Ullrich.

Figure 5. DNA aligned sequences of rat NT-3 (SEQ ID Nall) and human (SEQ ID N213) genes. The predicted translation start site is indicated by &quot; PREPRO- &quot; and the expected start of the mature NT-3 is indicated by &quot; MATURE- &quot;. Rare rat (SEQ ID No 12) and human (SEQ ID NO: 14) NT-3 proteins have identical amino acid sequences, while the prepro regions differ in 11 positions, which are underlined. The figure shown is from PCT International Publication No. WO 91/03569, published March 21, 1991.

Figure 6. DNA fragment 3 (SEQ ID N215 and 16) used in the chimeric NT-3 / BDNF construct corresponding to 35 amino acids of the NT-3 prepro region (SEQ ID Na17).

Figure 7. Western blot analysis of conditioned media from CHO cell clones expressing the original prepro BDNF (range 2-11) or chimeric prepro NT-3 / BDNF (range 12-19). Lane 1 was loaded with 450 ng of mature, purified human BDNF. Lane 20 was loaded with pre-stained low molecular weight BRL markers. Lane 8 and Lane 16 represent non-transfecting clones of each transfection.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel chimeric prepro or prepro peptides comprising bioactive neurotrophic factors and the use of these precursors and their nucleic acid sequences for the production of proteins or peptides having one or more activities biological properties of a neurotrophin. The chimeric prepro molecules provided by the present invention contain a heterologous prepro region fused to a mature neurotrophin sequence or portion thereof or biologically derived

74 586 6526-056-118. The mature neurotrophin sequences that may be used in accordance with the present invention are those of the NGF / BDNF family of homologous molecules including but not limited to NGF, BDNF, NT-3 and NT-4. Similarly, the prepro regions may be derived from the neurotrophin molecules of the NGF / BDNF family including, but not limited to, NGF, BDNF NT-3 and NT-4. The invention is based in part on the finding that chimeric prepro proteins or prepro peptides comprising the prepro region of nerve growth factor and the mature portion of brain derived neurotrophic factor (NGF / BDNF prepro) or region prepro of NT-3 and the mature portion of brain-derived neurotrophic factor (NT-3 / BDNF prepro) are processed more efficiently by a eukaryotic host cell than homologous prepro brain-derived neurotrophic factor (BDNF prepro). It is further based on the finding that stably amplified and transfected eukaryotic host cells expressing prepro NGF / BDNF or NT-3 / BDNF prepro segregate only the mature form of BDNF to the media. Post-translational processing of the prepro homologue BDNF is highly inefficient. In contaste, a member of the same neurotrophin gene family, NGF, is processed efficiently. Only the mature bioactive form of NGF is secreted into the host cell media following transient transfection. The problem of BDNF processing persisted in the generation of stable host cell lines for the production of mature bioactive BDNF. The present invention provides a novel solution to this processing problem by expression of chimeric constructs, which in a specific embodiment contains the long region of the prepro-NGF fused to the frame ("in frame") with the mature BDNF and in another specific embodiment contains the region prepro of NT-3 fused to the mature BDNF frame. The present invention provides nucleic acids encoding chimeric neurotrophic prepro proteins or prepro peptides and methods for the expression of these chimeric neurotrophic proteins and peptides by the use of such nucleic acids.

Expression of nucleic acids encoding chimeric neurotrophic prepro proteins or prepro peptides according to the present invention provides significant advantages over the use of nucleic acids encoding prepro proteins or neurotrophic prepro peptides, homologues. The production of prepro proteins or chimeric neurotrophic prepro peptides provides enhanced levels of bioactive neurotrophic factor expression. This increased level of expression should in addition provide better purification schemes of bioactive neurotrophic factor, insofar as the contaminated unprocessed forms of the expressed neurotrophic factors are not present in the crude supernatants.

5.1. THE EXPRESSION PRODUCTS OF THE PRESENT INVENTION

Bioactive proteins obtainable in accordance with the present invention are mature neurotrophic factors which are members of the neurotrophin gene family, or biologically active moieties or derivatives thereof. The term &quot; biologically active &quot; as used herein refers to the ability to express one or more biological activities of the full length mature neurotrophin. These neurotrophins include, but are not limited to, mature BDNF, NT-3, NGF and NT-4 and to other members which are identified by methods used to determine members of the neurotrophin gene family (eg using molecular probes generated by PCR, corresponding to regions of homology within the family - see PCT Publication WO 91/03569).

DNA sequences encoding various neurotrophin proteins that can be expressed using the present invention are available. See Ullrich et al. (Nature 303, 821 (1983), Publication E.P. 121,338, issued Oct. 10, 1984) with respect to the coding sequences of hNGF and, e.g., Meier et al. (EMBO J. 5: 1489 (1986)) and Schwarz et al. (J. Neurochem., 52: 1203 (1989)) over NGF cDNAs for several other species? ATCC plasmid strain phBDNF-C-1 (Accession Na 4068) relative to a cDNA clone of hBDNF and, e.g., Leibrock et al., relative to porcine BDNF cDNA; and the strain of 74 586 6526-056-118 -15-

plasmid ATCC pC8-hN3 (Pl) (Access Na 40765) to a human NT-3 cDNA clone and Maisonpierre et al. (Science 247: 1446 (1990)) and Hohn et al. (Nature 344: 339 (1990)) with respect to coding sequences of several other species. Cloning of human NT-3 genes (Rosenthal et al., Neuron 4: 767 (1990)) as well as rat (Maisonpierre et al.) Has been reported. In addition, the nucleotide and amino acid sequences for BDNF are described in PCT Publication WO 91/03568, published March 21, 1991 and copending U.S. Patent Application Serial No. 570,657, filed at 20 of August 1990; the nucleotide and amino acid sequences for NT-3 are described in PCT publication WO / 03569 published March 21, 1991 and co-pending U.S. Serial No. 570,189, filed Aug. 20, 1990). In addition, the NDNA (SEQ ID Nal-8), NGF (SEQ ID NO: 9-10), and NT-3 (SEQ ID Nall-14) are shown in Figures 3, 4 and 5, respectively, of the present invention.

