Growth factors nsg28, nsg30, and nsg32

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from Danish Application No. PA 2004 01162, filed Jul. 30, 2004, Danish Application No. PA 2005 00034, filed Jan. 7, 2005, Danish Application No. PA 2004 01164, filed Jul. 30, 2004, Danish Application No. PA 2005 00036, filed Jan. 7, 2005, Danish Application No. PA 2004 01166, filed Jul. 30, 2004, and Danish Application No. PA 2005 00038, filed Jan. 7, 2005. The entire content of each of these prior applications is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to the field of therapeutic use of proteins, genes and cells, in particular to the therapy based on the biological function of secreted therapeutic proteins, NsG28, NsG30, and NsG32 in particular for the treatment of disorders of the nervous system. The invention also relates to bioactive NsG28, NsG30, and NsG32 polypeptide fragments and the corresponding encoding DNA sequences.

BACKGROUND ART

Extracellular proteins play important roles in, among other things, the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, e.g., growth, proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the immediate environment. This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or signalling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment.

Disorders such as Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple and amyotrophic lateral sclerosis, stroke, schizophrenia, epilepsy and peripheral neuropathy and associated pain affect millions of people. It is the loss of normal neuronal function, which produces the behavioral and physical deficits which are characteristic of each of the different neurological disorders. In addition to chronic and acute neurodegenerative disorders, the aging process, physical trauma to the nervous system, and metabolic disorders may result in the loss, dysfunction, or degeneration of neural cells accompanied by the associated behavioral and physical deficits. Many of these diseases are today incurable, highly debilitating, and traditional drug therapies often fail. There is thus a great medical need for new therapeutic proteins that are disease modifying and not only for symptomatic use.

Several secreted factors with expression in the nervous system or associated target areas have important therapeutic uses in various neurological indications associated with reduction or loss of neuronal functions. E.g. NGF is a candidate for treatment of Alzheimer's disease, Neublastin (Artemin) a candidate for treatment of peripheral neuropathy, and GDNF is a candidate for treatment of Parkinson's Disease.

SUMMARY OF THE INVENTION

The present invention relates to various aspects and uses of Nerve Survival and Growth factor 28 (NsG28), Nerve Survival and Growth Factor 30 (NsG30), and Nerve Survival and Growth factor 32 (NsG32).

In a first aspect the invention relates to an isolated polypeptide for medical use, said polypeptide comprising an amino acid sequence selected from the group consisting of:

a) the amino acid sequence selected from the group consisting of SEQ ID No. 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, and 16 (or a subgroup thereof);

b) a sequence variant of the amino acid sequence selected from the group consisting of SEQ ID No. 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, and 16 (or a subgroup thereof), wherein the variant has at least 70% sequence identity to said SEQ ID No.; and

c) a biologically active fragment of at least 50 contiguous amino acids of any of a) through b).

The invention also relates to an isolated polypeptide for medical use, said polypeptide comprising an amino acid sequence selected from the group consisting of:

a) the amino acid sequence selected from the group consisting of SEQ ID No. 18, 19, 20, 21, 23, 24, and 25 (or a subgroup thereof);

b) a sequence variant of the amino acid sequence selected from the group consisting of SEQ ID No. 18, 19, 20, 21, 23, 24, and 25 (or a subgroup thereof), wherein the variant has at least 70% sequence identity to said SEQ ID No.; and

c) a biologically active fragment of at least 50 contiguous amino acids of any of a) through b).

The invention also relates to an isolated polypeptide for medical use, said polypeptide comprising an amino acid sequence selected from the group consisting of:

a) the amino acid sequence selected from the group consisting of SEQ ID No. 27, 28, 29, 31, 32, 33, 34, 36, 38, 40, and 41 (or a subgroup thereof);

b) a sequence variant of the amino acid sequence selected from the group consisting of SEQ ID No. 27, 28, 29, 31, 32, 33, 34, 36, 38, 40, and 41 (or a subgroup thereof), wherein the variant has at least 70% sequence identity to said SEQ ID No.; and

c) a biologically active fragment of at least 50 contiguous amino acids of any of a) through b).

In a further aspect, the invention relates to an isolated nucleic acid molecule for medical use comprising a nucleotide sequence selected from the group consisting of:

a) a nucleotide sequence coding for a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID No. 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, and 16 (or a subgroup thereof);

b) a nucleotide sequence coding for a sequence variant of the amino acid sequence selected from the group consisting of SEQ ID No. 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, and 16 (or a subgroup thereof), wherein the variant has at least 70% sequence identity to said SEQ ID No.;

c) a nucleotide sequence coding for a biologically active fragment of at least 50 contiguous amino acids of any of a) through b);

d) a nucleotide sequence selected from the group consisting of SEQ ID No. 2 and 8;

e) a nucleotide sequence having at least 70% sequence identity to a coding sequence selected from the group consisting of the coding sequence of SEQ ID No. 2 and 8;

f) a nucleic acid sequence of at least 150 contiguous nucleotides of a coding sequence selected from the group consisting of the coding sequence of SEQ ID No. 2 and 8;

g) the complement of a nucleic acid capable of hybridising with a nucleic acid molecule having the sequence of the coding sequence of SEQ ID No.: 2 and 8 under conditions of high stringency;

h) the nucleic acid sequence of the complement of any of the above.

The invention also relates to an isolated nucleic acid molecule for medical use comprising a nucleotide sequence selected from the group consisting of:

a) a nucleotide sequence coding for a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID No. 18, 19, 20, 21, 23, 24, and 25 (or a subgroup thereof);

b) a nucleotide sequence coding for a sequence variant of the amino acid sequence selected from the group consisting of SEQ ID No. 18, 19, 20, 21, 23, 24, and 25 (or a subgroup thereof), wherein the variant has at least 70% sequence identity to said SEQ ID No.;

c) a nucleotide sequence coding for a biologically active fragment of at least 50 contiguous amino acids of any of a) through b);

d) a nucleotide sequence selected from the group consisting of SEQ ID No. 17 and 22;

e) a nucleotide sequence having at least 70% sequence identity to a coding sequence selected from the group consisting of the coding sequence of SEQ ID No. 17 and 22;

f) a nucleic acid sequence of at least 150 contiguous nucleotides of a coding sequence selected from the group consisting of the coding sequence of SEQ ID No. 17 and 22;

g) the complement of a nucleic acid capable of hybridising with a nucleic acid molecule having the sequence of the coding sequence of SEQ ID No.: 17 and 22 under conditions of high stringency;

h) the nucleic acid sequence of the complement of any of the above.

The invention also relates to an isolated nucleic acid molecule for medical use comprising a nucleotide sequence selected from the group consisting of:

a) a nucleotide sequence coding for a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID No. 27, 28, 29, 31, 32, 33, 34, 36, 38, 40, and 41 (or a subgroup thereof);

b) a nucleotide sequence coding for a sequence variant of the amino acid sequence selected from the group consisting of SEQ ID No. 27, 28, 29, 31, 32, 33, 34, 36, 38, 40, and 41 (or a subgroup thereof), wherein the variant has at least 70% sequence identity to said SEQ ID No.;

c) a nucleotide sequence coding for a biologically active fragment of at least 50 contiguous amino acids of any of a) through b);

d) a nucleotide sequence selected from the group consisting of SEQ ID No. 26, 30, 35, 37, and 39 (or a subgroup thereof);

e) a nucleotide sequence having at least 70% sequence identity to a coding sequence selected from the group consisting of the coding sequence of SEQ ID No. 26, 30, 35, 37, and 39 (or a subgroup thereof);

f) a nucleic acid sequence of at least 150 contiguous nucleotides of a coding sequence selected from the group consisting of the coding sequence of SEQ ID No. 26, 30, 35, 37, and 39 (or a subgroup thereof);

g) the complement of a nucleic acid capable of hybridising with a nucleic acid molecule having the sequence of the coding sequence of SEQ ID No.: 26, 30, 35, 37, or 39 under conditions of high stringency; and

h) the nucleic acid sequence of the complement of any of the above.

In a further aspect, the invention relates to a vector comprising a nucleic acid molecule of any of the invention.

In a further aspect the invention relates to an isolated host cell transformed or transduced with a vector of the invention.

In a further aspect the invention relates to a packaging cell line capable of producing an infective virus particle, said virus particle comprising a Retroviridae derived genome comprising a 5′ retroviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide sequence encoding a polypeptide of the invention, an origin of second strand DNA synthesis, and a 3′ retroviral LTR.

In a further aspect the invention relates to an implantable biocompatible cell device, the device comprising:

i) a semipermeable membrane permitting the diffusion of a polypeptide or a virus vector of the invention; and

ii) a composition of cells according to the invention or a packaging cell line according to the invention.

In a further aspect, there is provided a pharmaceutical composition comprising

i) a polypeptide of the invention; or

ii) an isolated nucleic acid sequence of the invention; or

iii) an expression vector of the invention; or

iv) a composition of host cells according to the invention; or

v) a packaging cell line according to the invention; or

vi) an implantable biocompatible cell device according to the invention; and

vii) a pharmaceutically acceptable carrier.

In a further aspect, the invention relates to the use of

i) a polypeptide of the invention; or

ii) an isolated nucleic acid sequence of the invention; or

iii) an expression vector of the invention; or

iv) a composition of host cells according to the invention;

v) an implantable biocompatible cell device according to the invention; or

vi) a packaging cell line according of the invention;

for the manufacture of a medicament.

Furthermore, the invention relates to a method of treatment of a pathological condition in a subject comprising administering to an individual in need thereof a therapeutically effective amount of:

i) a polypeptide of the invention; or

ii) an isolated nucleic acid sequence of the invention; or

iii) an expression vector of the invention; or

iv) a composition of host cells according to the invention; or

v) an implantable biocompatible cell device according to the invention; or

vi) a packaging cell line according to the invention.

In a further aspect, the invention relates to the use of

i) a polypeptide of the invention; or

ii) an isolated nucleic acid sequence of the invention; or

iii) an expression vector of the invention; or

iv) a composition of host cells according to the invention; or

v) an implantable biocompatible cell device according to the invention;

as a male contraceptive.

In a further aspect the invention relates to a method of expanding a composition of mammalian cells, comprising administering to said composition the polypeptide of the invention; or transducing/transfecting the cells with the expression vector of the invention.

In a still further aspect, the invention relates to a method of differentiating a composition of mammalian cells, comprising administering to said composition the polypeptide of the invention; or transducing/transfecting the cells with the expression vector of the invention.

The invention also relates to an antibody capable of binding to a polypeptide of the invention.

Furthermore, the invention relates to an immunoconjugate comprising the antibody of the invention and a conjugate selected from the group consisting of: a cytotoxic agent such as a chemotherapeutic agent, a toxin, or a radioactive isotope; a member of a specific binding pair, such as avidin or streptavidin or an antigen; an enzyme capable of producing a detectable product.

The invention also provides an isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No. 6, 11, and 15 (or a subgroup thereof), and variants of said polypeptides, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15 of the amino acid residues in the sequence are so changed.

The invention also provides an isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No 7, 12, and 16 (or a subgroup thereof), and variants of said polypeptides, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15 of the amino acid residues in the sequence are so changed.

Furthermore, the invention relates to an isolated polypeptide having the amino acid sequence of SEQ ID No. 20, and variants of said polypeptide, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15 of the amino acid residues in the sequence are so changed.

The invention, in a further aspect relates to an isolated polypeptide having the amino acid sequence of SEQ ID No 21, and variants of said polypeptides, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15 of the amino acid residues in the sequence are so changed.

In a further aspect, the invention relates to an isolated polypeptide having an amino acid sequence of SEQ ID No. 33 and variants of said polypeptide, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 10 of the amino acid residues in the sequence are so changed.

