Monoclonal antibody against utricular epithelium

A non-naturally occurring peptide derived from EGF-like domains of NDF/heregulin protein isoforms is used to stimulate the proliferation of cells in the sensory epithelium of the inner ear. A monoclonal antibody against adult rat utricular epithelium is also described.

FIELD OF THE INVENTION 
This invention relates to the NDF/heregulin protein family, and more 
specifically to the use of a derivative peptide to stimulate the 
proliferation of sensory epithelial cells of the inner ear for the 
treatment of vestibular disorders. The invention also relates to 
monoclonal antibodies raised against adult rat utricular epithelium. 
BACKGROUND OF THE INVENTION 
The NDF/heregulins are a known family of molecules which stimulate the 
tyrosine phosphorylation of the erbB2/Her2 protooncogene product p185; see 
Peles et al., Cell, Volume 69, pages 1-14 (1992); Wen et al., Cell, Volume 
69, pages 559-572 (1992); Holmes et al., Science, Volume 256, pages 
1205-1210 (1992); and Bacus et al., Cancer Research, Volume 53, pages 
5251-5261 (1993). Thought at first to be ligands for erbB2/Her2, the 
NDF/heregulins are now known to bind to and stimulate the kinase activity 
of erbB3/Her3 and erbB4/Her4; see Plowman et al., Nature, Volume 366, 
pages 473-475 (1993); Kita et al., FEBS Letters, Volume 349, pages 139-143 
(1994); and Carraway et al., Journal of Biological Chemistry, Volume 269, 
pages 14303-14306 (1994). The NDF/heregulin family is considered to also 
include ARIA and glial growth factor (GGF); see, respectively, Falls et 
al., Cell, Volume 72, pages 801-805 (1993), and Marchionni et al., Nature, 
Volume 362, pages 312-316 (1993). 
The NDF/heregulins are transmembrane glycoproteins which, in the main, 
possess an EGF-like domain in the extracellular portion that may function 
as a receptor recognition site; Wen et al., Cell, above. The original 
group of NDF/heregulins comprise at least ten isoforms, which can be 
divided into two groups, called alpha (.alpha.) and beta (.beta.), based 
on differences in the EGF-like domain. Analysis suggests that the various 
isoforms are generated by alternative gene splicing and perform distinct 
tissue-specific functions in vivo; Wen et al., Molecular and Cellular 
Biology, Volume 14, Number 3, pages 1909-1919 (1994). 
Several functions of the NDF/heregulins have been identified, including the 
induction of either mitogenesis or differentiation in mammary cells (Peles 
et al., Cell, above); the stimulation of muscle acetylcholine receptors 
(Falls et al., above); and the mitogenesis of Schwann cells (Marchionni et 
al., above). 
Kita et al. in Biochemical and Biophysical Research Communications, Volume 
210, Number 2, pages 441-451 (1995), describe a biologically active 
52-amino acid peptide which is based on the sequence of the EGF-like 
domain of NDF-.alpha.2 and is produced by chemical synthesis. The peptide 
is shown to stimulate tyrosine phosphorylation of Her2, Her3 and Her4, and 
to induce morphological changes in breast cancer cells. 
U.S. Pat. No. 5,670,342 (issued Sep. 23, 1997) describes chemically 
synthesized peptides which are derived from the sequence of the EGF-like 
domain of NDF and are active in promoting the growth of both Schwann cells 
and colon epithelial cells. One of these peptides is a "hybrid" 
(.alpha./.beta.) molecule composed of sequence from the EGF-like domains 
of both the alpha(.alpha.) and the beta(.beta.) forms of NDF. See, also, 
U.S. Pat. No. 5,686,415 (issued Nov. 11, 1997), as well as pending 
applications Ser. No. 08/761,038 and Ser. No. 08/761,762, both filed Dec. 
5, 1996. 
SUMMARY OF THE INVENTION 
The present invention comprises the use of a peptide of following sequence 
as a growth stimulant for sensory epithelial cells of the inner ear: 
SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCQPGFTGARCQNYVMAS 
(SEQ ID NO: 1) 
This peptide has been described previously in U.S. Pat. No. 5,670,342 and 
U.S. Pat. No. 5,686,415 as a hybrid form derived from the EGF-like domains 
of NDF-.alpha. and NDF-.beta.. However, the usefulness of this molecule as 
a growth stimulant for sensory epithelial cells of the utricle in the 
inner ear, which is demonstrated in the working examples below, has not 
been previously recognized. Because all of the vestibular organs (e.g., 
utricle, semicircular canal, etc.) have similar cellular organization, it 
is reasonable to assume that they will respond in the same way to contact 
and treatment with the present peptide. Thus, the peptide is expected to 
be useful to treat vestibular disorders such as, for example, loss of 
balance due to utricular degeneration or disease in mammals, including 
humans. The peptide may also be useful to treat hearing loss in mammals, 
including humans, which is attributable to the degeneration of inner ear 
hair cells, i.e., by regenerating such hair cells in association with 
sensory epithelium. 
