U.S. patent application number 10/321717 was filed with the patent office on 2004-01-01 for detection and treatment methods for type i diabetes.
This patent application is currently assigned to Trustees of Dartmouth College. Invention is credited to Griffin, Ann C., Hickey, William F..
Application Number | 20040002113 10/321717 |
Document ID | / |
Family ID | 23038902 |
Filed Date | 2004-01-01 |
United States Patent
Application |
20040002113 |
Kind Code |
A1 |
Griffin, Ann C. ; et
al. |
January 1, 2004 |
Detection and treatment methods for type I diabetes
Abstract
Proinsulin peptide compounds that modulate an immunological
response by T cells of Type I diabetic subjects are disclosed. The
proinsulin peptide compounds of the invention are preferably
derived from a region of proinsulin that spans the junction between
the B chain and C peptide of proinsulin. Pharmaceutical
compositions comprising the proinsulin peptide compounds are also
disclosed. An immunological response to a proinsulin peptide
compound of the invention can be used as an indicator of Type I
diabetes in a subject. Accordingly, the invention provides
diagnostic assays for Type I diabetes using the proinsulin peptide
compounds. Methods for inhibiting the development or progression of
Type I diabetes in a subject by administering a proinsulin peptide
compound are also disclosed.
Inventors: |
Griffin, Ann C.; (Hanover,
NH) ; Hickey, William F.; (Lyme, NH) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Trustees of Dartmouth
College
|
Family ID: |
23038902 |
Appl. No.: |
10/321717 |
Filed: |
December 17, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10321717 |
Dec 17, 2002 |
|
|
|
08472701 |
Jun 6, 1995 |
|
|
|
6509165 |
|
|
|
|
08472701 |
Jun 6, 1995 |
|
|
|
08272220 |
Jul 8, 1994 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
530/326; 530/327; 530/328 |
Current CPC
Class: |
C07K 14/70539 20130101;
C07K 14/62 20130101; A61P 27/02 20180101; A61P 15/00 20180101; A61P
3/08 20180101; A61P 9/00 20180101; A61K 38/00 20130101; G01N
2333/62 20130101; A61P 17/00 20180101; Y10S 530/868 20130101; A61P
27/16 20180101; A61P 7/06 20180101; G01N 33/5091 20130101; A61P
29/00 20180101; Y10S 530/806 20130101; A61P 1/00 20180101; A61P
13/02 20180101; A61P 37/00 20180101 |
Class at
Publication: |
435/7.1 ;
530/326; 530/327; 530/328 |
International
Class: |
G01N 033/53; C07K
007/08 |
Goverment Interests
[0002] Work described herein was supported at least in part under
grants RO1-NS-27321 and T32-AI07363 awarded by the National
Institutes of Health. The U.S. government therefore may have
certain rights in this invention.
Claims
1. A method for detecting an indicator of Type I diabetes in a
subject, comprising a) obtaining a biological sample from the
subject; b) contacting the sample with a proinsulin peptide
compound which stimulates an immunological response by T cells of
Type I diabetic subjects; and c) detecting an immunological
activity in the sample against the proinsulin peptide compound as
an indicator of Type I diabetes in the subject.
2. The method of claim 1, wherein the compound is identical or
substantially similar to a region of proinsulin that spans the
junction between the B chain and the C peptide of proinsulin.
3. The method of claim 1, wherein the compound is derived from
human proinsulin.
4. The method of claim 1, wherein the compound comprises an amino
acid sequence comprising the formula:
8 Y.sub.1-(Xaa)n-(Ser/Thr)-Pro-Lys-(Ser/Thr)-Arg-Arg-
(Glu/Asp)-(Zaa)m-Y.sub.2
wherein Xaa and Zaa, which may or may not be present, represent
amino acid residues, n and m are integers from 1 to 15, Y.sub.1 is
hydrogen or an amino-derivative group and Y.sub.2 is hydrogen or a
carboxy-derivative group.
5. The method of claim 4, wherein the compound is 10-35 amino acids
in length.
6. The method of claim 4, wherein the compound is 10-20 amino acids
in length.
7. The method of claim 4, wherein the compound is 12-17 amino acids
in length.
8. The method of claim 4, wherein the compound comprises an amino
acid sequence:
9 Y.sub.1-Gly-Phe-Phe-Tyr-(Ser/Thr)-Pro-Lys-(Ser/Thr)-
Arg-Arg-(Glu/Asp)-Ala-Glu-(Glu/Asp)-Leu-Gln-Val- Gly-Y.sub.2
9. The method of claim 1, wherein the detected immunological
activity is a T cell response to the proinsulin peptide
compound.
10. The method of claim 9, wherein the T cell response is
proliferation.
11. The method of claim 9, wherein the T cell response is cytokine
production.
12. The method of claim 1, wherein the detected immunological
activity is antibody binding to the proinsulin peptide
compound.
13. A method for inhibiting the development or progression of Type
I diabetes in subject, comprising administering to the subject a
proinsulin peptide compound which modulates an immunological
response by T cells of Type I diabetic subjects.
14. The method of claim 13, wherein the compound is identical or
substantially similar to a region of proinsulin that spans the
junction between the B chain and the C peptide of proinsulin.
15. The method of claim 13, wherein the agent is a compound derived
from human proinsulin.
16. The method of claim 13, wherein the compound comprises an amino
acid sequence comprising the formula:
10 Y.sub.1-(Xaa)n-(Ser/Thr)-Pro-Lys-(Ser/Thr)-Arg-Arg-
(Glu/Asp)-(Zaa)m-Y.sub.2
wherein Xaa and Zaa, which may or may not be present, represent
amino acid residues, n and m are integers from 1 to 15, Y.sub.1 is
hydrogen or an amino-derivative group and Y.sub.2 is hydrogen or a
carboxy-derivative group.
17. The method of claim 16, wherein the compound is 10-35 amino
acids in length.
18. The method of claim 16, wherein the compound is 10-20 amino
acids in length.
19. The method of claim 16, wherein the compound is 12-17 amino
acids in length.
20. The method of claim 16, wherein the compound comprises an amino
acid sequence:
11 Y.sub.1-Gly-Phe-Phe-Tyr-(Ser/Thr)-Pro-Lys-(Ser/Thr)-
Arg-Arg-(Glu/Asp)-Ala-Glu-(Glu/Asp)-Leu-Gln-Val- Gly-Y.sub.2
21. The method of claim 13, wherein a tolerogenic amount of the
compound is administered to the subject.
22. The method of claim 14, wherein the compound comprises a
modified form of a proinsulin peptide that spans the junction
between the B chain and the C peptide of proinsulin.
23. The method of claim 13, wherein the compound is administered in
a pharmaceutically acceptable carrier.
24. The method of claim 13, wherein the compound is administered
intravenously.
25. The method of claim 13, wherein the compound is administered
subcutaneously.
26. The method of claim 13, wherein the compound is administered
orally.
27. The method of claim 13, wherein the compound is administered
intramuscularly intraperitoneally.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/272,220, filed Jul. 8, 1994, pending, the
entire contents of which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] Type I, or insulin-dependent, diabetes mellitus (also
referred to herein as DM-I) is known to occur spontaneously in
humans, rats and mice (Castao, L and Eisenbarth, G. (1990) Ann.
Rev. Immunol. 8:647-679). There is a genetic susceptibility to DM-I
associated with certain haplotypes of Class II antigens of the
major histocompatability complex (MHC), i.e., HLA-DR3, -DR4 and
-DQ3.2 in humans (see e.g., Platz. P. et al. (1981) Diabetologia
21:108-115; Todd, J. et al. (1987) Nature 329:599-604); RT1.sup.u
in Bio-Breeding (BB) rats (see e.g., Colle, E. (1990) Clin.
Immunol. & Immunopathol. 57:1-9; Parfrey, N. A. et al. (1989)
Crit. Rev. Immunol. 9:45-65) and H-2.sup.g7 in non-obese diabetic
(NOD) mice (see e.g., Kikutani, H. and Makino, S. in Adv. Immunol.
(Dixon, F. J., ed.), pp. 285-323, New York, N.Y.: Academic Press,
Inc., 1992). The pathology of DM-I consists of the progressive
inflammatory infiltration of pancreatic islets (i.e., insulitis)
containing immunocytes targeted specifically to insulin-secreting
.beta.-cells (see e.g., Bottazzo, G. F. et al. (1985) N. Eng. J.
Med. 313:353-360; Foulis, A. K. et al. (1991) J. Pathol.
165:97-103; Hanenberg, H. et al. (1991) Diabetologia 32:126-134).
This pathology develops over an indeterminate period of time
(months to years).
[0004] It has become clear that the development of Type I diabetes
occurs as a result of a complex relationship involving genetic
predisposition, environmental influences, and additional undefined
co-factors. In attempting to understand the pathogenesis of DM-I,
the most elusive pieces of information have been the definition of
the inciting autoantigen(s), and whether cellular or
humoral-mediated autoreactivity is the primary event. Serum
autoantibodies directed against islet cell cytoplasm and surface
antigens (i.e., ICA, ICSA), insulin (IAA) and glutamic acid
decarboxylase (GAD) can be found in pre-diabetic and newly
diagnosed diabetic humans and animals (see e.g., Rowley, M. et al.
