Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-07-11
2004-01-27
Ewoldt, Gerald R. (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S810000, C435S975000, C436S506000, C436S800000, C436S804000, C436S807000, C436S808000
Reexamination Certificate
active
06682897
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel peptides being epitopes of the human 60 KDa heat shock protein (hsp60) and to pharmaceutical compositions comprising them for the diagnosis and treatment of insulin-dependent diabetes mellitus (IDDM).
BACKGROUND OF THE INVENTION
Type I diabetes, or IDDM, is an autoimmune disease caused by T cells that attack and destroy the insulin-producing &bgr;-cells located in the islets of the pancreas (Castano and Eisenbarth, 1990). The autoimmune process culminating in IDDM begins and progresses without symptoms. The disease surfaces clinically only when the cumulative loss of &bgr;-cells exceeds the capacity of the residual &bgr;-cells to supply insulin. Indeed, the collapse of glucose homeostasis and clinical IDDM is thought to occur only after 80-90% of the &bgr;-cells have been inactivated by the immune system. Thus, patients who can be identified as suffering from IDDM are bound to be in an advanced stage of autoimmune destruction of their &bgr;-cells. Moreover, diagnosis of incipient, pre-clinical diabetes by the detection of immunological markers of &bgr;-cell autoimmunity can be made only after the onset of the autoimmune process. Therefore, the therapeutic quest is to find a safe, specific and effective way to turn off an autoimmune process that is already well underway.
The present inventors have examined this question before by studying the spontaneous diabetes developing in mice of the NOD strain, which is considered to be a faithful model of human IDDM (Castano and Eisenbarth, 1990). NOD mice develop insulitis around 4 weeks of age, which begins as a mild peri-islet infiltrate and progresses to severe intra-islet inflammation. Hyperglycemia, which attests to insulin insufficiency, begins in the females in our colony at about 14-17 weeks of age. Bad 35-40 weeks of age, almost all the female NOD mice have developed severe diabetes and most die in the absence of insulin treatment. Male NOD mice have a lower incidence of disease, for reasons that are not clear. The diabetes of NOD mice has been shown to be caused by autoimmune T cells (Bendelac et al., 1987).
T cell reactivity and autoantibodies to various antigens have been detected in human IDDM patients as well as in NOD mice (Elias, 1994), and it is not clear whether immunity to any single one of the possible target antigens is the primary cause of the disease. Beyond the question of causation is the question of therapy.
It has been demonstrated that the initiation of the autoimmune process in NOD mice can be prevented by subjecting the mice, before the onset of diabetes, to various manipulations such as restricted diet, viral infections, or non-specific stimulation of the immune system (Bowman et al., 1994). NOD diabetes is also preventable by induction of immunological tolerance in pre-diabetic mice to the antigen glutamic acid decarboxylase (Kaufman et al., 1993; Tisch et al., 1993).
Insulin dependent diabetes mellitus (IDDM) developing spontaneously in NOD female mice has been associated with immune reactivity to a variety of self-antigens (Bach, 1994). Notable among these antigens is the p277 peptide from the sequence of the mammalian 60 kDa heat shock protein (hsp60) molecule. This corresponds to residues 437-460 in the human hsp60 molecule (Elias et al 1991, Israel Patent Application No. 94241, PCT patent publication WO90/10449). The human p277 peptide has the following sequence:
Val-Leu-Gly-Gly-Gly-Cys-Ala-Leu-Leu-Arg-Cys-Ile-Pro-Ala-Leu-Asp-Ser-Leu-Thr-Pro-Ala-Asn-Glu-Asp (a.a. 437-460 of SEQ ID NO:1).
Pre-diabetic NOD mice manifest spontaneous, diabetogenic T cell responses to hsp60 and to the human (2) or mouse variants of the p277 peptide (3). The mouse and human peptides differ by 1 amino acid and are immunologicaly cross-reactive (3). Some non-diadetes prone strains of mice, such as C57BL/6, develop transient hyperglycemia and insulitis when immunized to p277 covalently conjugated to a foreign immunogenic carrier molecule (4). And mice of the C57BL/KsJ strain develop spontaneous T-cell responses to hsp60 and to p277 after treatment with a very low dose of the &bgr;-cell toxin streptozotocin (STZ) that induces autoimmune diabetes (5).
