Neublastin neurotrophic factors

Details Neublastin neurotrophic factors Neublastin neurotrophic factors

2000-09-15

2004-05-11

Kunz, Gary (Department: 1647)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S300000

Reexamination Certificate

active

06734284

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to neurotrophic factor polypeptides, nucleic acids encoding neurotrophic factor polypeptides, and antibodies that bind specifically to neurotrophic factors.
BACKGROUND
Neurotrophic factors are naturally-occurring proteins which promote survival, maintain phenotypic differentiation, prevent degeneration, and enhance the activity of neuronal cells and tissues. Neurotrophic factors are isolated from neural tissue and from non-neural tissue that is innervated by the nervous system, and have been classified into functionally and structurally related groups, also referred to as families, superfamilies, or subfamilies. Among the neurotrophic factor superfamilies are the fibroblast growth factor, neurotrophin, and transforming growth factor-&bgr; (TGF-&bgr;) superfamilies. Individual species of neurotrophic factors are distinguished by their physical structure, their interaction with their composite receptors, and their affects on various types of nerve cells. Classified within the TGF-&bgr; superfamily (Massague, et al,. 1994
, Trends in Cell Biology,
4:172-178) are the glial cell line-derived neurotrophic factor ligands (“GDNF”; WO 93/06116, incorporated herein by reference), which include GDNF, persephin (“PSP”; Milbrandt et al., 1998
, Neuron
20:245-253, incorporated herein by reference) and neurturin (“NTN”; WO 97/08196, incorporated herein by reference). The ligands of the GDNF subfamily have in common their ability to induce signalling through the RET receptor tyrosine kinase. These three ligands of the GDNF subfamily differ in their relative affinities for a family of neurotrophic receptors, the GFR&agr; receptors.
Due to the affects of neurotrophic factors on neuronal tissue, there remains a need to identify and characterise additional neurotrophic factors for diagnosing and treating disorders of the nervous system.
SUMMARY OF THE INVENTION
This invention relates to a novel neurotrophic factor herein called “neublastin,” or “NBN.” Neublastin is classified within the GDNF subfamily because it shares regions of homology with other GDNF ligands (see Tables 3 and 4, infra) and because of its ability to interact with RET (see, e.g., Airaksinen et al., Mol. Cell. Neuroscience, 13, pp. 313-325 (1999)), neublastin is a novel and unique neurotrophic factor. Unlike other GDNF ligands, neublastin exhibits high affinity for the GFR&agr;3-RET receptor complex and unique subregions in its amino acid sequence.
A “neublastin polypeptide,” as used herein, is a polypeptide which possesses neurotrophic activity (e.g., as described in Examples 6, 7, 8, and 9) and includes those polypeptides which have an amino acid sequence that has at least 70% homology to the human “neublastin” polypeptides set forth in AA
−95
-AA
105
of SEQ. ID. NO. 2, AA
1
-AA
105
of SEQ. ID. NO. 2, AA
−97
-AA
140
of SEQ. ID. NO. 4, AA
−41
-AA
140
of SEQ. ID. NO. 4 (“pro”), AA
1
-AA
140
of SEQ. ID. NO. 4, AA
−80
-AA
140
of SEQ. ID. NO. 9 (“wild type” prepro), AA
−41
-AA
140
of SEQ. ID. NO. 9 (pro), AA
1
-AA
140
of SEQ. ID. NO. 5 (mature 140AA), AA
1
-AA
116
of SEQ. ID. NO. 6 (mature 116AA), AA
1
-AA
113
of SEQ. ID. NO. 7 (mature 113AA), AA
1
-AA
140
of SEQ. ID. NO. 10 (mature 140AA), AA
1
-AA
116
of SEQ. ID. NO. 11 (mature 116AA), AA
1
-AA
113
of SEQ. ID. NO. 12 (mature 113AA), and variants and derivatives thereof. In addition, this invention contemplates those polypeptides which have an amino acid sequence that has at least 70% homology to the murine “neublastin” polypeptides set forth in AA
1
-AA
224
of SEQ. ID. NO. 16.
Preferably, the C-terminal sequence of the above identified neublastin polypeptides has an amino acid sequence as set forth in AA
72
-AA
105
of SEQ. ID. NO. 2 (i.e., AA
107
-AA
140
of SEQ. ID. NO. 9), more preferably AA
41
-AA
105
of SEQ. ID. NO. 2 (i.e., AA
76
-AA
140
of SEQ. ID. NO. 9), or the amino acid sequence set forth in AA