(1992) Diabetes 41:548-551; Palmer, J. et al. (1983) Science
222:1337-9; MacLaren, N. et al. (1988) Diabetes 38:534-538;
Baekkeskov, S. et al. (1990) Nature 347:151-156; Velleso, L. A. et
al. (1993) Diabetologia 36:39-46). Unfortunately, correlations
between antibody titers against these antigens and the clinical
onset of diabetes have not been successfully predictive. In
addition, a number of other .beta.-cell antigens become exposed as
islets are destroyed such as a 69 kd islet cell autoantigen (ICA69)
(Pietropaolo, M. et al. (1993) J. Clin. Invest. 92:359-371), 38 kd
and 62 kd insulin secretory granule proteins (Roep, B. O. et al.
(1991) Lancet 337:1439-1441; Brudzynski, K. et al. (1992) J.
Autoimm. 5:453-463) and proislets (Harrison, L. C. et al. (1992) J.
Clin. Invest. 89:1161-65; Harrison, L. et al. in Advances in
Endocrinology & Metabolism (Mazzaferri, E. L. et al., ed.), pp.
35-94, St. Louis, Mo.: Mosby-Year Book, 1990). However, it is not
clear whether the cellular and/or humoral immune responses to these
antigens are the cause or simply a consequence of ongoing islet
cell damage. In short, the immunologic nature of the pathogenic
mechanism and the exact antigen(s) inducing the diabetogenic attack
have yet to be elucidated.
[0005] Over one half million people in the United States suffer
from insulin-dependent diabetes. Prior to 1921, people who
developed DM-I were not expected to live much more than a year
after diagnosis. Afflicted individuals suffered from clinical signs
of chronic hyperglycemia (e.g., excessive thirst and urination,
rapid weight loss) as a consequence of abnormal carbohydrate
metabolism. Once insulin was purified and administered, the
life-expectancy of diabetics increased dramatically. However, DM-I
is a chronic disease that requires life-long treatment to prevent
acute illness and to reduce the risk of long-term complications.
Restrictive diets and daily insulin injections can be burdensome
for patients, thus reducing compliance, and even with treatment
complications such as cataracts, retinopathy, glaucoma, renal
disease and circulatory disease are prevalent.
[0006] Accordingly, more effective treatments for Type I diabetes
are needed, in particular therapies that address the autoimmune
basis of the disease, rather than merely treating the symptoms.
Additionally, given that the "pre-diabetic" phase of DM-I is long
in duration and clinically asymptomatic, one important opportunity
for therapeutic intervention falls during this period. However,
effective diagnostic assays that can identify people in this
pre-diabetic phase are lacking. Methods that would enable
identification of early or mounting .beta.-cell abnormalities in
individuals predisposed to diabetes are needed and would allow
treatment early in the disease process, which may help to avert
life-long insulin dependence.
SUMMARY OF THE INVENTION
[0007] This invention pertains to proinsulin peptide compounds
which modulate an immunological response by T cells of Type I
diabetic subjects. In one embodiment, a proinsulin peptide compound
of the invention stimulates an immunological response by the T
cells. For example, humans with DM-I have greater numbers of
circulating T cells which respond to a specific proinsulin peptide
described herein than do non-diabetic control humans. Accordingly,
a subject's immunological responsiveness to a stimulatory
proinsulin peptide compound can be used as an indicator of DM-I. In
another embodiment, a proinsulin peptide compound of the invention
inhibits an immunological response by T cells of Type I diabetic
subjects. The invention further provides therapeutic and
preventative methods involving the use of the proinsulin peptide
compounds of the invention to inhibit or prevent T cell
responsiveness to proinsulin in Type I diabetic subjects.
[0008] In a preferred embodiment, the proinsulin peptide compound
that modulates an immunological response from T cells of Type I
diabetic subjects is derived from a region of proinsulin that spans
the junction between the B chain and the C peptide of proinsulin.
In another embodiment, the proinsulin peptide compound is a
modified form of a proinsulin peptide derived from this region.
Such modified forms include peptides that have amino acid
substitutions compared to the native proinsulin amino acid sequence
yet retain certain structural and functional features of the native
peptide. Other modified forms of the proinsulin peptide compounds
within the scope of the invention include peptides with
end-terminal or side chain covalent modifications and peptide
analogs and mimetics. Such modified proinsulin peptides can be
selected for altered properties of the peptide, e.g., stability,
solubility, immunogenicity, etc. The proinsulin peptide compounds
of the invention can be incorporated into pharmaceutical
compositions suitable for administration to a subject.
[0009] Another aspect of the invention pertain to a method for
detecting an indicator of Type I diabetes in a subject by detecting
an immunological activity against a proinsulin peptide compound of
the invention in a biological sample from the subjects.
[0010] Yet another aspect of the invention pertains to a method for
inhibiting the development or progression of Type I diabetes in a
subject by administering to the subject a proinsulin peptide
compound which modulates an immunological response by T cells of
Type I diabetic subjects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic diagram of the location of synthetic
peptide encompassing the MHC class II (RT1.B/D) binding motif
relative to the structure of proinsulin prior to enzymatic cleavage
into insulin (A+B chains linked by disulfide bonds) and C-peptide.
The binding motif spans amino acids of the B and C chains in
proinsulin containing one pair of basic (Arg-Arg) residues cleaved
by a site-specific endoprotease during processing.
[0012] FIGS. 2A-C are graphic representations of the proliferation
of T cell lines specific for GAD.sub.412 (panel A), GAD.sub.520
(panel B) and PI (panel C) peptides in the presence or absence of
anti-MHC class II antibody (for reference, mean cpm for GAD.sub.412
T cells incubated with APCs and medium=850.+-.297;
GAD.sub.520=74.+-.30; and PI=1763.+-.181).
[0013] FIG. 3 is a flow cytometric profile of PI peptide-specific T
cell line expression of cell surface antigens following 72 hour
stimulation with concanavalin A. Histograms and percent positive
expression for each primary antibody (in parentheses) were
generated by FACScan analysis of 10,000 cells.
DETAILED DESCRIPTION OF THE INVENTION
[0014] This invention pertains to proinsulin peptide compounds
which modulate an immunological response by T cells of Type I
diabetic subjects and to the use of such compounds to detect and
treat DM-I. (The terms Type I diabetes, insulin-dependent diabetes
and DM-I are used interchangeably throughout the application.) The
invention is based, at least on part, on the discovery that T cells
specific for a proinsulin peptide of the invention are present in
the circulation of humans with DM-I at a much greater frequency
than in the circulation of non-diabetic controls. Additionally, T
cell clones specific for a proinsulin peptide of the invention can
cause diabetes when transferred to naive, genetically appropriate
animals. These data are consistent with the proinsulin region from
which the peptide is derived being a specific autoantigen attacked
during the generation of DM-I. Accordingly, methods related to the
diagnosis, therapy and prevention of DM-I using the proinsulin
peptide compounds are provided.
[0015] I Proinsulin Peptide Compounds
[0016] One aspect of the invention pertains to proinsulin peptide
compounds which modulate an immunological response from T cells of
Type I diabetic subjects. The language "proinsulin peptide
compound" as used herein is intended to include peptides derived
from proinsulin (i.e., peptides having the amino acid sequence of a
region of native proinsulin) as well as modified forms of such
peptides. These modified forms include peptides having amino acid
substitutions compared to the native proinsulin sequence but which
retain certain structural and functional characteristics, peptides
having covalent modifications (e.g., end terminal or side chain
modifications), peptide analogs and mimetics and peptides derived
from other proteins that are homologous to proinsulin within the
region from which the peptide is derived.
[0017] The proinsulin peptide compounds of the invention are
capable of modulating an immunological response from T cells of
Type I diabetic subjects. The language "modulation" is intended to
include either stimulation or inhibition of immunological
responses. Accordingly, in various embodiments of the invention,
the peptide compound can be a "stimulatory peptide compound" (i.e.,
a compound that stimulates an immunological response from T cells
of Type I diabetic subjects) or an "inhibitory peptide compound"
(i.e., a compound that inhibits an immunological response from T
cells of Type I diabetic subjects). The language "inhibitory
peptide compound" is intended to include peptides that inhibit T
cell responses to a native proinsulin peptide (or other similar
stimulatory peptide compounds).
[0018] A schematic diagram of unprocessed (i.e., immature) and
processed (i.e., mature) forms of insulin is shown in FIG. 1. As
used herein, the language "proinsulin" refers to the immature form
of insulin that includes the amino acid residues of the B chain, C
peptide and A chain linked contiguously from the amino terminus to
the carboxy terminus of the protein (amino acid residues 25 to 110
in FIG. 1). Upon normal processing, proinsulin is cleaved at the
junction between the B chain and the C peptide, and between the
C-peptide and the A chain to generate mature B chain and A chain.
The language "preproinsulin" refers to the immature form of insulin
that, in addition to the proinsulin sequences, includes a leader
sequence linked contiguously to the amino-terminus of the B chain
(amino acid residues 1-24 in FIG. 1). Upon normal processing of
preproinsulin, the leader sequence is cleaved from the B chain.
Since the entire coding sequence of proinsulin is contained within
preproinsulin, it will be appreciated that peptides described
herein as being derived from a particular region of proinsulin may
also be derived from the equivalent region of preproinsulin.