In a further aspect, the invention relates to an isolated polypeptide having an amino acid sequence of SEQ ID No 34, and variants of said polypeptide, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 10 of the amino acid residues in the sequence are so changed.

Furthermore, the invention provides an isolated polynucleotide coding for a polypeptide described herein.

In a further aspect, the invention relates to a method of preventing apoptosis in a neuronal cell by contacting a neuronal cell with an affective amount of a polypeptide, nucleic acid, or vector described herein.

The invention relates to a method of treating a disorder characterized by neuronal apoptosis by administering to a subject (e.g., a human) having a disorder characterized by neuronal apoptosis a therapeutically affective amount of a polypeptide, nucleic acid, or vector described herein.

FIGURES

FIGS. 1A-1B. Clustal W (1.82) alignment of the mature human and mouse (_m) peptides of the growth factor family, Cys10. In the alignments, conserved regions are shown in bold and variable regions in grey. FIG. 1A shows an alignment based on the four most similar members of the family. FIG. 1B shows an alignment based on all five members of the family of growth factors.

FIG. 1C shows a phylogenetic tree of the human protein family. The tree was generated by using Multi-Way Alignment (Align Plus 5 version 5.03, Scientific and Educational Software) and BLOSUM62 as scoring Matrix. The branch length is proportional to the difference between the sequences. The corresponding full length protein sequences are shown in Table 1.

FIGS. 2A-2B. Output from signal peptide prediction server SignalP v.2.0 for human, NsG28 (SEQ ID No. 4). Graphs shown for neural network based method (panel A) and hidden Markov model method (panel B).

FIG. 3. Prediction output from the ProtFun 2.1 protein function prediction algorithm for human, mouse, and rat NsG28 (SEQ ID No. 4, 9, and 13).

FIGS. 4A-4B. Output for human NsG30 (SEQ ID No. 18) from signal peptide prediction server SignalP v.2.0. Graphs shown for neural network based method (panel A) and hidden Markov model method (panel B).

FIG. 5. Prediction output from the ProtFun 2.1 protein function prediction algorithm for human and mouse NsG30 (SEQ ID No 18 and 23). Odds above 1 are shown in bold.

FIG. 6. Prediction of start codon using Human NsG32a cDNA. In the lines below the sequence, the predicted start codon is indicated by the letter “i” (initiation), other instances of “ATG” by the letter “N” (non-start). The dots (“.”) are placeholders for all the other sequence elements.

FIG. 7. Prediction of start codon using Mouse NsG32a cDNA. In the lines below the sequence, the predicted start codon is indicated by the letter “i” (initiation), other instances of “ATG” by the letter “N” (non-start). The dots (“.”) are placeholders for all the other sequence elements.

FIGS. 8A-8B. Output for human NsG32a (SEQ ID NO:27) from signal peptide prediction server SignalP v.2.0. Graphs shown for neural network based method (panel A) and hidden Markov model method (panel B).

FIGS. 9A-9B. Output for human NsG32b (SEQ ID No. 31) from signal peptide prediction server SignalP v.2.0. Graphs shown for neural network based method (panel A) and hidden Markov model method (panel B).

FIG. 10. Prediction output from the ProtFun 2.1 protein function prediction algorithm for human NsG32a (SEQ ID No 27 and 28) and NsG32b (SEQ ID No 31).

FIG. 11. Clustal W (1.82) alignment of human NsG28a (SEQ ID No 4) to mouse, (SEQ ID No 9) and rat (SEQ ID No 13) full length polypeptides.

FIG. 12. Clustal W (1.82) alignment of human NsG30 (SEQ ID No. 18) to mouse NsG30 (SEQ ID No 23) and partial rat NsG30 (SEQ ID No. 25). The signal sequence is shown in bold.

FIG. 13A. Clustal W (1.82) alignment of human NsG32a (SEQ ID No. 27) to mouse NsG32a (SEQ ID No 36), and rat NsG32a (SEQ ID No. 41). The signal sequence is shown in bold.

FIG. 13B. Clustal W (1.82) alignment of human NsG32b (SEQ ID No. 31) to mouse NsG32b (SEQ ID No 39). The signal sequence is shown in bold.

FIG. 14. Upper panel shows relative expression of NsG28, as measured by quantitative RT-PCR, (relative to tissue with the lowest expression) assuming same amounts of cDNA were synthesized from equal amounts of total RNA used for the cDNA step. Lower panel shows the relative expression of NsG28, as measured by quantitative RT-PCR, normalised to 1-microglobulin (relative to tissue with the lowest normalized expression). Results should be interpreted with caution as β₂-microglobulin expression levels vary between some tissues.

FIG. 15. Upper panel shows relative expression of NsG28, as measured by quantitative RT-PCR, (relative to tissue with the lowest expression) assuming same amounts of cDNA were synthesized from equal amounts of poly (A) RNA used for the cDNA step. Lower panel shows relative expression of NsG28, as measured by quantitative RT-PCR, normalised to β₂-microglobulin (relative to tissue with the lowest normalized expression).

FIG. 16 in the upper panel shows the relative expression of NsG30, as measured by quantitative RT-PCR, (relative to tissue with the lowest expression) assuming same amounts of cDNA were synthesised from equal amounts of total RNA used for the cDNA step. FIG. 16 shows in the lower panel the relative expression of NsG30, as measured by quantitative RT-PCR, normalised to β₂-microglobulin (relative to tissue with the lowest normalized expression). Results should be interpreted with caution as 32-microglobulin expression levels vary between some tissues.

FIG. 17 in the upper panel shows the relative expression of NsG30, as measured by quantitative RT-PCR, (relative to tissue with the lowest expression) assuming same amounts of cDNA were synthesised from equal amounts of poly (A) RNA used for the cDNA step. FIG. 17 in the lower panel shows the relative expression of NsG30, as measured by quantitative RT-PCR, normalised to %-microglobulin (relative to tissue with the lowest normalized expression).

FIG. 18 shows in the upper panel the relative expression of human NsG32, as measured by quantitative RT-PCR, (relative to tissue with the lowest expression) assuming same amounts of cDNA were synthesized from equal amounts of total RNA used for the cDNA step. FIG. 18 shows in the lower panel, the relative expression of human NsG32 normalised to β₂-microglobulin (relative to tissue with the lowest normalized expression). Results should be interpreted with caution as β₂-microglobulin expression levels vary between some tissues.

FIG. 19 shows in the upper panel the relative expression of human NsG32, as measured by quantitative RT-PCR, (relative to tissue with the lowest expression) assuming same amounts of cDNA were synthesized from equal amounts of poly (A) RNA used for the cDNA step. FIG. 19 shows in the lower panel the relative expression of human NsG32 normalised to β₂-microglobulin (relative to tissue with the lowest normalized expression).

FIG. 20. Human NsG28 cDNA (SEQ ID No. 2) and encoded polypeptide (NsG28a, SEQ ID No. 4). The start codon for NsG28a and the alternative start codon for NsG28 (SEQ ID No. 3) are shown in bold.

FIG. 21. Mouse NsG28 cDNA (SEQ ID No. 8) and encoded polypeptide (SEQ ID No. 9).

FIGS. 22A-22C show the relative expression of mGAPDH (panel A) as measured by quantitative RT-PCR (relative to tissue with the lowest expression) assuming same amounts of cDNA were synthesized from equal amounts of total RNA used for the cDNA step. In panel B and C is shown the expression of mALDH1A1 (panel B) and mOTX2 (panel C) has been normalised to the expression of mGADPH and is shown relative to the tissue with the lowest normalised expression. The expression is shown for regions of the developing mouse brain. For details, see example 13. Legend: dorsal forebrain (DFB), ventral forebrain (VFB), ventral mesencephalon (VM), dorsal mesencephalon (DM), spinal cord (SC), cortex (CTX), medial and lateral ganglionic eminences (MGE/LGE), cerebellum (Cb). E10.5, E11.5, and E13.5: embryo days (or days post conception) 10.5, 11.5 and 13.5 respectively. P1: one day post-natum. Ad: adult.

FIG. 23 shows the relative expression of mGDNF in regions of the developing mouse brain. The expression is measured by quantitative RT-PCR normalised to the expression of mGAPDH relative to tissue with the lowest normalised expression. For details, see Example 13. Legend as in FIGS. 22A-22C.

FIG. 24 shows the relative expression of mNsG28 in regions of the developing mouse brain. The expression is measured by quantitative RT-PCR normalised to the expression of mGAPDH relative to tissue with the lowest normalised expression. For details, see example 13. Legend as in FIGS. 22A-22C.

FIG. 25. Results of the PC12 viability assay described in Example 5. Data shown are means ±SEM (n=6) from a representative experiment, and * indicates a significant difference from cells transduced with cDNA for EGFP (P<0.05, one way ANOVA, Dunnett's Method). LV-EGFP: lentivirus EGFP transduced PC12 cells. LV-NsG30: PC12 cells transduced with human full length NsG30.

FIG. 26. Human NsG30 cDNA (SEQ ID No.17) and encoded protein (SEQ ID No. 18).

FIG. 27. Mouse NsG30 cDNA (SEQ ID No 22) and encoded protein (SEQ ID No. 23).

FIG. 28 shows the relative expression of mNsG30 in regions of the developing mouse brain. The expression is measured by quantitative RT-PCR normalised to the expression of mGAPDH relative to tissue with the lowest normalised expression. For details, see Example 13. Legend as in FIGS. 22A-22C.

FIG. 29. Gene structure of human NsG32 leading to two splice variants called NsG32a, and NsG32b.

FIG. 30A. Human NsG32a cDNA (SEQ ID No. 26) and encoded polypeptide (SEQ ID No. 27).

FIG. 30B. Human NsG32a cDNA (SEQ ID No. 26) and encoded polypeptide (SEQ ID No. 28).

FIG. 31. Mouse NsG32a cDNA (SEQ ID No. 35) and encoded polypeptide (SEQ ID No 36).

FIG. 32. Human NsG32b cDNA (SEQ ID No.30) and encoded polypeptide (SEQ ID No. 31).

FIG. 33. Mouse NsG32b cDNA (SEQ ID No. 37) and encoded polypeptide (SEQ ID No. 38).

FIG. 34. Effect of human NsG32a and b on PC12 survival in serum-free medium. For additional details see Example 5. Cells were seeded in collagen-coated 48-well plates, 2×10⁴cells/well in growth medium. The following day, cells were transduced by incubation overnight with 105 transducing units virus/well (MOI=5) in the presence of 5 μg/ml polybrene. After transduction, medium was changed to serum-free DMEM (Invitrogen) and cell survival was then assayed after four days using the MTS assay. Data shown are means ±SEM (n=6) from a representative experiment, and * indicates a significant difference from cells transduced with cDNA for EGFP (P<0.05, one way ANOVA, Dunnett's Method). EGFP: lentivirus EGFP transduced PC12 cells. NsG32a: PC12 cells transduced with human full length NsG32a coding for a protein with the sequence of SEQ ID No. 28. NsG32b: PC12 cells transduced with human full length NsG32b.

FIG. 35 shows the relative expression of mNsG32a in regions of the developing mouse brain. The expression is measured by quantitative RT-PCR normalised to the expression of mGAPDH relative to tissue with the lowest normalised expression. For details, see Example 13. Legend as in FIGS. 22A-22C.

FIG. 36. MTS measurements in CGN cultures from p9 mouse (DIV6) 2 days after change of medium to serum-free medium with high potassium (“25-S”) or a depolarizing level of potassium, 5 mM KCl with or without 100 ng/ml IGF1 (“5-S+I” and “5-S”, respectively). Control wells are left unchanged (“U”) in medium with serum and 25 mM potassium. MTS reduction for each condition is compared between non-transduced control cells and cells transduced with rLV-NsG28, rLV-NsG32a and rLV-EGFP, respectively.