An additional aspect of the invention comprises monoclonal antibodies 
raised against adult rat utricular epithelium, which are especially useful 
in study and research with such organs and tissue, e.g., because of their 
applicability for differential staining.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention contemplates the use of isolated peptide having the 
primary structural conformation (i.e., continuous sequence of amino acid 
residues) of SEQ ID NO: 1 (see also FIG. 1). The term "purified and 
isolated" herein means substantially free of unwanted substances so that 
the peptide is useful the described intended purpose, i.e., as an agent 
for causing the proliferation of vestibular sensory epithelial cells and 
growth of vestibular sensory epithelium. 
The usefulness of the peptide of SEQ ID NO: 1 as a mitogenic agent for 
vestibular sensory epithelial cells lends itself to the therapeutic 
treatment of balance disorders, such as vertigo or other chronic or acute 
types of dysequilibrium or dizziness attributable to vestibular organ 
degeneration and malfunction. 
The mitogenic activity of the peptide of SEQ ID NO: 1 on the vestibular 
sensory epithelium of the mammalian inner ear suggests that it may also be 
useful to regenerate hair cells, which are critical for hearing. Thus, the 
peptide may be beneficial for treating hearing loss associated with 
deteriorated or damaged inner ear hair cells, and such applications are 
included within the therapeutic treatments made possible by the present 
invention. 
The peptide of SEQ ID NO: 1 can be prepared by the use of chemical 
synthesis procedures. Alternatively, the peptide can be the product of 
recombinant expression of exogenous cDNA (obtained by DNA synthesis), the 
nucleic acid molecule of SEQ ID NO: 2, for the example, in a prokaryotic 
or eukaryotic host (e.g., bacterial, yeast, higher plant, insect or 
mammalian cells in culture). 
Suitable methods of chemical synthesis are well known to the skilled 
artisan; see Engels et al., Angew. Chem. Intl. Ed., Volume 28, pages 
716-734 (1989). Such methods include the phosphotriester, phosphoramidite 
and H-phosphonate methods for nucleic acid synthesis. A preferred method 
involves polymer supported synthesis using standard phosporamidite 
chemistry. 
Merely by way of example, the peptide of SEQ ID NO: 1 can be prepared 
directly from the method of fluorenylmethoxycarbonyl/t-butyl-based solid 
phase chemistry method described in U.S. Pat. No. 5,670,342, followed by 
folding into active product in a buffered solution composed of TRIS, EDTA 
and glutathione, and purification. 
If recombinant methods of production for the peptide are used, the product 
such of expression in typical yeast (e.g., Saccharomyces cerevisiae), 
insect, or prokaryote (e.g., E. coli) host cells will, like chemical 
synthesis, also be free of association with any mammalian proteins. The 
product of expression in vertebrate (e.g., non-human mammalian such as COS 
or CHO, and avian) cells is typically free of association with any human 
proteins. The peptide may also include an initial methionine amino acid 
residue (at position -1 with respect to the first amino acid residue of 
the mature peptide). 
Variant nucleic acid molecules having sequences which differ from SEQ ID 
NO: 2 but still encode the peptide of SEQ ID NO: 1 may be produced. Such 
variants would also include those containing nucleotide substitutions 
accounting for codon preference in the host cell being employed for 
expression. 
The nucleic acid molecule of SEQ ID NO: 2, or a variant thereof encoding 
the peptide of SEQ ID NO: 1, can be inserted into any suitable expression 
vector using standard ligation techniques. The vector is selected to be 
functional in the particular host employed (i.e., the vector is compatible 
with the host cell machinery, such that expression of the nucleic acid 
encoding the peptide can occur). The peptide may be expressed in 
prokaryotic, yeast, insect (baculovirus systems) or eukaryotic cells as 
the host. 