[0019] Preferred proinsulin peptide compounds of the invention are
derived from a region of proinsulin that spans the junction between
the B chain and the C peptide of proinsulin. A peptide derived from
such a region is illustrated schematically in FIG. 1 (labeled
"synthetic PI peptide"). The complete nucleotide and amino acid
sequences of human preproinsulin are shown in SEQ ID NOs: 1 and 2,
respectively (see also Bell, G. et al. (1979) Nature 282:525-527;
Sures, I. et al. (1980) Science 208:57-59). Using the amino acid
sequence numbering of SEQ ID NO: 2, the B chain of human proinsulin
corresponds to amino acid residues 25-54 and the C-peptide
corresponds to amino acid residues 57-87 (the dipeptide of residues
55-56 is cleaved from proinsulin during processing). Accordingly,
as used herein, a region of proinsulin that "spans the junction
between the B chain and the C peptide of proinsulin" refers to a
region encompassing amino acid residues of both the B chain and C
peptide, including at least the junctional residues 30-33. A
preferred region spanning this junction encompasses amino acid
residues from about position 47 to about position 63.
[0020] In one embodiment of the invention, the proinsulin peptide
compound that modulates an immunological response from T cells of
Type I diabetic subjects is a "stimulatory proinsulin peptide
compound". The language a "stimulatory proinsulin peptide compound"
is intended to include peptide compounds that stimulate T cell
responses, such as T cell proliferation and/or cytokine production.
Preferably, the stimulatory proinsulin peptide compound is
identical to a region of proinsulin that spans the junction between
the B chain and the C peptide of proinsulin, as described above. A
particularly preferred stimulatory peptide compound is derived from
human proinsulin. In one embodiment, the peptide comprises an amino
acid sequence:
1
Y.sub.1-(Xaa)n-(Ser/Thr)-Pro-Lys-(Ser/Thr)-Arg-Arg-(Glu/Asp)-(Zaa-
)m-Y.sub.2 (SEQ ID NO: 3)
[0021] wherein (Xaa) and (Zaa), which may or may not be present,
represent amino acid residues, n and m are integers from 1 to 15,
Y.sub.1 is hydrogen or an amino-derivative group and Y.sub.2 is
hydrogen or a carboxy-derivative group. Preferably, the peptide is
about 10-35 amino acids in length (i.e., n+m=3 to 28). More
preferably, the peptide is about 10-20 amino acids in length (i.e.,
n+m=3 to 13). Even more preferably, the peptide is about 12-17
amino acids in length (i.e., n+m=5 to 10).
[0022] In a particularly preferred embodiment, the stimulatory
peptide compound derived from human proinsulin comprises an amino
acid sequence:
Y.sub.1-Gly-Phe-Phe-Tyr-(Ser/Thr)-Pro-Lys-(Ser/Thr)-Arg-Arg-(Glu/Asp)-Ala-
-Glu-(Glu/Asp)-Leu-Gln-Val-Gly-Y.sub.2 (SEQ ID NO: 4),
corresponding to amino acid residues 47 to 64 of human
preproinsulin as shown in SEQ ID NO: 2.
[0023] In addition to peptide compounds derived from human
proinsulin, stimulatory proinsulin peptide compounds from the
equivalent region of proinsulin from other species are encompassed
by the invention. For example, a proinsulin peptide from the
equivalent region (e.g., residues 47-63) of rat proinsulin I
comprises an amino acid sequence:
Gly-Phe-Phe-Tyr-(Ser/Thr)-Pro-Lys-(Ser/Thr)-Arg-Arg-(Glu/Asp)-Val-Glu-(Gl-
u/Asp)-Pro-Gln-Val (SEQ ID NO: 5). A stimulatory proinsulin peptide
from the equivalent region of rat proinsulin II comprises an amino
acid sequence:
Gly-Phe-Phe-Tyr-(Ser/Thr)-Pro-Met-(Ser/Thr)-Arg-Arg-(Glu/Asp)-V-
al-Glu-(Glu/Asp)-Pro-Gln-Val (SEQ ID NO: 6). (The complete
nucleotide and amino sequences of the rat preproinsulin I and II
genes are disclosed in Lomedico, P. et al. (1979) Cell 18:545-558).
The amino acid sequences of proinsulins of other species are also
known in the art and can be used to design similar stimulatory
proinsulin peptides identical to regions spanning the B chain and
C-peptide junction of proinsulin (e.g., Perler, F. et al. (1980)
Cell 20:555-566 disclose the sequence of the chicken preproinsulin
gene; Watt V. M. et al. (1985) J. Biol. Chem. 260: 10926-29
disclose the sequence of the guinea pig preproinsulin gene).
[0024] Proinsulin peptide compounds of the invention can be
prepared by any suitable method for peptide synthesis, including
chemical synthesis and recombinant DNA technology. Preferably, the
peptides are chemically synthesized. Methods for chemically
synthesizing peptides are well known in the art (see e.g.,
Bodansky, M. Principles of Peptide Synthesis, Springer Verlag,
Berlin (1993) and Grant, G. A (ed.). Synthetic Peptides: A User's
Guide, W. H. Freeman and Company, New York (1992). Automated
peptide synthesizers are commercially available (e.g., Advanced
Chem Tech Model 396; Milligen/Biosearch 9600). Methods for
preparing peptides by recombinant expression in a host cell of DNA
encoding the peptide are also well known in the art (see e.g.,
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Laboratory Press).
[0025] In addition to proinsulin peptide compounds having an amino
acid sequence that is identical to a particular region of native
proinsulin (e.g., preferably the region spanning the B chain-C
peptide junction of proinsulin), the invention also encompasses
proinsulin peptide compounds that are "substantially similar" to a
native region of proinsulin. Peptide compounds that are
substantially similar to a native region of proinsulin may be
stimulatory (i.e., compounds that stimulate an immunological
response by T cells of Type I diabetic subjects) or, alternatively,
inhibitory (i.e., compounds that inhibit an immunological response
by T cells of a Type I diabetic subjects). Peptide compounds
described herein as being "substantially similar" to a particular
region of proinsulin (e.g., the region spanning the B chain-C
peptide junction of proinsulin) include peptides that retain
certain structural and functional features of the native peptide
yet differ from the native proinsulin amino acid sequence within
the particular region at one or more amino acid position (i.e., by
amino acid substitutions). For example, a stimulatory peptide that
is substantially similar to a native proinsulin peptide retains the
ability to stimulate T cell responses by T cells of Type I diabetic
subjects, whereas an inhibitory peptide that is substantially
similar to a native proinsulin peptide retains the ability to bind
to major histocompatibility complex (MHC) molecules but lacks the
ability to stimulate T cell responses by T cells of Type I diabetic
subjects.
[0026] It is well accepted in the art that antigenic peptides are
composed essentially of three categories of amino acid positions:
1) positions necessary for interaction of the peptide with MHC
molecules (referred to herein as "MHC contact residues"), 2)
positions necessary for interaction of the peptides with the T cell
receptor (TCR) complex to thereby stimulate T cell activation
(referred to herein as "TCR contact residues") and 3) "neutral"
positions that are not critical either for MHC contact or TCR
contact (see e.g., Rothbard, J. B. and Gefter, M. L. (1991) Ann.
Rev. Immunol. 9:527-565; Jorgensen, J. L. et al. (1992) Ann. Rev.
Immunol. 10:835-873; Rothbard, J. B. and Taylor, W. R. (1988) EMBO
J. 7:93-100; DeLisi C. and Berzofsky, J. (1985) Proc. Natl. Acad.
Sci. USA 82:7048; Berzofsky, J. et al. (1988) in Immunological
Reviews, pp. 5-31, Copenhagen, Denmark: Munksgaard; Margalit, H. et
al. (1987) J. Immunol. 138:2213-2229; O'Sullivan, D. et al. (1990)
J. Immunol. 145:1799-1808; Corr, M et al. (1993) J. Exp. Med.
178:1877-1892; Falk, K. et al. (1994) Immunogenetics 39:230-242;
Sidney, J. et al. (1994) J. Immunol. 152:4516-4525; Chicz, R. M. et
al. (1993) J. Exp. Med. 178:27-47; Kropshofer, H. et al. (1992) J.
Exp. Med. 175:1799-1803).
[0027] Accordingly, a stimulatory peptide compound substantially
similar to a native stimulatory proinsulin peptide can be selected
that has amino acid substitutions at one or more neutral position
but retains the critical MHC contact residues and TCR contact
residues such that the peptide retains both the capacity to bind
MHC molecules and the capacity to stimulate T cell responses.
Additionally or alternatively, the stimulatory peptide compound may
have amino acid substitutions at one or more positions involved in
MHC contact and/or TCR contact as long as the substitutions do not
alter the ability of the peptide to stimulate T cell responses
(e.g., conservative amino acid substitutions at the MHC and/or TCR
contact positions may be tolerated).
[0028] In contrast to the above-described modified stimulatory
peptide compounds, an inhibitory peptide compound substantially
similar to a native proinsulin peptide can be selected that retains
the capacity to bind to MHC molecules but lacks the capacity to
stimulate an immunological response by T cells of Type I diabetic
subjects. Thus, these inhibitory peptide compounds have amino acid
substitutions at critical TCR contact residues such that the
peptide cannot stimulate T cell responses but retains the critical
MHC contact residues (or has tolerated conservative amino acid
substitutions at the critical MHC contact positions) such that the
peptide can still bind MHC molecules. The inhibitory peptide
compounds may also have substitutions at neutral residues.