DEFINITIONS

NsG28, as used herein, refers to polypeptides having the amino acid sequences of substantially purified NsG28 obtained from any species, particularly mammalian, including chimpanzee, bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant. The term also refers to biologically active fragments of NsG28 obtained from any of these species, as well as to biologically active sequence variants of these and to proteins subject to posttranslational modifications.

NsG30, as used herein, refers to polypeptides having the amino acid sequences of substantially purified NsG30 obtained from any species, particularly mammalian, including chimpanzee, bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant. The term also refers to biologically active fragments of NsG30 obtained from any of these species, as well as to biologically active sequence variants of these and to proteins subject to posttranslational modifications.

NsG32, as used herein, refers to polypeptides having the amino acid sequences of substantially purified NsG32 obtained from any species, particularly mammalian, including chimpanzee, bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant. The term also refers to biologically active fragments of NsG32 obtained from any of these species, as well as to biologically active sequence variants of these and to proteins subject to posttranslational modifications. The term NsG32 refers to both splice variants of NsG32 unless one of the variants is specified.

Growth factor characteristics as used herein define sequence-related features similar to those of classical growth factors, which are secreted proteins acting on a target cell through a receptor to cause one or more of the following responses in the target cell: growth, proliferation, differentiation, survival, regeneration, migration, regain of function, improvement of function.

According to one embodiment of the invention, “treatment”, “therapy”, and “medical use” is intended to cover prophylaxis. “Treatment”, “therapy” and “medical use” may also cover inhibition of a disease or disorder, protection against a disease or disorder, and/or prevention (not absolute) of a disease or disorder. “Treatment”, “therapy” and “medical use” may also comprise curative, ameliorative, and/or symptomatic treatment, therapy and medical use.

An “allele” or “allelic sequence”, as used herein, is an alternative form of the gene encoding NsG28, NsG30, or NsG32. Alleles may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

A “deletion”, as used herein, refers to a change in the amino acid or nucleotide sequence and results in the absence of one or more amino acid residues or nucleotides.

An “insertion” or “addition”, as used herein, refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, as compared to the naturally occurring molecule.

The terms “specific binding” or “specifically binding”, as used herein, refers to the high affinity interaction between a protein or peptide and a binding molecule such as an antibody and a receptor or fragments thereof. The interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) of the protein recognized by the binding molecule. For example, if an antibody is specific for epitope “A”, the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labelled “A” and the antibody will reduce the amount of labeled A bound to the antibody.

The term “substantially purified”, as used herein, refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.

A “substitution”, as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

“Sequence identity”: The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTP programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.

In order to characterize the identity, subject sequences are aligned so that the highest order homology (match) is obtained. Based on these general principles the “percent identity” of two amino acid sequences may be determined using the BLASTP algorithm [Tatiana A. Tatusova, Thomas L Madden: Blast 2 sequences—a new tool for comparing protein and nucleotide sequences; FEMS Microbiol. Lett. 1999 174 247-250], which is available from the National Center for Biotechnology Information (NCBI) web site (http://www.ncbi.nlm.nih.gov), and using the default settings suggested here (i.e. Matrix=Blosum62; Open gap=11; Extension gap=1; Penalties gap x_dropoff=50; Expect=10; Word size=3; Filter on). The BLAST algorithm performs a two-step operation by first aligning two sequences based on the settings and then determining the % sequence identity in a range of overlap between two aligned sequences. In addition to % sequence identity, BLASTP also determines the % sequence similarity based on the settings.

In order to characterize the identity, subject sequences are aligned so that the highest order homology (match) is obtained. Based on these general principles, the “percent identity” of two nucleic acid sequences may be determined using the BLASTN algorithm [Tatiana A.

Tatusova, Thomas L Madden: Blast 2 sequences—a new tool for comparing protein and nucleotide sequences; FEMS Microbiol. Lett. 1999 174 247-250], which is available from the National Center for Biotechnology Information (NCBI) web site (http://www.ncbi.nlm.nih.gov), and using the default settings suggested here (i.e. Reward for a match=1; Penalty for a match=−2; Strand option=both strands; Open gap=5; Extension gap=2; Penalties gap x_dropoff=50; Expect=10; Word size=11; Filter on). The BLASTN algorithm determines the % sequence identity in a range of overlap between two aligned nucleotide sequences.

Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the FASTA sequence alignment software package (Pearson W R, Methods Mol Biol, 2000, 132:185-219).

Align calculates sequence identities based on a global alignment. Align0 does not penalise to gaps in the end of the sequences. When utilizing the ALIGN or Align0 program for comparing amino acid sequences, a BLOSUM50 substitution matrix with gap opening/extension penalties of −12/−2 is preferably used.

DETAILED DESCRIPTION OF THE INVENTION
I. The Cys10 Family of Growth Factors

The present invention in one aspect relates to medical use of NsG28, NsG30, and NsG32 and to bioactive fragments of NsG28, NsG30, and NsG32. NsG28, NsG30, and NsG32 are distinct members of a family of growth factors in the following referred to as the Cys10 family (referring to the 10 cysteines conserved among four of the five family members).

The five members of the family are referred to as NsG28, NsG29, NsG30, NsG31, and NsG32 (See Table 1). For NsG28, there are two alternative start codons in human beings, referred to as NsG28 and NsG28a. NsG31 exists as two different splice variants with different length. The long form NsG31-long (IPI00174927.1 REFSEQ_XP:XP_—209222 Tax_Id=9606 similar to hypothetical protein) differs from the other members of the family from position 89 to the C-terminal as a result of a frameshift. NsG32 also exists as two different splice variants, called NsG32a and NsG32b. For the NsG32a splice variant there are two possible start codons. The difference between NsG32a and NsG32b results in different signal peptides and also in different mature proteins, both of which are shown in FIG. 1B.

TABLE 1

Full length Cys10 proteins. The signal peptides are shown in bold.

The IPI reference numbers (IPI - The International Protein Index.

The European Bioinformatics Institute) refer to the accession

numbers of the sequences. The database version is shown in paren-

theses. The IPI databases were downloaded from

ftp://ftp.ebi.ac.uk/pub/databases/IPI/.

Human NsG28, >IPI00065186.1 (ver. 2.24)

MRSPRMRVCA KSVLLSHWLF LAYVLMVCCK LMSASSQHLR GHAGHHQIKQ GTCEVVAVHR

CCNKNRIEER SQTVKCSCFP GQVAGTTRAQ PSCVEASIVI QKWWCHMNPC LEGEDCKVLP

DYSGWSCSSG NKVKTTKVTR

Human NsG28a, >IPI00065186.1.modi N-terminal predicted using

NetStart (ver. 2.24)

MRVCAKSVLL SHWLFLAYVL MVCCKLMSAS SQHLRGHAGH HQIKQGTCEV VAVHRCCNKN

RIEERSQTVK CSCFPGQVAG TTRAQPSCVE ASIVIQKWWC HMNPCLEGED CKVLPDYSGW

SCSSGNKVKT TKVTR

Mouse NsG28a, >IPI00311118.3 (ver. 1.23)

MRVCAKWVLL SRWLVLTYVL MVCCKLMSAS SQHLRGHAGH HLIKPGTCEV VAVHRCCNKN

RIEERSQTVK CSCFPGQVAG TTRAQPSCVE AAIVIEKWWC HMNPCLEGED CKVLPDSSGW

SCSSGNKVKT TKVTR

Human NsG29, >IPI00376089.1 (ver. 2.29)

MAMVSAMSWV LYLWISACAM LLCHGSLQHT FQQHHLHRPE GGTCEVIAAH RCCNKNRIEE

RSQTVKCSCL PGKVAGTTRN RPSCVDASIV IGKWWCEMEP CLEGEECKTL PDNSGWMCAT

GNKIKTTRIH PRT

Mouse NsG29, >IPI00380407.1 (ver. 1.20)

MAMVSAMSWA LYLWISACAM LLCHGSLQHT FQQHHLHRPE GGTCEVIAAH RCCNKNRIEE

RSQTVKCSCL PGKVAGTTRN RPSCVDASIV IGKWWCEMEP CLEGEECKTL PDNSGWMCAT

GNKIKTTRIH PRT

Human NsG30, >IPI00166553.1 (ver. 2.24)

MSKRYLQKAT KGKLLIIIFI VTLWGKVVSS ANHHKAHHVK TGTCEVVALH RCCNKNKIEE

RSQTVKCSCF PGQVAGTTRA APSCVDASIV EQKWWCHMQP CLEGEECKVL PDRKGWSCSS

GNKVKTTRVT H

Mouse NsG30, >IPI00338844.1 (ver. 1.22)

MNKRYLQKAT QGKLLIIIFI VTLWGKAVSS ANHHKAHHVR TGTCEVVALH RCCNKNKIEE

RSQTVKCSCF PGQVAGTTRA APSCVDASIV EQKWWCHMQP CLEGEECKVL PDRKGWSCSS

GNKVKTTRVT H

Human NsG31, >IPI00334480.1 (ver. 2.24)

MSERVERNWS TGGWLLALCL AWLWTHLTLA ALQPPTATVL VQQGTCEVIA AHRCCNRNRI

EERSQTVKCS CFSGQVAGTT RAKPSCVDAS IVLQRWWCQM EPCLPGEECK VLPDLSGWSC

SSGHKVKTTK VTR

Mouse NsG31, >IPI00380405.1 (ver. 1.22)

MERPTSNWSA GSWVLALCLA WLWTCPASAS LQPPTSAVLV KQGTCEVIAA HRCCNRNRIE

ERSQTVKCSC LSGQVAGTTR AKPSCVDASI VLQKWWCQME PCLLGEECKV LPDLSGWSCS _

SGHKVKTTKV TR

Human NsG31-long, >IPI00174927.1 (ver. 2.24)

MSERVERNWS TGGWLLALCL AWLWTHLTLA ALQPPTATVL VQQGTCEVIA AHRCCNRNRI

EERSQTVKCS CFSGQVAGTT RAKPSCVDDL LLAAHCARRD PRAALRLLLP QPPSSCRDGG

VRWSPACRGR SVRCSRTCRD GAAAVDTKSK PPRSHDSSWG SRPGQERLD

Human NsG32a, > IPI00239265.1.modi, N-terminal predicted using

NetStart (ver. 2.24)

MSSTFWAFMI LASLLIAYCS QLAAGTCEIV TLDRDSSQPR RTIARQTARC ACRKGQIAGT

TRARPACVDA RIIKTKQWCD MLPCLEGEGC DLLINRSGWT CTQPGGRIKT TTVS

Human NsG32b, >IPI00385234.1 (ver. 2.29)

MQLLKALWAL AGAALCCFLV LVIHAQFLKE GQLAAGTCEI VTLDRDSSQP RRTIARQTAR

CACRKGQIAG TTRARPACVD ARIIKTKQWC DMLPCLEGEG CDLLINRSGW TCTQPGGRIK

TTTVS

Human NsG32a alternative start codon, > IPI00239265.1. (ver. 2.24)

MAPSPRTGSR QDATALPSMS STFWAFMILA SLLIAYCSQL AAGTCEIVTL DRDSSQPRRT

IARQTARCAC RKGQIAGTTR ARPACVDARI IKTKQWCDML PCLEGEGCDL LINRSGWTCT

QPGGRIKTTT VS

Mouse NsG32a, >IPI00224493.1.modi N-terminal predicted using MetStart

(ver. 1.19)