The vectors used in any of the host cells to express the peptide may also 
contain a 5' flanking sequence (also referred to as a "promoter") and 
other expression regulatory elements operatively linked to the nucleic 
acid molecule (DNA) to be expressed, as well as enhancer(s), an origin of 
replication element, a transcriptional termination element, a complete 
intron sequence containing a donor and acceptor splice site, a signal 
peptide sequence, a ribosome binding site element, a polylinker region for 
inserting the nucleic acid encoding the peptide to be expressed, and a 
selectable marker element, as those skilled in the art will know. 
Insertion of the vector into the selected host cell (also referred to as 
"transformation" or "transfection") may be accomplished using known 
materials or methods such as calcium chloride, electroporation, 
microinjection, lipofection, or the DEAE-dextran method. 
The host cell, when cultured under suitable nutrient conditions, will 
synthesize the peptide, which can subsequently be collected by isolation 
from the culture medium (if the host cell secretes it into the medium) or 
directly from the host cell (if not secreted). For peptide situated in the 
host cell cytoplasm and/or nucleus, the host cells are typically first 
disrupted mechanically or with detergent to release the intracellular 
contents into a buffered solution. The peptide can then be collected from 
this solution. 
Selection of the host cell will depend in part on whether the host cell is 
able to "fold" the peptide into its native tertiary structure such that 
biologically active material is prepared by the cell. Even where the host 
cell does not synthesize the peptide in the proper conformation, the 
peptide may be "folded" after synthesis using appropriate chemical 
conditions. 
Suitable cells or cell lines may be mammalian cells, such as Chinese 
hamster ovary cells (CHO) or 3T3 cells. The selection of suitable 
mammalian host cells and methods for transformation, culture, 
amplification, screening and product production and purification are known 
in the art. Other suitable mammalian cell lines, are the monkey COS-1 and 
COS-7 cell lines, and the CV-1 cell line. Further exemplary mammalian host 
cells include primate cell lines and rodent cell lines, including 
transformed cell lines. Normal diploid cells, cell strains derived from in 
vitro culture of primary tissue, and primary explants are also suitable. 
Candidate cells may be genotypically deficient in the selection gene, or 
may contain a dominantly acting selection gene. Other suitable mammalian 
cell lines include, but are not limited to, mouse neuroblastoma N2A cells, 
HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, 
BHK or HaK hamster cell lines. 
Similarly useful as host cells are bacterial cells. For example, the 
various strains of E. coli (e.g., HB101, DH5.alpha., DH10, and MC1061) are 
well-known as host cells in the field of biotechnology. Various strains of 
B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., and 
the like may also be employed in this method. 
Host cells containing the vector may be cultured using standard media well 
known to the skilled artisan. The media will usually contain all of the 
nutrients necessary for the growth and survival of the transformed cells. 
Suitable media for culturing E. coli cells are, for example, Luria Broth 
(LB) and/or Terrific Broth (TB). Suitable media for culturing eukaryotic 
cells are RPMI 1640, MEM, DMEM, all of which may be supplemented with 
serum and/or growth factors as required by the particular cell line being 
cultured. A suitable medium for the culturing of insect cells is Grace's 
medium supplemented with yeastolate, lactalbumin hydrolysate and/or fetal 
calf serum, as necessary. 
Typically, an antibiotic or other compound useful for selective growth of 
the transformed cells is added as a supplement to the growth medium. The 
compound to be used will be dictated by the selectable marker element 
present on the plasmid with which the host cell has been transformed or 
transfected. For example, where the selectable marker element is kanamycin 
resistance, the compound added to the culture medium will be kanamycin. 
Purification of the peptide from solution can be accomplished using a 
variety of techniques. If the peptide has been synthesized such that it 
contains a "tag", it may essentially be purified in a one-step process by 
passing the solution through an affinity column where the column matrix 
has a high affinity for the tag or for the peptide directly (i.e., a 
monoclonal antibody specifically recognizing the peptide). Where, on the 
other hand, the peptide is prepared without a tag attached, and no 
antibodies are available, other well known procedures for purification can 
be used. Such procedures include, without limitation, ion exchange 
chromatography, molecular sieve chromatography, HPLC, native gel 
electrophoresis in combination with gel elution, and preparative 
isoelectric focusing. In some cases, it may be preferable to use more than 
one of these methods for complete purification. 
