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[IP] Islet cell "cure": INGAP

Below is a chopped version of the patent on the INGAP protein, that
has caused regeneration of islet cells in animals.  I did run accross
something elsewhere, that leads me to believe they have already given
some of this protein to humans, in the beginings of clinical trials
or experiments...

I did remove about 50% of the text, and there's still plenty here
that might be more confusing than helpfull, but there may be others
who can benefit from the details below...

For the full, uneditted patent, do a search for patent #5840531, on
http://www.uspto.gov   (US patent office web site)


 United States Patent                                            5,840,531
 Vinik ,   et al.                                        November 24, 1998
Ingap protein involved in pancreatic islet neogenesis


Cellophane wrapping (CW) of hamster pancreas induces proliferation of duct
epithelial cells followed by endocrine cell differentiation and islet
neogenesis. Using the mRNA differential display technique a cDNA clone
expressed in cellophane wrapped but not in control pancreata was identified.
Using this cDNA as a probe, a cDNA library was screened and a gene not
previously described was identified and named INGAP.

 Inventors: Vinik; Aaron I. (Norfolk, VA); Pittenger; Gary L. (Virgina
            Beach, VA); Rafaeloff; Ronit (Chesapeake, VA); Rosenberg;
            Lawrence (Montreal, CA); Duguid; William P. (Montreal, CA)
 Assignee:  MoGill University (CA); Eastern Virginia Medical School of the
            Medicine College of Hampton Roads (Norfolk, VA)
 Appl. No.: 709662
 Filed:     September 9, 1996

 U.S. Class:                       435/69.1; 435/252.3; 536/23.1; 536/23.5;
                                                       536/24.3; 424/185.1
 Intern'l Class:                                                C12N 015/00
 Field of Search:              425/69.1,252.3 536/23.1,23.5,24.3 424/185.1

-----snip ----


Pancreatic islets of Langerhans are the only organ of insulin production in
the body. However, they have a limited capacity for regeneration. This
limited regeneration capacity predisposes mammals to develop diabetes
mellitus. Thus there is a need in the art of endocrinology for products
which can stimulate the regeneration of islets of Langerhans to prevent or
ameliorate the symptoms of diabetes mellitus.

One model of pancreatic islet cell regeneration involves cellophane-wrapping
of the pancreas in the Syrian golden hamster (1). Wrapping of the pancreas
induces the formation of new endocrine cells which appear to arise from duct
epithelium (2-4). There is a need in the art to identify and isolate the
factor(s) which is responsible for islet cell regeneration.

-----snip ----


We now report the identification of a gene, INGAP, that shows striking
homology to the pancreatitis associated protein (PAP) family of genes
(7-11). The predicted protein shares the carbohydrate recognition domain
(CRD) of the calcium dependent C-type lectins as defined by Drickamer (12).
INGAP protein plays a role in stimulation of islet neogenesis, in
particular, in beta cell regeneration from ductal cells.

The cDNA sequence of a mammalian INGAP is provided in SEQ ID NO: 1. The
predicted amino acid sequence is shown in SEQ ID NO:2. These sequences were
determined from nucleic acids isolated from hamster, but it is believed that
other mammalian species will contain INGAP genes which are quite similar.
For example, human INGAP cDNA shares the entire sequence in SEQ ID NO:1 with
the hamster. The predicted amino acid sequence of human INGAP protein is
from 1 to 174 in SEQ ID NO:2. One would expect homologous genes to contain
at least about 70% identity. Closer species would be expected to have at
least about 75%, 80%, 85%, 90%, 95%, or even 99% identity. In contrast,
other family members of the calcium dependent C-type lectins contain at most
60% identity with INGAP.