Furthermore, a neurotrophin gene of any organism may be identified using regions of homology shared by any two members of the BNF / NGF / NT-3 / NT-4 family of molecules, using the methods set forth above. For example, and not as a limitation, a novel neurotrophin can be identified and cloned by the synthesis of BDNF / NGF / NT-3 / NT-4 degenerate oligonucleotides corresponding to highly conserved protein sequence segments between any two neurotrophins. These oligonucleotides can be used as initiators in polymerase chain reactions (PCR) with the cDNA template prepared from cells suspected to express the desired neurotrophin. The PCR products may then be used as probes to allow the cloning of complete cDNA and / or genomic genes, the sequences of which may be determined by standard methods. Novel neurotrophins may be identified by selection of those which contain, in addition to the sequences homologous to other known neurotrophins, sequences not homologous to other known neurotrophins i.e. at least six contiguous nucleotides in which at least two nucleotides differ

74 586 6526-056-118 -16-rem). Similarly, oligonucleotides corresponding to sequences of a neurotrophin in one species can be used in PCR to generate probes to allow the cloning of the neurotrophin gene from other species. NGF and BDNF are basic proteins of approximately 120 amino acids sharing about 50% amino acid sequence identity, including the absolute conservation of six cysteine residues which have been shown to form three disulfide bonds in active NGF (Bradshaw, A. , 1978, Ann Rev. Biochem 47, 191-216, Leibrock et al., 1989, Nature 341: 149-52). Comparison of the NGF sequences from evolutionarily divergent species has shown that the amino acids flanking these cysteine residues comprise the more conserved regions of the molecule (Meier et al., 1986 EMBO J. 5, 1489-93, Selby et al. 1987, J. Neurosci Res 18: 293-8). Surprisingly, these are also the most similar regions between BDNF and NGF (Leibrock et al., 1989, Nature 341: 149-52.

In a preferred aspect of the present invention, a mature human neurotrophin is produced by expression of a chimeric prepro molecule in accordance with the present invention. In a specific embodiment, the chimeric prepro molecule is encoded by a nucleic acid containing the long prepro region of the fused NGF in the frame (&quot; in frame &quot;) with the mature BDNF coding sequence. In another embodiment, the chimeric prepro molecule is encoded by a nucleic acid containing the prepro region of the fused NT-3 in the frame with the mature BDNF coding region. In yet another embodiment, the long prepro region of NGF is fused in the frame with the NT-3 coding region.

As discussed above f was not documented any distinct biological meaning between the &quot; long prepro region &quot; and &quot; short &quot; of the NGF precursor. In yet another specific aspect of the invention, the long prepro region &quot; or &quot; short &quot; in the construction of chimeric neurotrophic genes. One skilled in the art can utilize a short prepro region &quot; of the NGF or a long prepro region &quot; of the NGF in the construction of the

The present invention comprising a prepro region of NGF.

Mature neurotrophin molecules which can be expressed as chimeric prepro precursors according to the present invention also include sequences and fragments or derivatives, substantially equivalent that are biologically active.

For example, neurotrophin nucleic acid sequences may be altered by substitutions, additions or deletions that can provide functional molecules. Due to the degeneracy of the coding nucleotide sequences, other DNA sequences encoding substantially the same neurotrophin amino acid sequence may be used in the practice of the present invention. These include but are not limited to null-ocetide sequences comprising all portions of the neurotrophin genes that are altered by substituting different codons that encode a functionally equivalent amino acid residue within the sequence, thereby producing a silent shift. Likewise, the neurotrophin proteins or fragments or derivatives thereof of the invention include, but are not limited to, those that contain, as part of their primary amino acid sequence, altered sequences in which residues within the sequence are replaced by functionally amino acid residues resulting in a silent change. For example, one or more amino acid residues within the sequence may be replaced by 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 among other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Neutral polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively (basic) amino acids include arginine, lysine, and histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid. Also included within the scope of the invention are proteins or (fti). S / · &quot; di

Fragments of neurotrophin or derivatives thereof which are obtained by modification during or after translation, eg by glycosylation, proteolytic cleavage, binding to an antibody molecule or other cellular ligand, acetylation, phosphorylation , cleavage reduction, etc.

In addition, a given neurotrophin sequence may undergo mutation in vitro or in vivo. to create and / or destroy translation, initiation and / or termination sequences or to create variations in the coding regions and / or to form new restriction endonuclease sites or to destroy other existing ones, to further facilitate the in vitro modification. Any mutagenesis technique known in the art, including but not limited to in vitro site-directed mutagenesis (Hutchinson, et al., 1978, J. Biol. Chem. 253: 6551) may be used. use of TAB ® (Pharmacia) connectors, etc. The present invention also relates to the expression of nucleic acids encoding chimeric prepro neurotrophin molecules and to the recovery of the mature neurotrophin product.

5.2. CONSTRUCTION OF PREPRO OR PEPTID PROTEINS PREPRO. NEUROTROPHICS. CHEMISTRY

Nucleic acids encoding chimeric neurotrophic prepro proteins or prepro peptides may be constructed using standard recombinant DNA technology, for example, by restriction enzyme digestion and ligation of the nucleic acid sequences encoding the desired prepro and mature regions. Alternatively, nucleic acid sequences may be constructed using chemical synthesis, such as solid phase phosphoramidite technology. In preferred embodiments of the invention, polymerase chain reaction (PCR; Saiki et al., 1985, Science 230: 1350-1354) can be used to achieve &quot; splicing &quot; of nucleic acid sequences by overlap extension (Horton et al., 1989, Gene 77: 61-68) and thus produce nucleic acids encoding the chimeric neurotrophic prepro proteins or prepro peptides of the invention (see e.g.