[0029] Stimulatory or inhibitory peptides altered from the native
proinsulin sequence can be prepared by substituting amino acid
residues within a native proinsulin peptide and selecting peptides
with the desired stimulatory or inhibitory activity. For example,
amino acid residues of the proinsulin peptide can be systematically
substituted with other residues and the substituted peptides can
then be tested in standard assays for evaluating the effects of
such substitutions on immunological responses. Typically, to retain
functional activity, conservative amino acid substitutions are
made. As used herein, the language a "conservative amino acid
substitution" is intended to include a substitution in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art, including basic side
chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
.beta.-branched side chains (e.g., threonine, valine, isoleucine)
and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine). Other generally preferred substitutions
involve replacement of an amino acid residue with another reside
having a small side chain, such as alanine or glycine. For example,
a panel of proinsulin peptide analogs of the stimulatory rat
proinsulin peptide shown in SEQ ID NO: 5 is synthesized to contain
alanine substitutions throughout the peptide, such as the panel of
peptides shown below:
2 AAFYAPKSRREVEDPAA (SEQ ID NO: 7) AAFYTAKSRREVEDPAA (SEQ ID NO: 8)
AAFYTPASRREVEDPAA (SEQ ID NO: 9) AAFYTPKARREVEDPAA (SEQ ID NO: 10)
AAFYTPKSAREVEDPAA (SEQ ID NO: 11) AAFYTPKSRAEVEDPAA (SEQ ID NO: 12)
AAFYTPKSRRAVEDPAA (SEQ ID NO: 13) AAPYTPKSRREAEDPAA (SEQ ID NO: 14)
AAFYTPKSRREVADPAA (SEQ ID NO: 15) AAFYTPKSRREVEAPAA (SEQ ID NO:
16)
[0030] Studies with other antigenic peptides have indicated that
substitutions of non-critical amino acid residues with alanine
residues are well tolerated (e.g., the peptide retains the ability
to bind MHC molecules; see Jardetzky et al. (1990) EMBO J.
9:1797-1803). Amino acid substituted peptides can be prepared by
standard techniques, such as automated chemical synthesis, as
described further above.
[0031] The effect of the amino acid substitutions on the ability of
the peptide to bind MHC molecules and/or stimulate T cell responses
is tested in standard assays for evaluating immunological
responses. Preferably, proinsulin-specific T cell clones or
hybridomas are used in these assays. T cell clones can be prepared
as described in Examples 1 and 3. T cell hybridomas can be prepared
from the T cell clones by standard methods, for example by fusing a
proinsulin peptide-specific T cell clone with a
TcR.alpha..beta..sup.- thymoma (such as the BW5147 cell line) using
polyethylene glycol. Antigen specificity of the proinsulin-specific
T cells is monitored by routine stimulation with proinsulin peptide
and assay of antigen-specific, interleukin-2 (IL-2) production. In
a standard assay for IL-2 production, culture supernatants are
assayed for the presence of IL-2 in a bioassay using an IL-2
dependent cell line, such as HT-2 cells. The supernatant is added
to the HT-2 cells for 24 hours, followed by an additional 12-14
hour period of incubation in the presence of .sup.3H-thymidine
(.sup.3H-TdR), after which the cells are harvested and .sup.3H-TdR
uptake is measured. Other suitable assays for IL-2, such as an
enzyme linked immunosorbent assay, are well known in the art.
[0032] In one assay for evaluating amino acid-substituted peptides,
the ability of the substituted peptides to directly stimulate a
proinsulin peptide-specific T cell clones and/or hybridomas is
assessed. A proinsulin-specific T cell clone and/or hybridoma is
tested for stimulation by the panel of proinsulin peptide analogs
with amino acid substitutions, typically in a range of
concentrations between 0.5-20 .mu.g/ml. Stimulatory peptide
compounds that retain the ability to activate the
proinsulin-specific T cell clones and/or hybridoma are selected
based on their ability to stimulate IL-2 production by the clone or
hybridoma, as described above.
[0033] In another assay for evaluating amino acid-substituted
peptides, the ability of the substituted peptides to compete with
the wild-type proinsulin peptide for binding to an MHC molecule on
the surface of an antigen presenting cell is assessed. Each
substituted proinsulin peptide analog is pre-incubated with
irradiated antigen presenting cells (e.g., for 2 hours) prior to
the addition of wild-type proinsulin peptide and a
proinsulin-specific T cell clone or hybridoma Supernatants are
harvested after an appropriate period of time (e.g., 24 hours) and
assayed for the presence of IL-2, as described above. A competitive
inhibitor is identified by its ability to inhibit IL-2 production
by the T cell clone or hybridoma that is normally induced by the
stimulatory proinsulin peptide. Moreover, an inhibitory peptide
compound can be selected based upon the combined results of the
direct stimulation assay and the competitive inhibition assay, for
example, an inhibitory peptide compound is selected that lacks the
ability to directly stimulate T cell responses but retains the
ability to competitively inhibit T cell responses against the
native proinsulin peptide. The ability of wild-type and substituted
peptides to bind to MHC molecules also can be directly assessed
using labeled peptides and purified MHC molecules in binding assays
(e.g., equilibrium dialysis, column binding assays etc., for
example as described in Sette, A. (1987) Nature 328:395-399).
[0034] Based upon animal studies (described further in Examples 1
and 2), a predicted MHC binding motif of the preferred proinsulin
peptide compounds of the invention comprises an amino acid
sequence: (Ser/Thr)-Xaa-Xaa-Xaa-Xaa-Xaa-(Glu/Asp) (SEQ ID NO: 17),
wherein Xaa represent any amino acid. The language "MHC binding
motif" refers to a pattern of amino acid residues present within a
peptide (or region of a whole protein) that allow the peptide to
bind to the antigenic binding site of an MHC molecule. That is, the
binding motif for a particular MHC molecule defines the critical
amino acid residues of a peptide fragment that are necessary for
binding of the fragment to the MHC molecule. The region of
proinsulin spanning the B chain-C peptide junction (e.g., amino
acid residues 47-63), from which preferred proinsulin peptides of
the invention are derived, includes two of these predicted MHC
binding motifs: Thr51-Glu57 and Ser54-Asp60. The involvement of
these residues in MHC binding can be directly evaluated by
preparing a panel of proinsulin peptides containing amino acid
substitutions (e.g., alanine substitutions) at these positions. An
additional peptide composed entirely of alanines except for Thr51,
Ser54, Glu57 and Asp 60 can also be tested. Thus, an example of a
panel of peptides for evaluating the MHC binding motif of the
proinsulin peptides is listed below:
3 GFFYAPKSRRAVEDPQV (SEQ ID NO: 18) GFFYTPKARREVEAPQV (SEQ ID NO:
19) AAAATAASAAEAADAAA (SEQ ID NO: 20)
[0035] The activity of these peptides can be assessed in the direct
T cell stimulation assay and/or the competitive inhibition assay,
described above, to evaluate the involvement of the
(Ser/Thr)-(Glu/Asp) motif in the MHC binding ability of the
peptides.
[0036] In addition to amino acid-substituted proinsulin peptides,
the invention also encompasses proinsulin peptide compounds having
other modifications. For example, the amino-terminus or
carboxy-terminus of the peptide can be modified. The language
"amino-derivative group" (e.g., Y.sub.1 in the formula presented
above) is intended to include amino-terminal modifications of the
peptide compounds of the invention. Examples of N-terminal
modifications include alkyl, cycloalkyl, aryl, arylalkyl, and acyl
groups. A preferred N-terminal modification is acetylation. The
N-terminal residue may be linked to a variety of moieties other
than amino acids such as polyethylene glycols (such as
tetraethylene glycol carboxylic acid monomethyl ether),
pyroglutamic acid, succinoyl, methoxy succinoyl, benzoyl,
phenylacetyl, 2-, 3-, or 4-pyridylalkanoyl, aroyl, alkanoyl
(including acetyl and cycloalkanoyl e.g., cyclohexylpropanoyl),
arylakanoyl, arylaminocarbonyl, alkylaminocarbonyl,
cycloalkylaminocarbonyl, alkyloxycarbonyl (carbamate caps), and
cycloalkoxycarbonyl, among others.
[0037] The language "carboxy-derivative group" (e.g., Y.sub.2 in
the formula presented above) is intended to include
carboxy-terminal modifications of the peptide compounds of the
invention. Examples of modifications of the C-terminus include
modification of the carbonyl carbon of the C-terminal residue to
form a carboxyterminal amide or alcohol (i.e., as reduced form). In
general, the amide nitrogen, covalently bound to the carbonyl
carbon on the C-terminal residue, will have two substitution
groups, each of which can be hydrogen, alkyl or an alkylaryl group
(substituted or unsubstituted). Preferably the C-terminal is an
amido group, such as --CONH.sub.2, --CONHCH.sub.3,
--CONHCH.sub.2C.sub.6H.sub.5 or --CON(CH.sub.3).sub.2, but may also
be 2-, 3-, or 4-pyridylmethyl, 2-, 3-, or 4-pyridylethyl,
carboxylic acid, ethers, carbonyl esters, alkyl, arylalkyl, aryl,
cyclohexylamide, piperidineamide and other mono or disubstituted
amides. Other moieties that can be linked to the C-terminal residue
include piperidine-4-carboxylic acid or amide and cis- or trans-
4-amino-cyclohexanecarboxylic acid or amide.