The DNA sequence provided herein can be used to form vectors which will
replicate the gene in a host cell, and may also express INGAP protein. DNA
sequences which encode the same amino acid sequence as shown in SEQ ID NO:2
can also be used, without departing from the contemplation of the invention.
DNA sequences coding for other mammalian INGAPs are also within the
contemplation of the invention. Suitable vectors, for both prokaryotic and
eukaryotic cells, are known in the art. Some vectors are specifically
designed to effect expression of inserted DNA segments downstream from a
transcriptional and translational control site. One such vector for
expression in eukaryotic cells employs EBNA His, a plasmid which is
available commercially from InVitrogen Corp. The loaded vector produces a
fusion protein comprising a portion of a histidine biosynthetic enzyme and
INGAP. Another vector, which is suitable for use in prokaryotic cells, is
pcDNA3. Selection of a vector for a particular purpose may be made using
knowledge of the properties and features of the vectors, such as useful
expression control sequences. Vectors may be used to transform or transfect
host cells, either stably or transiently. Methods of transformation and
transfection are known in the art, and may be used according to suitability
for a particular host cell. Host cells may be selected according to the
purpose of the transfection. A suitable prokaryotic host is E. coli
DH5.alpha.. A suitable eukaryotic host is cos7, an African Green Monkey
kidney cell line. For some purposes, proper glycosylation of INGAP may be
desired, in which case a suitable host cell should be used which recognizes
the glycosylation signal of INGAP.

Probes comprising at least 10, 15, 20, or 30 nucleotides of contiguous
sequence according to SEQ ID NO:1 can be used for identifying INGAP genes in
particular individuals or in members of other species. Appropriate
conditions for hybridizations to same or different species' DNA are known in
the art as high stringency and low stringency, respectively. These can be
used in a variety of formats according to the desired use. For example,
Southern blots, Northern blots, and in situ colony hybridization, can be
used as these are known in the art. Probes typically are DNA or RNA
oligomers of at least 10, 15, 20, or 30 nucleotides. The probe may be
labeled with any detectable moiety known in the art, including radiolabels,
fluorescent labels, enzymes, etc. Probes may also be derived from other
mammalian INGAP gene sequences.

INGAP genes can be isolated from other mammals by utilizing the nucleotide
sequence information provided herein. (More laboriously, they can be
isolated using the same method described in detail below for isolation of
the hamster INGAP gene.) Oligonucleotides comprising at least 10 contiguous
nucleotides of the disclosed nucleotide sequence of INGAP are hybridized to
genomic DNA or cDNA of the mammal. The DNA may conveniently be in the form
of a library of clones. The oligonucleotides may be labelled with any
convenient label, such as a radiolabel or an enzymatic or fluorescence
label. DNA molecules which hybridize to the probe are isolated. Complete
genes can be constructed by isolating overlapping DNA segments, for example
using the first isolated DNA as a probe to contiguous DNA in the library or
preparation of the mammal's DNA. Confirmation of the identity of the
isolated DNA can be made by observation of the pattern of expression of the
gene in the pancreas when subjected to cellophane wrapping, for example.
Similarly, the biological effect of the encoded product upon pancreatic
ductal cells will also serve to identify the gene as an INGAP gene.

If two oligonucleotides are hybridized to the genomic DNA or cDNA of the
mammal then they can be used as primers for DNA synthesis, for example using
the polymerase chain reaction or the ligase chain reaction. Construction of
a full-length gene and confirmation of the identity of the isolated gene can
be performed as described above.

INGAP protein may be isolated according to the invention by inducing
mammalian pancreatic cells to express INGAP protein by means of
cellophane-wrapping. This technique is described in detail in reference no.
1 which is expressly incorporated herein. INGAP protein so produced may be
purified from other mammalian proteins by means of immunoaffinity
techniques, for example, or other techniques known in the art of protein
purification. An antibody specific for a mammalian INGAP is produced using
all, or fragments of, the amino acid sequence of an INGAP protein, such as
shown in SEQ ID NO: 2, as immunogens. The immunogens can be used to identify
and purify immunoreactive antibodies. Monoclonal or polyclonal antibodies
can be made as is well known in the art. The antibodies can be conjugated to
other moieties, such as detectable labels or solid support materials. Such
antibodies can be used to purify proteins isolated from mammalian pancreatic
cells or from recombinant cells. Hybridomas which secrete specific
antibodies for an INGAP protein are also within the contemplation of the