MSSTFWAFMI LASLLIAYCS QLAAGTCEIV TLDRDSSQPR RTIARQTARC ACRKGQIAGT

TRARPACVDA RIIKTKQWCD MLPCLEGEGC DLLINRSGWT CTQPGGRIKT TTVS

Mouse NsG32b, >IPI00128075.1 (ver. 1.20)

MQLLKALWAL AGAALCCFLV LVIHAQFLKE GQLAAGTCEI VTLDRDSSQP RRTIARQTAR

CACRKGQIAG TTRARPACVD ARIIKTKQWC DMLPCLEGEG CDLLINRSGW TCTQPGGRIK

TTTVS

Mouse NsG32a alternative start codon, >IPI00224493.1. (ver. 1.19)

MAPSPRTSSR QDATALPSMS STFWAFMILA SLLIAYCSQL AAGTCEIVTL DRDSSQPRRT

IARQTARCAC RKGQIAGTTR ARPACVDARI IKTKQWCDML PCLEGEGCDL LINRSGWTCT

QPGGRIKTTT VS

As can be seen from FIGS. 1A and 1B, the proteins are highly conserved between man and mouse. Corresponding proteins with very high conservation are found in rat, and there is also evidence of expression of proteins with similar conserved cysteine-pattern in distantly related species such as Drosophila melanogaster, Danio rerio, Caenorhabditis elegans, Xenopus laevis, and Caenorhabditis briggsae. In these species, predicted protein fragments can be found, which fragments over lengths of 50-110 amino acids have 80-90% sequence identity to Cys10 proteins. The extremely high degree of evolutionary conservation among these species points to important functions in maintaining normal cell function in the tissues where the genes are expressed. In Xenopus laevis and in C. briggsae only two members of the Cys10 family are found. These are most similar to NsG28 and NsG32 respectively. A calculation of sequence identity and construction of a phylogenetic tree also indicate that NsG28-NsG31 have similar features and are distinct from NsG32. As predicted from the high degree of identity among NsG28, NsG29, NsG30, and NsG31, these genes may have a common ancestor gene and some functional redundancy among NsG28, NsG29, NsG30 and NsG31 may therefore be possible. Together, this indicates that there are at least two distinct functions related to the Cys10 family and that NsG28 in higher animals has evolved into four distinct members.

The percent sequence identity calculated on the basis of the mature part of the human growth factors is shown in Table 2. NsG28-NsG31 share sequence identities above 60%. NsG32 only has 8 of the 10 conserved cysteines. NsG32 is approximately 40% identical to the other members of the family. None of the members of the Cys10 family have a significant sequence identity to any characterised proteins outside this family. NsG31-long only shows partial homology to the other family members due to the frameshift located after the 6^thcysteine.

TABLE 2

Percent sequence identity between human mature protein

sequences using Align0 (global alignment).

NsG31-

NsG29
NsG30
long
NsG31
NsG32a
NsG32b

NsG28a
67.6
77.4
33.8
73.6
41.1
44.9

NsG29
—
66.1
31.4
61.4
39.4
40.5

NsG30

—
36.1
72.8
44.1
46.1

NsG31-

—
48.2
19.9
20.5

long

NsG31

—
40.4
41.3

NsG32a

—
89.0

Scoring matrix Blosum50.

Gap penalty −12/−2.

The Clustal W (1.82) multiple sequence alignment of the five human and mouse sequences (FIG. 1B) allows for the identification of one family fingerprint: G-T-C-E-[V/I]-[V/I]-x(3)-R-x(5)-[R/K]-x(5)-Q-T-[V/A]-[K/R]-C-x-C-x(2)-G-x-[V/I]-A-G-T-T-R-x(2)-P-x-C-V-[D/E]-A-x-I-[V/I]-x(2)-[K/R]-x-W-C-x-M-x-PC-L-x-G-E-x-C-x(2)-L-x(4)-G-W-x-C-x(2-3)-G-x-[K/R]-[V/I]-K-T-T (SEQ ID NO:1).

It is believed that amino acids, which are fully or partly conserved in this family fingerprint, are important for biological activity, mainly because they are believed to be important for the secondary and/or tertiary structure of the proteins. Non-conserved residues (marked as x) can probably be substituted without affecting biological activity, in particular if the substitution is a conservative substitution or to a residue found at the same or corresponding position in another Cys10 protein. Only NgG32a does not have the initial glycine residue. The same core sequence with minor modifications can be found in Drosophila melanogaster, X. laevis, C. briggsae, and Danio rerio. The present inventors therefore in one embodiment predict that the core sequence from GTCE to KTT is the bioactive core sequence, which can be used to elicit the same biological effects as mature Cys10 proteins.

From the alignment it can be seen, that NsG32 only has 8 of the 10 cysteines, that are conserved in the other members of the family and that the mature NsG32 protein in the region, where the other members have a double cysteine, has a stretch of amino acids, which differs from all the other members of the family. Another Clustal W (1.82) alignment of only the most similar members of the family, NsG28-NsG31 (FIG. 1A) gives another family fingerprint with a higher degree of conservation across the sub-family: G-T-C-E-V-[V/I]-A-x-H-R-C-C-N-[K/R]-N-[R/K]-I-E-E-R-S-Q-T-V-K-C-S-C-x(2)-G-x-V-A-G-T-T-R-x(2)-P-S-C-V-[D/E]-A-x-I-V-x(2)-[K/R]-W-W-C-x-M-x-P-C-L-x-G-E-[E/D]-C-K-x-L-P-D-x(2)-G-W-x-C-x-[S/T]-G-x-K-[V/I]-K-T-T-[R/K] (SEQ ID NO:42).

In another embodiment, the present inventors predict that a NsG28-NsG31 polypeptide comprising this conserved (GTCEV---KTT[RK]) region can elicit the biological effects of mature Cys10 proteins. It is believed that amino acids, which are fully or partly conserved in this family fingerprint, are important for biological activity, mainly because they are believed to be important for the secondary and/or tertiary structure of the proteins. Non-conserved residues (marked as x) can probably be substituted without affecting biological activity, in particular if the substitution is a conservative substitution or to a residue found at the same or corresponding position in another Cys10 protein.

The conserved cysteines may participate in forming intra- and inter-molecular cysteine bridges and may thus play an important role in the secondary and tertiary structure of the proteins, just as is the case for other growth factors with a conserved cysteine pattern (e.g. the TGF-beta family). It is possible that Cys10 proteins may form both homo- and heterodimers.

The ClustalW alignments in FIGS. 1, 11, 12, 13A, and 13B can be used to identify those parts of the growth factors in which mutations can be made without substantially altering the biological function. In particular it is expected that a residue, which is not fully conserved among mouse, rat, and man (FIGS. 11, 12, 13A, and 13B) in the same Cys10 member can be replaced with a residue found at the same position in another species. Furthermore, it is expected that a residues that are non-conserved among the Cys10 members (FIGS. 1A and 1B) can be substituted with a residue found in another Cys10 member. In particular, it is possible that residues at positions that are non-conserved within the NsG28-NsG31 group can be substituted with each other.

Apart from sharing a high degree of sequence identity, all members and variants of the Cys10 family are predicted to belong to the gene ontology class, growth factor and/or hormone (ProtFun prediction, see Example 2). Expression of all five members seems to be restricted to the central and peripheral nervous system including the eye, but each individual member shows differential expression in sub-regions.

II. NsG28, NsG30, and NsG32
NsG28

NsG28a is a 135 amino acid secreted growth factor protein or hormone. The mouse (IPI00311118.3 version 1.23) and rat (IPI00214302.2 version 1.11) have full lengths of 135 and 134 amino acids and the % identities are 93.3 and 91.9, respectively. In the databases a longer version of NsG28 with a different start codon is also found (SEQ ID No 3). An analysis of the probability of start codons however indicates that the most likely start codon is the start codon leading to NsG28a (SEQ ID No 4). Furthermore, a corresponding start codon leading to a longer protein is not found in mouse. SEQ ID No 3 and 4 lead to the same mature protein after cleavage of the signal peptide.

Human NsG28a contains an N-terminal signal peptide sequence, which is predicted to be 29 amino acids long, and which is predicted to be cleaved at the sequence motif MSA-SS. This signal peptide cleavage site is predicted by the SignalP method (Nielsen et al., 1997) and the output graph shown in FIGS. 2A-2B. A signal peptide cleavage site is found at a similar location in the mouse and rat NsG28 polypeptide sequences at position 29. As it is known in the art, signal peptide processing is not always exactly as predicted and actual cleavage may vary from case to case. Thus, it is expected that the N-terminal of mature NsG28 may vary by one to two or three amino acids from the predicted cleavage site. The actual N-terminal of mature NsG28 can be verified experimentally by C-terminal tagging with e.g. a his-tag, subsequent purification using a poly-his specific antibody or purification on a Ni column, and finally N-terminal sequencing of the purified mature peptide.

Human NsG28 belongs to the category of proteins acting as hormones or growth factors. This notion is supported by predictions by the ProtFun protein function prediction server (Jensen et al., 2002 & 2003), which provides odds scores above 1 for these categories for both human, mouse and rat NsG28 as shown in FIG. 3.

Results of the quantitative expression analysis in human tissues are shown in FIGS. 14 and 15. The expression analyses show:

High to moderate expression in: Substantia Nigra, Retina, Spinal Cord, Thalamus, Corpus Callosum, and Brain.

Low expression in: Putamen, testis, cerebellum, Dorsal Root Ganglion, fetal liver, Hippocampus, Amygdala, and Caudate Nucleus.

Very low or no expression in: Pancreas, heart, muscle, and Pituitary gland.

Results of the quantitative expression analysis in the developing mouse brain (FIG. 24) shows that the expression peaks in P1 ventral mesencephalon (from which the substantia nigra develops) and P1 lateral and medial ganglionic eminences (from which the striatum develops), i.e., at around the time of terminal differentiation in these tissues. Expression in cerebellum also peaks at P1 and is much lower in the adult mouse cerebellum. This means that NsG28 is expressed during development, primarily in the ventral mesencephalon and the striatum during the time of terminal differentiation, strongly suggesting a survival and/or differentiation effect on dopaminergic neurons and striatal medium spiny neurons.

Unlike structural proteins, growth factors are involved in cell signalling and in various functions such as growth, proliferation, differentiation, survival, regeneration, migration, regain of function and improvement of function. Therefore, growth factors can be administered and be used to exert a therapeutic effect. Based on the tissue specific expression, and the fact that NsG28 is predicted to be a secreted growth factor, NsG28 is contemplated for use in treating disorders of the nervous system in general (based on the nervous system specific expression and the neuroprotective and antiapoptotic effect mediated by NsG28), in particular cerebellar disorders (based on the neuroprotective effect of NsG28 in cerebellar granule cells), Parkinson's disease (based on the expression in substantia nigra and thalamus; and the expression in mouse P1 ventral mesencephalon and striatum), Huntington's disease (based on expression in putamen and substantia nigra; and the expression in mouse P1 ventral mesencephalon and striatum), Spinal Cord injury (based on expression in the spinal cord), ALS (based on expression in the spinal cord), peripheral neuropathies (based on expression in dorsal root ganglion and spinal cord), multiple sclerosis (based on expression in corpus callosum), thalamic pain and essential tremor (based on expression in the thalamus), testicular disorders and male contraception (based on expression in the testicles), and retinopathies (based on expression in retina). The function for the various indications can be verified in in vitro and in vivo assays as described in the examples.

Likewise, expression of therapeutically relevant secreted growth factors including GDNF, NGF, and Neublastin (Artemin) is found in target areas of the neurological disorder they may be used to treat. Expression of GDNF (FIG. 23) is also found in target areas of Parkinson's Disease at around the time of terminal differentiation in this target area.