[0038] Moreover, modification of one or more side chains of
non-critical amino acid residues (e.g., "neutral" residues) may be
tolerated without altering the function of the peptide. A covalent
modification of an amino acid side chain or terminal residue may be
introduced into the peptide by reacting targeted amino acid
residues of the peptide with an organic derivative agent that is
capable of reacting with selected side chains or terminal residues.
Examples of typical side chain modifications are described further
below:
[0039] Cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole.
[0040] Histidyl residues are derivatized by reaction with
diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively
specific for the histidyl side chain. Parabromophenacyl bromide
also is useful; the reaction is preferably performed in 0.1M sodium
cacodylate at pH 6.0.
[0041] Lysinyl and amino terminal residues are reacted with
succinic or other carboxylic acid anhydrides. Derivatization with
these agents has the effect of reversing the charge of the lysinyl
residues. Other suitable reagents for derivatizing
.alpha.-amino-containing residues include imodoesters such as
methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride; trinitobenzenesulfonic acid; O-methylisourea;
2,4-pentanedione; and transaminase-catalyzed reaction with
glyoxylate.
[0042] Arginyl residues are modified by reaction with one or
several conventional reagents, among them phenylglyoxal,
2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
Derivatization of arginine residues requires that the reaction be
performed in alkaline conditions because of the high pK.sub.a of
the guanidine functional group. Furthermore, these reagents may
react with the groups of lysine as well as the arginine
epsilon-amino groups.
[0043] The specific modification of tyrosyl residues per se has
been studied extensively, with particular interest in introducing
spectral labels into tyrosyl residues by reaction with aromatic
diazonium compounds or tetranitromethane. Most commonly,
N-acetylimidizol and tetranitromethane are used to form O-acetyl
tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl
residues are iodinated using .sup.125I or .sup.131I to prepare
labeled proteins for use in radioimmunoassay, the chloramine T
method described above being suitable.
[0044] Carboxyl side groups (aspartyl or glutamyl) are selectively
modified by reaction with carbodiimides (R'--N--C--N--R') such as
1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or
1-ethyl-3-(4-azonia-4,4-demethylpentyl) carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0045] Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl residues.
Alternatively, these residues are deamidated under mildly acidic
conditions. Either form of these residues falls within the scope of
this invention.
[0046] Other modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl
residues, methylation of the .alpha.-amino groups of lysine,
arginine, and histidine side chains (T. E. Creighton (1983)
Proteins: Structure and Molecular Properties, W. H. Freeman &
Co., San Francisco, pp. 79-86).
[0047] The activity of covalently modified peptides (e.g.,
end-terminal or side chain modified peptides) can be evaluated in
the direct T cell stimulation assay and/or the competitive
inhibition assay, described above
[0048] The proinsulin peptide compounds of the invention also
include peptide analogs and peptide mimetics of native proinsulin
peptides. The language "peptide analog" or "peptide mimetic" refers
to a compound composed of linked residues such that the compound
mimics the structure of a native proinsulin peptide. A "residue"
refers to an amino acid or amino acid mimetic incorporated in the
peptide compound by an amide bond or amide bond mimetic. Approaches
to designing peptide mimetics and analogs are known in the art. For
example, see Farmer, P. S. in Drug Design (E. J. Ariens, ed.)
Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball. J. B.
and Alewood, P. F. (1990) J. Mol. Recognition 3:55; Morgan, B. A.
and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243-252; and
Freidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270.
[0049] An "amino acid mimetic" refers to a moiety, other than a
naturally occurring amino acid, that conformationally and
functionally serves as a substitute for a particular amino acid in
a peptide compound without adversely interfering to a significant
extent with the function of the peptide (e.g., interaction of the
peptide with an MHC molecule). In some circumstances, substitution
with an amino acid mimetic may actually enhance properties of the
peptide (e.g., interaction of the peptide with an MHC molecule).
Examples of amino acid mimetics include D-amino acids. Proinsulin
peptide compounds substituted with one or more D-amino acids may be
made using well known peptide synthesis procedures. The effect of
amino acid substitutions with D-amino acids can be tested using
assays as described above for substitutions of L-amino acids.
Studies with other antigenic peptides on the effect of D-amino acid
substitutions on the MHC binding ability of the peptides have shown
that single D-amino acid substitutions at critical MHC contact
positions typically decrease the affinity of the peptide for MHC
molecules but that D-amino acid substitutions made outside of these
positions are relatively well tolerated. See e.g., PCT Publication
No. WO 92/02543 by Gaeta et al.
[0050] The peptide analogs or mimetics of the invention include
isosteres. The term "isostere" as used herein refers to a sequence
of two or more residues that can be substituted for a second
sequence because the steric conformation of the first sequence fits
a binding site specific for the second sequence. The term
specifically includes peptide back-bone modifications (i.e., amide
bond mimetics) well known to those skilled in the art. Such
modifications include modifications of the amide nitrogen, the
.alpha.-carbon, amide carbonyl, complete replacement of the amide
bond, extensions, deletions or backbone crosslinks. Several peptide
backbone modifications are known, including .psi.[CH.sub.2S],
.psi.[CH.sub.2NH], .psi.[CSNH.sub.2], .psi.[NHCO],
.psi.[COCH.sub.2], and .psi.[(E) or (Z) CH.dbd.CH]. In the
nomenclature used above, .psi. indicates the absence of an amide
bond. The structure that replaces the amide group is specified
within the brackets. Other examples of isosteres include peptides
substituted with one or more benzodiazepine molecules (see e.g.,
James, G. L. et al. (1993) Science 260:1937-1942)
[0051] Other possible modifications include an N-alkyl (or aryl)
substitution (.psi.[CONR]), backbone crosslinking to construct
lactams and other cyclic structures, or retro-inverso amino acid
incorporation (.psi.[NHCO]). By "inverso" is meant replacing
L-amino acids of a sequence with D-amino acids, and by
"retro-inverso" or "enantio-retro" is meant reversing the sequence
of the amino acids ("retro") and replacing the L-amino acids with
D-amino acids. For example, if the parent peptide is Thr-Ala-Tyr,
the retro modified form is Tyr-Ala-Thr, the inverso form is
thr-ala-tyr, and the retro-inverso form is tyr-ala-thr (lower case
letters refer to D-amino acids). Compared to the parent peptide, a
retro-inverso peptide has a reversed backbone while retaining
substantially the original spatial conformation o the side chains,
resulting in a retro-inverso isomer with a topology that closely
resembles the parent peptide and is able to bind the selected MHC
molecule. See Goodman et al. "Perspectives in Peptide Chemistry"
pp.283-294 (1981). See also U.S. Pat. No.. 4,522,752 by Sisto for
further description of "retro-inverso" peptides
[0052] The modified forms of proinsulin peptides of the invention,
including L- or D-amino acid substitutions, covalent modification
of end termini or side chains, and peptide analogs and mimetics can
be selected for desired alterations of the physical or chemical
properties of the peptide, for example, increased stability,
solubility, bioavailability, increased or decreased immunogenicity,
etc.
[0053] Although the preferred peptide compounds of the invention
are derived from a region of proinsulin, or are modified forms of a
peptide derived from proinsulin, it will be appreciated by those
skilled in the art that peptides derived from other proteins that
are homologous to proinsulin within the region from which the
peptide is derived may also be useful for modulating immunological
responses by T cells of Type I diabetic subjects. For example,
peptide compounds of the invention may be derived from proteins
having a region that is homologous to the region of proinsulin
spanning the B chain-C peptide junction (e.g., amino acids 47-63 of
proinsulin). One hypothesis to explain the initiating pathological
event leading to an autoimmune response against an autoantigen is
that the autoantigen mimics an environmental or microbial antigen
to which the subject has been exposed. Thus, this environmental or
microbial antigen serves as the immunizing stimulus that activates
the autodestructive immune elements. Accordingly, the amino acid
sequence of the region of proinsulin spanning the B chain-C peptide
junction (e.g., amino acids 47-63 of proinsulin) can be compared to
that of other proteins (e.g., potential environmental or microbial
antigens) to identify proteins with a homologous region. Peptides
derived from such a homologous region of another protein, or
modified peptides thereof, modified as described herein, may also
be useful for modulating (e.g., stimulating or inhibiting)
immunological responses by T cells of Type I diabetic subjects.
[0054] II. Pharmaceutical Compositions
[0055] The proinsulin peptide compounds of the invention can be
formulated into compositions suitable for pharmaceutical
administration. The pharmaceutical composition typically includes a
proinsulin peptide (or modified form thereof as described above)
and a pharmaceutically acceptable carrier. In a preferred
embodiment, the pharmaceutical composition includes a proinsulin
peptide compound identical or substantially similar to a region of
proinsulin that spans the junction between the B chain and the C
peptide of proinsulin. Preferably, the proinsulin peptide is
derived from human proinsulin.