Host cells as described above can be used to produce a mammalian INGAP
protein. The host cells comprise a DNA molecule encoding a mammalian INGAP
protein. The DNA can be according to SEQ ID NO:1, or isolated from other
mammals according to methods described above. Host cells can be cultured in
a nutrient medium under conditions where INGAP protein is expressed. INGAP
protein can be isolated from the host cells or the nutrient medium, if the
INGAP protein is secreted from the host cells.

It has now been found that INGAP and fragments thereof are capable of
inducing and stimulating islet cells to grow. Moreover, they are capable of
inducing differentiation of pancreatic duct cells, and of allowing such
cells to avoid the apoptotic pathway. Thus many therapeutic modalities are
now possible using INGAP, fragments thereof, and nucleotide sequences
encoding INGAP. Therapeutically effective amounts of INGAP are supplied to
patient pancreata, to isolated islet cells, and to encapsulated pancreatic
islet cells, such as in a polycarbon shell. Suitable amounts of INGAP for
therapeutic purposes range from 1-150 .mu.g/kg of body weight or in vitro
from 1-10,000 .mu.g/ml. Optimization of such dosages can be ascertained by
routine testing. Methods of administering INGAP to mammals can be any that
are known in the art, including subcutaneous, via the portal vein, by local
perfusion, etc.

Conditions which can be treated according to the invention by supplying
INGAP include diabetes mellitus, both insulin dependent and non-insulin
dependent, pancreatic insufficiency, pancreatic failure, etc. Inhibition of
INGAP expression can be used to treat nesidioblastosis.

According to the present invention, it has now been found that a small
portion of INGAP is sufficient to confer biological activity. A fragment of
20 amino acids of the sequence of SEQ ID NO: 2, from amino acid #103-#122 is
sufficient to stimulate pancreatic ductal cells to grow and proliferate. The
effect has been seen on a rat tumor duct cell line, a hamster duct cell
line, a hamster insulinoma cell line, and a rat insulinoma cell line. The
analogous portions of other mammalian INGAP proteins are quite likely to
have the same activity. This portion of the protein is not similar to other
members of the pancreatitis associated protein (PAP) family of proteins. It
contains a glycosylation site and it is likely to be a primary antigenic
site of the protein as well. This fragment has been used to immunize mice to
generate monoclonal antibodies.

The physiological site of expression of INGAP has been determined. INGAP is
expressed in acinar tissue, in the exocrine portion of the pancreas. It is
not expressed in ductal or islet cells, i.e., the paracrine portion of the
pancreas. Expression occurs within 24-48 hours of induction by means of
cellophane wrapping.

Transgenic animals according to the present invention are mammals which
carry an INGAP gene from a different mammal. The transgene can be expressed
to a higher level than the endogenous INGAP genes by judicious choice of
transcription regulatory regions. Methods for making transgenic animals are
well known in the art, and any such method can be used. Animals which have
been genetically engineered to carry insertions, deletions, or other
mutations which alter the structure of the INGAP protein or regulation of
expression of INGAP are also contemplated by this invention. The techniques
for effecting these mutations are known in the art.

Diagnostic assays are also contemplated within the scope of the present
invention. Mutations in INGAP can be ascertained in samples such as blood,
amniotic fluid, chorionic villus, blastocyst, and pancreatic cells. Such
mutations identify individuals who are at risk for diabetes. Mutations can
be identified by comparing the nucleotide sequence to a wild-type sequence
of an INGAP gene. This can be accomplished by any technique known in the
art, including comparing restriction fragment length polymorphisms,
comparing polymerase chain reaction products, nuclease protection assays,
etc. Alternatively, altered proteins can be identified, e.g.,
immunologically or biologically.

