U.S. patent application number 10/124089 was filed with the patent office on 2003-09-04 for islet cell antigen 1851.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Hagopian, William A., Jelinek, Laura J., Kindsvogel, Wayne, LaGasse, James M., Sheppard, Paul O..
Application Number | 20030166067 10/124089 |
Document ID | / |
Family ID | 26684187 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166067 |
Kind Code |
A1 |
Kindsvogel, Wayne ; et
al. |
September 4, 2003 |
Islet cell antigen 1851
Abstract
A mammalian islet cell antigen polypeptide involved in the
development of insulin-dependent diabetes mellitus (IDDM) is
disclosed. This islet cell antigen polypeptide, 1851, was found to
contain regions of homology to the protein tyrosine phosphatase
family. Methods for diagnosis and treatment, including use in
immunoprecipitation assays and the induction of immune tolerance
using the recombinant mammalian polypeptides and antibodies
specific to mammalian islet cell antigen 1851 polypeptides are
presented.
Inventors: |
Kindsvogel, Wayne; (Seattle,
WA) ; Jelinek, Laura J.; (Seattle, WA) ;
Sheppard, Paul O.; (Granite Falls, WA) ; Hagopian,
William A.; (Seattle, WA) ; LaGasse, James M.;
(Seattle, WA) |
Correspondence
Address: |
Phillip B.C. Jones, J.D., Ph.D.
Patent Department
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
26684187 |
Appl. No.: |
10/124089 |
Filed: |
April 16, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10124089 |
Apr 16, 2002 |
|
|
|
08811481 |
Mar 5, 1997 |
|
|
|
6300093 |
|
|
|
|
60012927 |
Mar 6, 1996 |
|
|
|
60027540 |
Oct 15, 1996 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 435/326; 435/7.21; 530/350; 530/388.26;
536/23.5 |
Current CPC
Class: |
C12N 9/16 20130101; C07K
14/4713 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/69.1 ;
435/325; 435/320.1; 435/326; 435/7.21; 530/388.26; 530/350;
536/23.5 |
International
Class: |
G01N 033/567; C07H
021/04; C12P 021/02; C12N 005/06; C07K 014/435; C07K 016/40; C12N
005/06 |
Claims
We claim:
1. An isolated polynucleotide comprising a DNA segment encoding a
mammalian islet cell antigen polypeptide comprising an amino acid
sequence selected from the group consisting of: a) a polypeptide of
SEQ ID NO:16 from Leu, amino acid residue 636 to Gln, amino acid
residue 1012: b) a polypeptide of SEQ ID NO:22 from Leu, amino acid
residue 442 to Gln, amino acid residue 818; and c) allelic variants
of (a) or (b) wherein the polypeptide forms an immune complex with
an autoantibody from a patient at risk of or predisposed to develop
IDDM.
2. An isolated polynucleotide according to claim 1, wherein said
isolated polynucleotide encodes a mammalian islet cell antigen
polypeptide selected from the group consisting of: a) a polypeptide
of SEQ ID NO:16 from Phe, amino acid residue 612 to Gln, amino acid
residue 1012; b) a polypeptide of SEQ ID NO:22 from Phe, amino acid
residue 418 to Gln, amino acid residue 818; and c) allelic variants
of (a) or (b).
3. An isolated polynucleotide according to claim 1, wherein said
isolated polynucleotide encodes a mammalian islet cell antigen
polypeptide selected from the group consisting of: a) a polypeptide
of SEQ ID NO:16 from Ala, amino acid residue 1 to Gln, amino acid
residue 1012; b) a polypeptide of SEQ ID NO:22 from His, amino acid
residue 1 to Gln, amino acid residue 818; and c) allelic variants
of (a) or (b).
4. An isolated polynucleotide according to claim 1, wherein said
isolated polynucleotide is a DNA molecule selected from the group
consisting of: a) a DNA molecule comprising the coding sequence of
SEQ ID NO:15 from nucleotide 1909 to nucleotide 3039; b) a DNA
molecule comprising the coding sequence of SEQ ID NO:21 from
nucleotide 1325 to nucleotide 2455; c) allelic variants of (a) or
(b); and d) complements of polynucleotide molecules that
specifically hybridize to (a), (b) or (c).
5. An isolated polynucleotide according to claim 1, wherein said
isolated polynucleotide is a DNA molecule selected from the group
consisting of: a) a DNA molecule comprising the coding sequence of
SEQ ID NO:15 from nucleotide 1837 to nucleotide 3039; b) a DNA
molecule comprising the coding sequence of SEQ ID NO:21 from
nucleotide 2 to nucleotide 2455; c) allelic variants of (a) or (b);
and d) complements of polynucleotide molecules that specifically
hybridize to (a), (b) or (c).
6. An isolated polynucleotide according to claim 1, wherein said
isolated polynucleotide is a DNA molecule selected from the group
consisting of: a) a DNA molecule comprising the coding sequence of
SEQ ID NO:15 from nucleotide 4 to nucleotide 3039; b) a DNA
molecule comprising the coding sequence of SEQ ID NO:21 from
nucleotide 1254 to nucleotide 2455; c) allelic variants of (a) or
(b); and d) complements of polynucleotide molecules that
specifically hybridize to (a), (b) or (c).
7. An isolated polynucleotide according to claim 1 which encodes a
full length mammalian islet cell antigen polypeptide comprising the
sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg,
amino acid residue 738.
8. An isolated polynucleotide comprising a DNA segment encoding a
mammalian islet cell antigen polypeptide according to claim 1,
wherein said mammalian islet cell antigen polypeptide is a primate
islet cell antigen polypeptide.
9. A DNA construct comprising a first DNA segment encoding a human
islet cell antigen polypeptide operably linked to additional DNA
segments required for the expression of said first DNA segment,
wherein said first DNA segment encodes a human islet cell antigen
polypeptide comprising an amino acid sequence selected from the
group consisting of: a) SEQ ID NO:22 from Leu, amino acid residue
442 to Gln, amino acid residue 818; b) SEQ ID NO:22 from Phe, amino
acid residue 418, to Gln, amino acid residue 818; c) a polypeptide
of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid
residue 818; and d) allelic variants of (a), (b) or (c) wherein
said mammalian islet cell antigen polypeptide forms an immune
complex with an autoantibody from a patient at risk of or
predisposed to develop IDDM.
10. A DNA construct according to claim 9 herein said first DNA
segment comprises a nucleotide sequence selected from the group
consisting of: a) a DNA molecule comprising the coding sequence of
SEQ. ID NO:21 from nucleotide 1325 to nucleotide 2455; b) a DNA
molecule comprising the coding sequence of SEQ ID NO:21 from
nucleotide 1253 to nucleotide 2455; c) a DNA molecule comprising
the coding sequence of SEQ ID NO:21 from nucleotide 2 to nucleotide
2455; d) naturally occurring allelic variants of (a), (b) or (c);
and e) complements of polynucleotide molecules that specifically
hybridize to (a), (b), (c) or (d).
11. A DNA construct according to claim 9, wherein said first
segment encodes a full length mammalian islet cell antigen
comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino
acid residue 442 to Arg, amino acid residue 738.
12. A DNA construct comprising a first DNA segment encoding a
mammalian islet cell antigen according to claim 9, wherein said
mammalian islet cell antigen polypeptide is a primate islet cell
antigen polypeptide.
13. A host cell containing a DNA construct comprising a first DNA
segment encoding a mammalian islet cell antigen polypeptide
operably linked to additional DNA segments required for the
expression of said first DNA segment, wherein said first DNA
segment encodes a human islet cell antigen polypeptide comprising
an amino acid sequence selected from the group consisting of: a)
SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid
residue 818; b) SEQ ID NO:22 from Phe, amino acid residue 418, to
Gln, amino acid residue 818; c) a polypeptide of SEQ ID NO:22 from
His, amino acid residue 1, to Gln, amino acid residue 818; and d)
allelic variants of (a), (b) or (c) wherein said mammalian islet
cell antigen polypeptide forms an immune complex with an
autoantibody from a patient at risk of or predisposed to develop
IDDM.
14. A host cell according to claim 13, wherein said first DNA
segment comprises a nucleotide sequence selected from the group
consisting of: a) a DNA molecule comprising the coding sequence of
SEQ ID NO:21 from nucleotide 1325 to nucleotide 2455; b) a DNA
molecule comprising the coding sequence of SEQ ID NO:21 from
nucleotide 1253 to nucleotide 2455; c) a DNA molecule comprising
the coding sequence of SEQ ID NO:21 from nucleotide 2 to nucleotide
2455; d) naturally occurring allelic variants of (a), (b) or (c);
and e) complements of polynucleotide molecules that specifically
hybridize to (a), (b), (c) or (d).
15. A host cell according to claim 13, wherein said first DNA
segment encodes a full length mammalian islet cell antigen
polypeptide comprising the sequence of SEQ ID NO:22 from Leu, amino
acid residue 442 to Arg, amino acid residue 738.
16. A host cell containing a DNA construct comprising a first DNA
segment encoding a mammalian islet cell antigen polypeptide
according to claim 13, wherein said mammalian islet cell antigen
polypeptide is a primate islet cell antigen polypeptide.
17. An isolated mammalian islet cell antigen polypeptide comprising
an amino acid sequence selected from the group consisting of: a)
SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid
residue 818; b) SEQ ID NO:22 from Phe, amino acid residue 418, to
Gln, amino acid residue 818; c) a polypeptide of SEQ ID NO:22 from
His, amino acid residue 1, to Gln, amino acid residue 818; and d)
allelic variants of (a), (b) or (c) wherein said mammalian islet
cell antigen polypeptide forms an immune complex with an
autoantibody from a patient at risk of or predisposed to develop
IDDM.
18. An isolated mammalian islet cell antigen polypeptide according
to claim 17, wherein said isolated mammalian islet cell antigen
polypeptide is a full length mammalian islet cell antigen
polypeptide comprising the sequence of SEQ ID NO:22 from Leu, amino
acid residue 442 to Arg, amino acid residue 738.
19. An isolated mammalian islet cell antigen polypeptide according
to claim 17, wherein said mammalian islet cell antigen polypeptide
is a primate islet cell antigen polypeptide.
20. A method for producing a mammalian islet cell antigen
polypeptide comprising the steps of: culturing a host cell
containing a DNA construct comprising a first DNA segment operably
linked to additional DNA segments required for the expression of
said first DNA segment, wherein said first DNA segment encodes a
human islet cell antigen polypeptide comprising an amino acid
sequence selected from the group consisting of: a) SEQ ID NO:22
from Leu, amino acid residue 442 to Gln, amino acid residue 818; b)
SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid
residue 818; c) a polypeptide of SEQ ID NO:22 from His, amino acid
residue 1, to Gln, amino acid residue 818; and d) allelic variants
of (a), (b) or (c) wherein said mammalian islet cell antigen
polypeptide forms an immune complex with an autoantibody from a
patient at risk of or predisposed to develop IDDM; and isolating
said mammalian islet cell antigen polypeptide.
21. A method for producing a mammalian islet cell antigen
polypeptide according to claim 20, wherein said first DNA segment
comprises a nucleotide sequence selected from the group consisting
of: a) a DNA molecule comprising the coding sequence of SEQ ID
NO:21 from nucleotide 1325 to nucleotide 2455; b) a DNA molecule
comprising the coding sequence of SEQ ID NO:21 from nucleotide 1253
to nucleotide 2455; c) a DNA molecule comprising the coding
sequence of SEQ ID NO:21 from nucleotide 2 to nucleotide 2455; d)
naturally occurring allelic variants of (a), (b) or (c); and e)
complements of polynucleotide molecules that specifically hybridize
to (a), (b), (c) or (d).
22. A method for producing a mammalian islet cell antigen
polypeptide according to claim 20, wherein said first DNA segment
encodes a full length mammalian islet cell antigen polypeptide
comprising the amino acid sequence of SEQ ID NO:22 from Leu, amino
acid residue 442 to Arg, amino acid residue 738.
23. A method for producing a human islet cell antigen polypeptide
according to claim 20, wherein said host cell is a bacterial cell
or a cultured human cell.
24. A method for determining the presence of an autoantibody to a
human islet cell antigen polypeptide in a biological sample
comprising the steps of: contacting a biological sample with a
human islet cell antigen polypeptide comprising an amino acid
sequence selected from the group consisting of: a) SEQ ID NO:22
from Leu, amino acid residue 442 to Gln, amino acid residue 818; b)
SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acid
residue 818; c) a polypeptide of SEQ ID NO:22 from His, amino acid
residue 1, to Gln, amino acid residue 818; and d) allelic variants
of (a), (b) or (c), under conditions conducive to immune complex
formation, and detecting the presence of immune complex formation
between said human islet cell antigen polypeptide and said
autoantibody to a human islet cell antigen, thereby determining the
presence of an autoantibody to said human islet cell antigen in
said biological sample.
25. The method of determining the presence of an autoantibody to a
human islet cell antigen polypeptide according to claim 24, wherein
said human islet cell antigen polypeptide is detectably
labeled.
26. A method of determining the presence of an autoantibody to a
human islet cell antigen polypeptide according to claim 24, wherein
said human islet cell antigen polypeptide is a full length human
islet cell antigen polypeptide comprising the amino acid sequence
of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acid
residue 738.
27. A method for predicting the clinical course of IDDM in a
patient comprising: testing a biological sample from a patient for
the presence of human islet cell antigen polypeptide comprising an
amino acid sequence selected from the group consisting of: a) SEQ
ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid
residue 818; b) SEQ ID NO:22 from Phe, amino acid residue 418, to
Gln, amino acid residue 818; c) a polypeptide of SEQ ID NO:22 from
His, amino acid residue 1, to Gln, amino acid residue 818; and d)
allelic variants of (a), (b) or (c), wherein said polypeptide forms
an immune complex with an autoantibody from a patient at risk of or
predisposed to develop IDDM and classifying said patient for
clinical course of diabetes based on the presence or absence of
mammalian islet cell antigen polypeptides in said sample.
28. A method for predicting the clinical course of IDDM according
to claim 27, wherein said patient is further tested for one or more
additional predictive markers associated with risk of or protection
from IDDM.
29. A method for predicting the clinical course of IDDM according
to claim 27, wherein said predictive marker is an autoantibody to
an antigen selected from the group consisting of GAD65,
IA-2/ICA512, or insulin.
30. A method for predicting the clinical course of IDDM according
to claim 27, wherein said predictive marker is a genotype selected
from the group consisting of HLA DR and HLA DQ.
31. A method for predicting the clinical course of IDDM according
to claim 27, wherein said predictive marker is a polymorphic region
in the 5' flanking region of a human insulin gene.
32. A method of predicting the clinical course of IDDM according to
claim 27, wherein said human islet cell antigen polypeptide is a
full length human islet cell antigen polypeptide comprising the
amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue
442 to Arg, amino acid residue 738.
33. A method of treating a patient to prevent an autoimmune
response to a human islet cell antigen polypeptide, the method
comprising inducing immunological tolerance in said patient by
administering a human islet cell antigen polypeptide comprising an
amino acid sequence selected from the group consisting of: a) SEQ
ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid
residue 818; b) SEQ ID NO:22 from Phe, amino acid residue 418, to
Gln, amino acid residue 818; c) a polypeptide of SEQ ID NO:22 from
His, amino acid residue 1, to Gln, amino acid residue 818; and d)
allelic variants of (a), (b) or (c), that specifically binds a
human islet cell antigen receptor on immature or mature T or B
lymphocytes.
34. A method of treating a patient to prevent an autoimmune
response to a human islet cell antigen polypeptide according to
claim 33, wherein said human islet cell antigen polypeptide is a
full length human islet cell antigen polypeptide comprising the
amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue
442 to Arg, amino acid residue 738.
35. A probe which comprises an oligonucleotide of at least about 16
nucleotides, wherein said oligonucleotide is at least 85% identical
to a sequence of the human islet cell antigen DNA sequence of SEQ
ID NOs: 15 or 21.
36. An isolated antibody which specifically binds to a human islet
cell antigen polypeptide, wherein said human islet cell antigen
polypeptide comprising an amino acid sequence selected from the
group consisting of: a) SEQ ID NO:22 from Leu, amino acid residue
442 to Gln, amino acid residue 818; b) SEQ ID NO:22 from Phe, amino
acid residue 418, to Gln, amino acid residue 818; c) a polypeptide
of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid
residue 818; and d) allelic variants of (a), (b) or (c).
37. An isolated antibody according to claim 36, wherein said
isolated antibody is a monoclonal antibody.
38. An isolated antibody according to claim 36, wherein said human
islet cell antigen polypeptide is a full length human islet cell
antigen polypeptide comprising the amino acid sequence of SEQ ID
NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue
738.
39. A hybridoma which produces a monoclonal antibody which
specifically binds to a human islet cell antigen polypeptide,
wherein said human islet cell antigen polypeptide comprises an
amino acid sequence selected from the group consisting of: a) SEQ
ID NO:22 from Leu, amino acid residue 442 to Gln, amino acid
residue 818; b) SEQ ID NO:22 from Phe, amino acid residue 418, to
Gln, amino acid residue 818; c) a polypeptide of SEQ ID NO:22 from
His, amino acid residue 1, to Gln, amino acid residue 818; and d)
allelic variants of (a), (b) or (c).