In addition to being involved in the expression of the disease, peptide p277 appears to be functional in healing the autoimmune process: Subcutaneous administration of p277 in incomplete Freund adjuvant (IFA; mineral oil) led to arrest of disease progression in young NOD mice (2) or in 12-17 week old NOD mice with advanced insulitis (6, 7). Both the human (6, 7) and mouse (3) variants of p277 were effective. NOD mice transgenic for the mouse hsp60 gene on an MHC class II promoter showed down-regulation of their spontaneous T-cell proliferative response to p277 and a significant proportion of the mice were spared the development of diabetes (8). Moreover, administration of p277 to C57BL/KsJ mice aborted the development of autoimmune diabetes in mice that had received earlier a very low dose of STZ; treatment of these mice with a peptide of the GAD65 molecule was not effective (9).
Variants of the p277 peptide in which one or both cysteine residues at positions 6 and 11 were replaced by valine residues, designated as p277(Val
6
), p277(Val
11
) and p277(Val
6
-Val
11
), respectively, were described in corresponding Israel Patent Application No. 112094, and shown to be as active as p277 in the treatment of diabetes.
It is an object of the present invention to provide additional peptides of human hsp60, such peptides being useful for diagnosis and treatment of IDDM.
SUMMARY OF THE INVENTION
In a study of fragments and peptides of the human hsp60 molecule, it was unexpectedly found that IDDM patients and NOD mice are responsive to other hsp60 T-cell epitopes that may be used for diagnosis and therapy of IDDM. These epitopes, by themselves or in conjunction with p277 or a p277 variant selected from p277(Val
6
), p277(Val
11
) and p277(Val
6
-Val
11
), may improve the efficacy of the treatment.
These new peptides are identified in Table 1.
TABLE 1
Hsp60 Synthetic Peptides and Their Sequence
Residue nos.
Amino acid sequence
Peptides
of SEQ ID NO:1
(one letter code)
p3
31-50
KFGADARALMLQGVDLLADA
p10
136-155
NPVEIRRGVMLAVDAVIAEL
p11
151-170
VIAELKKQSKPVTTPEEIAQ
p12
166-185
EEIAQVATISANGDKEIGNI
p14
195-214
RKGVITVKDGKTLNDELEII
p18
255-274
QSIVPALEIANAHRKPLVIIA
p20
286-305
LVLNRLKVGLQVVAVKAPGF
p24
346-365
GEVIVTKDDAMLLKGKGDKA
p29
421-440
VTDALNATRAAVEEGIVLGG
p30
436-455
IVLGGGCALLRCIPALDSLT
p32
466-485
EIIKRTLKIPAMTIAKNAGV
p35
511-530
VNMVEKGIIDPTKVVRTALL
p39
343-366
GKVGEVIVTKDDAM
Other peptides of hsp60, SEQ ID NO:1, including those designated p278 (corresponding to positions 458-474 of SEQ ID NO:1 in the human hsp60 sequence), p19 (corresponding to positions 271-290 of SEQ ID NO:1 in the human hsp60 sequence), and p21 (corresponding to positions 301-320 of SEQ ID NO:1 in the human hsp60 sequence were shown not to be as effective. It is noted that the amino terminus of p278 overlaps with the effective p277 peptide by three residues (NED) and the carboxy terminus of p278 overlaps with the effective p32 peptide by residues (EIIKRTLKI). Thus the remaining 11 residues of p32 are critical (PAMTIAKNAGV).
The present invention thus relates to the peptides identified in Table 1, and salts and functional derivatives thereof.
It is further an object of the present invention to provide methods and kits for the early diagnosis of IDDM using the peptides of the invention. In the course of developing IDDM, animals express hsp60 molecules, or molecules which are cross-reactive therewith, which find their way into the blood and urine of the animals. They also express antibodies and T cells directed specifically to such molecules. Thus, the presence of hsp60 (or molecules which are cross-reactive therewith) or antibodies or T cells specific thereto in blood or urine, serves as an assay for the detection of the IDDM process before the destruction of beta cells is completed and the individual is doomed to life-long diabetes.
The presence or incipi
Abulafia Rivka
Bockova Jana
Cohen Irun R.
Elias Dana
Ewoldt Gerald R.
Winston & Strawn LLP
Yeda Research and Development Co. Ltd.
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