The present invention also contemplates the use of INGAP antisense
constructs for treating nesidioblastosis, a condition characterized by
overgrowth of .beta. cells. The antisense construct is administered to a
mammal having nesidioblastosis, thereby inhibiting the overgrowth of .beta.
cells. An antisense construct typically comprises a promoter, a terminator,
and a nucleotide sequence consisting of a mammalian INGAP gene. The INGAP
sequence is between the promoter and the terminator and is inverted with
respect to the promoter as it is expressed naturally. Upon expression from
the promoter, an mRNA complementary to native mammalian INGAP is produced.

Immunological methods for assaying INGAP in a sample from a mammal are
useful, for example, to monitor the therapeutic administration of INGAP.
Typically an antibody specific for INGAP will be contacted with the sample
and the binding between the antibody and any INGAP in the sample will be
detected. This can be by means of a competitive binding assay, in which the
incubation mixture is spiked with a known amount of a standard INGAP
preparation, which may conveniently be detectably labeled. Alternatively, a
polypeptide fragment of INGAP may be used as a competitor. In one particular
assay format, the antibodies are bound to a solid phase or support, such as
a bead, polymer matrix, or a microtiter plate.

According to the present invention, pancreatic duct cells of a mammal with
pancreatic endocrine failure can be removed from the body and treated in
vitro. The duct cells typically comprise .beta. cell progenitors. Thus
treatment with a preparation of a mammalian INGAP protein will induce
differentiation of the .beta. cell progenitors. The duct cells are contacted
with a preparation of a mammalian INGAP protein substantially free of other
mammalian proteins. The treated cells can then used as an autologous
transplant into the mammal from whom they were derived. Such an autologous
treatment minimizes adverse host versus graft reactions involved in

INGAP protein can also be used to identify those cells which bear receptors
for INGAP. Such cells are likely to be the .beta. cell progenitors, which
are sensitive to the biological effects of INGAP. INGAP protein can be
detectably labeled, such as with a radiolabel or a fluorescent label, and
then contacted with a population of cells from the pancreatic duct. Cells
which bind to the labeled protein will be identified as those which bear
receptors for INGAP, and thus are .beta. cell progenitors. Fragments of
INGAP can also be used for this purpose, as can immobilized INGAP which can
be used to separate cells from a mixed population of cells to a solid
support. INGAP can be immobilized to solid phase or support by adsorption to
a surface, by means of an antibody, or by conjugation. Any other means as is
known in the art can also be used.

Kits are provided by the present invention for detecting a mammalian INGAP
protein in a sample. This may be useful, inter alia, for monitoring
metabolism of INGAP during therapy which involves administration of INGAP to
a mammal. The kit will typically contain an antibody preparation which is
specifically immunoreactive with a mammalian INGAP protein. The antibodies
may be polyclonal or monoclonal. If polyclonal they may be affinity purified
to render them monospecific. The kit will also typically contain a
polypeptide which has at least 15 consecutive amino acids of a mammalian
INGAP protein. The polypeptide is used to compete with the INGAP protein in
a sample for binding to the antibody. Desirably the polypeptide will be
detectably labeled. The polypeptide will contain the portion of INGAP to
which the antibody binds. Thus if the antibody is monoclonal, the
polypeptide will successfully compete with INGAP by virtue of it containing
the epitope of the antibody. It may also be desirable that the antibodies be
bound to a solid phase or support, such as polymeric beads, sticks, plates,