40. A hybridoma according to claim 39, wherein said human islet
cell antigen polypeptide is a full length human islet cell antigen
polypeptide comprising the amino acid sequence of SEQ ID NO:22 from
Leu, amino acid residue 442 to Arg, amino acid residue 738.
41. A diagnostic kit for use in detecting an autoantibody to
pancreatic .beta.-islet cells, comprising a container containing a
human islet cell antigen polypeptide wherein said human islet cell
antigen polypeptide comprises an amino acid sequence selected from
the group consisting of: a) SEQ ID NO:22 from Leu, amino acid
residue 442 to Gln, amino acid residue 818; b) SEQ ID NO:22 from
Phe, amino acid residue 418, to Gln, amino acid residue 818; c) a
polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln,
amino acid residue 818; and d) allelic variants of (a), (b) or (c),
wherein said polypeptide forms an immune complex with an
autoantibody from a patient at risk of or predisposed to develop
IDDM, and one or more containers containing additional
reagents.
42. A pharmaceutical composition which comprises a human islet cell
antigen polypeptide, wherein said human islet cell antigen
polypeptide comprises an amino acid sequence selected from the
group consisting of: a) SEQ ID NO:22 from Leu, amino acid residue
442 to Gln, amino acid residue 818; b) SEQ ID NO:22 from Phe, amino
acid residue 418, to Gln, amino acid residue 818; c) a polypeptide
of SEQ ID NO:22 from His, amino acid residue 1, to Gln, amino acid
residue 818; and d) allelic variants of (a), (b) or (c), wherein
said polypeptide forms an immune complex with an autoantibody from
a patient at risk of or predisposed to develop IDDM in combination
with a pharmaceutically acceptable carrier or vehicle.
43. A method for monitoring the disease state in a patient
comprising: testing a biological sample from a patient for the
presence of mammalian islet cell antigen post-translationally
modified polypeptides; determining the concentration of said
polypeptides; and correlating levels of said polypeptides in said
sample with the disease state in a patient.
44. A method for monitoring the disease state in a patient
according to claim 43 wherein said human islet cell antigen
post-translationally modified polypeptide comprise the sequence of
SEQ ID NO:22 from His, amino acid residue 1 to Glu, amino acid
residue 227.
45. A method for monitoring the disease state in a patient
according to claim 43 wherein said biological sample is plasma or
serum.
46. A method for monitoring the disease state in a patient
comprising: exposing T cells to islet cell antigen 1851 peptides;
detecting T cell reactivity; and correlating T cell reactivity with
disease state.
47. A method for monitoring the disease state according to claim 46
wherein said T cells are from peripheral blood mononuclear cells
from a prediabetic patient.
48. A method for monitoring the disease state according to claim 46
wherein said disease state is conversion from prediabetes to
diabetes.
Description
BACKGROUND OF THE INVENTION
[0001] Insulin-dependent diabetes mellitus (IDDM) is a disease
resulting from the autoimmune destruction of the insulin-producing
.beta.-cells of the pancreas. Studies directed at identifying the
autoantigen(s) responsible for .beta.-cell destruction have
generated several candidates, including poorly characterized islet
cell antigens (ICA) (Bottazzo et al., Lancet 2: 1279-83, 1974),
insulin (Palmer et al., Science 222: 1337-39, 1983), glutamic acid
decarboxylase (GAD) (Baekkeskov et al., Nature 298: 167-69, 1982;
Baekkeskov et al., Nature 347: 151-56, 1990), and a 64 kD islet
cell antigen that is distinct from GAD and that which yields 37 kD
and 40 kD fragments upon trypsin-digestion (Christie et al.,
Diabetes 41: 782-87, 1992).
[0002] Detection of specific autoantigens in prediabetic
individuals has been used as a predictive marker to identify,
before clinical onset and significant .beta.-cell loss has
occurred, those at greater risk of developing IDDM (Gorsuch et al.,
Lancet 2: 1363-65, 1981; Baekkeskov et al., J. Clin. Invest. 79:
926-34, 1987; Johnstone et al., Diabetologia 32: 382-86, 1989;
Ziegler et al., Diabetes 38: 1320-25, 1989; Baekkeskov et al.,
Nature (Lond) 347: 151-56, 1990; Bonifacio et al., Lancet 335:
147-49, 1990; and Bingley et al. Diabetes 43: 1304-10, 1994).
[0003] Antibodies to the 40 kD, and more particularly the 37 kD,
ICA fragments are detected when clinical onset of IDDM is imminent
and are found to be closely associated with IDDM development
(Christie et al., Diabetes 41: 782-87, 1992). Diabetic sera
containing antibodies specific to the 40 kD fragment were recently
found to bind to the intracellular domain of the protein tyrosine
phosphatase, IA-2/ICA512 (Lu et al., Biochem. Biophys. Res. Comm.
204: 930-36, 1994; Lan et al., DNA Cell Biol. 13: 505-14, 1994;
Rabin et al., J. Immunol. 152: 3183-88, 1994; Payton et al., J.
Clinc. Invest. 96: 1506-11, 1995; and Passini et al., Proc. Natl.
Acad. Sci. USA 92: 9412-16, 1995). Antibodies specific to the 37 kD
fragment are thought to bind either to a posttranslational in vivo
modification of IA-2/ICA512 or a different, but probably related,
protein precursor (Passini et al., ibid.).
[0004] ICA 512 was initially isolated as an autoantigen from an
islet cell cDNA library, and was subsequently shown to be related
to the receptor-linked protein tyrosine phosphatase family (Rabin
et al., ibid.). ICA 512 was later found to be identical to a mouse
and human protein tyrosine phosphatase, IA-2, isolated from brain
and insulinoma cDNA libraries (Lu et al., ibid.; and Lan et al.,
ibid.).
[0005] Detection of diabetes-associated autoantigens, especially
combinations of autoantigens, genotypes, such as HLA DR and HLA DQ,
and loci, such as the polymorphic region in the 5' flanking region
of the insulin gene; in prediabetic individuals have been shown to
be useful predictive markers of IDDM, see for example, Bell et al.,
(Diabetes 33:176-83, 1984); Sheehy et al., (J. Clin. Invest.
83:830-35, 1989); and Bingley et al., (Diabetes 43: 1304-10, 1994).
There is therefore a need in the art for autoantigens that would
serve to improve detection and diagnosis of IDDM. The present
invention fulfills this need by providing novel autoantigens as
well as related compositions and methods. The autoantigens of the
present invention represent a new .beta.-cell antigen. The present
invention also provides other, related advantages.
SUMMARY OF THE INVENTION
[0006] The present invention provides an isolated polynucleotide
which forms an immune complex with an autoantibody from a patient
at risk of or predisposed to develop IDDM, comprising a DNA segment
encoding a mammalian islet cell antigen polypeptide of SEQ ID NO:16
from Leu, amino acid residue 636 to Gln, amino acid residue 1012.
The invention also provides a mammalian islet cell antigen
polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to
Gln, amino acid residue 818. The invention also provides allelic
variants of these polypeptides. Within one aspect of the invention,
the isolated polynucleotide encodes a mammalian islet cell antigen
polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418, to
Gln, amino acid residue 818. Within another aspect of the
invention, the isolated polynucleotide encodes a mammalian islet
cell antigen polypeptide of SEQ ID NO:16 from Phe, amino acid
residue 612, to Gln, amino acid residue 1012. The invention further
provides allelic variants of these polypeptides. Within another
aspect, the isolated polynucleotide encoding a polypeptide of SEQ
ID NO:16 from Ala, amino acid residue 1, to Gln, amino acid residue
1012. Within another aspect, the isolated polynucleotide encoding a
polypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln,
amino acid residue 818. The invention further provides allelic
variants of these polypeptides. Within another aspect, the isolated
polynucleotide is a DNA molecule comprising a coding sequence
corresponding to SEQ ID NO:21 from nucleotide 1325 to nucleotide
2455. In still another aspect, the DNA molecule comprises a coding
sequence corresponding to SEQ ID NO:15 from nucleotide 1909 to
nucleotide 3039. The invention also provides allelic variants of
these molecules. The invention further provides complements of
polynucleotide molecules which specifically hybridize to these
molecules. In yet another aspect, the isolated polynucleotide is a
DNA molecule comprising a coding sequence corresponding to SEQ ID
NO:21 from nucleotide 1254 to nucleotide 2455. Within another
aspect, the isolated polynucleotide is a DNA molecule comprising a
coding sequence corresponding to SEQ ID NO:15 from nucleotide 1837
to nucleotide 3039. The invention also provides allelic variants of
these molecules. The invention further provides complements of
polynucleotide molecules which specifically hybridize to these
molecules. In still another aspect, the DNA molecule comprises a
coding sequence corresponding to SEQ ID NO:15 from nucleotide 4 to
nucleotide 3039. In still another aspect, the DNA molecule
comprises a coding sequence corresponding to SEQ ID NO:21 from
nucleotide 2 to nucleotide 2455. The invention also provides
allelic variants of these molecules. The invention further provides
complements of polynucleotide molecules which specifically
hybridize to these molecules. The invention also provides an
isolated polynucleotide molecule which encodes a complete coding
sequence of a mammalian islet cell antigen polypeptide comprising
the sequence of SEQ ID NO:22 from Leu, amino acid residue 442 to
Arg, amino acid residue 738. The invention also provides mammalian
islet cell antigens that are primate islet cell antigens.
[0007] The invention also provides DNA constructs comprising a
first DNA segment encoding a human islet cell antigen polypeptide
operably linked to additional DNA segments required for the
expression of the first DNA segment. The invention further provides
a first DNA segment that is an isolated polynucleotide molecule
encoding a human islet cell antigen polypeptide comprising the
amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue
442 to Gln, amino acid residue 818. The invention also provides a
first DNA segment that is an isolated polynucleotide molecule
encoding a human islet cell antigen polypeptide comprising the
amino acid sequence of SEQ ID NO:22 from Leu, amino acid residue
442 to Gln, amino acid residue 818. Within another aspect, the
invention provides a first DNA segment that is an isolated
polynucleotide molecule encoding a human islet cell antigen
polypeptide comprising the amino acid sequence of SEQ ID NO:22 from
His, amino acid residue 1, to Gln, amino acid residue 818. The
invention further provides host cells containing such DNA
constructs, as well as methods for producing human islet cell
antigen polypeptides comprising the steps of culturing such host
cell and isolating the human islet cell antigen polypeptide.
[0008] The invention further provides isolated mammalian islet cell
antigen polypeptides, wherein said isolated mammalian islet cell
antigen polypeptide forms an immune complex with an autoantibody
from a patient at risk of or predisposed to develop IDDM comprising
the amino acid sequence of SEQ ID NO:22 from Leu, amino acid
residue 442 to Gln, amino acid residue 818. The invention further
provides isolated mammalian islet cell antigen polypeptides
comprising the amino acid sequence of SEQ ID NO:16 from Leu, amino
acid residue 636 to Gln, amino acid residue 1012.
[0009] The invention also provides isolated polypeptides of SEQ ID
NO:16 from Phe, amino acid residue 612 to Gln, amino acid residue
1012. The invention also provides isolated polypeptides of SEQ ID
NO:22 from Phe, amino acid residue 418, to Gln, amino acid residue
818. The invention further provides isolated polypeptides of SEQ ID
NO:16 from Ala, amino acid residue 1 to Gln, amino acid residue
1012. The invention also provides isolated polypeptides of SEQ ID
NO:22 from His, amino acid residue 1, to Gln, amino acid residue
818. The invention further provides allelic variants of these
polypeptides. The invention still further provides an isolated
polypeptide which is a full length mammalian islet cell antigen
protein comprising the sequence of SEQ ID NO:22 from Leu, amino
acid residue 442 to Arg, amino acid residue 738. The invention also
provides mammalian islet cell antigens that are primate islet cell
antigens.
[0010] Within yet another aspect of the invention is provided a
method for determining the presence of an autoantibody to a human
islet cell antigen polypeptide in a biological sample, comprising
the steps of contacting the biological sample with the human islet
cell antigen polypeptide, which comprises an amino acid sequence
selected from the group consisting of a polypeptide of SEQ ID NO:22
from Leu, amino acid residue 442 to Gln, amino acid residue 81, a
polypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to
Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 from
His, amino acid residue 1 to Gln, amino acid residue 818, and
allelic variants thereof, under conditions conducive to immune
complex formation, and detecting the presence of immune complex
formation between the human islet cell antigen polypeptide and the
autoantibody to a human islet cell antigen, thereby determining the
presence of autoantibodies to the human islet cell antigen in the
biological sample. The invention further provides human islet cell
antigen polypeptides that are detectably labeled.
[0011] Within a further embodiment the invention provides a method
for predicting the clinical course of diabetes in a patient,
comprising testing a biological sample from a patient for the
presence of human islet cell antigen polypeptides comprising the
amino acid sequence selected from the group consisting of a
polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to
Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe,
amino acid residue 418 to Gln, amino acid residue 818, a
polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln,
amino acid residue 818, and allelic variants thereof, wherein the
polypeptide forms an immune complex with an autoantibody from a
patient at risk of or predisposed to develop IDDM, and classifying
the patient for clinical course of diabetes based on the presence
or absence of human islet cell antigens in the sample. The
invention further provides a method of predicting the clinical
course of IDDM by testing one or more additional predictive markers
associated with risk of or protection from IDDM. The invention
provides methods of predicting the clinical course where the
predictive marker is an autoantibody to an antigen selected from
the group consisting of GAD65, IA-2/ICA512 or insulin. The
invention also provides methods wherein the predictive marker is a
genotype selected from the group consisting of HLA DR and HLA DQ.
The invention also provides methods wherein the predictive marker
is a polymorphic region in the 5' flanking region of a human
insulin gene.
[0012] The invention also provides a method for treating a patient
to prevent an autoimmune response to a human islet cell antigen
polypeptide comprising inducing immunological tolerance in the
patient by administering a mammalian islet cell antigen polypeptide
comprising the amino acid sequence selected from the group
consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid
residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID
NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue
818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1
to Gln, amino acid residue 818, and allelic variants thereof, that
specifically binds a human islet cell antigen receptor on immature
or mature T or B lymphocytes.
[0013] The invention also provides oligonucleotide probes of at
least about 16 nucleotides, wherein which the oligonucleotide is at
least 85% homologous to a sequence of the mammalian islet cell
antigen DNA sequence of SEQ ID Nos:15 or 21.
[0014] The invention further provides isolated antibodies which
specifically bind to human islet cell antigen polypeptides which
comprise the amino acid sequence selected from the group consisting
of a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442
to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22 from
Phe, amino acid residue 418 to Gln, amino acid residue 818, a
polypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln,
amino acid residue 818, and allelic variants thereof. Within
another aspect, the invention provides monoclonal antibodies.
Within yet another aspect, the invention provides a hybridoma which
produces the monoclonal antibody.
[0015] The invention also provides a diagnostic kit for use in
detecting autoantibodies to pancreatic .beta.-islet cells,
comprising a container containing an islet cell antigen polypeptide
comprising an amino acid sequence selected from the group
consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid
residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID
NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue
818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1
to Gln, amino acid residue 818, and allelic variants thereof,
wherein the polypeptide forms an immune complex with autoantibodies
from a patient at risk of or predisposed to develop IDDM, and one
or more containers containing additional reagents.
[0016] Within another embodiment of the invention is provided a
pharmaceutical composition comprising an islet cell antigen
comprising an amino acid sequence selected from the group
consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acid
residue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID
NO:22 from Phe, amino acid residue 418 to Gln, amino acid residue
818, a polypeptide of SEQ ID NO:22 from His, amino acid residue 1
to Gln, amino acid residue 818, and allelic variants thereof, in
combination with a pharamceutically acceptable carrier or
vehicle.
[0017] Within a further embodiment of the invention is provided a
method for monitoring the disease state in a patient comprising
testing a biological sample from a patient for the presence of
human islet cell antigen post-translationally modified
polypeptides, determining the concentration of the peptides and
correlating the peptide levels in the sample with the disease state
in the patient. The invention provides that the human islet cell
antigen post-translationally modified polypeptide comprises the
sequence of SEQ ID NO:22 from His, amino acid residue 1 to Glu,
amino acid residue 227. The invention further provides that the
biological sample is plasma or serum.
[0018] Within yet a further embodiment, the invention provides a
method for monitoring the disease state in a patient comprising
exposing T cells to islet cell antigen 1851 peptides, detecting T
and correlating T cell reactivity with disease state. The invention
provides that the T cells are from peripheral blood mononuclear
cells from a prediabetic patient. The invention further provides
that the disease state is conversion from prediabetes to
diabetes.
DETAILED DESCRIPTION OF THE INVENTION
[0019] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
to be used hereinafter:
[0020] Allelic variant--Any of two or more alternative forms of a
gene occupying the same chromosomal locus. Allelic variation arises
naturally through mutation, and may result in phenotypic
polymorphism within populations. Gene mutations can be silent (no
change in the encoded polypeptide) or may encode polypeptides
having altered amino acid sequence. The term allelic variant is
also used herein to denote a protein encoded by an allelic variant
of a gene.