One may modify the peptide of SEQ ID NO: 1 to create a fusion molecule with 
another peptide sequence. For example, if one desired to "tag" the peptide 
with an immunogenic peptide, one could construct a DNA which would result 
in such fusion product. The tag may be at the N-terminus or the 
C-terminus. An example is a "FLAG-tag" version of the peptide. This type 
of "tagging" is useful to bind the peptide using reagents, such as 
antibodies, which are selective for the tag. Such binding may be for 
detection of the location or amount of peptide, or for peptide capturing 
processes where, for example, an affinity column is used to bind the tag, 
and thus the desired peptide. Other types of detectable labels, such as 
radioisotopes, light-emitting (e.g., fluorescent or phosporescent 
compounds), enzymatically cleavable, detectable antibody (or modification 
thereof), or other substances may be used for such labeling of the 
peptide. 
To enhance functional properties, the peptide may also be derivatized by 
the attachment of one or more other chemical moieties. Such chemical 
moieties may be selected from among various water soluble polymers. The 
polymer should be water soluble so that the peptide to which it is 
attached is miscible in an aqueous environment, such as a physiological 
environment. The water soluble polymer may be selected from the group 
consisting of, for example, polyethylene glycol, copolymers of ethylene 
glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl 
alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, 
ethylene/maleic anhydride copolymer, polyamino acids (either homopolymers 
or random or non-random copolymers; see further below regarding fusion 
molecules), and dextran or poly(n-vinyl pyrolidone)polyethylene glycol, 
propylene glycol homopolymers, polypropylene oxide/ethylene oxide 
co-polymers, polyoxyethylated polyols, polystyrenemaleate and polyvinyl 
alcohol. Polyethylene glycol propionaldehyde may have advantages in 
manufacturing due to its stability in water. 
Fusion peptides in accordance with the present invention may be prepared by 
attaching polyamino acids to the peptide of SEQ ID NO: 1. For example, the 
polyamino acid may be a carrier protein which serves to increase the 
circulation half life of the peptide. The polyamino acid should be one 
which does not create a neutralizing antigenic response, or other adverse 
in vivo response. The polyamino acid may be selected from the group 
consisting of serum album (such as human serum albumin), an antibody or 
portion thereof (such as an antibody constant region, sometimes called 
"F.sub.c ") or other polyamino acids. The location of attachment of the 
polyamino acid may be at the N-terminus of the peptide moiety, or other 
place, and also may be connected by a chemical "linker" moiety to the 
peptide. 
The polymer may be of any molecular weight, and may be branched or 
unbranched. For polyethylene glycol, the preferred molecular weight is 
between about 2 kilodaltons (kDa) and about 100 kDa (the term "about" 
indicating that in preparations of polyethylene glycol, some molecules 
will weigh more, some less, than the stated molecular weight) for ease in 
handling and manufacturing. Other sizes may be used, depending on the 
desired therapeutic profile (e.g., the duration of sustained release 
desired, the effects, if any on biological activity, the ease in handling, 
the degree or lack of antigenicity and other known effects of the 
polyethylene glycol). 
The number of polymer molecules so attached may vary, and one skilled in 
the art will be able to ascertain the effect on function. One may 
mono-derivatize, or may provide for a di-, tri-, tetra- or some 
combination of derivatization, with the same or different chemical 
moieties (e.g., polymers, such as different weights of polyethylene 
glycols). The proportion of polymer molecules to peptide molecules will 
vary, as will their concentrations in the reaction mixture. In general, 
the optimum ratio (in terms of efficiency of reaction in that there is no 
excess unreacted peptide or polymer) will be determined by factors such as 
the desired degree of derivatization (e.g., mono, di-, tri-, etc.), the 
molecular weight of the polymer selected, whether the polymer is branched 
or unbranched, and the reaction conditions. 
The chemical moieties should be attached to the peptide with consideration 
of effects on functional or antigenic domains of the peptide. There are a 
number of attachment methods available to those skilled in the art. See, 
for example, European Patent No. 0 401 384 (coupling PEG to G-CSF), and 
Malik et al., Experimental Hematology, Volume 20, pages 1028-1035 (1992) 
(reporting pegylation of GM-CSF using tresyl chloride). By way of 
illustration, polyethylene glycol may be covalently bound through amino 
acid residues via a reactive group, such as a free amino or carboxyl 
group. Reactive groups are those to which an activated polyethylene glycol 
molecule (or other chemical moiety) may be bound. The amino acid residues 
having a free amino group may include lysine residues and the N-terminal 
amino acid residue. Those having a free carboxyl group may include 
aspartic acid residues, glutamic acid residues, and the C-terminal amino 
acid residue. Sulfhydryl groups may also be used as a reactive group for 
attaching the polyethylene glycol molecule(s) or other chemical moiety. 