The therapeutic effect of NsG28 may be mediated through an effect on growth, proliferation, regeneration, regain of function, improvement of function, survival, migration, and/or differentiation of targeted cells.

One verified biological function of NsG28 is a neuroprotective effect against potassium deprivation-induced apoptosis in cerebellar granule cells (FIG. 36). Similarly, other members of the Cys10 family, NsG30, NsG32a, and NsG32b, have also demonstrated a survival enhancing effect in a cell line with neuronal potential (PC12 assay described in Example 5 herein) and in cerebellar granule cells (NsG32a; Example 7). These assays test the ability of the factors to protect cerebellar granule cells (Example 7) or a neuronal cell line (Example 5) against apoptotic cell death. Apoptotic cell death contributes to neuronal cell loss in the adult nervous system causing various neurological disorders like ischemic stroke, neurodegenerative diseases or brain traumata (Becker & Bonni, Prog Neurobiol, 2004 January; 72(1):1-25). A secreted growth factor capable of protecting neuronal cells (e.g., cerebellar cells) against apoptotic cell death is therefore a candidate for treating neurological disorders (e.g., disorders associated with the Cerebellum). The ability of a secreted growth factor to promote survival under conditions leading to apoptosis is an indication that this factor has a similar effect in other neuronal cell types of the central and/or peripheral nervous system.

NsG30

NsG30 is a 131 amino acid secreted growth factor protein or hormone. The mouse (IPI00338844.1 version 1.22) and rat (IPI00205786.1 version 1.3) homologues have are 131 and 95 (partial sequence) amino acids long and the % identities are 96.9 and 71.0, respectively.

Human NsG30 contains a N-terminal signal peptide sequence of 30 amino acids which is cleaved at the sequence motif VSS-AN. This signal peptide cleavage site is predicted by the SignalP method (Nielsen et al., 1997) and the output graph shown in FIGS. 4A-4B. A signal peptide cleavage site is found at a similar location in the mouse NsG30 at position 30.

As it is known in the art, signal peptide processing is not always exactly as predicted and actual cleavage may vary from case to case. Thus, it is expected that the N-terminal of mature NsG30 may vary by one to two or three amino acids from the predicted cleavage site. The actual N-terminal of mature NsG30 can be verified experimentally by C-terminal tagging with e.g. a his-tag, subsequent purification using a poly-his specific antibody or purification on a Ni column, and finally N-terminal sequencing of the purified mature peptide.

Human NsG30 belongs to the category of proteins acting as hormones or growth factors. This notion is supported by predictions by the ProtFun protein function prediction server (Jensen et al., 2002 & 2003), which provides scores above 1 for both these categories as shown in FIG. 5.

Results of the quantitative expression analysis in human tissues (Example 4a) are shown in FIGS. 16 and 17. The expression analyses show:

High to moderate expression in: Brain, Substantia Nigra, Hippocampus, Amygdala, and Thalamus

Intermediate and low expression in: Putamen, Retina, Spinal Cord, Dorsal Root Ganglion, testis, cerebellum, Corpus Callosum, Caudate Nucleus, and Pituitary

Very low or no expression in: Foetal liver, pancreas, muscle, Heart The expression pattern of NsG30 in the developing mouse CNS (FIG. 28) resembles that of NsG28. There is high expression in the ventral mesencephalon and the striatum during the latter part of the development during the early postnatal period. This relatively restricted regional and temporal expression pattern indicates a role in the differentiation and termination of the projections between the VM and the striatum (LGE/MGE). Similar to GDNF (and NsG28) a therapeutic effect on neurons involved in Huntingdon's and Parkinson's diseases is strongly indicated. Expression in the cortex, cerebellum, and dorsal mesencephalon also peaks in the developing mouse CNS during the early postnatal period (P1).

Furthermore, gene chip experiments (Example 4B) have shown that human NsG30 is expressed at significant levels in the developing human mesencephalon. This expression is indicative of a potential therapeutic use in the treatment of Parkinson's Disease. Unlike structural proteins, growth factors are involved in cell signalling and in various functions such as growth, proliferation, differentiation, survival, regeneration, migration, regain of function and improvement of function. Therefore, growth factors can be administered and be used to exert a therapeutic effect. Based on the tissue specific expression, and the fact that NsG30 is predicted to be a secreted growth factor, NsG30 is contemplated for use in treating disorders of the nervous system in general (based on the nervous system specific expression), in particular Parkinson's disease (based on the expression in substantia nigra, thalamus, and the developing human mesencephalon; and the developing mouse mesencephalon and striatum), Huntington's disease (based on expression in putamen, substantia nigra, and thalamus; and the developing mouse striatum), Alzheimer's disease (based on expression in hippocampus and amygdala), tremor including central tremor (based on expression in thalamus), thalamic pain (based on expression in the thalamus), cerebellar disorders (based on expression in adult human cerebellum and the developing mouse cerebellum), Spinal Cord injury (based on expression in the adult human spinal cord and the developing mouse spinal cord), ALS (based on expression in the adult human spinal cord and the developing mouse spinal cord), peripheral neuropathies (based on expression in dorsal root ganglion), retinopathies (based on expression in retina), testicular disorders (based on expression in testes). The function for the various indications can be verified in in vitro and in vivo assays as described in the examples.

Likewise, expression of therapeutically relevant secreted growth factors including GDNF, NGF, and Neublastin (Artemin) is found in target areas of the neurological disorder they may be used to treat.

The therapeutic effect of NsG30 may be mediated through an effect on growth, proliferation, regeneration, regain of function, improvement of function, survival, migration, and/or differentiation of targeted cells.

One verified biological function of NsG30 is a neuroprotective effect against starvation induced apoptosis in PC12 (pheochromocytoma) cells. Pheochromocytomas are tumors with characteristics of immature and adult chromaffin cells of the adrenal medulla. Chromaffin cells, sensory and sympathetic neurons in addition to pigment cells (melanocytes) are derived from a common precursor cell in the neural crest. Its differentiation into the specific lineages is highly dependent on external signals including secreted factors.

PC12 is a clonal cell line, which was originally established from a transplantable rat adrenal medullary pheochromocytoma (Greene and Tischler, 1976 Proc. Natl. Acad. Sci. U.S.A. 73, 2424). PC-12 cells are available from ATCC (American Type Culture Collection; accession number CRL-1721). PC12 cells are considered to be the pluripotent chromaffin precursor cell as it possesses the ability to differentiate to mature chromaffin cells, sympathetic neurons, as well as melanocytes depending on the culture conditions. PC12 cells have been widely used as a model system for studies of neuronal differentiation and survival. In serum-containing medium PC12 cells proliferate, whereas addition of certain neurotrophic factors including NGP induces differentiation of PC12 cells into a neuronal phenotype very similar to sympathetic neurons. In serum-free medium, PC12 cells will become apoptotic and die unless supplied with certain growth factors, hormones or small molecules that can act as survival factors.

The factors capable inducing differentiation and survival in PC12 cells including one of the neurotrophins (NGF) and a member of the secretin/glucagon/VIP family (PACAP) also display a similar activity in both the peripheral and central nervous system indicating that receptors and response systems expressed in PC12 cells are shared with many other neuronal cells.

NGF is an important differentiation and survival factor for responsive sympathetic and sensory neurons in addition to cholinergic neurons in the basal forebrain. PACAP promotes the differentiation of nascent dorsal root ganglion (DRG) neurons in that it increases both the number of neural-marker-positive cells and axonogenesis without affecting the proliferation of neural progenitor cells (Nielsen et al., Mol Cell Neurosci. 2004 April; 25(4):629-41). PACAP also show similar activities in neuronal populations in the CNS (Vaudry et al., Proc Natl Acad Sci USA. 2002 Apr. 30; 99(9):6398-403; Dicicco-Bloom et al., Ann N Y Acad. Sci. 1998 Dec. 11; 865:274-89).

Apoptotic cell death contributes to the neuronal cell loss in the adult nervous system causing various neurological disorders like ischemic stroke, neurodegenerative diseases or brain traumata (Becker and Bonni, Prog Neurobiol. 2004 January; 72(1):1-25). A secreted growth factor capable of protecting neuronal cells against apoptotic cell death is therefore a candidate for treating disorders of the nervous system in general and neurodegenerative disorders in particular. Thus, the ability of a secreted factor to induce neurite outgrowth and/or to promote survival in under conditions leading to apoptosis is an indication that this factor has a similar effect in other neuronal cell types of the central and/or peripheral nervous system and that this factor is a candidate for treating nervous system disorders, in particular neurodegenerative disorders.

NsG32

Two splice variants of NsG32 are found (FIG. 29). One is referred to as NsG32a, and the other splice variant is referred to as NsG32b. The amino acid sequence for human NsG32b is set forth in SEQ ID No 31. The identical amino acid sequence for mouse NsG32b is set forth in SEQ ID No 38. The difference between NsG32a and b on the polypeptide level is found in the signal sequence. From amino acid no 32 of human NsG32b, it is identical to human NsG32a. The difference in signal sequence also results in a difference in predicted cleavage, so that the mature NsG32b proteins have a longer N-terminal than NsG32a. Because of the longer N-terminal, mature NsG32b is more similar to the other members of the Cys10 protein family than mature NsG32a (see FIG. 1B) and thus appears the most probable splice variant.

NsG32a (SEQ ID No 27) is a 114 amino acid secreted protein. The mouse and rat homologues both have full lengths of 114 amino acids and 100% sequence identity to human NsG32a (FIG. 13A). Two alternative start codons can be found for both mouse and human NsG32a. These, however result in the same mature protein after cleavage of the signal peptide. The start codons for human and mouse NsG32a have been predicted using the program NetStart (A. G. Pedersen and H. Nielsen, 1997) and these predictions are shown in FIGS. 6 and 7, respectively. The prediction shows that the start codon corresponding to the short version of NsG32a is the most likely start codon.

Human and mouse NsG32b are identical (FIG. 13B).

Human NsG32a contains an N-terminal signal peptide sequence of 25 amino acids, which is cleaved at the sequence motif AAG-TC. This signal peptide cleavage site is predicted by the SignalP method (Nielsen et al., 1997) and the output graph shown in FIGS. 8A-8B.

Human NsG32b also contains an N-terminal signal peptide sequence of 25 amino acids, which is cleaved at the sequence motif IHA-QF. This signal peptide cleavage site is predicted by the SignalP method (Nielsen et al., 1997) and the output graph shown in FIGS. 9A-9B.

As it is known in the art, signal peptide processing is not always exactly as predicted and actual cleavage may vary from case to case. Thus, it is expected that the N-terminal of mature NsG32 may vary by one to two or three amino acids from the predicted cleavage site. The actual N-terminal of mature NsG32 can be verified experimentally by C-terminal tagging with e.g. a his-tag, subsequent purification using a poly-his specific antibody or purification on a Ni column, and finally N-terminal sequencing of the purified mature peptide.

NsG32a belongs to the category of proteins acting as hormones or growth factors. This notion is supported by predictions by the ProtFun protein function prediction server (Jensen et al., 2002 & 2003), which provides odds scores above 1 for the hormone as well as growth factor category as shown in FIG. 10. Human NsG32b also provides odds scores above 1 for the hormone ontology class.

The results of the quantitative RT-PCR expression analyses in human tissues are shown in FIGS. 18 and 19,

High expression was found in: Cerebellum, Substantia Nigra, Brain, Drosal Root Ganglion, Spinal Cord, Putamen, Foetal Brain, Hippocampus, Amygdala, Thalamus, Corpus Callosum, and Caudate Nucleus

Low expression was found in: Prostate, Placenta, Small Intestines, Foetal liver, Stomach, Lung, Heart, Uterus, Skeletal muscle, Colon, Pancreas, Trachea, Thymus, Spleen, and pituitary. Expression has also been found in the developing human mesencephalon (Example 4b).