[0056] As used herein the term "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0057] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration. For
example, solutions or suspensions used for parenteral, intradermal,
or subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0058] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0059] Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., proinsulin peptide or
derivative thereof) in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated above,
as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0060] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0061] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These may be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No. 4,522,811.
For example, liposome formulations may be prepared by dissolving
appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine,
stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and
cholesterol) in an inorganic solvent that is then evaporated,
leaving behind a thin film of dried lipid on the surface of the
container. An aqueous solution of invariant chain protein or
peptide is then introduced into the container. The container is
then swirled by hand to free lipid material from the sides of the
container and to disperse lipid aggregates, thereby forming the
liposomal suspension.
[0062] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of individuals.
[0063] In one embodiment, the pharmaceutical composition comprises
a tolerogenic amount of a proinsulin peptide compound of the
invention. As used herein, the language a "tolerogenic amount" is
intended to include an amount of peptide compound sufficient to
induce unresponsiveness to the peptide compound, or a related
peptide or a protein from which the peptide is derived (e.g.,
proinsulin), in a subject. While not intending to be limited by
mechanism, unresponsiveness to the compound may result from
induction of anergy in T cells specific for the compound, deletion
(e.g., destruction) of T cells specific for the antigen or
induction of T suppressor cell circuits. The tolerogenic amount of
a compound necessary to induce unresponsiveness in a subject is
likely to vary depending upon the particular form of peptide
compound used, the route of administration, the state of disease in
the subject, etc. Animal models accepted in the art as models of
human Type I diabetes (e.g., the Biobreeding rat or the NOD mouse)
can be used to test particular peptide compounds, routes of
administration etc., to determine appropriate tolerogenic amounts
of the peptide compounds of the invention.
[0064] III. Methods for Detecting an Indicator of DM-I in a
Subject
[0065] The proinsulin peptide compounds of the invention can be
used in assays to detect an indicator of Type I diabetes in a
subject. As described in further detail in Example 3, biological
samples from human Type I diabetic patients exhibit increased
immunological activity directed against a proinsulin peptide of the
invention than do biological samples from non-diabetic control
humans. Accordingly, immunological activity directed against a
stimulatory proinsulin peptide compound of the invention can be
detected as an indicator of Type I diabetes in a subject.
[0066] The invention provides a method for detecting an indicator
of Type I diabetes in a subject, comprising
[0067] a) obtaining a biological sample from the subject;
[0068] b) contacting the sample with a proinsulin peptide compound
that stimulates an immunological response by T cells of Type I
diabetic subjects; and
[0069] c) detecting an immunological activity in the sample against
the proinsulin peptide compound as an indicator of Type I diabetes
in the subject.
[0070] Proinsulin peptide compounds suitable for use in the method
include the stimulatory compounds described in detail hereinbefore.
Preferably, the proinsulin peptide compound is identical or
substantially similar to a region of proinsulin that spans the
junction between the B chain and the C peptide of proinsulin, as
described above. For detection of an indicator of DM-I in
biological samples from humans, the proinsulin peptide compound
preferably is derived from human proinsulin.
[0071] Typically, the biological sample used in the method is a
blood sample from the subject, or a subfraction thereof, such as
nucleated cells (e.g., lymphocytes) or serum, although any suitable
biological sample that may contain an immunological activity can be
used. Blood samples, or other biological samples, can be obtained
from a subject by standard techniques. Furthermore, blood samples
can be fractionated (e.g., to obtain nucleated cells, serum, etc.)
by standard techniques.
[0072] In one embodiment of the method, the immunological activity
that is detected is a T cell response to the proinsulin peptide
compound. For example, peripheral blood mononuclear cells, or
purified T cells, from a blood sample from the subject can be
cultured in the presence of the compound. After a sufficient period
of time in which to elicit a T cell response to the compound, the
responsiveness of the T cells to the compound is determined. T cell
responsiveness can be assessed, for example, by measuring T cell
proliferation (e.g., by standard tritiated thymidine uptake) or
production of cytokines (described further below).
[0073] Prior to or alternative to measuring T cell responsiveness,
proinsulin-specific T cell clones can be prepared from the
biological sample and quantitated (e.g., see Example 3). The
frequency of proinsulin- specific T cells in the biological sample
can be determined by limiting dilution analysis (e.g., as described
in Sabbaj, S et al. (1992) J. Clin. Immunol. 12:216-224).
Alternatively, the frequency of proinsulin specific T cells can be
determined using the hypoxanthine guanine phosphoribosyltransferase
(hprt) clonal assay (as described in Allegretta, M. et al. (1990)
Science 247:719-721; and Lodge, P. et al. (1994) Neurology
44(Suppl. 2):A147). A key feature of this clonal assay technique is
that T cells detected by this system are presumed to have undergone
multiple cycles of in vivo stimulation in response to the antigen
of interest prior to cloning. Thus, it is considered to reflect an
accurate picture of T cell specificity and clonal activity in the
subject by utilizing a test that can be performed in vitro. The
results can be compared to non-diabetic controls.
[0074] Alternative or in addition to measuring T cell
proliferation, T cell cytokine production can be measured as an
indicator of immunological activity against the proinsulin peptide
compound. T cell cytokine production can be assayed, for example,
by a standard enzyme linked immunosorbent assay (ELISA) or
radioimmunoassay (RIA) specific for the particular cytokine (e.g.,
proinflammatory cytokines such as interleukin-2 [IL-2],
interferon-.gamma. [IFN-.gamma.] and tumor necrosis
factor/lymphotoxin [TNF/LT]). In a nonlimiting example of the
method, bulk cultures (5.times.10.sup.6 cells) of peripheral blood
mononuclear cells from the subject are initiated in an appropriate
medium containing the proinsulin peptide. Seven days after the
initiation of the bulk cultures, IL-2 (as a T cell growth factor)
is added and then renewed every 3 to 4 days. At 14 days post
initiation of the culture, cells are harvested and adjusted to
2.times.10.sup.6 cells/ml in fresh medium lacking IL-2 for 48
hours. After 48 hours, supernatants are harvested to be assayed for
the presence of cytokines. The cultures can be maintained, for
example, up to 4 weeks, with the supernatants periodically
monitored for cytokine production. The supernatants can be frozen
until assayed. Kits for assaying cytokine levels are commercially
available (e.g., an ELISA for IL-2 is available from Genzyme; an
ELISA for TNF/LT is available from R & D Systems; an RIA for
IFN-.gamma. is available from Centocor). Again, the results can be
compared to non-diabetic controls.
[0075] In another embodiment of the method of detecting an
indicator of Type I diabetes in a subject, the immunological
activity that is detected by the method is antibody binding to the
preproinsulin peptide compound. A biological sample containing
immunoglobulin (e.g., serum) can be obtained from the subject and
contacted with the proinsulin peptide compound to determine whether
antibodies specific for the proinsulin peptide are present in the
sample. Standard methods can be used to detect the presence of
antibodies specific for the proinsulin peptide compound, such as
ELISAs and RIAs.
[0076] IV. Methods for Inhibiting the Development or Progression of
DM-I in a Subject
[0077] Another aspect of the invention pertains to methods for
inhibiting the development or progression of Type I diabetes in a
subject comprising administering to the subject a proinsulin
peptide compound of the invention which modulates an immunological
response by T cells of Type I diabetic subjects. The subject may
suffer from Type I diabetes, may be in a "pre-diabetic" phase of
the disease or may be susceptible to development of the disease
(e.g., the subject may have a genetic predisposition to Type I
diabetes). While not intending to be limited by mechanism, the
development or progression of DM-I in subjects can be inhibited
using peptide compounds as described herein by, for example,
depleting pathogenic T cells, inducing anergy in pathogenic T cells
or stimulating specific suppressor circuits to inhibit the
progression of islet cell destruction.
[0078] In one embodiment, a stimulatory proinsulin peptide compound
of the invention, as described in detail above, is administered to
a subject to inhibit the development or progression of Type I
diabetes. Studies in other autoimmune systems have demonstrated
that peptides that stimulate antigen-specific T cell response in
vitro can be used to induce T cell tolerance in vivo. For example,
in experimental autoimmune encephalitis (EAE), a modified peptide
of myelin basis protein that retains the ability to bind to MHC
molecules has an increased ability to stimulate T cells in vitro
can prevent EAE induction when administered before or after the
onset of the disease (see e.g., Wraith, D. C. et al. (1989) Cell
59:247-255; Smilek, D. et al. (1991) Proc. Natl. Acad. Sci. USA
88:9633-9637). Moreover, peripheral T cell tolerance to the major
cat allergen Fel d I can be induced by subcutaneous of Fel d I
peptides expressing immunodominant T cell epitopes (see e.g.,
Briner, T. J. et al. (1993) Proc. Natl. Acad. Sci. USA
90:7608-7612). Accordingly, a stimulatory proinsulin peptide of the
invention can be used to modulate responsiveness the responsiveness
of T cells in Type I diabetic subjects by administering a
tolerogenic amount of the peptide and/or by administering the
peptide by a tolerogenic route of administration. Preferred
tolerogenic routes of administration are subcutaneous injection
(see e.g., Briner et al., supra), oral administration (see e.g.,
Whitacre, C. et al. (1991) J. Immunol. 147:2155-2163) and
intrathymic injection (see e.g., Posselt, A. M. et al. (1992)
Science 256:1321-1324).