Pharmaceutical compositions containing a mammalian INGAP protein may be used
for treatment of pancreatic insufficiency. The composition may alternatively
contain a polypeptide which contains a sequence of at least 15 consecutive
amino acids of a mammalian INGAP protein. The polypeptide will contain a
portion of INGAP which is biologically active in the absence of the other
portions of the protein. The polypeptide may be part of a larger protein,
such as a genetic fusion with a second protein or polypeptide.
Alternatively, the polypeptide may be conjugated to a second protein, for
example, by means of a cross-linking agent. Suitable portions of INGAP
proteins may be determined by homology with amino acids #103 to #122 of SEQ
ID NO:2, or by the ability of test polypeptides to stimulate pancreatic duct
cells to grow and proliferate. As is known in the art, it is often the case
that a relatively small number of amino acids can be removed from either end
of a protein without destroying activity. Thus it is contemplated within the
scope of the invention that up to about 10% of the protein can be deleted,
and still provide essentially all functions of INGAP. Such proteins have at
least about 130 amino acids, in the case of hamster INGAP.

The pharmaceutical composition will contain a pharmaceutically acceptable
diluent or carrier. A liquid formulation is generally preferred. INGAP may
be formulated at different concentrations or using different formulants. For
example, these formulants may include oils, polymers, vitamins,
carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking
agents. Preferably carbohydrates include sugar or sugar alcohols such as
mono-, di-, or polysaccharides, or water soluble glucans. The saccharides or
glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose,
xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta
cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcellulose,
or mixtures thereof. Sucrose is most preferred. Sugar alcohol is defined as
a C.sub.4 to C.sub.8 hydrocarbon having an --OH group and includes
galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol.
Mannitol is most preferred. These sugars or sugar alcohols mentioned above
may be used individually or in combination. There is no fixed limit to
amount used as long as the sugar or sugar alcohol is soluble in the aqueous
preparation. Preferably, the sugar or sugar alcohol concentration is between
1.0 w/v % and 7.0 w/v %, more preferable between 2.0 and 6.0 w/v %.
Preferably amino acids include levorotary (L) forms of carnitine, arginine,
and betaine; however, other amino acids may be added. Preferred polymers
include polyvinylpyrrolidone (PVP) with an average molecular weight between
2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular
weight between 3,000 and 5,000. It is also preferred to use a buffer in the
composition to minimize pH changes in the solution before lyophilization or
after reconstitution, if these are used. Most any physiological buffer may
be used, but citrate, phosphate, succinate, and glutamate buffers or
mixtures thereof are preferred. Preferably, the concentration is from 0.01
to 0.3 molar. Surfactants can also be added to the formulation.

Additionally, INGAP or polypeptide portions thereof can be chemically
modified by covalent conjugation to a polymer to increase its circulating
half-life, for example. Preferred polymers, and methods to attach them to
peptides, are shown in U.S. Pat. Nos. 4,766,106, 4,179,337, 4,495,285, and
4,609,546. Preferred polymers are polyoxyethylated polyols and polyethylene
glycol (PEG). PEG is soluble in water at room temperature and has the
general formula: R(O--CH.sub.2 --CH.sub.2).sub.n O--R where R can be
hydrogen, or a protective group such as an alkyl or alkanol group.
Preferably, the protective group has between 1 and 8 carbons, more
preferably it is methyl. The symbol n is a positive integer, preferably
between 1 and 1,000, more preferably between 2 and 500. The PEG has a
preferred average molecular weight between 1000 and 40,000, more preferably
between 2000 and 20,000, most preferably between 3,000 and 12,000.
Preferably, PEG has at least one hydroxy group, more preferably it is a
terminal hydroxy group. It is this hydroxy group which is preferably
activated to react with a free amino group on the inhibitor.

After the liquid pharmaceutical composition is prepared, it is preferably
lyophilized to prevent degradation and to preserve sterility. Methods for
lyophilizing liquid compositions are known to those of ordinary skill in the
art. Just prior to use, the composition may be reconstituted with a sterile
diluent (Ringer's solution, distilled water, or sterile saline, for example)
which may include additional ingredients. Upon reconstitution, the
composition is preferably administered to subjects using those methods that
are known to those skilled in the art.