[0021] Biological sample--A sample that is derived from or contains
cells, cell components or cell products, including, but not limited
to, cell culture supernatants, cell lysates, cleared cell lysates,
cell extracts, tissue extracts, blood plasma, serum, and fractions
thereof, from a patient.
[0022] Complements of polynucleotide molecules--Polynucleotide
molecules having a complementary base sequence and reverse
orientation as compared to a reference sequence. For example, the
sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
[0023] Immune Complex Formation--A noncovalently bound molecule
formed between an antigen and an antibody specific for that
antigen, resulting in an extensively cross-linked mass. Conditions
conducive to complex formation are known in the art and easily
adaptable by those skilled in art, for example, the degree of
complex formation is in proportion to the relative amounts of
available antigen and antibody. Such complexes can be used, for
example, to identify and/or quantify the presence of either antigen
or antibody in a biological sample, identify and characterize
particular antibodies in tissues and cells, or to stimulate an
immune response.
[0024] Isolated--When applied to a protein the term "isolated"
indicates that the protein is found in a condition other than its
native environment, such as apart from blood and animal tissue. In
a preferred form, the isolated protein is substantially free of
other proteins, particularly other proteins of animal origin. It is
preferred to provide the proteins in a highly purified form, i.e.
greater than 95% pure, more preferably greater than 99% pure. When
applied to a polynucleotide molecule the term "isolated" indicates
that the molecule is removed from its natural genetic milieu and is
thus free of other extraneous or unwanted coding sequences, and is
in a form suitable for use within genetically engineered protein
production systems. Such isolated molecules are those that are
separated from their natural environment and include cDNA and
genomic clones. Isolated DNA molecules of the present invention are
free of other genes with which they are ordinarily associated and
may include naturally occurring 5' and 3' untranslated regions such
as promoters and terminators, the identification of such will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316: 774-78, 1985).
[0025] Operably linked--Indicates that the segments are arranged so
that they function in concert for their intended purposes, e.g.,
transcription initiates in the promoter and proceeds through the
coding segment to the terminator.
[0026] The DNA sequences encoding the polypeptides of the present
invention were unexpectedly identified during screening of a
primate islet cell cDNA library, and human insulinoma cDNA, for
autoantigens toward human diabetic sera. Analysis of the macaque
cDNA clones revealed a unique, previously unknown islet cell
antigen which contained regions of homology to the protein tyrosine
phosphatase family, especially the protein tyrosine phosphatase
IA2/ICA512. This novel islet cell antigen has been designated 1851
or ICA512.beta..
[0027] The present invention provides islet cell antigen
polypeptides which are .beta.-cell autoantigens. These autoantigens
were reactive with human prediabetic and diabetic sera. The
invention also provides methods for using the islet cell antigen
polypeptides for the detection, diagnosis, and treatment of
IDDM.
[0028] Representative islet cell antigen polypeptides of the
present invention comprise the amino acid sequences in SEQ ID
NOs:4, 16 or 22 and/or are encoded by polynucleotide sequences
comprising the sequences of SEQ ID NOs:3, 15 and 21 and form an
immune complex with autoantibodies from a patient at risk of or
predisposed to develop IDDM. The islet cell antigen polypeptides of
the present invention are preferably from mammals, especially
primates including humans. Preferred polypeptides of the present
invention include isolated polypeptides selected from the group
consisting of a polypeptide of SEQ ID NO:2 from Leu, amino acid
residue 265, to Gln amino acid residue 641. The invention also
provides polypeptides of SEQ ID NO:2 from Glu, amino acid residue
1, to Gln, amino acid residue 641. The invention further provides
macaque polypeptides of SEQ ID NO:16 from Ala, amino acid residue 1
to Gln, amino acid residue 1012 and human polypeptides of SEQ ID
NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue
818 and SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino
acid residue 818. The invention further provides allelic variants
and isolated sequences that are substantially identical to the
representative polypeptide sequences of SEQ ID NOs:2, 16 and 22 and
their species homologs. The term "substantially identical" is used
herein to denote proteins having 50%, preferably 60%, more
preferably 70%, and most preferably at least 80%, sequence identity
to the representative sequences shown in SEQ ID NO:2, 16 or 22 or
its species homologs. Within preferred embodiments, such proteins
will be at least 90% identical, and most preferably 95% or more
identical, to SEQ ID NO:2, 16 or 22 or their species homologs.
[0029] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:
603-616, 1986; Pearson and Lipman, Proc. Natl. Acad. Sci. USA
85:2444-2448, 1988; and Henikoff and Henikoff, Proc. Natl. Acad.
Sci. USA 89:10915-10919, 1992. Briefly, two amino acid sequences
are aligned to optimize the alignment scores using a gap opening
penalty of 10, a gap extension penalty of 1, and the "blosum 62"
scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1
(amino acids are indicated by the standard one-letter codes). The
percent identity of the optimum alignment is then calculated
as:
1 TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0
6 D -2 2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -2 0 0 2 -4 2 5 G 0
-2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3
-4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1
-3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 -2 -1 5 F -2 -3 -3 -3 -2 -3
-3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S
1 -1 2 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2
-2 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4
-3 -2 11 Y -2 -2 -2 -3 -2 -2 -2 -3 2 -1 -1 -2 -2 3 -3 -2 -2 2 7 V 0
-3 -3 -3 -2 -2 -2 -3 -3 3 2 -2 1 -1 -2 -2 0 -3 -1 4
[0030] 1 Total number of identical matches [ length of the longer
sequence plus the number of gaps introduced into the longer
sequence in order to align the two sequences ] .times. 100
[0031] Substantially identical proteins are characterized as having
one or more amino acid substitutions, deletions or additions. These
changes are preferably of a minor nature, that is conservative
amino acid substitutions (see Table 2) and other substitutions that
do not significantly affect the folding or activity of the protein;
small deletions, typically of one to about 30 amino acids;
amidation of the amino- or carboxyl-terminal; and small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine
residue, a small linker peptide of up to about 20-25 residues, or a
small extension that facilitates purification, such as a
poly-histidine tract, an antigenic epitope or a binding domain.
See, in general, Ford et al., Protein Expression and Purification
2: 95-107, 1991, which is incorporated herein by reference.
2TABLE 2 Conservative amino acid substitutions Basic: arginine
lysine histidine Acidic: glutamic acid aspartic acid Polar:
glutamine asparagine Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine
serine threonine methionine
[0032] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244, 1081-85, 1989). In
the latter technique, single alanine mutations are introduced at
every residue in the molecule, and the resultant mutant molecules
are tested for biological activity (e.g. protein tyrosine
phosphatase activity, Strueli et al., EMBO J. 9: 2399-407, 1990, or
binding to autoantibodies in prediabetic or diabetic sera) to
identify amino acid residues that are critical to the activity of
the molecule. Sites of ligand-receptor interaction can also be
determined by analysis of crystal structure as determined by such
techniques as nuclear magnetic resonance, crystallography or
photoaffinity labeling. See, for example, de Vos et al., Science
255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992;
Wlodaver et al., FEBS Lett. 309:59-64, 1992.
[0033] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or
Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-156, 1989).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a protein, selecting for
functional protein, and then sequencing the mutagenized proteins to
determine the spectrum of allowable substitutions at each position.
These methods allow the rapid determination of the importance of
individual amino acid residues in a protein of interest, and can be
applied to proteins of unknown structure.
[0034] The present invention further provides isolated
polynucleotide molecules encoding islet cell antigen polypeptides
which form immune complexes with autoantibodies from a patient at
risk of or predisposed to develop IDDM. Useful polynucleotide
molecules in this regard include mRNA, genomic DNA, cDNA and
synthetic DNA. For production of recombinant islet cell antigen
polypeptides, cDNA is preferred. The invention provides an isolated
polynucleotide molecule wherein the molecule is a DNA molecule
comprising a coding sequence corresponding to SEQ ID NO:1 from
nucleotide 795 to nucleotide 1922. The invention also provides a
DNA molecule comprising a coding sequence corresponding to SEQ ID
NO:1 from nucleotide 1 to nucleotide 2168. The invention also
provides a DNA molecule comprising a coding sequence corresponding
to nucleotide 4 to nucleotide 3039 of SEQ ID NO: 15. The invention
also provides DNA molecules from nucleotide 1325 to nucleotide
2455, from nucleotide 1254 to nucleotide 2455 and from nucleotide 2
to nucleotide 2544 of SEQ ID NO:21. The invention also provides
allelic variants of the sequences shown in SEQ ID NOs:1, 15 or 21,
and polynucleotide molecules that specifically hybridize to allelic
variants. Such polynucleotide molecules will hybridize to the
representative DNA sequences of SEQ ID NOs:1, 15, 21 or their
allelic variants under stringent conditions (Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor, N.Y., 1989). As used herein, the term "stringent
conditions" refers to hybridizing conditions that employ low ionic
strength and high temperature for washing, for example, 0.015 M
NaCl/0.0015 M sodium citrate/0.1% SDS at 50.degree. C.; employ
during hybridization a denaturing agent such as formamide, for
example, 50% (vol/vol) formamide with 0.1% polyvinylpyrrolidone/50
mM sodium citrate at 42.degree. C.; or employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075M sodium pyrophosphate,
5.times.Denhardt's solution, sonicated salmon sperm DNA (50 g/ml),
0.1% SDS, and 10% dextran sulfate at 42.degree. C., with washes at
42.degree. C. in 0.2.times.SSC and 0.1% SDS. Such hybridizable
polynucleotide molecules would include genetically engineered or
synthetic variants of the representative islet cell antigen
polynucleotide sequence, SEQ ID NO: 1, and polynucleotide molecules
that encode one or more amino acid substitutions, deletions or
additions, preferably of a minor nature, as discussed above.
Genetically engineered variants may be obtained by using
oligonucleotide-directed site-specific mutagenesis, by use of
restriction endonuclease digestion and adapter ligation, polymerase
chain reaction (PCR), or other methods well established in the
literature (see for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989,
and Smith et al., Genetic Engineering: Principles and Methods,
Plenum Press, 1981; which are incorporated herein by reference). In
addition, hybridizable polynucleotide molecules may encompass
sequences containing degeneracies in the DNA code wherein
host-preferred codons are substituted for the analogous codons in
the representative sequences of SEQ ID NOs: 1, 15 and 21.
[0035] Analysis of the representative cDNA sequences of SEQ ID
NO:1, 15 and 21 and their representative polypeptide sequences of
SEQ ID NO:2, 16 and 22, show that they contain regions of homology
to transmembrane protein tyrosine phosphatases. Comparison of the
human protein tyrosine phosphatase IA-2/ICA512 cDNA and amino acid
sequences with those of 1851 suggests that the coding region of
macaque 1851 is missing amino-terminal sequence corresponding to
approximately 1 amino acid and human 1851 is missing approximately
200 amino acid residues of the amino terminus. To recover the 5'
region, cDNA libraries from different tissues can be screened to
obtain a full length cDNA, which encodes a full length mammalian
islet cell antigen polypeptides. Another option for obtaining the
complete coding sequence comprises using 5' RACE (Rapid
Amplification cDNA Ends) PCR. RACE is an art recognized PCR-based
method for amplifying the 5' ends of incomplete cDNAs, a frequent
occurrence in cDNA cloning. To obtain the 5' portion of a cDNA, PCR
is carried out on specially prepared cDNA which contains unique
anchor sequences, using anchor primers provided with the 5' RACE
reagents available from, for example, Clontech, Palo Alto, Calif.
and a 3' primer based on known sequence. The 5'-RACE-Ready cDNA can
be purchased commercially (Clontech), or prepared according to
known methods. A secondary PCR reaction can then be carried out
using the anchor primer and a nested 3' primer, according to known
methods. Once a full-length cDNA is obtained, it is expressed and
analyzed for overall structural similarity to known protein
tyrosine phosphatases, and examined for features such as a
continuous open reading frame flanked by translation initiation and
termination sites and a potential signal sequence.
[0036] Transmembrane, or receptor-linked, protein tyrosine
phosphatases consist of a conserved cytoplasmic domain which may
have one or two (tandemly duplicated) catalytic regions, a single
transmembrane domain, a highly variable extracellular domain and a
signal peptide. These structural features suggest that
receptor-linked protein tyrosine phosphatases would be capable of
binding ligand and transducing external signal, but no ligands as
of yet have been identified. Based on the representative amino acid
sequence of SEQ ID NOs:2 and 15, the macaque 1851 polypeptide has
an approximately 611 amino acid extracellular domain, from Ala,
amino acid residue 1 to Lys, amino acid residue 611 of SEQ ID
NO:16, containing a post translational modification dibasic site,
at amino acid residue 423-424, or a tribasic site at amino acid
residues 422-424; a 24 amino acid transmembrane domain comprising
amino acid residue 241 to amino acid residue 265 of SEQ ID NO:2 or
Phe, amino acid residue 612 to Cys, amino acid residue 635 of SEQ
ID NO:16 and an approximately 375 amino acid cytoplasmic domain
comprising the amino acid residue 265 to amino acid residue 640 of
SEQ ID NO:2 or Leu, amino acid residue 636 to Gln, amino acid
residue 1012 of SEQ ID NO:16. The representative amino acid
sequence of the human islet cell antigen 1851 (SEQ ID NO:22) has
417 amino acids of an extracellular domain, from His, amino acid
residue 1 to Lys, amino acid residue 417 of SEQ ID NO:22; a 24
amino acid residue transmembrane domain, from Phe, amino acid
residue 418 to Cys, amino acid residue 441, of SEQ ID NO:22; and a
376 amino acid cytoplasmic domain, from Leu, amino acid residue 442
to Gln, amino acid residue 818 of SEQ ID NO:22.
[0037] The cytoplasmic domain of 1851 contains many regions that
are conserved between members of the protein tyrosine phosphatase
family. Within the cytoplasmic domain of protein tyrosine
phosphatases is a catalytic region of about 230 amino acids, which
contains a highly conserved catalytic core segment of approximately
11 amino acid residues (VHCXAGXXRXG SEQ ID NO:13) where the first
three X's are any amino acid, the fourth X is S or T, and the
cysteine appears to be essential to the catalytic mechanism
(Fischer et al., Science 253: 401-06). The catalytic core sequence
of the representative macaque 1851 polypeptide sequences of SEQ ID
Nos:2 and 16 and human 1851 polypeptide sequence represented by SEQ
ID NO:22 differs from other members of the protein tyrosine
phosphatase family in that alanine has been replaced by aspartic
acid and the second variable amino acid (X) is alanine. 1851, like
IA-2/ICA512, has a single catalytic region. Deletion of C-terminal
amino acids from the intracellular domain of human islet cell
antigen 1851 reduced reactivity with new onset IDDM sera,
suggesting this region may play a role in defining an autoantibody
epitope. Removal of the C-terminal 27 amino acids decreased
reactivity from 19/53 sera (36%) to 10/53 sera (19%), a 47%
decrease. Removal of the C-terminal 80 amino acids decreased
reactivity further to 9/53 sera (17%), a 53% decrease, and removal
of the C-terminal 160 amino acids abolished all recognition by all
53 new onset IDDM sera. This is similar to the reports of one of
two described intracellular IA-2/ICA512 autoantibody epitopes
(Bonifacio et al., J. Immunol. 155:5419-426, 1995). That human
islet cell antigens 1851 and human IA-2/ICA512 are each
precipitated by sera that do not precipitate the other suggests
that each antigen has unique autoantibody epitopes, which is
consistent with previous findings regarding the 37 kD and 40 kD
tryptic fragments (Payton et al., J. Clin. Invest. 96:1506-11,
1995). A comparison between the overall human and macaque islet
cell antigen 1851 nucleotide and amino acid sequences shows a 96.2%
nucleotide identity and a 94.6% amino acid identity, in particular
there was 97% identity within the nucleotide sequence and 98.9%
identity within the amino acid sequence of the corresponding
cytoplasmic domains, 100% identity within the transmembrane domain.
There is 77% amino acid identity within the cytoplasmic domain
between the claimed human (SEQ ID NO:22) and macaque (SEQ ID NO:16)
islet cell antigen 1851 sequences and the reported human
IA-2/ICA512 sequences (Lan et al., ibid.; and Rabin et al., ibid.).
Between the full length macaque islet cell antigen 1851 sequence
(as represented in SEQ ID Nos:15 and 16) and rat phogrin sequences
(Wasmeier and Hutton, J. Biol. Chem. 271:18161-70, 1996) there was
less homology, 75.5% identity within the nucleotide sequence and
69.9% identity within the amino acid sequence.
[0038] In contrast, there is little homology in the extracellular
regions of transmembrane protein tyrosine phosphatases. Some
contain Ig-like and/or fibronectin type III repeats (Streuli et
al., J. Exp. Med. 168: 1523, 1988; Hariharan et al., Proc. Natl.