[0079] Additionally, a stimulatory peptide compounds of the
invention, such as a modified proinsulin peptide or a modified
peptide derived from an environmental or microbial antigen
homologous to proinsulin may be useful for vaccinating individuals
who are susceptible or predisposed to development of DM-I. Suitable
peptide compounds, in an appropriate vehicle, can be administered
to a susceptible individual to deplete autoaggressive immunological
elements and/or to produce a protective immune response the
modified peptide compound which does not crossreact with self
tissue.
[0080] In another embodiment, an inhibitory peptide compound of the
invention as described in detail above, is administered to a
subject to inhibit the development or progression of Type I
diabetes. Studies in other autoimmune systems have demonstrated
that inhibitory peptides, such as MHC blocking peptides (i.e.,
peptides that retain the ability to bind MHC molecules but which do
not stimulate T cell responses) can be used to prevent and/or treat
autoimmune responses. Successful examples of this approach include
the treatment of EAE (see e.g., Wauben, M. H. et al. (1992) J. Exp.
Med. 176:667-677; Sakai, K. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:9470-9474), adjuvant arthritis (Wauben et al., supra) and
diabetes in the NOD mouse (see e.g., Hurtenbach, U. et al. (1993)
J. Exp. Med. 177:1499-1504).
[0081] A peptide compound of the invention is administered to a
subject in a biologically compatible form suitable for
pharmaceutical administration in vivo, by which is meant that the
form of the compound is one in which any toxic effects are
outweighed by the therapeutic effects of the agent. The term
"subject" is intended to include living organisms which are
susceptible to Type I diabetes, e.g., mammals and in particular,
humans. Administration of a compound as described herein can be in
any pharmacological form including a therapeutically active amount,
alone or in combination with another therapeutic compound, and a
pharmaceutically acceptable carrier.
[0082] Administration of a therapeutically effective amount of a
compound of the invention is defined as an amount, at dosages and
for periods of time, sufficient to achieve the desired result. For
example, the desired result can include inhibition of at least one
symptom of the DM-I, slowing or halting the progression of the
disease or other clinically desirable result. A therapeutically
effective amount of the compound may vary according to factors such
as the disease state, age, sex, and weight of the individual, and
the ability of the compound to elicit a desired response in the
individual. Dosage regimens may be adjusted to provide the optimum
therapeutic response. For example, the composition may be
administered at once, or several divided doses may be administered
daily for a period of time. The dose may be proportionally reduced
as indicated by the exigencies of the therapeutic situation. The
concentration of active compound in the composition will depend on
absorption, inactivation, and excretion rates of the compound as
well as other factors known to those of skill in the art. It is to
be noted that dosage values will also vary with the severity of the
condition to be alleviated. It is to be further understood that for
any particular subject, specific dosage regimens should be adjusted
over time according to the individual need and the professional
judgment of the person administering or supervising the
administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed composition.
[0083] The compound can be administered for prophylactic and/or
therapeutic treatments. In therapeutic applications, the compound
is administered to a subject already suffering from the disease in
an amount sufficient to alleviate or at least partially arrest the
symptoms of the disease and/or its complications. An amount
adequate to accomplish this is referred to as a "therapeutically
effective dose". Amounts effective for this use may vary widely,
but nonlimiting examples of therapeutic systemic dosages for the
compounds described herein are those ranging from 0.1 mg to about
2,000 mg of peptide per day for a 70 kg subject, with dosages of
from about 0.5 mg to about 700 mg of peptide per day being more
typical. In prophylactic applications, the compound is administered
to a subject susceptible or otherwise at risk for the disease in an
amount sufficient to enhance the subject's own immunoregulatory
capabilities. Such an amount is referred to herein as a
"prophylactically effective dose". Again, amounts effective for
this use may vary widely, but nonlimiting examples of prophylactic
systemic dosages for the compounds described herein are those
ranging from 0.1 mg to about 500 mg of peptide per day for a 70 kg
subject, with dosages of from about 0.5 mg to about 200 mg of
peptide per day being more typical.
[0084] The compound may be administered in a convenient manner
suitable to achieve the desired result. For example, in one
embodiment, the agent is administered intravenously. In another
embodiment, the agent is administered orally. In yet other
embodiments, the agent is administered subcutaneously,
intrathymically, intramuscularly or intraperitoneally. Depending on
the route of administration, the agent may be coated in a material
to protect the agent from the action of enzymes, acids and other
natural conditions which may inactivate the compound and/or to
deliver the compound in a slow-release formulation.
[0085] The effectiveness of particular peptide compounds, dosages,
carriers, routes of administration and the like can be evaluated in
well characterized animal models of human Type I diabetes, wherein
positive results in these animal models are predictive of efficacy
in humans. Preferred animal models include the Biobreeding rat (see
e.g., Parfrey, N. A. et al. (1989) Crit. Rev. Immunol. 9:45-65;
Logothetopoulous, L. et al. (1984) Diabetes 33:33-36) and the NOD
mouse (see e.g., Kikutani, H. et al., in Adv. Immunol. (F. J.
Dixon, ed.), pp. 285-323, New York, N.Y.:Academic Press, 1992).
[0086] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are hereby incorporated by
reference.
EXAMPLE 1
Proinsulin Peptide-Specific T Cell Clones Induce Insulitis
[0087] Type I diabetes (also referred to as insulin-dependent
diabetes mellitus), an autoimmune disease which occurs in humans
and animals, is characterized by the destruction of
insulin-secreting islet .beta.-cells of the pancreas.
Epidemiological studies in man have documented a strong genetic
predisposition linked to HLA-DR3, -DR4 and -DQ3.2 class II alleles
of the human MHC (see e.g., Wolf, E. et al. (1983) Diabetologia
24:224-230; Platz, P. et al. (1981) Diabetologia 21:108-115;
Warram, J. et al. (1994) in Joslin's Diabetes Mellitus, R. Kahn and
G. Weir (eds.), Philadelphia, Pa., Lea & Febiger, pp. 201-215;
and Tait, B. D. et al. (1991) Bailliere's Clin. Endocrinol. &
Metab. 5:211-228). This suggests a significant association between
pathogenic pancreatic autoantigen(s) and antigen presentation to T
lymphocytes in the context of these specific MHC class II
molecules.
[0088] In this example, a rat MHC class II (RT1.B.sup.1) binding
motif was used to predict potentially autoreactive CD4.sup.+ T cell
epitopes in two islet .beta.-cell constituents: the enzyme glutamic
acid decarboxylase (GAD) and the insulin precursor hormone,
proinsulin (PI). Seventeen-amino acid long peptide fragments of GAD
and PI containing the binding motif were synthesized and used to
generate peptide-specific, MHC Class II-restricted, CD4.sup.+ T
cell lines from DA(RP) rats (MHC type
RT.1A.sup.uB/D.sup.1E/C.sup.a). Once established, the
DA(RP)-derived T cell lines specific for rat islet GAD and PI were
adoptively transferred to naive DA(RP) rats. At 10 days
post-transfer, insulitis had developed in rats receiving
proinsulin-specific T cells, while no insulitis was observed in
pancreases of rats receiving GAD-specific T cells. The pathogenic
PI peptide-specific T cells are CD4.sup.+/CD8.sup.- and secrete
TH.sub.1-like cytokines in response to antigen. Thus, this example
provides a novel, antigen-specific model of autoimmune insulitis by
T cells specific for a peptide fragment of proinsulin.
[0089] The following methodologies were used in this example:
[0090] Materials and Methods
[0091] Animals. DA(RP) rats, with the MHC type
RT1.A.sup.uB.sup.1D.sup.1E/- C.sup.a, were used in these studies.
This rat strain develops neither spontaneous insulitis nor
diabetes. DA(RP) strain rats were originally obtained from Dr.
Heinz Kunz (Department of Pathology, University of Pittsburgh,
Pittsburgh, Pa.), and a breeding colony was maintained in the
Animal Research Facility of the Dartmouth Medical School, Lebanon,
N.H. Both male and female DA(RP) rats were used in experiments
between the ages of 50-70 days. All procedures and animal care were
in accordance with the National Institutes of Health guidelines on
laboratory animal welfare.
[0092] MHC Class II Binding Motif Peptide Synthesis. Sequences of
the PI and GAD peptides containing the binding motif were
synthesized by standard F-moc chemistry using the RapidAmide
Multiple Peptide Synthesis (RaMPS) system (NEN-Dupont, Wilmington,
Del.). Given that MHC Class II-restricted T cells typically
recognize peptides between 13-25 amino acids in length (Zamvil, S.
et al. (1986) Nature 324:258-260; Wraith, D. C. et al. (1989) Cell
59:247-255), GAD and PI peptides were extended by four to six amino
acids on each end of the motif, and synthesized to yield 17-amino
acid long peptides (designated GAD.sub.412, GAD.sub.520, and PI).
Moreover, peptides were N-terminally acetylated to encourage
.alpha.-helix formation and MHC interaction (Mains, R. et al.
(1983) Trends Neurosci. 6:229-235).