The results of the quantitative RT-PCR in the developing mouse CNS are shown in FIG. 35. The expression peaks in the early postnatal ventral mesencephalon and the early postnatal medium and lateral ganglionic eminences (from which the striatum develops). This corresponds to the period of differentiation and terminal differentiation of the projections between the VM and the striatum.

Unlike structural proteins, growth factors are involved in cell signalling and in various functions such as growth, proliferation, differentiation, survival, regeneration, migration, regain of function and improvement of function. Therefore, growth factors can be administered and be used to exert a therapeutic effect. Based on the tissue specific expression, and the fact that NsG32 is predicted to be a secreted growth factor, NsG32 is contemplated for use in treating disorders of the nervous system in general (based on the nervous system-specific expression), in particular Parkinson's disease (based on the expression in human substantia nigra and thalamus, and the differential expression in the developing mouse VM and striatum), Alzheimer's disease (based on expression in amygdala and hippocampus), Huntington's disease (based on expression in human putamen, substantia nigra, and thalamus, and the differential expression in the developing mouse striatum and VM), cerebellar disorders including ataxia (based on expression in cerebellum), peripheral neuropathies (based on expression in dorsal root ganglion), thalamic pain and essential tremor (based on expression in the thalamus), multiple sclerosis (based on expression in corpus callosum and spinal cord), and amyotrophic lateral sclerosis and spinal cord injury (based on expression in the spinal cord). The function for the various indications can be verified in in vitro and in vivo assays as described in the examples.

Likewise, expression of therapeutically relevant secreted growth factors including GDNF, NGF, and Neublastin (Artemin) is found in target areas of the neurological and/or testicular disorder they may be used to treat.

The therapeutic effect of NsG32 may be mediated through an effect on growth, proliferation, regeneration, regain of function, improvement of function, survival, migration, and/or differentiation of targeted cells.

One verified biological function of both splice variants of NsG32 is a neuroprotective effect against starvation-induced apoptosis in PC12 (pheochromocytoma) cells (FIG. 34). Pheochromocytomas are tumors with characteristics of immature and adult chromaffin cells of the adrenal medulla. Chromaffin cells, sensory and sympathetic neurons in addition to pigment cells (melanocytes) are derived from a common precursor cell in the neural crest. Its differentiation into the specific lineages is highly dependent on external signals including secreted factors.

An additional verified biological function of NsG32a is a neuroprotective effect against potassium deprivation-induced apoptosis in cerebellar granule cells (FIG. 36). A secreted growth factor capable of protecting neuronal cells against apoptotic cell death is therefore a candidate for treating neurological disorders (e.g., disorders associated with the Cerebellum). Thus, the ability of a secreted factor to induce neurite outgrowth and/or to promote survival in under conditions leading to apoptosis is an indication that this factor has a similar effect in other neuronal cell types of the central and/or peripheral nervous system and that this factor is a candidate for treating nervous system disorders, in particular neurodegenerative disorders.

III. NsG28, NsG30, and NsG32 Polypeptides
NsG28 Polypeptides

In addition to full-length NsG28, substantially full-length NsG28, and to truncated forms of NsG28, the present invention provides for biologically active variants of the polypeptides. An NsG28 polypeptide or fragment is biologically active if it exhibits a biological activity of naturally occurring NsG28. It is to be understood that the invention relates to substantially purified NsG28 as herein defined.

One biological activity is the ability to compete with naturally occurring NsG28 in a receptor-binding assay. Another biological activity is the ability to bind to an antibody, which is directed at an epitope, which is present on naturally occurring NsG28.

Biologically active variants may also be defined with reference to one or more of the other in vitro and/or in vivo biological assays described in the examples.

A preferred biological activity is the ability to elicit substantially the same response as in the PC12 assay described in the Examples. In this assay PC12 cells are transduced with full length human NsG28 coding sequence (FIG. 20). By substantially the same response in the PC12 assay is intended that the number of neurite bearing cells is at least 10% of the number obtained in Example 5 (transduction with full length human NsG28), more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%. The PC12 assay may also be used to document the percentage improvement in survival over a control treatment. Substantially the same response in this context means an activity resulting in at least 10% of the improvement obtained in Example 5, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%. The biological activity of a fragment or variant of NsG28 may also be higher than that of the naturally occurring NsG28.

Specific fragments of NsG28 include polypeptides having a sequence selected from the group consisting of SEQ ID No. 6, 11, and 15 (or a subgroup thereof), and sequence variants of said polypeptides, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 10 of the amino acid residues in the sequence are so changed. These isolated polypeptides constitute NsG28 polypeptides spanning from the first to the last of the 10 conserved cysteine residues of the Cys10 family. It is believed that the biological activity mainly resides in this part of the protein, and that the sequence with all the ten cysteines is required for correct folding of the protein. Preferably any changes in these peptides are to residues marked in the alignment of FIG. 1A as unconserved, weakly conserved, or strongly conserved. More preferably any changed amino acids are selected from those designated as unconserved, weakly conserved or strongly conserved in FIG. 11. Preferably any changed amino acid is changed to a residue found at the same or corresponding position in another Cys10 protein (see FIGS. 1A and 1B), more preferably to a residue found at the same or corresponding position in an NsG28 sequence from another species, such as the species shown in FIG. 11. In a preferred embodiment, less than 8 amino acids have been changed, more preferably less than 5 amino acids, more preferably 1 or 2 amino acids, more preferably no amino acids have been changed. These truncated NsG28 polypeptides may have up to 5 additional C and/or N-terminal amino acids selected from those of the mature NsG28 polypeptides, i.e. fragments up to AA₁₄-AA₉₈of SEQ ID No 5, fragments up to AA₁₄-AA₉₈of SEQ ID No. 10, and fragments up to AA₁₄-AA₉₈of SEQ ID No. 14.

Further specific polypeptides are selected from the group consisting of SEQ ID No 7, 12, and 16 (or a subgroup thereof), and sequence variants of said polypeptides, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 10 of the amino acid residues in the sequence are so changed. These isolated polypeptides constitute the NsG28 core sequence, which is conserved in the Cys10 subfamily shown in FIG. 1A. Preferably any changes in these peptides are to residues marked in the alignment of FIG. 1A as unconserved, weakly conserved, or strongly conserved. More preferably any changed amino acids are selected from those designated as unconserved, weakly conserved or strongly conserved in FIG. 11. Preferably any changed amino acid is changed to a residue found at the same or corresponding position in another Cys10 protein (see FIGS. 1A and 1B), more preferably to a residue found at the same or corresponding position in an NsG28 sequence from another species, such as the species shown in FIG. 11. In a preferred embodiment, less than 8 amino acids have been changed, more preferably less than 5 amino acids, more preferably 1 or 2 amino acids, more preferably no amino acids have been changed. These truncated NsG28 polypeptides may have up to 5 additional C and/or N-terminal amino acids selected from those of the mature NsG28 polypeptides, i.e. fragments up to AA₁₂-AA₁₀₆of SEQ ID No 5, fragments up to AA₁₂-AA₁₀₆of SEQ ID No. 10, and fragments up to AA₁₂-AA₁₀₅of SEQ ID No. 14.

Variants can differ from naturally occurring NsG28 in amino acid sequence or in ways that do not involve sequence, or in both ways. Variants in amino acid sequence (“sequence variants”) are produced when one or more amino acids in naturally occurring NsG28 is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid. Particularly preferred variants include naturally occurring NsG28 or biologically active fragments of naturally occurring NsG28 whose sequences differ from the wild type sequence by one or more conservative and/or semi-conservative amino acid substitutions, which typically have minimal influence on the secondary and tertiary structure and hydrophobic nature of the protein or peptide. Variants may also have sequences, which differ by one or more non-conservative amino acid substitutions, deletions or insertions, which do not abolish the NsG28 biological activity. The Clustal W alignments in FIGS. 1A, 1B, and 11 can be used to predict which amino acid residues can be substituted without substantially affecting the biological activity of the protein.

Substitutions within the following group (Clustal W, ‘strong’ conservation group) are to be regarded as conservative substitutions within the meaning of the present invention

-STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.

Substitutions within the following group (Clustal W, ‘weak’ conservation group) are to be regarded as semi-conservative substitutions within the meaning of the present invention

-CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY.

Other variants within the invention are those with modifications which increase peptide stability. Such variants may contain, for example, one or more nonpeptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: variants that include residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids such as beta or gamma amino acids and cyclic variants. Incorporation of D-instead of L-amino acids into the polypeptide may increase its resistance to proteases. See, e.g., U.S. Pat. No. 5,219,990. Splice variants are specifically included in the invention.

When the result of a given substitution cannot be predicted with certainty, the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of biological activity.

Variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with human, murine or rat NsG28 (SEQ ID NO: 3, 4, 9, and 13). More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

Variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 5, 10, and 14 (or a subgroup thereof). More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

Variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with a polypeptide having the sequence of SEQ ID NO: 6, 11, or 15. More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

Variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with a polypeptide having the sequence of SEQ ID NO: 7, 12, or 16, More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In a preferred embodiment the sequence identity of the variant NsG28 is determined with reference to a human NsG28 polypeptide (SEQ ID No 3, 4, 5, 10, and 14).

For the purposes of determining homology the minimum length of comparison sequences will generally be at least 8 amino acid residues, usually at least 12 amino acid residues. For the purposes of the present invention, the percent sequence identity is preferably calculated in a range of overlap of at least 25 amino acids, more preferably at least 30 amino acids, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 55, more preferably at least 60, such as at least 70, for example at least 80, such as at least 90, the range being determined by BLASTP under default settings.

In one embodiment the percent sequence identity is calculated using global alignment (GAP or Align), so that the variant and SEQ ID sequences are aligned, the total number of identical amino acid residues calculated and divided by the length of the SEQ ID NO.

In one embodiment, a variant NsG28 comprises a naturally occurring allelic variant of the sequence selected from the group consisting of SEQ ID No 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, and 16 (or a subgroup thereof). Said allelic variant sequence may be an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID No 2 and 8.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 3, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 4, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 5, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 6, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 7, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 9, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 10, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 11, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 12, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 13, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 14, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 15, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 16, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, a variant NsG28 at corresponding positions comprises the residues marked in FIG. 11 as fully conserved (*), more preferably a variant NsG28 also comprises at corresponding positions the residues marked in FIGS. 11, 12, 13A, and 13B as strongly conserved (:strongly conserved groups include: STA, NEQK, NHQK, NEDQ, QHRK, MILV, MILF, HY FYW), more preferably a variant NsG28 also comprises at corresponding positions the residues marked in FIGS. 11, 12, 13A, and 13B as less conserved (less conserved groups include: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHK, NEQHRK, VLIM, HFY). In particular, it is contemplated that the conserved cysteines (FIG. 1B) preferably are located at corresponding positions in a variant NsG28.

NsG30 Polypeptides

In addition to full-length NsG30, substantially full-length NsG30, and to truncated forms of NsG30, the present invention provides for biologically active variants of the polypeptides. An NsG30 polypeptide or fragment is biologically active if it exhibits a biological activity of naturally occurring NsG30. It is to be understood that the invention relates to substantially purified NsG30 as herein defined.

One biological activity is the ability to compete with naturally occurring NsG30 in a receptor-binding assay. Another biological activity is the ability to bind to an antibody, which is directed at an epitope, which is present on naturally occurring NsG30.

Biologically active variants may also be defined with reference to one or more of the other in vitro and/or in vivo biological assays described in the examples.