Acad. Aci. USA 88: 11266, 1991); others have glycosylated segments
(Sap et al., Proc. Natl. Acad. Sci. USA 87:6112, 1990; and Krueger
et al., EMBO J. 9: 3241, 1990) and a conserved cysteine-rich region
(Tonks et al., J. Biol. Chem. 265: 10674-80, 1990) (Lan et al.
ibid.). There is 31% identity between macaque islet cell antigen
1851 (as represented by SEQ ID NO:15) and IA-2/ICA512 (Lan et al.,
ibid.; and Rabin et al., ibid.) within the extracellular
domain.
[0039] The tissue distribution of human islet cell antigen 1851 is
generally neuroendocrine. Northern analysis showed strong
hybridization to human mRNA from brain and pancreas and weaker
hybridization in spinal cord, thyroid, adrenal and GI tract. In
situ hybridization using macaque tissues further localized
pancreatic and adrenal expression to islets and adrenal medulla,
respectively. Northern blot analysis of rat phogrin showed
expression in brain, pancreas and .alpha. and .beta.cell tumor
lines (Wasmeier and Hutton, ibid.); mouse IA-2.beta. in brain,
pancreas, stomach and in insulinoma and glucagomoma cell lines (Lu
et al., Proc. Natl. Acad. Sci. USA 93:2307-11, 1996); human IA-2 in
brain, pituitary and pancreas, four insulinoma cell lines and a
glioblastoma cell line (Lan et al., ibid.); and human ICA512, brain
and pancreas (Rabin et al., ibid.).
[0040] Limited trypsinization of IA-2/ICA512 and human islet cell
antigen 1851 yielded a 40 kD IA-2/ICA512 fragment and a 37 kD islet
cell antigen 1851 fragment. These correspond to the 37 kD and 40 kD
tryptic fragments described by Christie et al. (J. Exp. Med.
172:789-94, 1990), Payton et al. (J. Clin. Invest. 96:1506-11,
1995), Bonifacio et al. (J. Immunol. 155:5419-26, 1995), Lu et al.
(Proc. Natl. Acad. Sci. USA 93:2307-11, 1996) and Wasmeier and
Hutton (ibid.).
[0041] Members of the protein tyrosine phosphatase family have been
shown to display alternative mRNA splicing (Moeller et al., WO
94/21800; Hall et al., J. Immunol. 141: 2781-87, 1988; Johnson et
al., J. Biol. Chem. 264: 6220-29, 1989; Streuli and Saito, EMBO J.
8: 787-96, 1989; Matthews et al., Proc. Natl. Acad. Sci. USA 87:
4444-48, 1990; Walton and Dixon, Ann. Rev. Biochem. 62: 101-20,
1993; and Pan et al., J. Biol. Chem. 268: 19284-91, 1993).
Alternative splicing may be important in autoantibody recognition;
"inappropriate" splicing could lead to autoimmunity by activating T
cells, for example.
[0042] The invention provides isolated DNA molecules that are
useful in producing recombinant islet cell antigens. As will be
evident to one skilled in the art, each individual domain or
combinations of the domains may be prepared synthetically or by
recombinant DNA techniques for use in the present invention. Thus,
the present invention provides the advantage that islet cell
antigens are produced in high quantities that may be readily
purified using methods known in the art (see generally; Scopes,
Protein Purification, Springer-Verlag, NY, 1982). Alternatively,
the proteins of the present invention may be synthesized following
conventional synthesis methods, such as the solid-phase synthesis
method of Barany and Merrifield (in The Peptides. Analysis,
Synthesis, Biology Vol. 2, Gross and Meienhofer, eds, Academic
Press, NY, pp. 1-284, 1980), by partial solid-phase techniques, by
fragment condensation or by classical solution addition.
[0043] DNA molecules of the present invention can be isolated using
standard cloning methods such as those described by Maniatis et al.
(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,
1982; which is incorporated herein by reference), Sambrook et al.,
(Molecular Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor, N.Y., 1989), or Mullis et al. (U.S. Pat. No.
4,683,195) which are incorporated herein by reference.
Alternatively, the coding sequences of the present invention can be
synthesized using standard techniques that are well known in the
art, such as by synthesis on an automated DNA synthesizer.
[0044] The sequence of a polynucleotide molecule encoding a
representative islet cell antigen polypeptide is shown in SEQ ID
NOs: 1, 15 and 21 and the corresponding amino acid sequences are
shown in SEQ ID NOs: 2, 16 and 22. Those skilled in the art will
recognize that these sequences correspond to one allele of either
the macaque or human gene, and that allelic variation is expected
to exist. Allelic variants of the DNA sequence shown in SEQ ID NO:
1, 15 and 21 including those containing silent mutations and those
in which mutations result in amino acid sequence changes, are
within the scope of the present invention, as are proteins which
are allelic variants of SEQ ID NO: 2, 16 and 22.
[0045] The macaque sequence disclosed herein is useful for
isolating polynucleotide molecules encoding islet cell antigen
polypeptides from other species ("species homologs"). In
particular, the macaque cDNA was used to conduct a sequence search
for a human homolog. A match was found as an expressed sequence tag
(EST) from a human fetal brain library submitted to the Genbank
database (GenBank ID: TO361, clone ID: HFBCV88). This 127 amino
acid polypeptide, SEQ ID NO:5, had homology to a region of the
cytoplasmic domain of M1.18.5.1 (SEQ ID NO:2) and was used to
design PCR primers to clone a 1.1 kD cytoplasmic portion (SEQ ID
NOs:6 and 7) of the human 1851 sequence, as described in the
examples below. Other preferred species homologs include mammalian
homologs such as bovine, canine, porcine, ovine, and equine
proteins. Methods for using sequence information from a first
species to clone a corresponding polynucleotide sequence from a
second species are well known in the art. See, for example, Ausubel
et al., eds., Current Protocols in Molecular Biology, John Wiley
and Sons, Inc., NY, 1987.
[0046] DNA molecules of the present invention or portions thereof
may be used as probes, for example, to directly detect 1851
sequences in cells or biological samples. Such DNA molecules are
generally synthetic oligonucleotides, but may be generated from
cloned cDNA or genomic sequences and will generally comprise at
least about 16 nucleotides, more often from about 17 nucleotides to
about 25 or more nucleotides, sometimes 40 to 60 nucleotides, and
in some instances a substantial portion or even the entire 1851
gene or cDNA. The synthetic oligonucleotides of the present
invention have at least 85% identity to a representative macaque or
human 1851 DNA sequence (SEQ ID Nos:1, 15 and 21) or their
complements. For use as probes, the molecules are labeled to
provide a detectable signal, such as with an enzyme, biotin, a
radionuclide, fluorophore, chemiluminescer, paramagnetic particle,
etc., according to methods known in the art. Probes of the present
invention may also be used in diagnostic methods to detect
autoantibodies in diabetic and prediabetic sera.
[0047] DNA molecules used within the present invention may be
labeled and used in a hybridization procedure similar to the
Southern or dot blot. As will be understood by those skilled in the
art, conditions that allow the DNA molecules of the present
invention to hybridize to the representative DNA sequence of SEQ ID
NO:1, 15 or 21 or their allelic variants may be determined by
methods well known in the art (reviewed, for example, by Sambrook
et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor, N.Y., 1989; which is incorporated herein by
reference). Those skilled in the art will be capable of varying
hybridization conditions (i.e. stringency of hybridization) of the
DNA molecules as appropriate for use in the various procedures by
methods well known in the literature (see, for example, Sambrook et
al., ibid., pages 11.45-11.53). The higher the stringency of
hybridization, the lower the number of mismatched sequences
detected. Alternatively, lower stringency will allow related
sequences to be identified.
[0048] Alternatively, allelic variants may be identified using DNA
molecules of the present invention and, for example, the polymerase
chain reaction (PCR) (disclosed by Saiki et al., Science 239: 487,
1987; Mullis et al., U.S. Pat. No. 4,686,195; and Mullis et al.,
U.S. Pat. No. 4,683,202) to amplify DNA sequences, which are
subsequently detected by their characteristic size on agarose gels
or which may be sequenced to detect sequence abnormalities.
[0049] DNA molecules encoding the islet cell antigen polypeptides
of the present invention may be inserted into DNA constructs. As
used within the context of the present invention a DNA construct is
understood to refer to a DNA molecule, or a clone of such a
molecule, either single- or double-stranded, which has been
modified through human intervention to contain segments of DNA
combined and juxtaposed in a manner that would not otherwise exist
in nature. DNA constructs of the present invention comprise a first
DNA segment encoding an islet cell antigen polypeptide operably
linked to additional DNA segments required for the expression of
the first DNA segment. Within the context of the present invention,
additional DNA segments will generally include promoters and
transcription terminators, and may further include enhancers and
other elements. One or more selectable markers may also be
included. DNA constructs useful for expressing cloned DNA segments
in a variety of prokaryotic and eukaryotic host cells can be
prepared from readily available components or purchase from
commercial suppliers.
[0050] In general, a DNA sequence encoding a protein of the present
invention is operably linked to a transcription promoter and
terminator within a DNA construct. The construct will commonly
contain one or more selectable markers and one or more origins of
replication, although those skilled in the art will recognize that
within certain systems selectable markers may be provided on
separate vectors, and replication of the exogenous DNA may be
provided by integration into the host cell genome. Selection of
promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0051] In one embodiment the first DNA segment is an isolated
polynucleotide molecule encoding a mammalian islet cell antigen
polypeptide comprising the amino acid sequence of SEQ ID NO:4,
wherein the polypeptide forms an immune complex with autoantibodies
from a patient at risk of or predisposed to IDDM. In another
embodiment, the first DNA segment is an isolated polynucleotide
encoding a polypeptide of SEQ ID NO:2 from Leu, amino acid residue
265 to Gln, amino acid residue 641. In another embodiment, the
first DNA segment is an isolated polynucleotide encoding a
polypeptide of SEQ ID NO:2 from Ser, amino acid residue 1, to Gln,
amino acid residue 641.
[0052] Within yet another embodiment, the first DNA segment is an
isolated polynucleotide encoding a polypeptide of SEQ ID NO:22 from
Leu, amino acid residue 442 to Gln, amino acid residue 818. In
another embodiment, the first DNA segment is an isolated
polynucleotide encoding a polypeptide of SEQ ID NO:16 from Ala,
amino acid residue 1 to Gln, amino acid residue 1012.
[0053] The proteins of the present invention can be produced in
genetically engineered host cells according to conventional
techniques. Suitable host cells are those cell types that can be
transformed or transfected with exogenous DNA and grown in culture,
and include bacteria, fungal cells, and cultured higher eukaryotic
cells. Techniques for manipulating cloned DNA molecules and
introducing exogenous DNA into a variety of host cells are
disclosed by Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, and Ausubel et al., ibid., which are
incorporated herein by reference.
[0054] To direct a protein of the present invention into the
secretory pathway of the host cells, a secretory signal sequence
(also known as a leader sequence, prepro sequence or pre sequence)
is provided in the expression vector. The secretory signal sequence
is joined to the DNA sequence encoding a protein of the present
invention in the correct reading frame. Secretory signal sequences
are commonly positioned 5' to the DNA sequence encoding the protein
of interest, although certain signal sequences may be positioned
elsewhere in the DNA sequence of interest (see, e.g., Welch et al.,
U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
The secretory signal sequence may be that normally associated with
a protein of the present invention, or may be from a gene encoding
another secreted protein.
[0055] Cultured mammalian cells are also preferred hosts within the
present invention. A preferred vector system for use in the present
invention is the pZCEP vector system as disclosed by Jelineck et
al., Science, 259: 1615-16, 1993. Methods for introducing exogenous
DNA into mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et
al., eds., Current Protocols in Molecular Biology, John Wiley and
Sons, Inc., NY, 1987), and cationic lipid transfection using
commercially available reagents including the Boehringer Mannheim
Transfection-Reagent (N-[1-(2,3-Dioleoyloxy)propyl]-N-
,N,N-trimethyl ammoniummethylsulfate; Boehringer Mannheim,
Indianapolis, Ind.) or LIPOFECTIN.about. reagent
(N-[1-(2,3-Dioleyloxy)propyl]-N,N,N-tr- imethylammonium chloride
and dioeleoyl phosphatidylethanolamine; GIBCO-BRL, Gaithersburg,
Md.) using the manufacturer-supplied directions, which are
incorporated herein by reference. The production of recombinant
proteins in cultured mammalian cells is disclosed, for example, by
Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat.
No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and
Ringold, U.S. Pat. No. 4,656,134, which are incorporated herein by
reference. Preferred cultured mammalian cells include the COS-1
(ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL
1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham
et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary
(e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell
lines are known in the art and available from public depositories
such as the American Type Culture Collection, Rockville, Md. In
general, strong transcription promoters are preferred, such as
promoters from SV-40 or cytomegalovirus.
[0056] Prokaryotic cells can also serve as host cells for use in
carrying out the present invention. Particularly preferred are
strains of the bacteria Escherichia coli, although Bacillus and
other genera are also useful. Techniques for transforming these
hosts and expressing foreign DNA sequences cloned therein are well
known in the art (see, e.g., Sambrook et al., ibid.). When
expressing the proteins in bacteria such as E. coli, the protein
may be retained in the cytoplasm, typically as insoluble granules,
or may be directed to the periplasmic space. In the former case,
the cells are lysed, and the granules are recovered and denatured
using, for example, guanidine isothiocyanate. The denatured protein
is then refolded by diluting the denaturant. In the latter case,
the protein can be recovered from the periplasmic space in a
soluble form.
[0057] Fungal cells are also suitable as host cells. For example,
Saccharomyces ssp., Hansenula polymorpha, Schizosaccharomyces
pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago
maydis, Pichia pastoris, Pichia guillermondii, Pichia methanolica,
and Candida maltosa transformation systems are known in the art.
See, for example, Kawasaki, U.S. Pat. No. 4,599,311, Kawasaki et
al., U.S. Pat. No. 4,931,373, Brake, U.S. Pat. No. 4,870,008; Welch
et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No.
4,845,075, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986
and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be
utilized according to the methods of McKnight et al., U.S. Pat. No.
4,935,349, which is incorporated herein by reference. Methods for
transforming Acremonium chrysogenum are disclosed by Sumino et al.,
U.S. Pat. No. 5,162,228, which is incorporated herein by
reference.
[0058] Other higher eukaryotic cells can also be used as hosts,
including insect cells, plant cells and avian cells. Transformation
of insect cells and production of foreign proteins therein is
disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and Bang et
al., U.S. Pat. No. 4,775,624, which are incorporated herein by
reference. The use of Agrobacterium rhizogenes as a vector for
expressing genes in plant cells has been reviewed by Sinkar et al.,
J. Biosci. (Bangalore) 11:47-58, 1987.
[0059] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. Selection is
carried out in the presence of a neomycin-type drug, such as G-418
or the like. Selection systems may also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate.
[0060] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell.
[0061] The recombinant islet cell antigen polypeptides expressed
using the methods described herein are isolated and purified by
conventional procedures, including separating the cells from the
medium by centrifugation or filtration, precipitating the
proteinaceous components of the supernatant or filtrate by means of
a salt, e.g. ammonium sulfate, purification by a variety of
chromatographic procedures, e.g. ion exchange chromatography or
affinity chromatography, or the like. Methods of protein
purification are known in the art (see generally, Scopes, R.,
Protein Purification, Springer-Verlag, NY (1982), which is
incorporated herein by reference) and may be applied to the
purification of the recombinant proteins of the present invention.
Substantially pure recombinant islet cell antigen polypeptides of
at least about 50% is preferred, at least about 70-80% more
preferred, and 95-99% or more homogeneity most preferred,
particularly for pharmaceutical uses. Once purified, partially or
to homogeneity, as desired, the recombinant islet cell antigen
polypeptides may then be used diagnostically, therapeutically, etc.
as further described below.
[0062] Recombinant 1851 polypeptides can also be produced by
expressing islet cell antigen DNA fragments, such as fragments
generated by digesting an islet cell antigen cDNA at convenient
restriction sites. The isolated recombinant polypeptides or
cell-conditioned media are then assayed for activity as described
in the examples below. Alternatively, the proteins of the present
invention may be synthesized following conventional synthesis
methods such as the solid-phase synthesis using the method of
Barany and Merrifield (in The Peptides. Analysis, Synthesis,
Biology Vol. 2, Gross and Meienhofer, eds, Academic Press, NY, pp.
1-284, 1980, which are incorporated herein by reference), by
partial solid-phase techniques, by fragment condensation or by
classical solution addition. Short polypeptide sequences, or
libraries of overlapping peptides, usually from about 6 up to about
35 amino acids, which correspond to selected islet cell antigen
polypeptide regions can be readily synthesized and then screened in
screening assays designed to identify peptides having a desired
activity, such as domains which are responsible for or contribute
to binding activity, immunodominant epitopes (particularly those
recognized by autoantibodies), and the like.
[0063] Although the use of recombinant 1851 polypeptides is
preferred within the methods of the present invention, 1851
polypeptides may also be prepared from cells that naturally produce
1851 protein (such as islet cells). For example, 1851 polypeptides
may be prepared from islet cells by isolation of a membrane
fraction. This 1851-enriched fraction is then used to detect
autoantibodies to 1851 in prediabetic and diabetic sera.