74 586 6526-056-118

Section 6, below).

In a preferred aspect, the novelty acids of the invention are produced by the use of two separate PCR reactions, each with a different template. As an illustration, if an XY chimera is desired, one first template PCR, for example X, is first performed using a fully homologous probe with X and a probe with an X homologous region and a Y homologous region. The PCR reaction is then isolated and used as a probe in a second PCR reaction, with Y as a template and a second probe fully homologous with Y.

It may further be desirable to incorporate useful restriction endonuclease sites into the primers.

In addition, chimeric neurotrophic factors can be produced by PCR in a single step using three oligonucleotide primers. For example, a nucleic acid encoding at least a portion of the desired prepro region (X) may be ligated to a nucleic acid sequence encoding a mature, neurotrophic protein or peptide (Y) creating three oligonucleotide primers, one of which corresponds to a portion of the sequence X (the &quot; X-primer &quot;), another corresponds to a portion of the Y sequence (the &quot; Y-primer &quot;) and a third containing a portion of the sequences X and Y (&quot;;). These three oligonucleotides can be combined in one step PCR, it being desirable for primers X and Y to be present in greater amounts than the XY primer, for example at an X: Y: XY ratio of about 100: 1: 100. [The template used in the PCR may be a mixture of nucleic acids encoding the desired prepro region and the mature neurotrophic protein or peptide]. The position of the binding sites is determined by the bridged nucleotide (e.g. the XY primer).

The amplification conditions commonly used in the art, for example, 1 minute at about 94 ° C, 2 minutes at about 43 ° C and 3 minutes at about 72 ° C for 35 cycles, using standard solutions and methods of PCR. The PCR fragment

The resultant can then be purified on gel electrophoresis, digested with the appropriate restriction endonuclease and ligated into a suitable cloning vector.

Additional processes for constructing the chimeras of the present invention will be readily apparent to those skilled in the art.

The DNA reaction products can be cloned using any method known in the art. Any number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmid cosmids or modified viruses, but the vector systems must be compatible with the host cell used. These vectors include but are not limited to bacteriophages such as lambda derivatives or plasmids such as pBR322, pUC or Bluescriptâ "¢ (Stratagene) plasmid derivatives. Recombinant molecules can be introduced into the host cells by transformation, transfection, infection, electroporation, etc.

5.3.EXCESSION OF NUCLEIC ACIDS ENCODING PROTEINS PREPRO OR PÉPTIDOS PREPRO. NEUROTROPHICS. The nucleotide sequence encoding a prepro, neurotrophic, chimeric, prepro protein or peptide can be ligated into a suitable expression vector, i.e. a vector containing the elements necessary for the transcription of the cloned qimeric DNA sequence. The required transcription and translation signals may also be provided by one of the neurotrophin genes and / or its flanking regions corresponding to the prepro protein or neurotrophic, chimeric, prepro peptide. A variety of eukaryotic-vector host systems may be used for expression of the chimeric, cloned DNA sequence and the resulting mRNA transcript. These include, but are not limited to mammalian cell systems infected with virus, vaccinia virus, adenovirus, etc.), transfected with other vectors, containing chromosomally integrated nucleic acids of the invention, etc., but the system used host cell must have the appropriate cellular machinery to process the prepro chimera in the

Mature neurotrophin. The expression elements of the vectors vary in their potencies and specificities. Depending on the host-vector system used, any of a number of suitable transcription and translation elements may be used.

Any of the previously described methods for inserting DNA fragments into a vector can be used to construct expression vectors containing a sequence encoding a chimeric, neurotrophic, prepro or prepro peptide consisting of the appropriate transcription / translation control signals upstream of the chimeric DNA sequences. These processes may include recombinant and synthetic DNA techniques in vitro and in vivo recombination (genetic recombination). Expression of nucleic acid sequences encoding chimeric, neurotrophic, prepro protein or prepro peptide can be regulated by a second nucleic acid sequence so that the chimeric, neurotrophic, prepro protein or prepro peptide is expressed in a host transformed with the molecule of recombinant DNA. For example, the expression may be controlled by any promoter / enhancer element known in the art to be active in mammalian cells. Promoters that can be used to control expression of chimeric neurotrophic factor include, but are not limited to, the cytomegalovirus (CMV) promoter, the SV40 early promoter region (Bernoist and chambom, 1981, Nature 290: 304-310). to the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), to the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad Sci USA 78: 1414-1445), to the regulatory sequences of the metallothionine gene (Brinster et al., 1982, Nature 296: 39-42) and to the following transcriptional control animal regions, which exhibit tissue specificity and were used in transgenic animals: control region of the elastase I gene that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, Cold Spring Harbor Symp., Quant. Biol. 5 (3: 399-409; MacDonald, 1987, Hepatology 2: 425-515), an insulin gene control region that is active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115 -122) region of 74 586 6526-056-118 -22-

control of the immunoglobulin gene that is active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538: Alexander et al., 1987, Mol. Cell: Biol. 7: 1436-1444) mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cell cells (Leder et al., 1986, Cell 45: 485-495) , albumin gene control region that is active in the liver (Pinkert et al., 1987, Genes and Devei. 1: 268-276), alpha-fetoprotein gene control region that is active in the liver (Krumlauf et al. al. 1985, Mol. Cell Biol. 5: 1639-1648: Hammer et al., 1987, Science 235: 53-581: alpha 1-antitrypsin gene control region that is active in the liver (Kelsey et al. al., 1987, Genes and Devei, 1: 161-171) beta globin gene control region that is active in myeloid cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al. 1986, Cell 46: 89-94; control region of the protein gene in the basic myelin that is active in oligodendrocytes in the brain (Readhead et al. f 1987, Cell 48: 703-712); a myosin light chain gene control region that is active in skeletal muscle (Sani, 1985, Nature 314: 283-286) and a control region of the release gonadotropic hormone gene that is active in the hypothalamus (Mason et al. 1986, Science 234: 1372-1378).