The following examples are not intended to limit the scope of the invention,
but merely to exemplify that which is taught above.


Example 1

This example describes the cloning and isolation of a cDNA encoding a novel,
developmentally regulated, pancreatic protein.

We hypothesized that a unique locally produced factor(s) is responsible for
islet cell regeneration. Using the recently developed mRNA differential
display technique (5,6) to compare genes differentially expressed in
cellophane wrapped (CW) versus control pancreata (CP) allowed us to identify
a cDNA clone (RD19-2) which was uniquely expressed in cellophane wrapped

A cDNA library was constructed from mRNA isolated from cellophane wrapped
hamster pancreas using oligo d(T) primed synthesis, and ligation into pcDNA3
vector (Invitrogen). The number of primary recombinants in the library was
1.2.times.10.sup.6 with an average size of 1.1 kb. The cDNA library was
screened for clones of interest using high density colony plating
techniques. Colonies were lifted onto nylon membranes (Schleicher & Schuell)
and further digested with proteinase K (50(g/ml). Treated membranes were
baked at 80.degree. C. for 1 hour and hybridized at 50.degree. C. for 16-18
hours with 1-5.times.10.sup.6 cpm/ml of >(.sup.32 P!-dCTP(Dupont-New England
Nuclear) radiolabeled RD 19-2 probe. Colonies with a positive hybridization
signal were isolated, compared for size with Northern mRNA transcript, and
sequenced to confirm identity with the RD 19-2 sequence.

Example 2

This example compares the sequence of INGAP to other proteins with which it
shares homology.

The nucleotide sequence of the hamster INGAP clone with the longest cDNA
insert was determined. As shown in FIG. 1 the hamster cDNA comprises 747
nucleotides (nt), exclusive of the poly(A) tail and contains a major open
reading frame encoding a 175 amino acid protein. The open reading frame is
followed by a 3'-untranslated region of 206nt. A typical polyadenylation
signal is present 11nt upstream of the poly(A) tail. The predicted INGAP
protein shows structural homology to both the PAP/HIP family of genes which
is associated with pancreatitis or liver adenocarcinoma (7-11) and the
Reg/PSP/lithostatine family of genes (13,15) which has been shown to
stimulate pancreatic beta-cell growth (14) and might play a role in
pancreatic islet regeneration. Comparison of the nucleotide sequence and
their deduced amino acids between hamster INGAP and rat PAP-I shows a high
degree of homology in the coding region (60 and 58% in nucleotide and amino
acid sequences, respectively). The predicted amino acid sequence of the
hamster INGAP reveals 45% identity to PAP II and 50% to PAP III both of
which have been associated with acute pancreatitis, and 54% to HIP which was
found in a hepato-cellular carcinoma. INGAP also shows 40% identity to the
rat Reg/PSP/lithostatine protein (FIG. 2). Reg is thought to be identical to
the pancreatic stone protein (PSP) (15,16) or pancreatic thread protein
(PTP) (17). The N-terminus of the predicted sequence of INGAP protein is
highly hydrophobic which makes it a good candidate for being the signal
peptide which would allow the protein to be secreted. Similar to PAP/HIP but
different from the Reg/PSP/lithostatine proteins a potential N-glycosylation
site is situated at position 135 of the INGAP sequence. Unique to INGAP is
another potential N-glycosylation site situated at position 115. INGAP also
shows a high degree of homology (12/18) (FIG. 2) with a consensus motif in
members of the calcium-dependent (C-type) animal lectin as determined by
Drickamer including four perfectly conserved cysteines which form two
disulfide bonds (12). Two extra cysteines found at the amino-terminus of
INGAP (FIG. 2) are also present in Reg/PSP and PAP/HIP. However, it is not
clear what the biological significance might be.