[0064] Islet cell antigen polypeptides produced according to the
present invention can be used diagnostically, in the detection and
quantitation of autoantibodies in a biological sample, that is, any
sample derived from or containing cells, cell components or cell
products, including, but not limited to, cell culture supernatants,
cell lysates, cleared cell lysates, cell extracts, tissue extracts,
blood plasma, serum, and fractions thereof. By means of having
islet cell antigen polypeptides which specifically bind to
autoantibodies in prediabetic and diabetic sera, the presence or
absence of such autoantibodies can be determined, and the
concentration of such autoantibodies in an individual can be
measured. This information can then be used to monitor the
progression or regression of the potentially harmful autoantibodies
in individuals at risk of, or with a predisposition to develop
IDDM, and would be useful for predicting the clinical course of the
disease in a patient. The assay results can also find use in
monitoring the effectiveness of therapeutic measures for treatment
of IDDM or related diseases.
[0065] As will be recognized by those skilled in the art, numerous
types of immunoassays are available for use in determining the
presence of autoantibodies. For instance, direct and indirect
binding assays, competitive assays, sandwich assays, and the like,
as are generally described in, e.g., U.S. Pat. Nos. 4,642,285;
4,376,110; 4,016,043; 3,879,262; 3,852,157; 3,850,752; 3,839,153;
3,791,932; and Harlow and Lane, Antibodies, A Laboratory Manual,
Cold Spring Harbor Publications, N.Y., 1988, each incorporated
herein by reference. In one assay format, autoantibodies directed
to the polypeptides of the present invention are quantified
directly by measuring the binding of autoantibodies in a biological
sample to recombinant or synthetic islet cell antigen polypeptides.
The biological sample is contacted with at least one islet cell
antigen polypeptide of the invention under conditions conducive to
immune complex formation. The immune complexes formed between the
islet cell antigen polypeptide and the antibodies are then
detected, and the presence and quantity of autoantibodies can then
be used to diagnose or direct treatment of IDDM. The immune
complexes can be detected by means of antibodies that bind to the
islet cell antigen of the present invention or by labeling the
polypeptide as described below. Separation steps (e.g., washes) may
be necessary in some cases to distinguish specific binding over
background. In another format, the serum level of a patient's
autoantibodies to the islet cell antigen polypeptides in serum can
be measured by competitive binding with labeled or unlabeled
antibodies to the islet cell antigen polypeptides of the present
invention. Unlabeled 1851 polypeptides can be used in combination
with labeled antibodies that bind to human antibodies or to islet
cell antigens. Alternatively, the islet cell antigen polypeptide
can be directly labeled. A wide variety of labels can be employed,
such as radionuclides, particles (e.g., gold, ferritin, magnetic
particles, red blood cells), fluors, enzymes, enzyme substrates,
enzyme cofactors, enzyme inhibitors, ligands (particularly
haptens), chemiluminescers, biotin and other compounds that provide
for the detection of the labeled polypeptide or protein. For
example, an 1851 polypeptide can be radiolabeled using conventional
methods such as in vitro transcription and translation.
Radiolabeled 1851 polypeptide is combined with patient serum under
conditions suitable for immune complex formation. Immune complexes
are then separated, such as by binding to protein A. Precipitated
1851 polypeptides are then quantitated by conventional methods,
such as gel electrophoresis, fluorography, densitometry or by
direct counting of immunoprecipitated, radiolabeled antigen. The
amount of 1851 polypeptide precipitated by test sera can be
statistically compared to mean counts precipitated by healthy
control sera, each measured separately. In an alternative format,
an 1851 polypeptide antigen, labeled with biotin, is combined with
patient serum under conditions suitable for immune complex
formation. The serum is then transferred to a protein A-coated
container, such as a well of an assay plate, and the container is
allowed to stand so that immune complexes can form. The container
is then washed, and streptavidin, conjugated to a suitable enzyme
(e.g. alkaline phosphatase), is added. A chromogenic substrate is
then added, and the presence of 1851 polypeptide autoantibodies in
the sample is indicated by a color change. Additional assay formats
will be evident to those skilled in the art.
[0066] Thus, autoantibodies to islet cell antigen polypeptides can
be identified and, if desired, extracted from a patient's serum by
binding to 1851 polypeptides of the present invention. The islet
cell antigen polypeptides may be attached, e.g., by adsorption, to
an insoluble or solid support, such as ELISA microtiter well,
microbead, filter membrane, insoluble or precipitable soluble
polymer, etc. to function as an affinity resin. The captured
autoantibodies can then be identified by several methods. For
example, antisera or monoclonal antibodies to the antibodies can be
used. These antisera or monoclonal antibodies are typically
non-human in origin, such as rabbit, goat, mouse, etc. These
anti-antibodies can be detected directly if attached to a label
such as .sup.125I, enzyme, biotin, etc., or can be detected
indirectly by a labeled secondary antibody made to specifically
detect the anti-antibody.
[0067] The diagnostic methods of the present invention can be used
in conjunction with other known assays and diagnostic techniques
(see for example, WO 95/07464, incorporated herein by reference in
its entirety). Such other assays and techniques include measurement
of body mass index (BMI), defined as the quotient of the patient's
weight in kg divided by the square of height in meters; C-peptide
level (Heding, Diabetologia 11: 541-548 (1975); Landin-Olsson et
al., Diabetologia 33: 561-568 (1990)); or one or more additional
diabetes-associated autoantibodies, genotypes or loci. A low BMI
(i.e. less than about 25) in combination with other indicators is
suggestive of type I diabetes. BMI is thus a useful indicator for
distinguishing type I from type II diabetes. C-peptide level can be
measured using standard methods, such as that of Heding (ibid.), in
which insulin and proinsulin are removed from serum and C-peptide
is measured in the resulting insulin-free fraction
radioimmunologically.
[0068] The islet cell antigen polypeptides of the current invention
can also be used to assess T cell reactivity, as a method for
monitoring the disease state in a patient. Mammalian islet cell
antigen 1851 peptides will generally comprise at least about 12
amino acids, and more often from about 15 amino acids to about 20
or more amino acids. In some instances, a substantial portion or
domain or even the entire 1851 protein, can be used to assess T
cell reactivity in peripheral blood mononuclear cells (PBMNCs) from
prediabetics. Methods for detecting such in vitro activity are
known in the art, including a proliferation assay measuring
.sup.3H-thymidine incorporation, analysis of activation markers,
such as CD69, or measuring cytokine production, such as IL-2.
Correlations can be drawn between T cell reactivity to islet cell
antigen 1851 and conversion from prediabetes to diabetes. This
correlation would be consistent with the appearance of
autoantibodies to islet cell antigen peptides late in prediabetes
(Christie et al., Diabetes 43:1254-59, 1994).
[0069] Mammalian cells, such as COS cells or L cells, may also be
transfected with appropriate Class I or Class II alleles specific
for the islet cell antigen of the present invention. Such MHC
molecules may be soluble or membrane bound, and the 1851 antigenic
polypeptide may be recombinantly tethered to the N-terminal region
of the .alpha. or .beta. chain using a flexible linker containing,
for example, repeating glycine residues separated by a serine
residue, such that the antigenic peptide binds to the MHC molecule
and is properly presented to the T cell. Alternatively, the
antigenic peptide may be exogenously loaded into the MHC peptide
binding grove. The MHC-antigenic peptide complex can then be used
to assess the reactivity of peripheral blood T cells derived from
prediabetic or diabetic patients. This reactivity may be assessed
by methods known in the art, such as .sup.3H thymidine
incorporation, cytokine production or cytolysis. Alternatively,
islet cell antigen expressed in microorganisms can be "fed" to
peripheral blood mononuclear cells (PBMN). The antigen-fed cells
can then be used to stimulate peripheral blood T cells derived from
diabetics or prediabetics.
[0070] The islet cell antigen polypeptides are also contemplated to
be advantageous for use as immunotherapeutics to induce
immunological tolerance or nonresponsiveness (anergy) to 1851
polypeptide autoantigens in patients predisposed or already
mounting an immune response to 1851 polypeptide autoantigens of the
islet .beta.-cells. This therapy can take the form of autoantigenic
1851 peptides bound to an appropriate MHC Class I or Class II
molecule as described above. The therapy can also be in the form of
oral tolerance (Weiner et al., Nature 376: 177-80, 1995), or IV
tolerance, for example. The use of polypeptide antigens in
suppression of autoimmune disease is disclosed by Wraith, et al.,
(Cell 59: 247-55, 1989). Tolerance can be induced in patients,
although conditions for inducing such tolerance will vary according
to a variety of factors. In a neonate, tolerance can be induced by
parenteral injection of an islet cell antigenic polypeptide, either
with recombinant polypeptide or synthetic antigen, or more
conveniently by oral administration in an appropriate formulation.
The precise amount of administration, its mode and frequency of
dosages will vary.
[0071] To induce immunological tolerance to the islet cell
autoantigens in an adult susceptible to or already suffering from a
islet cell antigen related disease such as IDDM, the precise
amounts and frequency of administration will also vary, for adults
about 1 to 1,000 mg/kg can be administered by a variety of routes,
such as parenterally, orally, by aerosols, intradermal injection,
etc. For neonates the doses will generally be higher than those
administered to adults; e.g. 100 to 1,000 mg/kg.
[0072] The islet cell antigen 1851 polypeptides will typically be
more tolerogenic when administered in a soluble form rather than an
aggregrated or particulate form. Persistence of an islet cell
antigen polypeptide of the invention is generally needed to
maintain tolerance in an adult, and thus may require more frequent
administration of the antigen, or its administration in a form
which extends the half-life of the islet cell antigen. See for
example, Sun et al. (Proc. Natl. Acad. Sci. USA 91: 10795-99,
1994).
[0073] The islet cell antigen polypeptides described herein are
also contemplated to be advantageous for use as immunotherapeutics
in treating longer term IDDM patients that have been identified by
autoantibody testing at the time of clinical non-insulin dependent
diabetes mellitus (NIDDM) diagnosis. Intervention in these patients
may be especially effective, perhaps due to the slowly progressive
nature of their .beta. cell destruction. Since the numbers of such
patients is nearly the same as those with classical childhood IDDM,
there is a need for such therapeutic intervention (Hagopian et al.,
J. Clin. Invest. 91:368-74; 1993; Harris and Robbins, Diabetes Care
17:1337-40, 1994; and Kobayashi et al., Diabetes 45:622-26,
1996).
[0074] The N-terminal domain of islet cell antigen 1851 is expected
to be inside the insulin secretory granule. The islet cell antigen
polypeptides of the current invention contain post translational
modification sites within the N-terminal domain. A dibasic site or
tribasic site at amino acid residues 228-230 (Arg-Lys-Lys) in SEQ
ID NO:22 and amino acid residues 422-424 (Arg-Lys-Lys) in SEQ ID
NO:16 could result in cleavage of a 420 amino acid
post-translationally modified mammalian islet cell antigen
polypeptide from the islet cell antigen 1851 polypeptide. All or
part of this cleaved polypeptide may be released from the .beta.
cell via either the constitutive secretory pathway for granule halo
components, or via the regulated pathway involved in insulin
release. Detection and quantitation of post translationally
modified polypeptides in a biological sample (that is, any sample
derived from or containing cells, cell components or cell products,
including, but not limited to, cell culture supernatants, cell
lysates, cleared cell lysates, cell extracts, tissue extracts,
blood plasma, serum, and fractions thereof) can be used
diagnostically to monitor disease state in a patient. The presence
or absence of such polypeptides in prediabetic and diabetic sera
can be determined, for example by radioimmunoassay, and the
concentration of such polypeptides in such an individual serum
sample can be measured. This information can then be used, for
example, to monitor insulin secretory activity, such as .beta. cell
insulin secretory rates; or to indicate altered .beta. cell
physiology associated with cellular stress as in an immune attack.
Peptide levels could be an indicator of .beta. cell distress or
.beta. cell death, and would be useful for predicting the disease
state in a patient. Alternatively, the peptides herein function
serve in paracrine or endocrine signaling to other islet cells or
remote cells in other organs. The assay results can also find use
in monitoring the effectiveness of therapeutic measures for
treatment of IDDM or related diseases. In a preferred embodiment, a
post-translationally modified mammalian islet cell antigen
polypeptide comprises the sequence of SEQ ID NO:22 from His, amino
acid residue 1 to Glu, amino acid residue 227. In another preferred
embodiment the biological sample is blood.
[0075] The present invention also relates to a pharmaceutical
composition comprising an islet cell antigen polypeptide of the
present invention, together with a pharmaceutically acceptable
carrier or vehicle, such as saline, buffered saline, water or the
like. Formulations may further include one or more excipients,
preservatives, solubilizers, etc. Methods of formulation are well
known in the art and are disclosed, for example, in Remington's
Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton
Pa., 1990, which is incorporated herein by reference. Therapeutic
doses will generally be in the range of 0.1 to 100 .mu.g/kg of
patient weight, with the exact dose determined by the clinician
according to accepted standards, taking into account the nature and
severity of the condition to be treated, patient traits, etc.
Determination of dose is within the level of ordinary skill in the
art. In general, a therapeutically effective amount of an islet
cell antigen polypeptide of the present invention is an amount
sufficient to produce a clinically significant reduction in
.beta.-cell loss or a delay of clinical onset of IDDM.
[0076] In a related aspect, the present invention provides
diagnostic kits for use with the recombinant or synthetic islet
cell antigen polypeptides of the present invention, in detecting
autoantibodies to pancreatic .beta.-islet cells. Thus, 1851
polypeptides may be provided, usually in lyophilized form, in a
container, either alone or in conjunction with additional reagents,
such as 1851-specific antibodies, labels, and/or anti-human
antibodies and the like. The 1851 polypeptides and antibodies,
which may be conjugated to a label or unconjugated, are included in
the kits with buffers, such as Tris phosphate, carbonate, etc.,
stabilizers, biocides, inert proteins, e.g., serum albumin, and the
like. Frequently it will be desirable to include an inert extender
or excipient to dilute the active ingredients, where the excipient
may be present in from about 1 to 99% of the total composition.
Where an antibody capable of binding to the islet cell antigen
polypeptide autoantibody or to the recombinant or synthetic 1851
polypeptide is employed in an assay, this will typically be present
in a separate vial.
[0077] Within one aspect of the present invention, islet cell
antigen polypeptides, including derivatives thereof, as well as
portions or fragments of these polypeptides, are utilized to
prepare antibodies for diagnostic or therapeutic uses which
specifically bind to islet cell antigen polypeptides. As used
herein, the term "antibodies" includes polyclonal antibodies,
monoclonal antibodies, antigen-binding fragments thereof such as
F(ab').sub.2 and Fab fragments, as well as recombinantly produced
binding partners. These binding partners incorporate the variable
regions from a gene which encodes a specifically binding monoclonal
antibody. Antibodies are defined to be specifically binding if they
bind to the islet cell antigen polypeptides with a K.sub.a of
greater than or equal to 10.sup.7/M. The affinity of a monoclonal
antibody or binding partner may be readily determined by one of
ordinary skill in the art (see, Scatchard, Ann. NY Acad. Sci. 51:
660-72, 1949).
[0078] Methods for preparing polyclonal and monoclonal antibodies
have been well described in the literature (see for example,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G. R.,
Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications,
CRC Press, Inc., Boca Raton, Fla., 1982, which is incorporated
herein by reference) As would be evident to one of ordinary skill
in the art, polyclonal antibodies may be generated from a variety
of warm-blooded animals such as horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, or rats, for example. The immunogenicity
of the islet cell antigen polypeptide may be increased through the
use of an adjuvant such as Freund's complete or incomplete
adjuvant. A variety of assays known to those skilled in the art may
be utilized to detect antibodies which specifically bind to an
islet cell antigen. Exemplary assays are described in detail in
Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold
Spring Harbor Laboratory Press, 1988. Representative examples of
such assays include: concurrent immunoelectrophoresis,
radio-immunoassays, radio-immunoprecipitations, enzyme-linked
immuno-sorbent assays, dot blot assays, inhibition or competition
assays, and sandwich assays.
[0079] Additional techniques for the preparation of monoclonal
antibodies may be utilized to construct and express recombinant
monoclonal antibodies. Briefly, mRNA is isolated from a B cell
population and used to create heavy and light chain immunoglobulin
cDNA expression libraries in a suitable vector such as the
.lambda.IMMUNOZAP(H) and .lambda.IMMUNOZAP(L) vectors, which may be
obtained from Stratogene Cloning Systems (La Jolla, Calif.). These
vectors are then screened individually or are co-expressed to form
Fab fragments or antibodies (Huse et al., Science 246: 1275-81,
1989; Sastry et al., Proc. Natl. Acad. Sci. USA 86: 5728-32, 1989).
Positive plaques are subsequently converted to a non-lytic plasmid
which allows high level expression of monoclonal antibody fragments
in E. coli.
[0080] Binding partners such as those described above may also be
constructed utilizing recombinant DNA techniques to incorporate the
variable regions of a gene which encodes a specifically binding
antibody. The construction of these proteins may be readily
accomplished by one of ordinary skill in the art (see for example,
Larrick et al., Biotechnology 7: 934-38, 1989; Reichmann et al.,
Nature 322: 323-27, 1988 and Roberts et al. Nature 328: 731-34,
1987). Once suitable antibodies or binding partners have been
obtained, they may be isolated or purified by many techniques well
described in the literature (see for example, Antibodies: A
Laboratory Manual, ibid.). Suitable techniques include protein or
peptide affinity columns, HPLC or RP-HPLC, purification on protein
A or protein G columns or any combination of these techniques.