Preferred for therapeutic manufacturing purposes is attachment at an amino 
group, such as at the N-terminus or to a lysine group. Attachment at 
residues important for receptor binding should be avoided if receptor 
binding is important. 
One may specifically desire N-terminally chemically modified derivatives. 
Using polyethylene glycol as an illustration, one may select from a 
variety of polyethylene glycol molecules (by molecular weight, branching, 
etc.), the proportion of polyethylene glycol molecules to peptide 
molecules in the reaction mixture, the type of pegylation reaction to be 
performed, and the method of obtaining the selected N-terminally pegylated 
peptide. The method of obtaining the N-terminally pegylated preparation 
(i.e., separating this moiety from other monopegylated moieties if 
necessary) may be by purification of the N-terminally pegylated material 
from a population of pegylated peptide molecules. Selective N-terminal 
chemical modification may be accomplished by reductive alkylation which 
exploits the differential reactivity of different types of primary amino 
groups (lysine versus the N-terminal) available for derivatization in a 
particular protein. See PCT application WO 96/11953, published Aug. 25, 
1996. 
Any of the above mentioned derivatives may be used in the method of this 
invention. 
The peptide of SEQ ID NO: 1 or a derivative thereof can be formulated into 
a composition suitable for administration by injection, or for oral, 
pulmonary, nasal, transdermal, or other forms of administration. Included 
within the invention are pharmaceutical compositions comprising effective 
amounts of the peptide or derivative product together with 
pharmaceutically acceptable diluents, preservatives, solubilizers, 
emulsifiers, adjuvants and/or carriers. By "effective amount" is meant an 
amount sufficient to produce a measurable biological (e.g., mitogenic) 
effect on the treated sensory epithelial cells or tissue. Such 
compositions include diluents of various buffer content (e.g., Tris-HCl, 
acetate, phosphate), pH and ionic strength; additives such as detergents 
and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants 
(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., 
Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, 
mannitol); incorporation of the material into particulate preparations of 
polymeric compounds such as polylactic acid, polyglycolic acid, etc., or 
into liposomes. See, for example, PCT application WO 96/29989 (Collins et 
al.), published Oct. 3, 1996. Hyaluronic acid may also be used, and this 
may have the effect of promoting sustained duration in the circulation. 
Such compositions may influence the physical state, stability, rate of in 
vivo release, and rate of in vivo clearance of the present proteins and 
derivatives. See, for example, Remington's Pharmaceutical Sciences, 18th 
Edition, Mack Publishing Co., Easton, Pa., pages 1435-1712 (1990). The 
compositions may be prepared in liquid form, or may be in dried powder, 
such as lyophilized form. Implantable sustained release formulations are 
also contemplated, as are transdermal formulations. 
The peptide may be chemically modified so that oral delivery of the 
derivative is enhanced. Generally, the chemical modification contemplated 
for the present purposes is the attachment of at least one moiety to the 
peptide itself, where this moiety permits (a) inhibition of proteolysis 
and (b) uptake into the blood stream from the stomach or intestine. Also 
desired is the increase in overall stability of the peptide and increase 
in circulation time in the body. See PCT application WO 95/21629, 
Habberfield, "Oral Delivery of Chemically Modified Proteins" (published 
Aug. 17, 1995), and U.S. Pat. No. 5,574,018 (Habberfield et al.), issued 
Nov. 12, 1996. 
Nasal delivery of the peptide of SEQ ID NO: 1 (or derivative) is also 
contemplated. Nasal delivery allows the passage of the peptide (or 
derivative) to the blood stream directly after administering the 
therapeutic product to the nose, without the necessity for deposition of 
the product in the lung. Formulations for nasal delivery include those 
with absorption enhancing agents, such as dextran or cyclodextran. 
If desired, the peptide or derivative may also be administered systemically 
in a sustained release formulation or preparation. Suitable examples of 
sustained release preparations include semipermeable polymer matrices in 
the form of shaped articles, for example, films or microcapsules. 
Sustained release matrices include polyesters, hydrogels, polylactides 
(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma 
ethyl-L-glutamine (Sidman et al, Biopolymers, Volume 22, pages 547-556, 
1983), poly(2-hydroxyethyl-methacrylate) (Langer et al., Journal of 
Biomedical Materials Research, Volume 15, pages 167-277, 1981, and Langer, 
Chemical Technology, Volume 12, pages 98-105, 1982), ethylene vinyl 
acetate (Langer et al., above) or poly-D(-)-3-hydroxybutyric acid. 