A preferred biological activity is the ability to elicit substantially the same response as in the PC12 assay described in the Examples. In this assay, PC12 cells are transduced with full length human NsG30 coding sequence (FIG. 26). By substantially the same response in the PC12 assay is intended that the number of neurite bearing cells is at least 10% of the number obtained in Example 5 (transduction with full length human NsG30), more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%. The PC12 assay may also be used to document the percentage improvement in survival over a control treatment. Substantially the same response in this context means an activity resulting in at least 10% of the improvement obtained in Example 5, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%. The biological activity of a fragment or variant of NsG30 may also be higher than that of the naturally occurring NsG30.

Specific fragments of NsG30 include polypeptides having the sequence of SEQ ID No. 20, and sequence variants of said polypeptide, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 10 of the amino acid residues in the sequence are so changed. These isolated polypeptides constitute NsG30 polypeptides spanning from the first to the last of the 10 conserved cysteine residues of the Cys10 family. It is believed that the biological activity mainly resides in this part of the protein, and that the sequence with all the ten cysteines is required for correct folding of the protein. Preferably any changes in these peptides are to residues marked in the alignment of FIG. 1A as unconserved, weakly conserved, or strongly conserved. More preferably any changed amino acids are selected from those designated as unconserved, weakly conserved or strongly conserved in FIG. 12. Preferably any changed amino acid is changed to a residue found at the same or corresponding position in another Cys10 protein (see FIGS. 1A and 1B), more preferably to a residue found at the same or corresponding position in an NsG30 sequence from another species, such as the species shown in FIG. 12. In a preferred embodiment, less than 8 amino acids have been changed, more preferably less than 5 amino acids, more preferably 1 or 2 amino acids, more preferably no amino acids have been changed. These truncated NsG30 polypeptides may have up to 5 additional C and/or N-terminal amino acids selected from those of the mature NsG30 polypeptides, i.e. fragments up to AA₉-AA₉₃of SEQ ID No 19 or 24.

Further specific fragments are polypeptides having the sequence of SEQ ID No 21, and sequence variants of said polypeptide, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 10 of the amino acid residues in the sequence are so changed. These isolated polypeptides constitute the NsG30 core sequence, which is conserved in the Cys10 subfamily shown in FIG. 1A. Preferably any changes in these peptides are to residues marked in the alignment of FIG. 1A as unconserved, weakly conserved, or strongly conserved. More preferably any changed amino acids are selected from-hose designated as unconserved, weakly conserved or strongly conserved in FIG. 12. Preferably any changed amino acid is changed to a residue found at the same or corresponding position in another Cys10 protein (see FIGS. 1A and 1B), more preferably to a residue found at the same or corresponding position in an NsG30 sequence from another species, such as the species shown in FIG. 12. In a preferred embodiment, less than 8 amino acids have been changed, more preferably less than 5 amino acids, more preferably 1 or 2 amino acids, more preferably no amino acids have been changed. These truncated NsG30 polypeptides may have up to 5 additional C and/or N-terminal amino acids selected from those of the mature NsG30 polypeptides, i.e. fragments up to AA₇-AA₁₀₁of SEQ ID No 19 or 24.

Variants can differ from naturally occurring NsG30 in amino acid sequence or in ways that do not involve sequence, or in both ways. Variants in amino acid sequence (“sequence variants”) are produced when one or more amino acids in naturally occurring NsG30 is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid. Particularly preferred variants include naturally occurring NsG30, or biologically active fragments of naturally occurring NsG30, whose sequences differ from the wild type sequence by one or more conservative and/or semi-conservative amino acid substitutions, which typically have minimal influence on the secondary and tertiary structure and hydrophobic nature of the protein or peptide. Variants may also have sequences, which differ by one or more non-conservative amino acid substitutions, deletions or insertions, which do not abolish the NsG30 biological activity. The Clustal W alignments in FIGS. 1A, 1B, and 12 can be used to predict which amino acid residues can be substituted without substantially affecting the biological activity of the protein.

Substitutions within the following group (Clustal W, ‘strong’ conservation group) are to be regarded as conservative substitutions within the meaning of the present invention

-STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.

Substitutions within the following group (Clustal W, ‘weak’ conservation group) are to be regarded as semi-conservative substitutions within the meaning of the present invention

-CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM,

Variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with human, murine or rat NsG30 (SEQ ID NO: 18, 23, and 25). More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

Variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 19 and 24. More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In a preferred embodiment the sequence identity of the variant NsG30 is determined with reference to a human NsG30 polypeptide (SEQ ID No 18, 19, 20, and 21).

In one embodiment, a variant NsG30 comprises a naturally occurring allelic variant of the sequence selected from the group consisting of SEQ ID No 18, 19, 20, 21, 23, 24, and 25 (or a subgroup thereof). Said allelic variant sequence may be an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID No 17 and 22.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 18, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 19, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 20, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 21, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 23, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 24, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 25, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, a variant NsG30 at corresponding positions comprises the residues marked in FIG. 12 as fully conserved (*), more preferably a variant NsG30 also comprises at corresponding positions the residues marked in FIG. 12 as strongly conserved (strongly conserved groups include: STA, NEQK, NHQK, NEDQ, QHRK, MILV, MILF, HY FYW), more preferably a variant NsG30 also comprises at corresponding positions the residues marked in FIG. 12 as less conserved (less conserved groups include: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHK, NEQHRK, VLIM, HFY). In particular, it is contemplated that the conserved cysteines (FIG. 1B) preferably are located at corresponding positions in a variant NsG30.

NsG32 Polypeptides

In addition to full-length NsG32, substantially full-length NsG32, mature NsG32, and to truncated forms of NsG32, the present invention provides for biologically active variants of the polypeptides. An NsG32 polypeptide or fragment is biologically active if it exhibits a biological activity of naturally occurring NsG32. It is to be understood that the invention relates to substantially purified NsG32 as herein defined.

One biological activity is the ability to compete with naturally occurring NsG32 in a receptor-binding assay. Another biological activity is the ability to bind to an antibody, which is directed at an epitope, which is present on naturally occurring NsG32. Biologically active variants may also be defined with reference to one or more of the other in vitro and/or in vivo biological assays described in the examples.

Preferably, biological activity of naturally occurring NsG32 is the activity of human or mouse NsG32b having the amino acid sequence of SEQ ID No. 30 and 37, respectively.

A preferred biological activity is the ability to elicit substantially the same response as in the PC12 assay described in the Examples. In this assay PC12 cells are transduced with full-length human NsG32a or NsG32b coding sequence. By substantially the same response in the PC12 assay is intended that the number of neurite bearing cells is at least 10% of the number obtained in Example 5 (transduction with full length human NsG32a or NsG32b CDS), more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%. The PC12 assay may also be used to document the percentage improvement in survival over a control treatment. Substantially the same response in this context means an activity resulting in at least 10% of the improvement obtained in Example 5, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%. The biological activity of a fragment or variant of NsG32 may also be higher than that of the naturally occurring NsG32.

Specific fragments of NsG32 include polypeptides having the of SEQ ID No. 34, and sequence variants of said polypeptide, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 10 of the amino acid residues in the sequence are so changed. These isolated polypeptides constitute NsG32 polypeptides spanning from the first to the last of the 8 Cys10 conserved cysteine residues found in NsG32. It is believed that the biological activity mainly resides in this part of the protein, and that the sequence with all the eight cysteines is required for correct folding of the protein. Preferably any changes in these peptides are to residues marked in the alignment of FIG. 1A as unconserved, weakly conserved, or strongly conserved. Preferably any changed amino acid is changed to a residue found at the same or corresponding position in another Cys10 protein (see FIGS. 1A and 1B). In a preferred embodiment, less than 8 amino acids have been changed, more preferably less than 5 amino acids, more preferably 1 or 2 amino acids, more preferably no amino acids have been changed. These truncated NsG32 polypeptides may have up to 5 additional C and/or N-terminal amino acids selected from those of the mature NsG32 polypeptides, i.e. fragments up to AA₈-AA₉₂of SEQ ID No 32.

Further specific fragments include polypeptides having the sequence of SEQ ID No 33, and sequence variants of said polypeptides, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 10 of the amino acid residues in the sequence are so changed. These isolated polypeptides constitute a core fragment of NsG32b corresponding to the core sequence, which is conserved in the Cys10 subfamily shown in FIG. 1A. Preferably any changes in these peptides are to residues marked in the alignment of FIG. 1A as unconserved, weakly conserved, or strongly conserved. Preferably any changed amino acid is changed to a residue found at the same or corresponding position in another Cys10 protein (see FIG. 1B). In a preferred embodiment, less than 8 amino acids have been changed, more preferably less than 5 amino acids, more preferably 1 or 2 amino acids, more preferably no amino acids have been changed. These truncated NsG32b polypeptides may have up to 5 additional C and/or N-terminal amino acids selected from those of the mature NsG32b polypeptides, i.e. fragments up to AA₆-AA₁₀₀of SEQ ID No 32.

Variants can differ from naturally occurring NsG32 in amino acid sequence or in ways that do not involve sequence, or in both ways. Variants in amino acid sequence (“sequence variants”) are produced when one or more amino acids in naturally occurring NsG32 is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid. Particularly preferred variants include naturally occurring NsG32, or biologically active fragments of naturally occurring NsG32, whose sequences differ from the wild type sequence by one or more conservative and/or semi-conservative amino acid substitutions, which typically have minimal influence on the secondary and tertiary structure and hydrophobic nature of the protein or peptide. Variants may also have sequences, which differ by one or more non-conservative amino acid substitutions, deletions or insertions, which do not abolish the NsG32 biological activity. The Clustal W alignment in FIG. 1B can be used to predict which amino acid residues can be substituted without substantially affecting the biological activity of the protein.

Substitutions within the following group (Clustal W, ‘strong’ conservation group) are to be regarded as conservative substitutions within the meaning of the present invention

-STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.

Substitutions within the following group (Clustal W, ‘weak’ conservation group) are to be regarded as semi-conservative substitutions within the meaning of the present invention

-CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY.

Other variants within the invention are those with modifications, which increase peptide stability. Such variants may contain, for example, one or more nonpeptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: variants that include residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids such as beta or gamma amino acids and cyclic variants. Incorporation of D-instead of L-amino acids into the polypeptide may increase its resistance to proteases. See, e.g., U.S. Pat. No. 5,219,990. Splice variants are specifically included in the invention.

Variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with human or mouse NsG32a (SEQ ID NO: 27, 28, and 36). More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

Variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 27, 28, and 31 (or a subgroup thereof). More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%. These polypeptides constitute full-length NsG32 polypeptides.

Preferred variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 31 and 32. More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%. These polypeptides constitute full length NsG32b and mature NsG32b.

Variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with a polypeptide having the sequence of SEQ ID NO: 29 and 32. More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%. These polypeptides constitute mature human NsG32 proteins.

Variants within the scope of the invention in one embodiment include proteins and peptides with amino acid sequences having at least 60 percent identity with a polypeptide having the sequence of SEQ ID NO: 27, 28, 29, 31, 32, 33, and 34. More preferably the sequence identity is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%. These polypeptides constitute human NsG32 proteins.

In one embodiment, a variant NsG32 comprises a naturally occurring allelic variant of the sequence selected from the group consisting of SEQ ID No 27, 28, 29, 31, 32, 33, 34, 36, 38, 40, and 41 (or a subgroup thereof). Said allelic variant sequence may be an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID No 26, 30, 35, 37, and 39 (or a subgroup thereof).

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 27, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 28, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 29, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 31, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 32, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 33, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 34, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In one embodiment, the variants include proteins comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO 41, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%.

In particular, it is contemplated that the eight conserved cysteines (FIG. 1B) preferably are located at corresponding positions in a variant NsG32.