Within the context of the present invention, the term "isolated" as
used to define antibodies or binding partners means "substantially
free of other blood components."
[0081] Antibodies of the present invention may be produced by
immunizing an animal, a wide variety of warm-blooded animals such
as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and
rats can be used, with a recombinant or synthetic islet cell
antigen polypeptide or a selected portion thereof (e.g., a
peptide). For example, by selected screening one can identify a
region of the islet cell antigen polypeptide such as that
predominantly responsible for recognition by anti-islet cell
antigen polypeptide antibodies, or a portion which comprises an
epitope of a islet cell antigen polypeptide variable region, which
may thus serve as a islet cell antigen polypeptide-specific marker.
Antibody producing cells obtained from the immunized animals are
immortalized and screened, or screened first for, e.g., the
production of antibody which inhibits the interaction of the
anti-islet cell antigen polypeptide autoantibody with the islet
cell antigen polypeptide and then immortalized. As the generation
of human monoclonal antibodies to a human antigen, such as an 1851
polypeptide, may be difficult with conventional immortalization
techniques, it may be desirable to first make non-human antibodies
and then transfer via recombinant DNA techniques the antigen
binding regions of the non-human antibodies, e.g. the F(ab')2 or
hypervariable regions, to human constant regions (Fc) or framework
regions to produce substantially human molecules. Such methods are
generally known in the art and are described in, for example, U.S.
Pat. No. 4,816,397, and EP publications 173,494 and 239,400, which
are incorporated herein by reference.
[0082] Alternatively, one may isolate DNA sequences which encode a
human monoclonal antibody or portions thereof that specifically
bind to islet cell antigen polypeptides by screening a DNA library
from human B cells according to the general protocol outlined by
Huse et al., Science 246: 1275-81, 1989, incorporated herein by
reference, and then cloning and amplifying the sequences which
encode the antibody (or binding fragment) of the desired
specificity.
[0083] In another aspect of the invention, the mammalian islet cell
antigen polypeptides can be used to clone T cells which have
specific receptors for the islet cell antigen polypeptide. Once the
islet cell antigen polypeptide specific T cells are isolated and
cloned using techniques generally available to the skilled artisan,
the T cells or membrane preparations thereof can be used to
immunize animals to produce antibodies to the islet cell antigen
polypeptide receptors on T cells. The antibodies can be polyclonal
or monoclonal. If polyclonal, the antibodies can be murine,
lagomorph, equine, ovine, or from a variety of other mammals.
Monoclonal antibodies will typically be murine in origin, produced
according to known techniques, or human, as described above, or
combinations thereof, as in chimeric or humanized antibodies. The
anti-islet cell antigen polypeptide receptor antibodies thus
obtained can then be administered to patients to reduce or
eliminate T cell subpopulations which recognize and participate in
the immunological destruction of islet cell antigen polypeptide
bearing cells in an individual predisposed to or already suffering
from a disease, such as IDDM. Further, the islet cell antigen
polypeptide T cell receptors can thus be identified, cloned and
sequenced, and receptor polypeptides synthesized which bind to the
islet cell antigen polypeptides and block recognition of the islet
cell antigen polypeptide-bearing cells, thereby impeding the
autoimmune response against host islet cells. Howell et al.
(Science 246: 668-70, 1989) have demonstrated that T cell receptor
peptides can block the formation of the tri-molecular complex
between T cells, autoantigen and major histocompatibilty complex in
an autoimmune disease model.
[0084] Antibodies and binding partners of the present invention may
be used in a variety of ways. The tissue distribution of the islet
cell antigen, for example, may be determined by incubating tissue
slices with a labeled monoclonal antibody which specifically binds
to the islet cell antigen polypeptides, followed by detection of
the presence of the bound antibody. Labels suitable for use within
the present invention are well known in the art and include, among
others, fluorescein, isothiocyanate, phycoerythrin, horseradish
peroxidase, and colloidal gold. The antibodies of the present
invention may also be used for the purification of the islet cell
antigen polypeptides of the present invention. The coupling of
antibodies to solid supports and their use in purification of
proteins is well known in the literature (see for example, Methods
in Molecular Biology, Vol. 1, Walker (Ed.), Humana Press, New
Jersey, 1984, which is incorporated by reference herein in its
entirety). Antibodies of the present invention may be used as a
marker reagent to detect the presence of islet cell antigen
polypeptides on cells or in solution. Such antibodies are also
useful for western analysis or immunoblotting, particularly of
purified cell secreted material. Polyclonal, affinity purified
polyclonal, monoclonal and single chain antibodies are suitable for
use in this regard. In addition, proteolytic and recombinant
fragments and epitope binding domains can be used herein. Chimeric,
humanized, veneered, CDR-replaced, reshaped or other recombinant
whole or partial antibodies are also suitable.
[0085] The following examples are offered by way of illustration,
not by way of limitation.
EXAMPLES
Example 1
Synthesis of Macaque Islet Cell cDNA and Preparation of a Macaque
Islet Cell cDNA Library
[0086] Islets of Langerhans (.about.100,000) were isolated by
collagenase digestion and Ficoll density gradient centrifugation
from pancreas of Macaca nemestrina (obtained from the University of
Washington Primate Center, Seattle, Wash.). These cells were then
flash frozen in liquid nitrogen and stored at -80.degree. C. until
use. Total RNA from the islets was isolated according to the method
of Chirgwin et al., Biochemistry 18: 52-94, 1994, incorporated
herein by reference, using polytron homogenization in guanidinium
thiocynate and LiCl centrifugation. Poly(A)+ RNA was isolated using
oligo d(T) cellulose chromatography (Aviv and Leder, Proc. Natl.
Acad. Sci. USA 69: 1408-12, 1972).
[0087] First strand cDNA was synthesized from two-time poly
d(T)-selected liver poly(A).sup.+RNA. Ten microliters of a solution
containing 10 .mu.g of liver poly(A).sup.+RNA was mixed with 2
.mu.l of 20 pmole/.mu.l first strand primer ZC3747 (SEQ ID NO:8)
and 4 .mu.l of diethylpyrocarbonate-tr- eated water. The mixture
was heated at 65.degree. C. for 4 minutes and cooled by chilling on
ice.
[0088] The first strand cDNA synthesis was initiated by the
addition of 8 .mu.l of 5.times. SUPERSCRIPT buffer (GIBCO BRL,
Gaithersburg, Md.), 4 .mu.l of 100 mM dithiothreitol, and 2.0 .mu.l
of a deoxynucleotide triphosphatate solution containing 10 mM each
of dATP, dGTP, dTTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology
Inc., Piscataway, N.J.) to the RNA-primer mixture. The reaction
mixture was incubated at 45.degree. C. for 4 minutes. After
incubation, 10.0 .mu.l of 200 U/.mu.l SUPERSCRIPT reverse
transcriptase (GIBCO BRL) was added. The efficiency of the first
strand synthesis was analyzed in a parallel reaction by the
addition of 10 .mu.Ci of .sup.32P-.alpha.dCTP to a 5 .mu.l aliquot
of the reaction mixture to label the reaction products. The first
strand synthesis reaction mixtures were incubated at 45.degree. C.
for 45 minutes followed by a 15 minute incubation at 50.degree. C.
Unincorporated nucleotides were removed from each reaction by
precipitating the cDNA in the presence of 8 .mu.g of glycogen
carrier, 2.5 M ammonium acetate and 2.5 volume ethanol. The
unlabeled cDNA was resuspended in 50 .mu.l water and used for the
second strand synthesis. The length of first strand cDNA was
assessed by resuspending the labeled cDNA in 20 .mu.l water and
determining the cDNA size by agarose gel electrophoresis.
[0089] Second strand synthesis was performed on the RNA-DNA hybrid
from the first strand synthesis reaction under conditions that
promoted first strand priming of second strand synthesis resulting
in DNA hairpin formation. A reaction mixture was prepared
containing 20.0 .mu.l of 5.times. polymerase I buffer (100 mM Tris,
pH 7.4, 500 mM KCl, 25 mM MgCl.sub.2, 50 MM
(NH.sub.4).sub.2SO.sub.4), 1.0 .mu.l of 100 mM dithiothreitol, 2.0
.mu.l of a solution containing 10 mM of each deoxynucleotide
triphosphate, 3.0 .mu.l 5 mM .alpha.-NAD, 1.0 .mu.l of 3 U/.mu.l E.
coli DNA ligase (New England Biolabs, Inc., Beverly, Mass.), 5.0
.mu.l of 10 U/.mu.l E. coli DNA polymerase (Gibco BRL) and 50.0
.mu.l of the unlabeled first strand DNA. A parallel reaction in
which a 10 .mu.l aliquot of the second strand synthesis was labeled
by the addition of 10 .mu.Ci of .sup.32P-.alpha.dCTP was used to
monitor the efficiency of second strand synthesis. The reaction
mixtures were incubated at room temperature for 5 minutes followed
by the addition of 1.5 .mu.l of 2 U/.mu.l RNase H (Gibco BRL) to
each reaction mixture. The reactions were incubated at 15.degree.
C. for 2 hours and 15 minutes, followed by a 15 minute incubation
at room temperature. The reactions were each terminated by serial
phenol/chloroform and chloroform/isoamylalcohol extractions. The
DNA from each reaction was precipitated in the presence of ethanol
and 2.5 M ammonium acetate. The DNA from the unlabeled reaction was
resuspended in 100 .mu.l water. The labeled DNA was resuspended and
electrophoresed as described above.
[0090] The single-stranded DNA in the hairpin structure was cleaved
using mung bean nuclease. The reaction mixture contained 20 .mu.l
of 10.times. Mung Bean Nuclease Buffer (Stratagene Cloning Systems,
La Jolla, Calif.), 16 .mu.l of 100 mM dithiothreitol, 54 .mu.l
water, 100 .mu.l of the second strand cDNA, and 10 .mu.l of a 1:10
dilution of Mung Bean Nuclease, final concentration 10.5 U/.mu.l
(Promega Corp., Madison, Wis.) in Stratagene MB dilution Buffer
(Stratagene Cloning Systems). The reaction was incubated at
37.degree. C. for 15 minutes, and the reaction was terminated by
the addition of 20 .mu.l of Tris-HCl, pH 8.0 followed by sequential
extractions with phenol/chloroform and chloroform/isoamylalcohol.
Following the extractions, the DNA was precipitated in ethanol and
resuspended in water.
[0091] The resuspended cDNA was blunt-ended with T4 DNA polymerase.
The cDNA, which was resuspended in a volume of 50 .mu.l of water,
was mixed with 50 .mu.l of 5.times. T4 DNA polymerase buffer. (250
mM Tris-HCl, pH 8.0, 250 mM KCl, 25 MM MgCl.sub.2), 3 .mu.l of 100
mM dithiothreitol, 3 .mu.l of a solution containing 10 mM of each
deoxynucleotide triphosphate, and 4 .mu.l of 1.0 U/.mu.l T4 DNA
polymerase (Boehringer Mannheim, Indianapolis, Ind.). After an
incubation at 10.degree. C. for 60 minutes, the reaction was
terminated by serial phenol/chloroform and
chloroform/isoamylalcohol extractions. The cDNA fragments less than
400 bp in length were removed by chromatography on a Clontech TE400
spin column (Clontech, Palo Alto, Calif.). The DNA was ethanol
precipitated and resuspended in 9 .mu.l of water. Based on the
incorporation of .sup.32P-dCTP, the yield of cDNA was estimated to
be 4 .mu.g from a starting mRNA template of 10 .mu.g.
[0092] Eco RI adapters (Pharmacia LKB Biotechnology Inc.,
Piscataway, N.J.) were added to the cDNA prepared above to
facilitate the cloning of the cDNA into a mammalian expression
vector. A 9 .mu.l aliquot of the cDNA and 975 pmole of the adapter
(15 .mu.l) were mixed with 3 .mu.l 10.times. ligase buffer (Promega
Corp.), 1 .mu.l 10 mM ATP, and 20 Units (2 .mu.l), of T4 DNA
ligase. (Promega Corp.). The reaction was incubated for 16 hours at
a temperature gradient of 4.degree. C. to 15.degree. C. The
reaction was terminated by the addition of 185 .mu.l water, 25
.mu.l REACT 2 buffer (Gibco BRL) followed by an incubation at
65.degree. C. for between 30 and 60 minutes. After incubation, the
reaction was terminated by serial phenol/chloroform and
chloroform/isoamylalcohol extractions and ethanol precipitation as
described above. Following centrifugation, the DNA pellet was
washed with 70% ethanol and was air dried. The pellet was
resuspended in 89 .mu.l of water.
[0093] To facilitate the directional insertion of the cDNA into a
mammalian expression vector, the cDNA was digested with Xho I,
resulting in a cDNA having a 5' Eco RI adhesive end and a 3' Xho I
adhesive end. The Xho I restriction site at the 3' end of the cDNA
was introduced through the ZC3747 primer (SEQ ID NO:8). The
restriction digestion was terminated by serial phenol/chloroform
and chloroform/isoamylalcohol extractions. The cDNA was ethanol
precipitated, and the resulting pellet was washed with 70% ethanol
and air-dried. The pellet was resuspended in 1.times. loading
buffer (10 mM phosphate buffer, pH 8.8, 5% glycerol, 0.125%
bromphenol blue).
[0094] The resuspended cDNA was heated to 65.degree. C. for 10
minutes, cooled on ice and electrophoresed on a 0.8% low melt
agarose gel (Seaplaque GTG Low Melt Agarose, FMC Corp., Rockland,
Me.) using a 1 Kb ladder (Gibco BRL) as size markers. The
contaminating adapters and by-product fragments below 600 bp in
size were excised from the gel. The electrodes were reversed, and
the cDNA was electrophoresed until concentrated near the lane
origin. The area of the gel containing the concentrated DNA was
excised, placed in a microfuge tube, and the approximate volume of
the gel slice was determined. An aliquot of TE (10 mM Tris HCl pH
7.4, 1 mM disodium ethylenediaminetetraacetate.2 H.sub.2O (EDTA))
equivalent to half the volume of the gel slice was added to the
tube, and the agarose was melted by heating to 65.degree. C. for
fifteen minutes. Following equilibration of the sample to
42.degree. C., approximately 5 units of .beta.-Agarase I (New
England Biolabs, Inc.) was added. The sample was incubated for 2
hours to digest the agarose. After incubation, a 0.1.times. volume
of 3M sodium acetate was added to the sample, and the mixture was
incubated on ice for fifteen minutes. After incubation, the sample
was centrifuged at 14,000.times.g for 10 minutes to remove the
undigested agarose. The cDNA in the supernatant was ethanol
precipitated. The cDNA pellet was washed with 70% ethanol, air
dried and resuspended in 37 .mu.l of water. The cDNA recovered from
the agarose gel was phosphorylated using T4 polynucleotide kinase.
The reaction consisted of 37 .mu.l cDNA, 5 .mu.l 10.times.
Stratagene Ligase Buffer (Stratagene Cloning Systems). Following a
5 minute incubation at 65.degree. C., the reaction was cooled to
room temperature where 5 .mu.l 10 mM ATP (Pharmacia) and 3 .mu.l T4
DNA polymerase (10 U/.mu.l, Stratagene) were added. The reaction
was incubated at 37.degree. C. for 45 minutes and at 65.degree. C.
for 10 minutes. The reaction was terminated by serial
phenol/chloroform extractions. The samples were chromatographed
through a Clontech TE400 spin column and were precipitated in the
presence of 2.5 M ammonium acetate. The cDNA was resuspended in 15
.mu.l of 2.5 mM Tris-HCl, pH 8.0, 0.25 mM EDTA.
[0095] The resulting Eco RI-Xho I cDNA library was cloned into the
E. coli vector pZCEP (Jelinek et al., Science 259: 1614-16, 1993).
Eco RI-Xho I linearized pZCEP was ligated with the Eco RI-Xho I
cDNA library. The resulting plasmids were electroporated into the
E. coli strain DH10B ELECTROMAX.about. (Gibco BRL). The library was
plated to obtain >5.times.10.sup.5 independent colonies and
aliquoted into 120 pools to give approximately 5,000 colonies per
pool. An aliquot of the cells from each pool was removed for use in
preparing plasmid DNA. The remaining cell mixtures were brought to
a final concentration of 15% glycerol, aliquoted and frozen at
-80.degree. C. Plasmid DNA was prepared from each pool and the
resulting plasmid DNA was digested with RNAse (Boehringer Mannheim)
according to the manufacturer's instructions. The RNAse reaction
was terminated by a phenol/chloroform/isoamylalcohol (24:24:1)
extraction, and the DNA was ethanol precipitated. The pools were
systematically screened as described in the examples below.
Example 2
Transfection of Macaque DNA into COS-7 Cells
[0096] Macaque DNA from each pool was transfected into COS-7 cells
(African Green Monkey Kidney cells, ATCC CRL 1651) using the method
essentially described by McMahan et al. (EMBO J. 10: 2821-32, 1991;
which is incorporated by reference herein in its entirety).