Example 3

This example demonstrates the temporal expression pattern of INGAP upon

In order to determine the temporal expression of the INGAP gene, total RNA
extracted from CP and CW pancreas was probed with the hamster INGAP cDNA
clone in Northern blot analysis. A strong single transcript of 900 bp was
detected (FIGS. 3A, 3B and 3C) 1 and 2 days after cellophane wrapping which
disappeared by 6 through 42 days and was absent from CP. INGAP mRNA is
associated with CW induced pancreatic islet neogenesis, since it is present
only after CW. It is not likely that the increased expression of INGAP is
associated with acute pancreatitis as is the case with the PAP family of
genes. During the acute phase of pancreatitis the concentrations of most
mRNAs encoding pancreatic enzymes including amylase are decreased
significantly (16,18). In contrast, in the CW model of islet neogenesis in
which high expression of INGAP has been detected, amylase gene expression
was simultaneously increased above normal (FIGS. 3A, 3B and 3C) rather than
decreased, suggesting that INGAP expression is not associated with
pancreatitis but rather with islet neogenesis. The cause of increased
amylase gene expression 1 and 2 days after CW is as yet unclear, and more
studies need to be done to elucidate this issue. It is unlikely though, that
the increase is associated with exocrine cell regeneration which occurs at a
later time after CW (19). Thus, INGAP protein plays a role in stimulation of
islet neogenesis, in particular, in beta cell regeneration from ductal

Example 4

This example describes the cloning and partial sequence of a human cDNA
encoding INGAP protein.

Human polyA.sup.+ RNA was isolated from a normal human pancreas using a
commercially available polyA.sup.+ extraction kit from Qiagen. Subsequently,
500 ng polyA.sup.+ RNA was used as a template for reverse transcription and
polymerase chain reaction (RT-PCR). The experimental conditions were set
according to the instructions in the RT-PCR kit from Perkin Elmer. Oligo
d(T) was used as the primer in reverse transcription. Primers corresponding
to nucleotides 4 to 23 and 610 to 629 in SEQ ID NO:1 were used as the
specific primers in the polymerase chain reaction. A 626 bp PCR fragment was
cloned using a TA cloning kit from Invitrogen. The human INGAP cDNA is 100%
identical to the hamster INGAP cDNA sequence in SEQ ID NO:1.

Example 5

This example demonstrates that synthetic peptides from INGAP play a role in
stimulation of islet neogenesis, and that at least one epitope coded by the
as yet unsequenced 120 bp segment of human INGAP is shared with hamster

A synthetic peptide corresponding to amino acids 104-118 in SEQ ID NO:2 of
the deduced hamster INGAP protein was used as an immunogen to raise
polyclonal antibodies in a rabbit. The antiserum was subsequently used in
immunohistochemistry assays using the avidin-biotin complex (ABC) method.
Cells in the peri-islet region in humans with neoislet formation stained
positively for INGAP demonstrating that human and hamster INGAP share a
common epitope between amino acids 104 to 118 in SEQ ID NO:2.

The same synthetic peptide was tested for its ability to stimulate .sup.3
H-thymidine incorporation into rat pancreatic tumor duct cells (ARIP) and
hamster insulinoma tumor cells (HIT). 10 .mu.Ci of .sup.3 H-thymidine at
80.4 Ci/mmole concentration was added to approximate 10.sup.6 cells cultured
in Ham's F-12K media. After 24 hrs, the cells were harvested and
solubilized. Differential precipitation of the nucleic acids with
trichloroacetic acid (TCA) was performed according to the procedure modified
by Rosenberg et al. and the .sup.3 H-thymidine proportion incorporated was
calculated. Addition of the synthetic peptide to ARIP in culture resulted in
a 2.4-fold increase in .sup.3 H-thymidine incorporation comparing to the
absence of the synthetic peptide in the culture. The synthetic peptide had
no effect on the control cell line HIT. This result strongly suggests that
INGAP plays a role in stimulating islet neogenesis.

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