Non-sequence modifications may include, for example, in vivo or in vitro chemical derivatisation of portions of naturally occurring NsG28, NsG30, or NsG32, as well as acetylation, methylation, phosphorylation, carboxylation, PEG-ylation, or glycosylation. Just as it is possible to replace substituents of the protein, it is also possible to substitute functional groups, which are bound to the protein with groups characterized by similar features. Such modifications do not alter primary sequence. These will initially be conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group.

Many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes such as glycosylation and other post-translational modifications, or by chemical modification techniques which are well known in the art. Among the known modifications which may be present in polypeptides of the present invention are, to name an illustrative few, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a polynucleotide or polynucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as, for instance, I. E. Creighton, Proteins-Structure and Molecular Properties, 2nd Ed., W. H. Freeman and Company, New York, 1993. Many detailed reviews are available on this subject, such as, for example, those provided by Wold, F., in Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp 1-12, 1983; Seifter et al., Meth. Enzymol. 182: 626-646, 1990 and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62, 1992.

In addition, the protein may comprise a protein tag to allow subsequent purification and optionally removal of the tag using an endopeptidase. The tag may also comprise a protease cleavage site to facilitate subsequent removal of the tag. Non-limiting examples of affinity tags include a polyhis tag, a GST tag, a HA tag, a Flag tag, a C-myc tag, a HSV tag, a V5 tag, a maltose binding protein tag, a cellulose binding domain tag. Preferably for production and purification, the tag is a polyhistag. Preferably, the tag is in the C-terminal portion of the protein.

The native signal sequence of NsG28, NsG30, or NsG32 may also be replaced in order to increase secretion of the protein in recombinant production in other mammalian cell types.

It will be appreciated, as is well known and as noted above, that polypeptides are not always entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslational events, including natural processing events and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translational natural processes and by entirely synthetic methods, as well and are all within the scope of the present invention.

Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a function of how it is made. For polypeptides made by expressing a cloned gene in a host, for instance, the nature and extent of the modifications in large part will be determined by the host cell's posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence. For instance, glycosylation often does not occur in bacterial hosts such as E. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins having native patterns of glycosylation, inter alia. Similar considerations apply to other modifications.

It will be appreciated that the same type of modification may be present to the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.

In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized by expressing a polynucleotide in a host cell.

Also included within the invention are agents, which specifically bind to a protein of the invention, or a fragment of such a protein. These agents include Ig fusion proteins and antibodies (including single chain, double chain, F_abfragments, and others, whether native, humanized, primatized, or chimeric). Additional descriptions of these categories of agents are in WO 95/16709, the disclosure of which is herein incorporated by reference.

Antibodies refer to intact molecules as well as fragments thereof, such as F_ab, F_(ab′), and F_v, which are capable of binding the epitopic determinant. Antibodies that bind NsG28, NsG30, or NsG32 polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal can be derived from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).

Humanised antibodies, as used herein, refer to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability. Humanised antibodies may be used therapeutically to treat conditions, where it is desirable to limit or block the action of NsG28, NsG30, or NsG32.

Also included within the scope of the present invention are immunoconjugates of antibodies and conjugates selected from the group consisting of: a cytotoxic agent such as a chemotherapeutic agent, a toxin, or a radioactive isotope; a member of a specific binding pair, such as avidin, or streptavidin, or an antigen; an enzyme capable of producing a detectable product. These immunoconjugates can be used to target the conjugates to cells expressing a NsG28, NsG30, or NsG32 receptor.

Specific antibodies to any NsG28, NsG30, or NsG32 are also useful in immunoassays to quantify the substance for which a given antibody has specificity. Specific antibodies to an NsG28, NsG30, or NsG32 may also be bound to solid supports, such as beads or dishes, and used to remove the ligand from a solution, either for use in purifying the protein or in clearing it from the solution. Each of these techniques is routine to those of skill in the immunological arts.

Also with the scope of the present invention are NsG28, NsG30, or NsG32 fusion proteins. An NsG28, NsG30, or NsG32 fusion protein can be used to allow imaging of tissues which express a receptor for NsG28, NsG30, or NsG32, or in the immunohistological or preparative methods described above for antibodies to an NsG28, NsG30, or NsG32. Fusion proteins encompassing an NsG28, NsG30, or NsG32 can be used to specifically target medical therapies against cells, which express an NsG28, NsG30, or NsG32 receptor.

IV. NsG28, NsG30, or NsG32 Nucleotide Sequences
NsG28 Nucleotide Sequences

The invention provides medical use of genomic DNA and cDNA coding for NsG28, including for example the nucleotide sequence of human, and mouse NsG28 cDNA (SEQ ID NO 2 and 8).

Variants of these sequences are also included within the scope of the present invention.

The invention relates to an isolated nucleic acid molecule for medical use comprising a nucleotide sequence selected from the group consisting of:

c) a nucleotide sequence coding for a biologically active fragment of at least 50 contiguous amino acids of any of a) through b);

d) a nucleotide sequence selected from the group consisting of SEQ ID No. 2 and 8;

e) a nucleotide sequence having at least 70% sequence identity to a coding sequence selected from the group consisting of the coding sequence of SEQ ID No. 2 and 8;

f) a nucleic acid sequence of at least 150 contiguous nucleotides of a coding sequence selected from the group consisting of the coding sequence of SEQ ID No. 2 and 8;

g) the complement of a nucleic acid capable of hybridising with a nucleic acid molecule having the sequence of the coding sequence of SEQ ID No.: 2 and 8 under conditions of high stringency;

h) the nucleic acid sequence of the complement of any of the above.

The nucleic acid molecule may comprise the nucleotide sequence of a naturally occurring allelic nucleic acid variant.

The nucleic acid molecule of the invention may encode a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant.

In one embodiment the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID No. 2 and 8.

Preferably the encoded polypeptide has at least 60% sequence identity to a sequence selected from the group consisting of SEQ ID No. 3, 4, 9, and 13 (or a subgroup thereof) preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably, 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, more preferably wherein the polypeptide has a sequence selected from the group consisting of said SEQ ID Nos.

In a preferred embodiment the encoded polypeptide has at least 60% sequence identity to a sequence selected from the group consisting of SEQ ID No. 3, 4, 5, 6, and 7 (or a subgroup thereof), preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably, 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, more preferably wherein the polypeptide has a sequence selected from the group consisting of said SEQ ID Nos. Said sequences constitute human NsG28.

In a preferred embodiment the encoded polypeptide has at least 60% sequence identity to a sequence selected from the group consisting of SEQ ID No. 5, 10, and 14 (or a subgroup thereof), preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably, 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, more preferably wherein the polypeptide has a sequence selected from the group consisting of said SEQ ID Nos. Said sequences constitute mature NsG28.

In a preferred embodiment the encoded polypeptide has at least 60% sequence identity to a sequence selected from the group consisting of SEQ ID No. 6, 11, and 15 (or a subgroup thereof), preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably, 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, more preferably wherein the polypeptide has a sequence selected from the group consisting of said SEQ ID Nos. Said sequences constitute NsG28 fragments.

In a preferred embodiment the encoded polypeptide has at least 60% sequence identity to a sequence selected from the group consisting of SEQ ID No. 7, 12, and 16 (or a subgroup thereof), preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably, 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, more preferably wherein the polypeptide has a sequence selected from the group consisting of said SEQ ID Nos. Said sequences constitute NsG28 fragments.

In a preferred embodiment the encoded polypeptide has at least 60% sequence identity to the sequence of SEQ ID No. 3, preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably, 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, more preferably wherein the polypeptide has a sequence selected from the group consisting of said SEQ ID No.

In a preferred embodiment the encoded polypeptide has at least 60% sequence identity to the sequence of SEQ ID No. 4, preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably, 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, more preferably wherein the polypeptide has a sequence selected from the group consisting of said SEQ ID No.

In a preferred embodiment the encoded polypeptide has at least 60% sequence identity to the sequence of SEQ ID No. 5, preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably, 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, more preferably wherein the polypeptide has a sequence selected from the group consisting of said SEQ ID No.

In a preferred embodiment the encoded polypeptide has at least 60% sequence identity to the sequence of SEQ ID No. 6, preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably, 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, more preferably wherein the polypeptide has a sequence selected from the group consisting of said SEQ ID No.

In a preferred embodiment the encoded polypeptide has at least 60% sequence identity to the sequence of SEQ ID No. 7, preferably at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably, 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, more preferably wherein the polypeptide has a sequence selected from the group consisting of said SEQ ID No.

SEQ ID No 6, 7, 11, 12, 15, and 16 represent the polypeptide sequences of NsG28 fragment polypeptides. For recombinant expression in a eukaryotic expression system, these are preferably ligated to an appropriate signal sequence to ensure that the NsG28 polypeptide is secreted from the cells.

In one aspect the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of

a) the nucleotide sequence selected from the group consisting of SEQ ID No. 2 and 8;

b) a nucleotide sequence having at least 70% sequence identity to a nucleotide sequence selected from the group consisting of the coding sequence of SEQ ID No. 2 and 8;

c) a nucleic acid sequence of at least 150 contiguous nucleotides of a sequence selected from the group consisting of the coding sequence of SEQ ID No. 2 and 8;

d) the complement of a nucleic acid capable of hybridising with nucleic acid having the sequence selected from the group consisting of the coding sequence of SEQ ID No. 2 and 8 under conditions of high stringency; and

e) the nucleic acid sequence of the complement of any of the above.

In one preferred embodiment, the isolated polynucleotide of the invention has at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, preferably at least 85%, more preferred at least 90%, more preferred at least 95%, more preferred at least 98% sequence identity to a polynucleotide sequence selected from the group consisting of the coding sequence of SEQ ID NO: 2 and 8.

The nucleotide sequence for nucleic acids coding for full length NsG28, for mature NsG28 and for fragments of NsG28 can be derived from FIGS. 20 and 21, which show the cDNA sequence and the translated peptides of human NsG28 (FIG. 20) and mouse NsG28 (FIG. 21). Specific fragments of these cDNA sequences include those coding for mature NsG28 and fragments of NsG28, including the nucleic acid molecule having the nucleotide sequence of nucleotides 466-888 of SEQ ID No. 2 (long human NsG28 CDS); the nucleic acid molecule having the nucleotide sequence of nucleotides 481-888 of SEQ ID No. 2 (human NsG28 CDS); the nucleic acid molecule having the nucleotide sequence of nucleotides 568-885 of SEQ ID No. 2 (human mature NsG28 CDS); the nucleic acid molecule having the nucleotide sequence of nucleotides 622-846 of SEQ ID No. 2 (human NsG28 cys1-cys10 fragment CDS); the nucleic acid molecule having the nucleotide sequence of nucleotides 616-876 of SEQ ID No. 2 (human NsG28 core fragment CDS); the nucleic acid molecule having the nucleotide sequence of nucleotides 206-613 of SEQ ID No. 8 (mouse NsG28 CDS); the nucleic acid molecule having the nucleotide sequence of nucleotides 293-610 of SEQ ID No. 8 (mouse mature NsG28 CDS); the nucleic acid molecule having the nucleotide sequence of nucleotides 347-571 of SEQ ID No. 8 (mouse NsG28 cys1-cys10 fragment CDS); and the nucleic acid molecule having the nucleotide sequence of nucleotides 341-601 of SEQ ID No. 8 (mouse NsG28 core fragment CDS).

NsG30 Nucleotide Sequences

The invention provides medical use of nucleic acids coding for NsG30, including for example the nucleotide sequence of human, and mouse NsG30 cDNA (SEQ ID NO 17 and 22).