191
-AA
224
of SEQ. ID. NO. 16.
Also, it is preferable that the neublastin polypeptide retain the 7 conserved Cys residues that are characteristic of the GDNF family and of the TGF-beta super family.
Preferably, the neublastin polypeptide has an amino acid sequence greater than 85% homology, most preferably greater than 95% homology, to the foregoing sequences (i.e., AA
−95
-AA
105
of SEQ. ID. NO. 2, AA
1
-AA
105
of SEQ. ID. NO. 2, AA
−97
-AA
140
of SEQ. ID. NO. 4, AA
1
-AA
140
of SEQ. ID. NO. 4, AA
−41
-AA
140
of SEQ. ID. NO. 4, AA
−80
-AA
140
of SEQ. ID. NO. 9 (“wild type” prepro), AA
−41
-AA
140
of SEQ. ID. NO. 9 (pro), AA
1
-AA
140
of SEQ. ID. NO. 5 (mature 140AA), AA
1
-AA
116
of SEQ. ID. NO. 6 (mature 116AA), AA
1
-AA
113
of SEQ. ID. NO. 7 (mature 113AA), AA
1
-AA
140
of SEQ. ID. NO. 10 (mature 140AA), AA
1
-AA
116
of SEQ. ID. NO. 11 (mature 116AA), AA
1
-AA
113
of SEQ. ID. NO. 12 (mature 113AA)), and AA
1
-AA
224
of SEQ. ID. NO. 16.
A “neublastin nucleic acid,” as used herein, is a polynucleotide which codes for a neublastin polypeptide. Accordingly, an isolated neublastin nucleic acid is a polynucleotide molecule having an open reading frame of nucleotide codons that, were it to be exposed to the appropriate components required for translation, would encode, or code for, a neublastin polypeptide. Neublastin nucleic acids of the invention may be RNA or DNA, e.g., genomic DNA, or DNA which is complementary to and/or transcribed from, a neublastin mRNA (“cDNA”). Thus, a neublastin nucleic acid of the invention further includes polynucleotide molecules which hybridize with specificity, under high stringency hybridization conditions, to a polynucleotide that codes for a neublastin polypeptide. This invention also relates to nucleic acid primers that are useful in identifying, isolating and amplifying polynucleotides that encode neublastin polypeptides, or fragments thereof. In certain embodiments of the invention, certain of these primers are neublastin-specific probes useful for hybridization to a neublastin nucleic acid, but not to nucleic acids coding for the other members of the GDNF family. By “specific”, “specificity”, or “specifically”, is meant an ability to hybridize with neublastin nucleic acid and inability to hybridize with non-neublastin nucleic acids, including an inability to hybridize to nucleic acids that code uniquely for the GDNF ligands (e.g., GDNF, persephin, and neurturin).
In another embodiment, a neublastin nucleic acid of the invention is one that is identified as being complementary to a polynucleotide that codes for a neublastin polypeptide, either by having a complementary nucleic acid sequence or demonstrating that it hybridizes with specificity at high stringency hybridization conditions to a polynucleotide that codes for neublastin. Particular neublastin nucleic acids include, without limitation, the nucleic acid sequences shown herein and designated SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 29 and SEQ ID NO: 30 as well as primers SEQ ID NOS: 17-28, 31 and 32. A neublastin nucleic acid of the invention further includes a unique subregion, or fragment, of a neublastin nucleic acid, including without limitation the nucleic acid fragments shown in FIG.
8
.
The neublastin nucleic acids of the invention may be used to express a neublastin polypeptide, e.g., by expressing a neublastin polypeptide in vitro, or by administering a neublastin nucleic acid to an animal for in vivo expression. Neublastin nucleic acids may be included within a nucleic acid vector, e.g., an expression vector or a cloning vector. A neublastin nucleic acid may, but need not of necessity, be maintained, reproduced, transferred, or expressed as part of a nucleic acid vector. A recombinant expression vector containing a neublastin polynucleotide sequence can be introduced into and/or maintained within a cell. Cells hosting a neublastin vector may be prokaryotic. Alternatively, a neublastin nucleic acid can be introduced into a eukaryotic cell, e.g., a eukaryotic cell that contains the app

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