Briefly, one day prior to transfection approximately
2.times.10.sup.5 COS-7 cells in 2 ml growth medium containing 10%
fetal bovine serum (Dulbecco's modified Eagle's medium (DMEM), 1%
L-glutamine, 1% PNS antibiotic mix (Gibco BRL), 25 mM Hepes, and 1
mM NaPyruvate) were plated on sterile, single-chamber slides (Nunc
AS, Roskilde, Denmark) that had been coated with 10 .mu.g/ml of
human fibronectin in PBS for 30 minutes at 37.degree. C. and washed
with phosphate buffered saline (PBS). For each pool to be tested,
1-2 .mu.g of macaque islet cell library pooled DNA was added into
100 .mu.l of serum free medium (SFM, F/DV medium, 10 mg/l
transferrin, 2 .mu.g/l selenium, 10 mg/l fetuin, 5 mg/l insulin, 1
L-glutamine, 25 mM Hepes, 1 mM NaPyruvate, and 0.1 mM NEAA). To
each DNA sample was added 100 .mu.l SFM containing 12 .mu.l
LipofectAMINE.about. (Gibco BRL). The transfection solution was
mixed by pipetting up and down and kept at room temperature for 15
to 45 minutes. To each mix was added 0.8 ml SFM which was then
gently added to the COS-7 cells which had been washed once with
SFM. The cells were incubated at 37.degree. C., 5% CO.sub.2 for 4-5
hours. One milliliter of growth medium containing 20% FBS was added
to each slide. Slides were incubated overnight at 37.degree. C., 5%
CO.sub.2. The spent medium was removed and replaced with 2 ml
growth medium containing 10% FBS and the cells incubated for 24 to
48 hours, preferably 48 hours, at 37.degree. C., 5% CO.sub.2.
Example 3
Diabetic Sera
[0097] Sera from two prediabetic subjects, EmWi and JoGr, were
selected for screening the islet cell cDNA library. Sera from both
subjects were characterized for autoantibodies to known .beta.-cell
antigens using techniques known in the art. The sera were tested
for GAD65 autoantibodies using an in vitro
transcription/translation assay (Grubin et al., Diabetologia 37:
344-50, 1994) followed by immunoprecipitation using radiolabeled
recombinant human GAD65 according to Hagopian et al., J. Clin.
Invest. 91: 368-74, 1993.
[0098] Recombinant radiolabeled GAD was expressed in the presence
of .sup.35S Methionine (Amersham Corp., Arlington Heights, Ill.)
using the Sp6 bacteriophage promoter and the TNT reticulocyte
lysate kit (Promega), according to manufacturer's direction.
.sup.35S Methionine incorporation was determined by precipitation
using trichloroacetic acid (TCA), and 25% or more incorporation was
considered acceptable. Radiolabeled antigen was stored at
-80.degree. C. until use.
[0099] Radiolabeled antigen was diluted 1:10 in immunoprecipitation
buffer (150 mM NaCl, 1% v/v Triton X-114 (Sigma Chemical Co., St.
Louis, Mo.), 0.05% Bovine serum albumin (Sigma), 10 mM benzamidine
(Sigma), and 10 mM HEPES pH 7.4). The antigen was incubated for
preclearing for 4 hours at 4.degree. C. with 50 .mu.l normal human
serum. Immunoglobulin was removed using 200 .mu.l Protein A
Sepharose beads (Pharmacia LKB Biotechnology Inc.) for 45 minutes.
The cleared supernatant was diluted to 50,000 TCA-precipitable
counts per minute (cpm) per 400 .mu.l immunoprecipitation buffer.
Four microliters of serum from diabetic or control patients was
separately incubated in duplicate with 400 .mu.l diluted antigen at
4.degree. C. overnight with mixing by gentle rotation.
Antigen-antibody complexes were precipitated by 16 .mu.l Protein A
Sepharose, and the pellet was washed 5 times in ice-cold wash
buffer which consisted of 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05%
BSA, and 0.25% Triton X-114. Antigen was dissociated from the
pellet by boiling in the presence of 2% SDS and 5%
.beta.-mercaptoethanol, and counted by scintillation counting in
scintillation fluid. Counts per minute reflect the level of
autoantibodies present in the sera to capture the antigen.
[0100] Autoantibodies to the protein tyrosine phosphatase
IA-2/ICA512 were detected as above using a radiolabeled cytoplasmic
domain of human IA-2/ICA512 (Lan et al., DNA Cell Biology 13:
505-14, 1994; and Hagopian et al., Autoimmunity 21: 61, 1995). The
complete cytoplasmic domain of human IA-2 was isolated by RT-PCR
from U87MG glioblastoma cells (ATCC M85). Briefly, total RNA was
prepared from 5.times.10.sup.7 glioblastoma cells which were
homogenized in 3.5 ml guanidine/LiCl followed by CsCl
centrifugation. First strand cDNA was synthesized using a
Superscript.about. Preamplification System (GIBCO BRL) according to
the manufacturer's directions. One and one half microliters of a
solution containing 5 .mu.g total U87MG RNA was mixed with 1 .mu.l
oligo dT solution and 11.5 .mu.l diethylpyrocarbonate-treated
water. The mixture was heated at 70.degree. C. for 10 minutes and
cooled by chilling on ice.
[0101] First strand cDNA synthesis was initiated by the addition of
2 .mu.l Superscript.about. II buffer, 2 .mu.l 0.1 M dithiothreitol,
1 .mu.l deoxynucleotide triphosphate solution containing 10 mM each
of dATP, dGTP, dTTP, and dCTP, and 1 .mu.l of 200 U/.mu.l
Superscript.about. II reverse transcriptase to the RNA-primer
mixture. The reaction was incubated at room temperature for 10
minutes followed by an incubation at 42.degree. C. for 50 minutes,
then 70.degree. C. for 15 minutes, then cooled on ice. The reaction
was terminated by addition of 1 .mu.l RNase H which was incubated
at 37.degree. C. for 20 minutes, then cooled on ice.
[0102] A 100 .mu.l PCR reaction mixture was then prepared
containing 20 .mu.l of first strand template, 8 .mu.l 10.times.
synthesis buffer, 3.3 .mu.M ZC8802 (SEQ ID NO:9, contains 5' Xho I
site and ATG), 5.4 .mu.M ZC8803 (SEQ ID NO:10, contains Eco RI site
following stop codon), 65 .mu.l dH.sub.2O and 1 wax bead
(AmpliWax.about., Perkin-Elmer Cetus, Norwalk, Conn.). Following an
initial cycle of 95.degree. C. for 2 minutes, 4.degree. C. for 10
minutes, 5 U Taq polymerase was added, and the reaction was
amplified for 30 cycles of 1 minute at 95.degree. C., 2 minutes at
55.degree. C. and 3 minutes at 72.degree. C. The reaction mixture
was then stored at 4.degree. C. The resulting 1.2 kb fragment (SEQ.
ID. No.30) was digested with Eco RI-Xho I, treated with RNAse, then
isolated by low melt agarose gel electrophoresis and ligated into
Eco RI-Xho I linearized pZCEP. Sera were screened for IA-2/ICA512
autoantibodies as described above for GAD autoantibodies.
[0103] Both EmWi and JoGr sera showed reactivity to IA-2/ICA512.
The sera were titered for IA-2/ICA512 reactivity on vector only
transfected COS-7 cells using techniques known in the art, see for
example, Greenbaum et al. (Diabetes 41: 1570-1574, 1992). The sera
were separately adsorbed with porcine insulin (Hoechst, 10 mg/ml)
and GAD (1 mg/ml) until reactivity was abolished in the respective
antibody assays. These sera were then retitered for IA-2/ICA512 as
above. JoGr had IA-2/ICA512 reactivity of 280 JDFU (Juvenile
Diabetes Foundation Units) which persisted at >130 JDFU after
adsorption. EmWi had IA-2/ICA512 reactivity of 140 JDFU which
persisted at >130 JDFU after adsorption. EmWi had the lowest
background staining and was therefore used for primary
screening.
[0104] Twenty milliliters of EmWi was diluted 1:1 in 0.1 M
NaPO.sub.4 buffer, pH 8.0 and incubated with an equal volume of
Protein A covalently linked to Sepharose beads (Zymed, South San
Francisco, Calif.) for affinity purification. After gentle mixing
for 45 minutes at 4.degree. C., the slurry was loaded onto a column
and washed with 10 column volumes of 0.1 M NaPO.sub.4 buffer, pH
8.0 and one column volume of 0.01 M NaPO.sub.4 buffer, pH 8.0,
before elution of immunoglobulins with 0.05 M Na citrate buffer, pH
3.5. Eluted immunoglobulins were immediately neutralized to pH 7.0
with 2 M Tris, pH 8.0. Eluted fractions were evaluated by
spectrophotometric absorption at 280 nM, and peak fractions were
pooled, aliquoted and flash frozen for storage at -80.degree. C.
Typically the concentration was 4 mg/ml IgG. COS-7 cells were grown
to confluence in 150 ml T-flasks, washed with PBS, fixed in 4%
paraformaldehyde, and permeabilized by freeze/thaw. The pooled sera
were diluted to 1 mg/ml in PBS and incubated with the permeabilized
COS-7 cell lysate overnight at 4.degree. C. Supernatant was cleared
at 100,000.times.g and aliquotted for storage at -80.degree. C. for
use in the binding assay.
Example 4
Binding Assay
[0105] The macaque DNA transformed COS-7 cells on single chamber
slides, from Example 2, were prepared for assay by removing spent
medium from the slides and washing the cells 3 times in PBS at room
temperature. The cells were fixed with 1 ml 50% ETOH/50% acetone
for 5 minutes at room temperature followed by two washes in PBS and
two washes in 1% bovine serum albumin (BSA) in PBS. The precleared
serum (EmWi) was diluted to 0.2 mg/ml in a 5% BSA in PBS solution,
and 500 .mu.l was added to each of the slides which were then
covered, wrapped in plastic wrap, and rocked gently on a rocker
overnight at room temperature.
[0106] The slides were then washed three times in a 1% BSA/PBS
solution, three minutes for each wash. Following the final wash,
the slides were blocked for 10 minutes with 1 ml 5% BSA/4% normal
goat serum (Sigma) in PBS at room temperature. The blocking buffer
was removed, and 500 .mu.l of 0.02 mg/ml biotinylated Protein A
(Amersham Corp., Arlington Heights, Ill.) in 5% BSA/4% normal goat
serum/PBS was added, followed by a 30 minute incubation at room
temperature. The slides were washed three times with 1% BSA/PBS,
three minutes for each wash, then 500 .mu.l streptavidin-gold
(Amersham) diluted 1:50 in 5% BSA/4% normal goat serum/PBS was
added to each slide. Following a 60 minute incubation at room
temperature the slides were washed three times in 1% BSA/PBS and
one final time in PBS. The slides were then fixed by adding 0.5 ml
of 9% formaldehyde/45% acetone in PBS for 30 seconds followed by
three, 3 minute washes in dH.sub.2O.
[0107] An equal volume of silver enhancement solution and initiator
(IntenSE.about. M Silver Enhancement Kit, Amersham) were mixed in a
15 ml conical tube, and 0.5 ml was added to each slide. The slides
were allowed to develop for 20 minutes or until the desired color
intensity was achieved. The slides were then rinsed twice for five
minutes in dH.sub.2O and air dried. A single positive pool (#18)
containing approximately 5,000 clones was found out of
approximately 50 pools screened using EmWi sera.
[0108] To isolate the positive clone(s) from pool #18, one 150 mm
plate was plated to give approximately 10,000 colonies from the #18
pool. Filter lifts were prepared using the methods essentially
described by Hanahan and Meselson (Gene 10: 63, 1980) and Maniatis
et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y., 1982), which are incorporated herein by reference in their
entirety. The hybridization probe was obtained by PCR amplification
of plasmid DNA from pool #18. Briefly, an aliquot of the plasmid
DNA from pool #18 was subjected to PCR amplification using
oligonucleotides ZC8802 and ZC8803 (SEQ ID NOS:9 and 10,
respectively). A 50 .mu.l reaction mixture was prepared containing
0.05 .mu.g of the plasmid DNA from pool #18; 20 pmole of ZC 8802
and ZC 8803 (SEQ ID NOS:9 and 10, respectively); 10 nmoles of each
deoxynucleotide triphosphate (Pharmacia); 4 .mu.l 10.times.
synthesis buffer (Boehringer Mannheim), and 2.5 U Taq polymerase
(Boehringer Mannheim). The PCR reaction was run for 24 cycles (1
minute at 94.degree. C., 1 minute at 56.degree. C., and 1 minute at
72.degree. C.). An approximately 1.1 Kb band was isolated on a low
melt agarose gel electrophoresis and random primed using the
MEGAPRIME.about. Kit (Amersham) according to the manufacturer's
instructions.
[0109] The filter was hybridized in a solution containing 6.times.
SSC, 0.1% SDS, 5.times. Denhardt's, 200 .mu.g/ml denatured, sheared
salmon sperm DNA, and 1.times.10.sup.5 cpm/ml of .sup.32P-labeled
PCR fragment. The filter was hybridized overnight at 65.degree. C.
The excess label was removed by two, 15 minute washes with 2.times.
SSC, 0.1% SDS at 65.degree. C. The filter was exposed to film
overnight at -80.degree. C. with two screens.
[0110] Eighteen positive colonies were detected. Six of these
colonies were cultured and subjected to a second round of filter
lifts as described above, and from this two positive clones were
identified. Restriction endonuclease analysis showed that both
contained an approximate 2 Kb insert. One clone, designated
M1.18.5.1, was sequenced, revealing a 2,170 bp coding region which
contained regions of homology to the protein tyrosine phosphatase
family, especially IA-2/ICA512. Comparison of the full length human
protein tyrosine phosphatase IA-2/ICA512 with M1.18.5.1 suggests
that the coding region of M1.18.5.1 is missing amino terminal
sequence corresponding to approximately 400 amino acids. The
partial nucleic acid sequence and deduced amino acid sequence of
M1.18.5.1 is shown in SEQ ID NO 1 and SEQ ID NO: 2.
[0111] M1.18.5.1 was re-transfected into COS-7 cells and assayed as
described above. In addition to the EmWi sera, the JoGr sera, which
had a high titer to IA-2, was added to the screen and both detected
M1.18.5.1.
Example 5
Isolation of Human Islet Cell Antigen 1851
[0112] The 2,170 nucleotide sequence from M1.18.5.1 (SEQ ID NO 1)
was used to conduct a sequence search for a human homolog. A match
was found in the GenBank database (GenBank ID: TO361, clone ID:
HFBCV88) submitted by The Institute of Genomic Research,
Gaithersburg, Md., as an expressed sequence tag (EST) from a human
fetal brain library (Stratagene Cloning Systems). HFBCV88
(EST24415.seq), a 127 amino acid polypeptide, SEQ ID NO:5, had
homology to a region of the cytoplasmic domain of M1.18.5.1. The
closest human DNA sequence to HFBCV88 is HSICA512, islet cell
antigen ICA-512.
[0113] An oligonucleotide primer (ZC10,011 SEQ ID NO:11) was made
to a conserved region between 1851 and HFBCV88 which differed from
the corresponding sequence of mouse and human IA-2/ICA512 in that
an arginine was substituted for a methionine. Combined with a 128
fold degenerate primer (ZC10,019 SEQ ID NO:14, AARGCNACNGTNGAYAAY,
wherein R is A or G, N is A, C, T, or G, and Y is C or T) which
lies just upstream of the transmembrane domain, in the
extracellular domain, a portion of the human homologue of M1.18.5.1
was identified in human insulinoma cDNA by PCR. Briefly, a PCR
reaction was performed in a 100 .mu.l final volume using 12.5 ng
Marathon-ready human insulinoma cDNA prepared according to
manufacturer's instruction (Marathon.about. cDNA Amplification Kit,
Clontech), 20 pmoles each of primers ZC 10,011 (SEQ ID NO:11) and
ZC 10,019 (SEQ ID NO:14), and the reagents provided in the
Marathon.about. PCR kit (Clontech) according to the manufacturer's
instructions. The reaction was amplified for 30 cycles (1 minute at
94.degree. C., 30 seconds at 60.degree. C., 5 minutes at 68.degree.
C.) followed by a 10 minute extension at 72.degree. C. An 800 bp
(WK11111, SEQ ID NO:32) and a 1,200 bp (WK121315, SEQ ID NO:34)
fragment were isolated by low melt agarose gel electrophoresis.
[0114] A 3'RACE Marathon PCR was also performed in a 50 .mu.l final
volume using 12.5 ng Marathon-ready human insulinoma cDNA, 10
pmoles each of primers ZC 10,177 (SEQ ID NO:12) the complement to
ZC 10,011, and AP-1 (adaptor primer, supplied with kit), and the
reagents provided in the 3'RACE Marathon.about. PCR kit (Clontech),
according to the manufacturer's instructions. The reaction was
amplified for 30 cycles (30 seconds at 94.degree. C., 30 seconds at
68.degree. C.). A 900 bp and a 2,000 bp (WK121111, SEQ ID NO:33)
fragment were isolated by low melt agarose electrophoresis.