Sustained-release compositions also may include liposomes, which can be 
prepared by any of several methods known in the art; see, for example, 
Epstein et al., Proceedings of the National Academy of Sciences USA, 
Volume 82, pages 3688-3692 (1985), and Hwang et al., National Academy of 
Sciences USA, Volume 77, pages 4030-4034 (1980). 
Typically, the peptide will be in highly purified form, and the therapeutic 
composition will normally be presterilized for use, such as by filtration 
through sterile filtration membranes. 
The amount of peptide that will be effective in vivo may vary. One skilled 
in the art will be able to ascertain effective dosages by administration 
and observing the resulting therapeutic effect. Preferably, the 
formulation of the peptide in a pharmaceutical composition will be such 
that between about 0.10 .mu.g/kg/day and 10 mg/kg/day will yield the 
desired therapeutic effect. The composition may be administered as a 
single dose, or as two or more doses (which may or may not contain the 
same amount of peptide) over time, or on a continuous basis. 
The invention is described in further detail with regard to the following 
working examples, which are not intended to be limiting. 
EXAMPLE 1 
Cell Proliferation in Primary Culture of Sensory Epithelium 
Sensory epithelial cells obtained from utricles in the inner ear of both 
seven day-old (infant) rats and six week-old (adult) rats were isolated 
with the use of thermolysin treatment; see Corwin et al., Abstracts of the 
Association for Research in Otolaryngology, Volume 18, page 87 (1995). All 
edges were trimmed away and the central portion of the epithelium was cut 
into quarters. Epithelial cells from the infant rats were cultured in 
DMEM/F12 with 10% FBS (Gibco BRL, Grand Island, N.Y.), and 3 micrograms 
per milliliter (.mu.g/ml) of mitosis tracer BrdU (Aldrich Chemicals, 
Milwaukee, Wis.) for seventy-two hours, with or without 50 nanograms per 
milliliter (ng/ml) of the peptide of SEQ ID NO: 1 or 50 ng/ml of 
recombinant derived FGF-10, recombinant derived FGF-16, recombinant 
derived ciliary-derived neurotrophic factor (CNTF), recombinant derived 
neurotrophic growth factor (NGF), recombinant derived glial-derived 
neurotrophic factor (GDNF), recombinant derived keratinocyte growth factor 
(KGF), or a control (no growth factor present). The experiment was ended 
by fixing in 4% paraformaldehyde for one hour. 
For the adult rats, the total time for cell culture was thirteen days, with 
the peptide of SEQ ID NO: 1 or other growth factor being added for the 
last five days and the BrdU (3 .mu.g/ml) for the last two days. The 
experiment was ended as above by fixing the explants in paraformaldehyde. 
After several washes, the explants were immunostained for calretinin (a 
vestibular organ hair cell marker) using rabbit antiserum (SWant Swiss 
Antibodies, Belinzona, Switzerland), diluted 1:10,000. The secondary 
antibody was goat anti-rabbit immunoglobulin conjugated to biotin, diluted 
1:1,000. Extensive washes were followed by incubating the explants with 
egg white avidin conjugated to Cy3, diluted 1:1,000. The explants were 
fixed again with paraformaldehyde as described above, then processed for 
BrdU immunostaining (a measure of cell proliferation) using standard 
immunostaining protocols. The mouse monoclonal ant-BrdU antibody was 
obtained from Caltag Laboratories of San Francisco, Calif. The secondary 
antibody, a goat anti-mouse conjugated to FITC, was obtained from 
Antibodies, Inc. of Davis, Calif. 
In the test group using cultured epithelia from infant rats, the number of 
BrdU-positive nuclei per piece of epithelium was found to be significantly 
higher in the peptide-treated (SEQ ID NO: 1) group, 397.+-.33, when 
compared to: the control (untreated) group (50.+-.7), the FGF-10-treated 
group (11.+-.4), the FGF-16-treated group (100.+-.16), the CNTF-treated 
group (23.+-.3), the NGF-treated group (50.+-.9), the GDNF-treated group 
(37.+-.13), and the KGF-treated group (89.+-.16). See FIG. 2 for a 
graphical representation of these results. 