A specific example of an expression vector that can be used is CDM8 (Seed, 1987, Nature 329: 840-842: Seed and Aruffo, 1987, Proc.Natl.Ac..Sci.USA, 84: 3365-3369; Aruffo and Seed, Proc Natl Acad Sci USA 84: 8573-8577); another example may be pCMX (see co-pending application Serial No. 678,408, filed March 28, 1991).

Expression vectors containing chimeric neurotrophic prepro protein or prepro protein inserts can be identified by three general approaches: (a) DNA-DNA hybridization, (b) the presence or absence of marker gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a foreign gene inserted into an expression vector can be detected by DNA-DNA hybridization using probes comprising sequences that are homologous to at least one

The present invention relates to the preparation of a chimeric neurotrophic prepro protein or prepro peptide gene. In the second approach, the recombinant / host vector system can be identified and selected based on the presence or absence of certain gene functions &quot; markers &quot; (e.g., thymidine kinase activity, phenotype transformation, etc.) caused by the insertion of foreign genes into the vector. For example, if the coding sequence of the chimeric neurotrophic prepro protein or prepro peptide is inserted into the vector marker gene sequence, the recombinants containing the chimeric insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. These assays may be based, for example, on the physical or functional properties of the neurotrophic factor gene product in bioassay systems as described infra. in Section 5.4.

Once a particular recombinant DNA molecule has been identified and isolated, various methods known in the art can be used to propagate them. Once appropriate host systems and growth conditions have been established, recombinant expression vectors can be propagated and prepared in quantity. Expression from certain promoters may be elevated in the presence of certain inducers; thus, the expression of genetically engineered, chimeric, neuroprophilic prepro or peptide prepro protein can be controlled. In addition, different host cells have specific and characteristic mechanisms for translation and for the processing and modification of post-translational (e .q .. glycosation, cleavage) of proteins. Appropriate cell lines or host systems should be chosen so as to ensure the necessary processing and removal of the prepro region by cleavage and for any desired modification. Mammalian host cells, such as monkey, human or bovine, are thus preferred.

74 586 6526-056-118 -24-

In specific embodiments of the invention, DNA encoding chimeric neurotrophins may be expressed in a CHO cell system according to the procedures set forth below. Once a recombinant that expresses the chimeric neurotrophin is identified, the mature qene product should be analyzed. This may be achieved by tests based on the physical or functional properties of the product. See Section 5.4. below.

Once the mature neurotrophic factor protein or peptide has been identified, it can be isolated and purified by standard procedures, including chromatography (ion exchange chromatography, affinity and size separation), centrifugation, differential solubility or by any another common technique for purifying proteins.

5.4. NEUROTROPIC FACTOR TESTS

The neurotrophin proteins and peptides produced according to the invention are capable of exhibiting one or more biological activities, including, but not limited to, neurotrophic activity, binding by neurotrophin antibodies, binding to related receptors, and the like. The term &quot; neurotrophic activity &quot; as used herein should be construed as referring to a biological effect on cells of the nervous system, including, but not limited to, neurons, astrocytes, glial cells, oligodendrocytes, the microglia and Schwann cells . The biological effect is a change in the structure and / or physiology of a nerve cell that does not occur in the absence of direct or indirect exposure to the chimeric neurotrophic factor. Examples of biological effects are the prolongation of survival, neurite outgrowth, maintenance of the development of differentiated functions (such as the expression of an enzyme, echoline-acetyltransferase or tyrosine hydroxylase), or otherwise death or cell senescence or dedifferentiation. The presence of neurotrophic activity may be determined using any assay known for such activity as well as systems which may be developed in the future. 74 586 6526-056-118 Systems

tissue culture bioassay systems using tissue explants, cells prepared from immortalized tissues or cell lines, for example derived from the brain, spinal cord or peripheral nervous system, as well as in vivo test systems. in which the neurotrophic factor can be administered to an animal; neurotrophic effects may be detected in this animal by performing chemical, histological or behavioral tests using said animal. In addition, a neurotrophic factor can be incorporated as a transgene into a non-human transgenic animal and its biological epoetins can be measured in said animal.

For example, but not as a limitation, neurotrophic activity can be measured using any of the well known bioassay systems, as follows: (i) dorsal root ganglia assay system, as described in Barde et al., 1980, Proc. Natl. Acad. Know. USA. 77: 1199-1203, which is hereby incorporated by reference in its entirety; (ii) nodal ganglia assay system, as described by Lindsay et al., 1985, Dev. Biol. 112: 319-328. which is hereby incorporated by reference in its entirety; (iii) sympathetic ganglia assay as described by Barde et al., 1982, EMBO J., 1: 549-553, which is hereby incorporated by reference in its entirety; (iv) spinal-cord neurons. Briefly, the spinal cord can be removed aseptically from a test animal, cut caudal to the spinal bulb and released from sensory ganglia and meninges. The medulla may then be subdivided into ventral and mediodorsal segments for separate cultures, and the tissues cut into small pieces and dissociated by trituration through a Pasteur pipette in a 50 percent DMEM (Gibco) mixture and 50 percent nutrient mixture of Ham F12 74 586 6526-056-118 -26-

(Gibco) supplemented with 33 mM glucose, 2 mM glutamine, 15 mM NaHC03, 10 mM HEPES, 25 μg / ml insulin, 100 μg / ml transferrin, 60 μg putrescine, 20 nM progesterone, 30 μM Na selenite, 5 μg / ml penicillin G, 0.5 μg / ml streptomycin and 2.5 μg / ml bovine serum albumin. The grinding can then be repeated twice and the supernatants can be pooled and filtered through a 40 μ Tetko filter. The dissociated ventral cells can then be plated on a culture plate coated with poly-D-lysine (10 μg / ml) at a density of 0.5 million cells per 35 mm dish. Dissociated mediodorsal cells can be plated at a density of 1.5 million cells per 35 mm plate coated with poly-D-lysine (10 μg / ml), poly-L-ornithine (10 μg / ml) or poly-L -ornithine plus laminin (5 μg / ml). (v) baseline forebrain cholinergic neuron assays (see PCT Publication WO 91/03568, published March 21, 1991); (vi) testing of ventricular mesencephalic dopaminergic neurons (see PCT Publication WO 91/03568, published March 21, 1991; and (vii) PC12 cell assays.