[0115] The 800 bp, (SEQ ID NO:32) 1,200 bp, (SEQ ID NO:34) and
2,000 bp (SEQ ID NO:33) PCR fragments were independently subcloned
into pCR1 (Invitrogen Inc., San Diego, Calif.), using the TA
Cloning Kit (Invitrogen Inc.) according to the manufacturer's
instructions. The resulting plasmids (11.1.1, 11.1.2, and 11.1.3,
respectfully) were used to transform E. coli XL-1 cells.
Transformants were screened for presence of insert, followed by
sequencing of the insert.
Example 6
Detection of Human Islet Cell Antigen Autoantibodies
[0116] An approximately 1.1 kb (SEQ ID NO:6) Eco RI-Hind III
cytoplasmic fragment of human islet cell antigen 1851 cDNA was
inserted into the vector pcDNAII (Invitrogen, San Diego, Calif.),
and designated IL1851-3. The resultant polypeptide was transcribed
and translated in vitro using a TNT Coupled. Reticulocyte Lysate
System (Promega), according the manufacturer's instructions.
[0117] The labeled, synthesized cytoplasmic portion of human islet
cell antigen 1851 was used to screen diabetic sera from six
patients, for the presence of autoantibodies. Protein A-Sepharose
immunoprecipitation, as described above, showed that sera from all
six reacted positively with the in vitro synthesized, human islet
cell antigen, and indicated that the major autoepitope is likely
present on this polypeptide.
[0118] Additional immunoprecipitation assays were performed with a
spectrum of serum samples, including 91 healthy control sera
(median age 22 years, range 1-49 years, 49% males and 51% females);
183 newly diagnosed IDDM patients sampled at onset (median age 11
years, 51% males and 49% females); and 60 first degree relatives of
type I diabetic patients sampled a mean of 2.0 years before onset
(median age 12 years, 58% males and 42% females). Parallel
autoantibody assays used the intracellular domain of IA-2/ICA512.
Immunoprecipitation assays were as described above. Briefly, 4
.mu.l of serum from diabetic or control patients were separately
incubated in duplicate with 400 .mu.l .sup.35S radiolabeled antigen
(cytoplasmic portion of human islet cell antigen 1851, SEQ ID NO:
6, in immunoprecipitation buffer (10 mM Hepes, 0.05% BSA, 150 mM
NaCl, 10 mM benzamidine, and 1% Triton X114)) at 4.degree. C.
overnight with mixing by gentle rotation (Hagopian et al., J.
Clinc. Invest. 91:368-74, 1995). Antigen-antibody complexes were
precipitated using 20 .mu.l Protein A Sepharose, and the pellet was
washed 3 times in ice-cold wash buffer (which consisted of 10 mM
HEPES pH 7.4, 150 mM NaCl, 0.25% BSA, and 0.25% Triton X-114) and
one cold water wash. Antigen was dissociated from the pellet by
boiling in the presence of 2% SDS and 5% .beta.-mercaptoethanol,
counted by scintillation counting in scintillation fluid, and the
results expressed as islet cell antigen 1851 index (Hagopian et
al., Diabetes 42:631-36, 1993). Counts per minute reflect the level
of autoantibodies present in the sera that can capture the antigen.
Assay cutoff was an index of 0.04, determined as the mean +3
standard deviations of 91 control sera. Assay sensitivity,
specificity, and positive predictive value were calculated
(Hagopian et al., ibid., 1995).
[0119] Immunoprecipitation assays revealed autoantibodies in 56/183
(30.6%) newly diagnosed IDDM patients, 28/60 (46.7%) first degree
relatives later progressing to clinical diabetes, but only 1/91
(1.1%) healthy control subject groups. For first degree relatives,
this represents a positive predictive value of 58% and a
sensitivity of 48%.
[0120] Of sera from 153 newly diagnosed patients, 83 (54%)
recognized IC-2/ICA512 and 48 (31%) recognized islet cell antigen
1851. Only 1/48 (2%) from the sera recognizing islet cell antigen
1851 did not precipitate IA-2/ICA512, but 35/83 (42%) from the sera
reactive with IA-2/ICA512 did not bind islet cell antigen 1851. Of
those positive for both antigens, reactivity to IA-2/ICA512 was
generally stronger than that to islet cell antigen 1851.
[0121] The intracellular domains of human islet cell antigen 1851
and IA-2/ICA512 were expressed and radiolabeled by in vitro
transcription and translation using a TNT Coupled Reticulocyte
Lysate System (Promega), according the manufacturer's instructions,
as described above. SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) and autoradiography of the resulting radiolabeled
polypeptide revealed, for human islet cell antigen 1851, a major
band of 46 kD and a minor band at 33 kD, both immunoprecipitated by
IDDM sera. Limited trypsin digest of the radiolabeled
immunoprecipitated intracellular fragment of macaque and human
islet cell antigen 1851 and IA-2/ICA512 was done using the method
of Christie et al. (J. Exp. Med. 172:789-94, 1990), followed by
SDS-PAGE and autoradiography, which revealed a 37 kD product from
both macaque and human islet cell antigen 1851. This product was
distinct from the 40 kD product produced by limited trypinization
of the intracellular domain of IA-2/ICA512.
[0122] In order to test whether IA-2/ICA512 autoantibodies
recognized only epitopes shared with islet cell antigen 1851, the
intracellular domain of IA-2/ICA512 was expressed in baby hamster
kidney cells (BHK cells). The 1.2 kb IA-2/ICA512 intracellular
fragment (SEQ ID NO:30) from Example 3 was ligated into pZEM219b
under the SV40 promoter (Busby et al., J. Biol. Chem. 266:15286-92,
1991) and cellular expression was determined by immunocytochemistry
using rabbit polyclonal antiserum to IA-2/ICA512 (Rabin et al., J.
Immunol. 152:3183-88, 1994). IA-2/ICA512-transfected BHK cells were
homogenized in homogenization buffer (0.25% Triton X-114, 10 mM
benzamidine). Using Western blotting, the concentration of
recombinant intracellular IA-2/ICA512 was estimated at 7 .mu.g/ml
of cell extract.
[0123] Immunoprecipitation assays, as described above, were done
using radiolabeled islet cell antigen 1851 in the presence of 0.5
.mu.g of unlabeled IA-2/ICA512 per microliter of islet cell antigen
1851 positive sera, as a competitor. Islet cell antigen 1851
autoantibodies not fully blocked by this amount of IA-2/ICA512 were
subjected to repeated immunoprecipitation assays using a 2.5 fold
increase of unlabeled IA-2/ICA512 as a competitor. As a control,
extracts from non-transfected BHK cells were used. Recombinant
intracellular IA-2/ICA512 fully blocked islet cell antigen 1851
reactivity in 29/53 islet cell antigen 1851 positive sera, while a
median of 21.4% (range 3%-55%) of original immunoreactivity was
retained in 24/53 sera. Increasing the IA-2/ICA512 concentration
did not reduce this residual immunoreactivity, suggesting that
unique islet cell antigen 1851 epitopes are being recognized in
certain sera.
Example 7
Cloning the Remaining 5' Sequence of Macaque and Human Islet Cell
Antigen 1851 cDNA
[0124] To obtain the remaining 5' macaque cDNA sequence one pool
(#12) from the macaque library described in Example 1 was plated at
10,000 colonies/150 mm plate. Filter lifts were prepared (Maniatis
et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y., 1982) and denatured with 0.5 M NaOH for four minutes,
neutralized with 1 M Tris pH 8.0 for 2 minutes followed by
renaturation with 1 M Tris pH 8.0/1.5 M NaCl for 2 minutes. Filters
were cross linked in a UV Stratalinker (1200 .mu.J) (Stratagene
Cloning Systems, La Jolla, Calif.). The filters were prehybridized
in 20 ml hybridization buffer (6.times. SSC, 0.5% SDS, 5.times.
Denhardts and 0.2 mg/ml boiled salmon sperm DNA) overnight at
65.degree. C. The filters were then hybridized in 20 ml
hybridization buffer containing 1.times.10.sup.6 cpm/ml
.sup..gamma.32P-ATP labeled hybridization probe (ZC10504 SEQ ID
NO:18) overnight at 65.degree. C. The labeled hybridization probe
was prepared by adding to a 5 .mu.l final volume 30 pmol oligo
ZC10504 (SEQ ID NO:18), T4 polynucleotide kinase buffer, 37.5 pmol
.sup..gamma.32P-ATP and 10 U T4 polynucleotide kinase The reaction
was incubated for 1 hour at room temperature and unincorporated ATP
was removed using a Stratagene push column according the
manufacturer's instructions (Stratagene Cloning Systems, La Jolla,
Calif.). Following the hybridization, excess unbound label was
removed from the filters with eight washes in 2.times. SSC/0.1 SDS
(2 times with 20 ml, 5 times with 30 ml and a final wash in 100 ml)
for 5 to 10 minutes at 65.degree. C. The filters were exposed to
film overnight at -80.degree. C.
[0125] Several positive colonies were detected. One of these
colonies was cultured from a replica plated colony and subjected to
sequence analysis. The clone, 12.10504.1, contained 2,736 bp coding
region (SEQ ID NO:23), containing the cytoplasmic and transmembrane
domains and extending the 5' end of the macaque extracellular
domain sequence (SEQ ID NO:1) by 609 bp.
[0126] 5' RACE PCR was used to generate the remaining 5' cDNA
fragments of macaque islet cell antigen 1851. To a 50 .mu.l final
volume was added 5 pmol of a vector-specific oligonucleotide primer
(ZC11197, SEQ ID NO:29), 5 pmol of a macaque specific primer
(ZC11654, SEQ ID NO:28), 1 ng macaque islet cell cDNA library from
Example 1, 40 mM dNTPs, TAQ Polymerase buffer and 1.25 U TAQ
Polymerase. A one minute denaturation at 94.degree. C. was followed
by 30 amplification cycles (30 seconds at 94.degree. C., 1 minute
at 60.degree. C., 2 minutes at 72.degree. C.) followed by a 6
minute extension at 72.degree. C.).
[0127] Four independent 5'RACE PCR reactions were run, each using a
different pool from the macaque library as template. Four fragments
were obtained, a 738 bp fragment (SEQ. ID. No. 24) extending the 5'
end by 246 bp; a 932 bp fragment (SEQ ID NO:25) extending the 5'
end by 193 bp; a 999 bp fragment (SEQ ID NO:26) extending the 5'
end by 68 bp and a 1011 bp fragment (SEQ ID NO:27) which contained
the remaining 5' sequence with the exception of the start
methionine. The fragments were isolated by agarose electrophoresis,
excised and separated from the agarose using the Qiagen Qiaquick
Gel Extraction System (Qiagen, Inc., Chatsworth, Calif.) according
to manufacturer's instruction. The fragments were subcloned into
pGEM-T (Promega Corp., Madison, Wis.), using the TA Cloning Kit
(Promega Corp.) according to the manufacturer's instructions. The
resulting plasmids pJML8, 7, 9 and 10 respectfully, were used to
transform E. coli DH10B cells. Transformants were screened for
presence of insert, followed by sequencing of the insert.
[0128] The 5' RACE fragments (SEQ ID Nos:23, 24, 25, 26 and 27)
contain overlapping segments and were aligned with the macaque
islet cell antigen 1851 sequence of SEQ ID NO:1 to give a full
length macaque islet cell antigen 1851 DNA sequence as represented
in SEQ ID NO:15. Comparison of the human protein tyrosine
phosphatase IA-2/ICA512 cDNA and amino acid sequences with those of
the macaque islet cell antigen 1851 cDNA and amino acid sequences
(SEQ ID NOs: 15 and 16) suggests that the coding region is missing
the start methionine.
[0129] A vector containing the full length macaque sequence can be
created using PCR. The macaque 5' RACE fragments (SEQ ID NOs: 23,
24, 25, 26 and 27) can be joined using PCR. A clone shown to
possess the complete coding sequence can then be digested with
convenient restriction sites and subcloned into a vector of choice.
Clones can be screened for correct insertion of the full length
sequence and subjected to DNA sequence analysis.
[0130] PCR using macaque derived primers was done to identify
remaining 5' cDNA sequence for the human islet cell antigen 1851
(SEQ ID NO:6). To a 50 .mu.l final volume was added 5 pmol each of
two gene-specific oligonucleotide primers ZC10504, SEQ ID NO:18 and
ZC11653, SEQ ID NO:17, 1 ng Marathon-ready insulinoma cDNA,
prepared according to manufacturer's instruction (Marathon.about.
cDNA Amplification Kit, Clontech), 40 mM dNTPs, TAQ Polymerase
buffer and 1.25 U TAQ Polymerase. The reaction was denatured at
94.degree. C. for one minute, amplified for 30 cycles (30 seconds
at 94.degree. C., 1 minute at 63.degree. C., 2 minutes at
72.degree. C.), followed by a 6 minute extension at 72.degree.
C.).
[0131] A 1263 bp fragment (SEQ ID NO:31) was isolated by agarose
electrophoresis. The isolated fragment was then excised and
subcloned into pGEM-T using the TA Cloning Kit (Promega, Corp.), as
described above. The clones were then analyzed for the presence of
insert, and those containing insert were subjected to DNA sequence
analysis. The human islet cell antigen 1851 fragments can be joined
using PCR to give the human sequence as represented in SEQ ID
NO:21. Clones can be screened for correct insertion of the
fragments and subjected to DNA sequence analysis. Comparison of the
human protein tyrosine phosphatase IA-2/ICA512 cDNA with that of
the human islet cell antigen 1851 sequences (SEQ ID NO:21 and 22)
suggests that the coding region is missing 5' sequence
corresponding to approximately 600 bp. Including the 3'
untranslated region, but not the 5' untranslated region, the
estimated mRNA size for the human sequence is 5 kb, which is
consistent with the 5.5 kb mRNA observed in Northern blots
discussed below. To obtain the remaining 5' human islet cell
antigen 1851 cDNA sequence, additional PCR or 5' RACE PCR reactions
can be performed as described above.
Example 8
Tissue Distribution
[0132] Human Multiple Tissue Northern Blots (MTN I, MTN II, and MTN
III; Clontech, Palo Alto, Calif.) were probed to determine the
tissue distribution of human islet cell antigen 1851 expression. A
38 nucleotide oligonucleotide sequence just external to the
transmembrane region of human islet cell antigen 1851, which is
distinct from the corresponding sequence of IA-2/ICA512 (SEQ ID
NO:18) was radioactively labeled with .gamma..sup.32P using a T4
nucleotide kinase (GIBCO BRL, Gaithersburg, Md.) according to the
manufacturer's specifications. ExpressHyb.about. (Clontech)
solution was used for prehybridization and as a hybridizing
solution for the Northern blots. Hybridization took place overnight
at 37.degree. C. using 5.times.10.sup.6 cpm/ml of labeled probe.
The blots were then washed three times at room temperature, once at
50.degree. C. for 30 minutes, once at 60.degree. C., in 6.times.
SSC, 0.1% SDS. A final wash at 68.degree. C. with 2.times. SSC,
0.05% SDS for 20 minutes was done prior to autoradiography. Two
transcript sizes were detected. A strong 5.5 kb band and a weaker
3.3 kb band were detected in brain, pancreas and prostate, with
lesser signals in spinal cord, thyroid, adrenal and GI tract. With
the exception of prostate, this represents the expected
neuroendocrine distribution.
[0133] In order to define tissue localization further, in situ
hybridization was performed on macaque pancreas, adrenal gland and
muscle. The 38 nucleotide islet cell antigen 1851 oligonucleotide
(SEQ ID NO:18), a 38 bp IC-2/ICA512 oligonucleotide (SEQ ID NO:19)
and a 30 bp insulin .beta.-chain probe for pancreatic islets
(Petersen et al., Diabetes 42:484-95, 1993) (SEQ ID NO:20) were
end-labeled with .sup.33P-dATP (New England Nuclear, Boston, Mass.)
using terminal deoxytransferase (GIBCO BRL) according to
manufacturer's instructions. Frozen sections (14 .mu.m) from
macaque pancreas, adrenal, pituitary and muscle were fixed in 4%
paraformaldehyde, followed by acetylation with acetic anhydride and
then delipidated in chloroform prior to use. Labeled probes (2
pmol/ml) were incubated on the sections overnight and then washed
in two changes of 1.times. SSC at 60.degree. C. for 30 minutes,
followed by dehydration in ethanol and apposition to
autoradiography film (Hyperfilm Betamax, Amersham Corp., Arlington
Heights, Ill.) for 2 to 6 days. The slides were then coated with
NTB2 Track emulsion (Eastman Kodak, Rochester, N.Y.) and exposed
for 12-18 days before development and counterstain with cresyl
violet. Images were captured using a Dage 72 CCD camera and a MCID
M2 imaging system (Imaging Research, Ontario, Canada). Strong
hybridization was detected in pancreatic islets and adrenal medulla
but not in muscle. The IA-2/ICA512 and the insulin .beta. chain
probes hybridized to islets.
[0134] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 0
0
* * * * *