The mitogenic activity of the peptide of SEQ ID NO: 1 was also compared 
with glial growth factor (GGF) using a similar protocol. This test was 
additionally used to evaluate whether the peptide of SEQ ID NO: 1 was 
active on sensory epithelium derived from adult (rather than just infant) 
rats. While treatment with the peptide of SEQ ID NO: 1 induced a 32.5-fold 
increase in proliferation of adult rat sensory epithelial cells over the 
control group, treatment with GGF only induced a 22.3-fold increase in the 
proliferation of such cells. See FIG. 3 for a graphical representation of 
these results. 
The effect of treatment with the peptide of SEQ ID NO: 1 in the primary 
cell culture of infant rat sensory epithelium is also shown very clearly 
in FIG. 4A and 4B. As can be seen, there is a very noticeable increase in 
the presence of BrdU-positive nuclei (shown as lighter colored dots) in 
the supporting cells which have been treated with the peptide of SEQ ID 
NO: 1 (FIG. 4A) versus the control (untreated) cells (FIG. 4B). Hair cells 
immunostained with calretinin are shown in the background as grayish 
spots. The proliferation of supporting cells is a necessary prerequisite 
for hair cell generation/regeneration in the vestibular and auditory 
organs. The effect observed here with the peptide of SEQ ID NO: 1 is very 
potent, and superior to any in vitro result reported in the literature so 
far for any other growth agent. Typically, cell culture conditions are not 
effective for generating new hair cells. The effect obtained here with 
supporting cells is very surprising and suggests that a positive effect 
will also be obtained in vivo which may lead to hair cell generation. 
EXAMPLE 2 
Comparison with Other NDF/heregulins 
Using the test procedure of Example 1, the peptide of SEQ ID NO: 1 was 
compared with members of the NDF-heregulin family in primary cultures of 
young rat utricular sensory epithelial cells, at a treatment concentration 
of 50 ng/ml in each case. The comparison molecules were as follows: 
recombinant rat NDF.alpha.2 (see Wen et al., Molecular and Cellular 
Biology, above), recombinant human NDF.alpha.2, and recombinant human 
NDF.beta.1 (See PCT application WO 94/28133, published Dec. 8, 1994, for a 
description of these two proteins). 
As can be seen from FIG. 5, the most potent effect was obtained with the 
peptide of SEQ ID NO: 1 ("Peptide"), which represented a 29-fold increase 
in induction of cell proliferation over the control. 
EXAMPLE 3 
Proliferation of Cells from Rodent Vestibular Organ 
An established utricular cell line was derived from a transgenic mouse, 
using the procedure described by Gu et al. in Abstracts of the Association 
for Research in Otolaryngology, Volume 21, page 16 (1998). The cell line 
was cultured at 37.degree. C. for three days in DMEM/F12 (Gibco BRL), 
containing N2 supplement (Sigma Chemical Company, St. Louis, Mo.) in a 
proportion of 1:100, under conditions restricting oncogene expression. For 
the treated group, fifty nanograms per milliliter of the peptide of SEQ ID 
NO: 1 were added twenty-four hours prior to fixation. Three micrograms per 
milliliter of BrdU (Aldrich Chemicals) were added for the final sixteen 
hours. 
Cell proliferation was measured by ELISA for BrdU. Briefly, cells were 
fixed with 4% paraformaldehyde (Amersham, Life Sciences, Cleveland, Ohio) 
for one hour at room temperature. After treatment with 2N HCl for twenty 
minutes, the cells were washed and then sequentially incubated for one 
hour with a monoclonal antibody against BrdU (Caltag Laboratories) and 
horse anti-mouse antibody conjugated to biotin (Vector Laboratories, 
Burlingame, Calif.). After washing, the cells were further incubated with 
Europium streptavidin (Wallac Inc., Gaithersburg, Md.) diluted 1:1,000 and 
processed for time-resolved fluorescence in accordance with the 
manufacturer's recommendations. The signal was read on a Victor 1420 
multi-label counter (Wallac Inc.). 
The ELISA reading from the peptide-treated group (SEQ ID NO: 1) was found 
to be 1.7 times higher than for the control (untreated) group. These 
results, taken together with those of Example 1, above, demonstrate that 
the peptide of SEQ ID NO: 1 is a potent stimulator of cells of the 
vestibular epithelium from both newborn and mature rodents. 
EXAMPLE 4 
Generation of Monoclonal Antibodies Against Sensory Epithelial Cells of 
Rodent Inner Ear 
The lack of a specific marker for sensory epithelium cells adds to the 
challenges associated with research on hair cell regeneration in the inner 
ear of mammals. Monoclonal antibodies against hair cells have been 
reported in the literature; Finley et al., Assoc. Res. Otolaryngol. 