6. EXAMPLE: CONSTRUCTION AND EXPRESSION OF NGF-PREPRO / BDNF MADURO CHIMMER

6.1. CONSTRUCTION OF CHEMICAL NUCLEIC ACID MOLECULES USING POLYMERASE CHAIN REACTION

Polymerase chain reaction cloning (PCR; Saiki et al., 1985, Science 230: 1350-1354) was used for the construction of a mature NGF / BDNF prepro chimera consisting of the long prepro form of mouse NGF fused to mature human BDNF sequence. 74 586 6526-056-118 -27-

PCR. The 5 'primer (5'-CTC-GTC-GAC-CGC-CGC-CAC-TCT-GAC-CCT-GCG-CGC-CGC-CGA-3') [SEQ ID Na17] encoded the first 7 amino acids of BDNF including two sites NaEL and BssH2, which were generated by modifying the use of the codons. The 3 'primer was a 3' pCDM8 oligo corresponding to a region downstream of the polylinker sequence at the 3 'end of the BDNF sequence in pC8hB (5'-CAA-AGA-TCC-TCT-AGA-GTC-G (C ) -3 ') [SEQ ID Na18]. The polylinker contains a NotI restriction site. These two primers were used in PCR with pC8hB DNA (hBDNF in pCDM8) as template. Five micrograms of pCShB were used with 500 ng of each of the primers for 5 cycles of PCR. The PCR product was digested with Nael and Notl simultaneously and a 365 bp digestion product was isolated by gel electrophoresis. The vector preparation was performed by digesting pC81mN (mouse NGF, long in pCDM8) with Eco47 and NotI and isolating the 4.6 kb vector fragment by gel electrophoresis. The 365 bp fragment was ligated into the Eco47 / NotI sites of pC81 mM. This binding resulted in a direct fusion within the prepro region of the mouse NGF with the mature BDNF coding region. The constructs were diagnostically tested by digestion with BssH2, evaluating the loss of the Eco47 site during subcloning and, finally, by DNA sequencing.

6.2.CHECKING OF CHEMICAL MOLECULES

CHO-DG44 cells were used to generate stable lines for the production of bioactive BDNF. CHO-DG44 cells (obtained from Dr. L. Chasin of Columbia University) do not have both copies of the dihydrofolate reductase gene (Urlaub and Chasin, 1980, Proc. Natl Acad Sci USA 77: 4216-4220). Stably transfected CHO-DG44 cell lines expressing BDNF were previously described (PCT International Publication WO 91/03568, published March 21, 1991). These lines were generated by transfection with pC8hB DNA encoding the human BDNF gene including the prepro region cloned into the pCDM8 expression vector. CHO-DG44 cells (1 x 106 cells / 100 mm dish) were transfected by the coprecipitation process with calcium phosphate with 20 μg of the NGF / BDNF chimera (pC81mN / B) together with 0.2 μg of plasmid p410 encoding a weakened dihydrofolate reductase (dhfr) gene. Forty-eight hours post-transfection, the cells were passed through selection media (Ham's F12 without hypoxanthine and thymidine containing 10% dialyzed fetal bovine serum and 1% each of penicillin and streptomycin; The HTH-resistant clones were treated as pools for methotrexate (MTX) amplification. Clones obtained with 0.05 μM MTX were also treated as pools for further amplification with 2.5 μM MTX. A single clone, selected first in 0.05 μM MTX and, by deposition, in 2.5 μ MT MTX (hence two amplification series) (DGC-N / B-2.5 # 23), which proved to be the largest producer of BDNF as assessed by bioactivity and metabolic labeling. The bioactivity was assessed by classifying the outward growth of embryonic chick dorsal root ganglia (DRG) neurites (E8) (Maisonpierre et al., 1990, Science 247: 1446-1451).

7. EXAMPLE: COMPARISON OF PROCESSING EFFICIENCY BETWEEN BDNF PREPRO. HOMOLOGO. AND THE CHEMERA NGF PREPRO / BDNF

Experiments were performed for the direct comparison of the processing and expression of prepro BDNF with the NGF prepro / BDNF chimera in CHO cells.

7.1 METABOLIC MARKING

CHO-DG44 cells stably transfected with pC8hD or pC81mN / B and amplified with 2.5 μM methotrexate were compared by metabolic labeling (Figure 1). For this labeling experiment, BDNF-expressing CHO-DG44 cells from the short preprocedure BDNF construct (cell line = DGZ1000-B-3-2.5) or from the prepro long NGF chimeric construct from mouse / BDNF (cell line = DGC-N /B-2.5-#23) were seeded at equal densities (2x105 cells / well in 6-well plate) 24 hours prior to labeling. Cells were then labeled with 35 S-cysteine and 35 S-methionine for 4 hours under serum-free conditions. Aliquots of 30 μΐ of

Supernatants from cells labeled by SDS polyacrylamide gel electrophoresis (15% gel) and the labeled proteins were transferred to nylon membranes and visualized by autoradiography. As seen in Figure 1, CHO-DG44 cells stably transfected with the human NGF gene in the pCDM8 expression vector expressed the mature form of NGF migrating to a molecular mass of approximately 12,300 (Figure 1, lane 2). Wild-type CHO-DG44 cells are shown in lane 1 as a control. Unprocessed proBDNF (31 kD), the propro portion of the processed proBDNF precursor (16 kD) and the mature form (14 kD) were preprocessed short BDNF protein in the stably transfected cell line DGZ1000-B-3-2.5 (obtained after similar selection in MTX and amplification as used for the DGC-N / B-2.5- # 23 cell line (Figure 1, lane 3). Only the mature, proteolytically processed form of BDNF (14 kD) in DGC-N / B-2.5- # 23, stably transfected with the long proNGF / BDNF chimeric construct (Figure 1, lane 4) was detected. No proNGF / BDNF was detected in the conditioned media of this cell line. We calculated, from the strength of mature BDNF labeling, that the DGC-N / B-2.5- # 23 cell line produces about five (5) fold more mature BDNF protein per cell relative to the DGZ1000-3-2.5 cell line made with short proBDNF construction.

7.2 Bioactivity

The bioactivity of the BDNF produced in the two CHO cell lines described above was compared. Crude supernatants were assayed with embryonic dorsal root ganglia (E8) and outgrowth of neurites was recorded. Consistent with metabolic labeling experiments, the DGC-N / B-2.5- # 23 cell line appeared to produce approximately five (5) times more mature BDNF relative to the DGZ1000-B-3-2.5 cell line. For example, maximum neurite outward growth was achieved with 10 μΐ of DGC-N / B-2.5- # 23 cell derived supernatant whereas 50 μΐ of DGZ1000-B-3-2.5 supernatant was required to achieve the maximal bioactivity of DRG (Figure 2). 74 586 6526-056-118

• sP &quot; ' 7.3

COMPARISON OF NGF EXPRESSION USING NGF PREPRO REGIONS COMPLETED AND SHORT

COS cells were transfected with prepro NGF containing the long region (1mNGFH) or short (&quot; smNGF &quot;) of NGF prepro with the mature NGF coding region. Culture supernatants were harvested 48 hours post-transfection and assayed in DRG explants, together with purified NGF and a supernatant of falsely transfected COS cells. The results using three different concentrations of each construct, as shown in Table 1, show significant bioactivity of NGF expressed in any of the short and long forms of the prepro region. TABLE 1

Effect of Various COS Cell Supernatants on DRG Explants SAMPLE DILUTION DRG (-) CONTROL NGF io ng / ml 0, 0, 0.55 + 5 + 5 + 5 + 5 + FALSE 10 0.5, 1, 1, 1, 250, 2, 2.5, 2, 1, 1, 1, 5, 2.5, 2.5 smNGF 10 μΐ 2, 3, 3, 3, 3.5 50 μΐ 5, 5, 5, 5 100 μΐ 5+, 5+, 5+, 5+, 5+ 250 μΐ 5+, 5+, 5+, 5+, 5+ lmNGF 10 μΐ 2, 2, 2, 2, 2 50 μΐ 4, 4, 4, 4, 4 100 μΐ 5, 5, 5, 5 250 μΐ 5, 5, 5, 5, 5 74 586

6526-056-118 -31-

7.4.CONCLUSIONS

It is concluded from these studies that the pro long portion of NGF is better suited for the processing of BDNF in CHO cells than the short pro portion of BDNF. The advantages of the mature proNGF / BDNF chimeric gene construct are to allow higher levels of BDNF expression on a per cell basis in mammalian cells. Additionally, it should allow better purification schemes for BDNF as no contaminant forms of unprocessed BDNF appear in the crude supernatants.

In addition, the use of the long or short prepro region of NGF results in the expression of biologically active NGF. This indicates that the long or short prepro region of NGF can be used in the construction of chimeric neurotrophic genes.

8. EXAMPLE: CONSTRUCTION AND EXPRESSION OF THE NT-3 PREPRO / BDNF CHIMERA

8.1. CONSTRUCTION OF CHEMICAL NUCLEIC ACID MOLECULES

A HindIII-XhoI DNA fragment containing the entire coding region of human prepro and mature BDNF was obtained from digestion of plasmid pC8hB with the corresponding restriction enzymes. Plasmid pC8hB was derived by cloning the human BDNF coding sequences, including the entire prepro region, into the pCDM8 expression vector (discussed supra). this fragment was ligated into pDSRa2 (see European Published Patent Application 90305433.6, EPO Publication 0398753A2, incorporated herein by reference in its entirety. Plasmid pDSRa2 had been pre-digested to make available the cloning sites δ'-ΗΐηάΙΙΙ and 3 / Sal I for attachment of the human BDNF containing fragment. The resulting plasmid was designated pDSRα2 (BDNF).

For generation of a chimeric plasmid with a prepro NT-3 sequence and a mature BDNF sequence, three DNA fragments were prepared as follows, and then ligated in a specific orientation. A 5'-HindIII / 3 / -NarI DNA fragment of 74 586 6526-056-118 -32-

approximately 400 bp containing the entire human prepro BDNF sequence was deleted by restriction enzyme digestion from the pDSRa2 expression plasmid (BDNF) described above. A DNA fragment recovered from this digestion contained the entire pDSRα2 expression vector and the mature human BDNF sequence, bounded by the 5'-HindIII and 3'-NariI sites (Nal labeled DNA fragment). A δ'-ΗίηάΙΙΙ / ''-SacII fragment of approximately 300 base pairs containing the prepro region of human NT-3 was obtained by digestion of the plasmid pC8hN3 with the corresponding restriction enzymes. Coding sequences, corresponding to 35 amino acid residues from the prepro region of NT-3, were deleted downstream of the SacII site as a consequence of digestion. Plasmid pC8hN3 was derived by cloning the human NT-3 coding sequences, including the entire prepro region, into the pCDM8 expression vector. The 300 bp 5-HindIII / 3 / -SacII fragment was labeled Na2 DNA fragment. Finally, the Na3 DNA fragment, which was an oligonucleotide linker synthesized to regenerate the previously missing amino acid residue, was prepared (Figure 6 and SEQ ID NO: 15-17). The linker also contained the half sites of the 5'-SacII and 3'-Narl restriction sites to promote binding to the N8s 1 and 2 DNA fragments described supra. This binding resulted in the expression vector pDSRa2 (NT-3 / BDNF), wherein the NT-3 prepro region (Ns2 fragment) is bound to the mature BDNF (Nal fragment) by the oligonucleotide linker (Na3 fragment; Figure 6 and SEQ ID NO 15).