Abstr., Volume 20, page 16 (1997) and Holley et al., J. Neurocytol., 
Volume 24, pages 853-864 (1997). However, none of these antibodies are 
specific to supporting cells in the mammalian vestibular organs. 
The following is a description of the preparation of four distinct 
monoclonal antibodies raised against rat utricular epithelia which 
specifically label supporting cells of the vestibular organs in the inner 
ear of the rodent. These antibodies constitute an additional aspect of the 
present invention. 
In this method, sensory epithelia were isolated from adult rat inner ear 
utricles by the thermolysin method; see above for description. Seventy 
pieces of epithelia were homogenized by ultrasound, and then emulsified 
with Freund's adjuvant (Sigma Chemical Company, St. Louis, Mo.). The 
emulsification product was injected intraperitoneallly into Balb/c mice on 
days 0, 29 and 53, prior to fusion of day 56. The fusion time was 
determined when the serum derived from bled mice showed high titer against 
the antigen (i.e., the utricle extract). Mouse splenocytes were harvested 
and then fused with HL-1 myeloma cells (Kohler and Milstein, Nature, 
Volume 256, pages 495-497 (1975). Screening for monoclonal antibodies was 
conducted by immunostaining on frozen 10-micron sections of rat utricle. 
Briefly, adult rat utricles were rapidly isolated and fixed with 4% 
paraformaldehyde for one hour. The tissues were transferred sequentially 
to 5%, 10%, 15% and 20% sucrose solutions in a phosphate buffer for twenty 
four hours each. The tissue was then embedded in 7.5% gelatine in the 20% 
sucroise solution for one hour at 37.degree. C. The tissues were then 
mounted in a cryomold (Polysciences, Inc. Warrington, Pa.) and frozen at 
-30.degree. C., and 10-micron sections were generated on six-well chamber 
slides (Roboz Instruments, Inc., Rockville, Md.) and immunostained using 
the manufacturer's recommended procedure. Over thirty hybridomas were 
chosen for selective staining of sensory epithelium. Double staining with 
calretinin and monoclonal antibodies on both cultured utricular epithelium 
and frozen rat utricle sections resulted in the selection of four 
monoclonal antibodies (labeled "SC-1", "SC-2", "SC-3" and "SC-4", 
respectively). 
Each of the monoclonal antibodies specifically stained the supporting 
cells, but with a characteristically different pattern. SC-1 stained the 
top portion of the supporting cells brightly, while gradually decreasing 
around the cell nuclei. SC-2 stained only the top portion of the 
supporting cells. SC-3 immunoreactivity was concentrated on the lower 
cytoplasmic portion of the supporting cells in neotal rat utricles, and 
migrated to the upper portion in adult utricles. SC-4 immunoreactivity was 
found mostly in the supporting cell apex of the adult utricle. SC-4 and 
SC-3 immunostaining was found in embryonic progenitors of supporting cells 
of the inner ear. 
A monoclonal antibody (SC-4) prepared as described above was deposited with 
the American Type Culture Collection (ATTC), 10801 University Boulevard, 
Manassas, Va. 20110, on Nov. 18, 1998, under accession number HB-12598. 
The invention described above is defined in the appended claims. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - &lt;160&gt; NUMBER OF SEQ ID NOS: 2 
- - &lt;210&gt; SEQ ID NO 1 
&lt;211&gt; LENGTH: 52 
&lt;212&gt; TYPE: PRT 
&lt;213&gt; ORGANISM: Human 
- - &lt;400&gt; SEQUENCE: 1 
- - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val 
Asn 
1 5 - # 10 - # 15 
- - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr 
20 - # 25 - # 30 
- - Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Al - #a Arg Cys Gln Asn Tyr 
35 - # 40 - # 45 
- - Val Met Ala Ser 
50 
- - - - &lt;210&gt; SEQ ID NO 2 
&lt;211&gt; LENGTH: 156 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Human 
- - &lt;400&gt; SEQUENCE: 2 
- - agccatcttg taaaatgtgc ggagaaggag aaaactttct gtgtgaatgg ag - 
#gggagtgc 60 
- - ttcatggtga aagacctttc aaacccctcg agatacttgt gcaagtgcca ac - 
#ctggattc 120 
- - actggagcaa gatgtcaaaa ctacgtaatg gccagc - # 
- # 156 
__________________________________________________________________________