8.2. EXPRESSION AND CHARACTERIZATION OF NT-3 / BDNF CHEMERA IN CHO CELLS

CHO-D (-) cells (ATCC accession number CCL 61) were used to generate stable lines for bioactive BDNF production. CHO-D (-) cells lack the gene encoding dihydrofolate reductase and are maintained in Dulbecco's modified Eagle's medium (D-MEM), supplemented with MEM non-essential amino acids, 1% each of penicillin and streptomycin, 10% fetal bovine serum, hypoxanthine and thymidine. CHO-D (-) cells (0.8 x 106 / plate 74 586 6526-056-118)

calcium phosphate using 2.5 μg of the NT-3 / BDNF chimeric construct [pDSRα2 (NT-3 / BDNF)] pre-linearized by restriction enzyme digestion Pvul. This vector (pDSRa2) encodes a mouse dihydrofolate reductase (dhfr) minigene which, when expressed, allows transfected CHO - (-) cells to overcome deficiency in the dhfr gene and become capable of growing in the absence of the nucleotides hypoxanthine and thymidine. The progenitor CHO-D (-) cells or cells unsuccessfully transfected by the vector pDSRa2 will not survive in the selection media, which has the maintenance medium composition described above, except that the fetal bovine serum is replaced with serum of bovine fetal dialysate and hypoxanthine and thymidine are omitted. Cells were trypsinized and seeded 48 hours post-transfection at 1 x 10 5 cells / 100 mm dish in selection media. Individual colonies were collected two weeks later using cloning cylinders. Each clone was expanded into 100 mm plates. When cultures reached confluency, the original serum-containing media were aspirated and replaced with 3 ml of serum-free media. The conditioned media (CM) was collected and loaded 50 μΐ each onto a 15% SDS-polyacrylamide gel and subjected to gel electrophoresis. Western blotting of the gel was performed with rabbit antiserum specific for mature BDNF. As shown in Figure 7, all clones expressing the original BDNF from pDSRa2 (BDNF) secreted multiple forms of unprocessed BDNF, in addition to the mature, processed form of BDNF. The ratio of unprocessed forms to processed form was about 2: 1. In contrast, all clones expressing chimeric NT-3 / BDNF from pDSRa2 (BDNF) secreted only the fully processed, mature form of BDNF without detectable partially processed precursors.

One liter of serum-free conditioned media from one of the chimeric NT-3 / BDNF clones was subjected to purification by passage through an S-Sepharose column followed by a Sephacryl S-200 size exclusion column. The analysis by

SDS-PAGE and determination of the amino acid sequence m and a homogeneous protein having a molecular weight of 14 kD (as predicted for mature human BDNF) had been obtained with an N sequence terminal sequence according to the N-terminal sequence of mature human BDNF. In addition, it was demonstrated by pinto dorsal root ganglia assay (described for chimeric NT-3 / BDNF, supra) that purified BDNF had complete biological activity.

8.3. EXPRESSION IN COS-BIOACTIVIDIDES OF BACTERIUM NT-3 / BDNF

COS-7 cells (ATCC accession number CRL 1651) were used as the transient expression system to test the production of bioactive BDNF. COS-7 cells are routinely maintained in D-MEM with 10% fetal bovine serum and 1% of the antibiotics penicillin and streptomycin. COS-7 cells (5x106 cells / ml) were transfected by electroporation at 1600 volts for 0.4 ms with individually 20 μg of each of pDSRa2, pDSRa2 (BDNF) and pDSRa2 (NT-3 / BDNF). Transfected COS-7 cells were plated at 2x106 cells / 60mm dish. Conditioned media was collected between 24 and 72 hours post-transfection. Bioactivity was assessed by grading the outgrowth of pinus dorsal root ganglia (E8) neurites (such as for the NGF / BDNF chimera). As shown in Table 2, clonal isolates Cl 1 and Cl 20 of chimeric pDSRa2 (NT-3 / BDNF) were approximately 5-fold more active than mature BDNF expressed from pDSRa2 (BDNF), the latter containing unprocessed pre-BDNF region. (SECTION TABLE 2)

74 586 6526-056-118 TABLE 2

Pinto DRG Explants Assay of Media Conditioned from COS Cells Transfected with Plasmid DNA 1- 1 DNA FOUND -Γ 1 Volume of 1-, Graduation of the 1 1 Med. to the outside | 1 I μ, Ι | of neuritis 1 1 | pDSRa2 1 1 10 o O ** o o 1 1 50 10, 0, 0.5, 0.5 | I 1 | pDSRa2 (BDNF) 1 1 10 1 1 | 0, 0.5, 0.5, 0.5, 0.5 | 1 I 50 | 1, 1/1 / 1/5, 1/5 | I 1 pDSRa 2 (NT-3 / BDNF), 1 IC 2 | 10 1 1 | 1, 1, 1/5, 1,5, 1,5 | 1 I 50 2.5, 2.5, 2.5, 2, 2 | | pDSRa2 (NT-3 / BDNF), 1 IC 20 | 10 1 1 | 1/1/2, 2, 2 | 1 1 50 50 2.5, 2.5, 2.5, 2.5, 2.5 J 11

8.4. CONCLUSIONS

These studies demonstrate that the substitution of the prepro region of BDNF with the prepro region of NT-3 facilitates proteolytic processing of the prepro region and significantly increases the net yield of mature BDNF. In addition, the cleavage site reconstituted between the prepro NT-3 and mature BDNF DNA sequences was accurately recognized by the host cell without any change in the NH 2 terminus of the mature, processed BDNF. As with the chimeric NGF / BDNF gene construct, the chimeric NT-3 / BDNF gene construct results in higher levels of BDNF processed at the base of a cell in mammalian cells and this should also allow for better purification schemes by eliminating and minimizing unprocessed contaminant forms.

The present invention is not to be limited in scope by the specific embodiments described herein. In fact, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying figures. These modifications are intended to fall within the scope of the appended claims. Various publications and patent applications are cited herein, and their disclosures are incorporated by reference in their entirety.

Lisbon, 13. 1992

By RE6ENER0N PHARMACEUTICALS, INC. and AMGEN, INC. = 0 FACTORY AGENT =

Claims (50)

Chimeric prepro protein or prepro peptide, characterized by comprising (a) a prepro region of a first neurotrophin and (b) an amino acid sequence substantially equivalent to the mature form of a second neurotrophin , wherein the first and second neurotrophins are different. Chimeric prepro protein or prepro peptide, characterized by comprising (a) a prepro region of a first neurotrophin and (b) a biologically active amino acid sequence substantially equivalent to a portion of the mature form of a second neurotrophin, wherein the first and second neurotrophins are different. The chimeric prepro protein according to claim 1, characterized in that the first and second neurotrophins are selected from the group consisting of nerve growth factor, brain derived neurotrophic factor and neurotrophin-3, The chimeric prepro protein according to claim 2 wherein the first and second neurotrophins are selected from the group consisting of nerve growth factor, brain derived neurotrophic factor and neurotrophin-3. The chimeric prepro or prepro peptide protein according to claim 1, characterized in that the prepro region is the long prepro region of nerve growth factor. The chimeric prepro or prepro peptide protein according to claim 2, characterized in that the prepro region is the long prepro region of nerve growth factor. Chimeric prepro protein or prepro peptide according to claim 1, characterized in that the prepro region is at 74 586 6526-056-118 -2 short prepro region of nerve growth factor Chimeric prepro or prepro peptide protein according to claim 2, characterized in that the prepro region is the short prepro region of nerve growth factor. Chimeric prepro protein or prepro peptide according to claim 1, characterized in that the first neurotrophin is neurotrophin-3. Chimeric prepro or prepro peptide protein according to claim 2, characterized in that the first neurotrophin is neurotrophin-3. Chimeric prepro or prepro peptide protein according to claim 1, characterized in that the first neurotrophin is brain derived neurotrophic factor. Chimeric prepro or prepro peptide protein according to claim 2, characterized in that the first neurotrophin is a neurotrophic factor derived from the brain. Chimeric prepro or prepro peptide protein according to claim 5 or 6, characterized in that the second neurotrophin is neurotrophic factor derived from the brain. Chimeric prepro or prepro peptide protein according to claim 7 or 8, characterized in that the second neurotrophin is brain derived neurotrophic factor. Chimeric prepro or prepro peptide protein according to claim 9 or 10, characterized in that the second neurotrophin is brain derived neurotrophic factor. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 1. 74 586 6526-056-118 -3- A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 2. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 3. 19. A nucleic acid molecule comprising a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 4. 20. A nucleic acid molecule comprising a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 5. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 6. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 7. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 8. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 9. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 10. A nucleic acid molecule characterized in that it comprises a nucleotide sequence encoding the protein prepro or the prepro peptide of claim 11. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 12. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 13. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 14. A nucleic acid molecule which comprises a nucleotide sequence encoding the chimeric prepro protein or prepro peptide of claim 15. A nucleic acid molecule according to claim 16 or 17, characterized in that it is a vector. A nucleic acid molecule according to claim 18 or 19, characterized in that it is a vector. Recombinant cell characterized in that it contains the vector of claim 31. A recombinant cell characterized in that it contains the vector of claim 32. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 1 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the mature form of the second neurotrophin. A process for the production of a neurotrophin characterized Comprising constructing a recombinant nucleic acid molecule cell of claim 2 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the portion of the mature form of the second neurotrophin. A process for producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 3 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the mature form gives the second neurotrophin. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 4 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the portion of the mature form of the second neurotrophin. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 5 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the mature form of the second neurotrophin. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 6 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the portion of the mature form of the second neurotrophin. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 7 under conditions such that the chimeric prepro protein or peptide is expressed and 74 586 cc; SW v ....., &gt; Processed by the cell so as to produce the mature form of the second neurotrophin. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 8 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the portion of the mature form of the second neurotrophin. A process for producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 9 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the mature form of the second neurotrophin. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 10 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the portion of the mature form of the second neurotrophin. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 11 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the mature form of the second neurotrophin. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 12 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the portion of the mature form of the second neurotrophin. 74 586 6526-056-118 A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 13 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the mature form of the second neurotrophin. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 14 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the mature form of the second neurotrophin. A method of producing a neurotrophin characterized by comprising culturing a recombinant cell containing the nucleic acid molecule of claim 15 under conditions such that the chimeric prepro protein or peptide is expressed and processed by the cell so as to produce the mature form of the second neurotrophin. A method according to claim 35 or 36 wherein the mature form of the second neurotrophin or portion thereof is capable of exhibiting neurotrophic activity. Lisbon, 13. By REGENERON PHARMACEUTICALS, INC. and AMGEN, INC. = 0 FACTORY AGENT =