[0093] Generation of Peptide-specific T Cell Lines and Adoptive
Transfer. Individual T cell lines were generated against
GAD.sub.412, GAD.sub.520 and PI peptides. To initiate T cell lines,
peptides were emulsified in complete Freund's adjuvant (CFA)
supplemented with 5 mg/ml H37RA (DIFCO, Detroit, Mich.) and
injected intradermally in the hind footpads of DA(RP) rats in a
final concentration of 200 .mu.g peptide. After nine days, rats
were sacrificed via Fluothane anesthesia (Ayerst Laboratories, New
York, N.Y.) to obtain popliteal lymph nodes. Lymph nodes were
mechanically dissociated with forceps into a single cell
suspension, washed two times in phosphate buffered saline (PBS),
and resuspended in initiation medium with 50 .mu.g/ml peptide.
Initiation/proliferation medium consisted of RPMI 1640, 1%
autologous rat serum, 5% NCTC-109 (Bio Whittaker Products,
Walkersville, Md.), 2 mM glutamine, 100 U/ml penicillin, 100
.mu.g/ml streptomycin, 100 .mu.g/ml fungizone (ICN Biomedicals,
Costa Mesa, Calif.), and 5.times.10.sup.-5 M 2-mercaptoethanol
(Sigma, St. Louis, Mo.). After three days, T-lymphoblasts were
collected by density centrifugation using Histopaque-1.077 (Sigma),
washed two times in PBS, and resuspended in medium containing 10%
fetal calf serum and 5% IL-2-rich supernate from concanavalin
A-stimulated spleen cells. Peptide-specific T cell lines were
allowed to come to a resting state 7-10 days after the initial in
vitro stimulation with peptide, then restimulated with irradiated
thymocytes (2000 rad) and 20 .mu.g/ml peptide.
[0094] For adoptive transfer studies, anti-GAD and anti-PI T cells
were stimulated with either peptide (20 .mu.g/ml) or the mitogenic
lectin concanavalin-A (5 .mu.g/ml) for 72 hr prior to intravenous
(i.v.) injection into naive recipients. Ten days following i.v.
transfer of peptide-specific T cells (ranging in concentrations
from 25-120.times.10.sup.6), rats were sacrificed under ether
anesthesia to obtain the pancreas. Tissue was fixed in 10%
formalin, embedded in paraffin, sectioned (6 microns), mounted and
stained with hematoxylin and eosin.
[0095] Antigen Specificity and MHC Restriction of Peptide-specific
T Cell Lines. Peptide-specific T cells were collected and
co-cultured in triplicate (5-7.times.10.sup.4/well) with or without
peptide (5-20 .mu.g/ml), irradiated (2000 rad) thymocytes
(5.times.10.sup.5/well) as antigen presenting cells (APC), OX-3,
OX-6 or OX-17 (anti-RT1.B and D) antibodies, or W3/25 (anti-CD4),
OX-8 (anti-CD8) antibodies for 72 hr, including a final 18 hr pulse
with .sup.3H-Thymidine (0.5 .mu.Ci/well). .sup.3H-Thymidine
incorporation was measured by liquid scintillation counting
(Wallac, Gaithersburg, Md.) and the results expressed as
stimulation indices.+-.one standard deviation (SD) for triplicate
cultures.
[0096] Cytokine Production Profile of Peptide-specific T cells.
Proinsulin-specific T cells were tested for cytokine production in
response to PI peptide. Proinsulin peptide specific levels of
interleukin 2 (IL-2) and interleukin 4 (IL-4) were measured in a
bioassay using IL-2-dependent HT-2 cells, and IFN-.gamma.
production was measured with an ELISA specific for rat IFN-.gamma.
(GIBCO BRL, Gaithersburg, Md.). Briefly, 100 .mu.l aliquots of
supernatant were collected from in vitro cultures of PI-specific T
cells, PI peptide, APC+anti-MHC Class II antibodies at 40 hr for
IL-2 and IL-4 assay, and 72 hr for IFN-.gamma.. Interleukin-2
containing supernates were cultured with 1.times.10.sup.3 HT-2
cells for 48 hr, including a pulse with .sup.3H-Thymidine for the
final 12 hr of culture. Interleukin-4 containing supernates were
cultured with 1.times.10.sup.3 HT-2 cells which had been
preincubated with an IL-2 receptor antibody (PC 61.5.3, ATCC,
Rockville, Md.). After 48 hr, HT-2 cells were harvested and
.sup.3H-Thymidine uptake measured by liquid scintillation
counting.
[0097] Flow Cytometric Analyses of Peptide-specific T Cell Lines.
PI-specific and GAD-specific T cells were stimulated with 20
.mu.g/ml peptide or 5 .mu.g/ml concanavalin A for 72 hr prior to
determinations of surface antigen expression. Samples of
1.times.10.sup.6 anti-PI T cells each were stained with primary
antibody (OX-19 (CD5); W3/25 (CD4); OX-8 (CD8); R7.3 (.alpha..beta.
T cell receptor); OX-3, OX-6, OX-17 (RT1.B and RT1.D); OX-22
(CD45RC); 12.5-20 .mu.g/ml protein from ascites; Harlan Bioproducts
for Science, Indianapolis, Ind.) for 30 min on ice, washed twice,
then incubated with fluorescein isothiocyanate (FITC)-conjugated
goat anti-mouse F(ab)'.sub.2 antibody (Cappel-Orginon Teknika, West
Chester, Pa.) for 30 minutes on ice, washed twice and fixed with 1%
paraformaldehyde/PBS. Control staining consisted of cells stained
with an IgG1 isotype control antibody (MOPC 21, Sigma, St. Louis,
Mo.) in place of primary antibody, in addition to unstained cells
and cells stained with secondary antibody only. Cells were analyzed
on a FacScan flow cytometer (Becton-Dickinson, Lincoln Park,
N.J.).
[0098] RINm5F Insulinoma Cells. RINm5F insulinoma cells (Gadzar, A.
et al. (1980) Proc. Natl. Acad. Sci USA 77:3519-3523) were obtained
from Dr. Walter Hsu, Iowa State University. RIN cells were cultured
in RPMI 1640 medium supplemented with 10% heat-inactivated fetal
calf serum (Hyclone), 100 .mu.g/ml streptomycin, 100 U/ml
penicillin, and 2 mM L-glutamine (Bio Whittaker). Exhausted RIN
cell supernatant was collected after 5 days of growth, 50-70%
confluency of RIN cells. Supernates were centrifuged, sterile
filtered and stored at -20.degree. C. until use in PI-specific T
cell assays. RIN cell supernate was added in a 20% v/v
concentration to PI-specific T cells in vitro.
[0099] Results
[0100] Proinsulin (PI) and GAD peptides containing the MHC Class II
binding motif were synthesized and used to generate T cell lines.
Table I lists the amino acid sequences of the synthetic PI and GAD
peptides. FIG. 1 represents a diagram of the proximal relationship
between the amino acid sequence of rat preproinsulin, the MHC Class
II binding motif, and the cleavage products of insulin and
C-peptide.
4TABLE I Amino Acid Sequences of Synthetic Peptides containing the
MHC Class II (RT1.B.sup.1) Binding Motif a) rat PROINSULIN .sub. *
* PROINSULIN I .sub. 47G F F Y T P K S R R E V E D P Q V.sub.63
(SEQ ID NO: 5) .sub. PROLNSULIN II .sub. 47G F F Y T P M S R R E V
E D P Q V.sub.63 (SEQ ID NO: 6) b) rat islet GAD.sub.412 .sub.412L
L Q C S A I L V K E K G I L Q G.sub.428 (SEQ ID NO: 21) c) rat
islet GAD.sub.520 .sub.520Y I P Q S L R G V P D S P E R R E.sub.536
(SEQ ID NO: 22) * represents cleavage site during processing of
proinsulin to insulin; Arg-Arg residues are released as a dipeptide
at the separation of the insulin B-chain and C-peptide.
[0101] Proinsulin-specific and GAD-specific T cell lines were
adoptively transferred to DA(RP) rats by i.v. tail vein injection.
Ten days following transfer of peptide-specific T cells, pancreases
were obtained and examined for signs of histopathology by staining
with hematoxylin and eosin. No detectable insulitis was observed in
pancreases of rats receiving GAD.sub.412- or GAD.sub.520-specific T
cells (range=3.0-12.0.times.10.sup.7 cells/rat) relative to
untreated pancreas. In contrast, the intravenous injection of
3.0-7.0.times.10.sup.7 T cells/rat specific for PI peptide caused
insulitis in all DA(RP) rats (n=18). The average inflammatory
involvement of islets per pancreas section/per rat was 40+6% (range
21-62% per pancreas section) by day 10 post-transfer of PI-specific
T cells. Within pancreas sections, the severity of islet
involvement ranged from no involvement to early peri-insular
inflammation, to marked insulitis. Immunohistochemical analysis
revealed that PI-induced insulitis consisted primarily of CD4+T
cells and macrophages, an infiltrate typical of delayed type
hypersensitivity and autoimmune reactions in the rat.
[0102] The phenotypic and functional characteristics of the GAD and
PI-specific T cell lines were investigated to determine antigen
specificity and MHC restriction. FIG. 2 shows .sup.3H-Thymidine
incorporation assay results for anti-GAD and anti-PI T cell lines
and their MHC Class II restriction patterns. The stimulation index
(S.I.) is defined as the mean cpm of the experimental sample
divided by the mean cpm of control wells (medium only).
.sup.3H-thymidine incorporation assays were replicated 2-3 times
for cells of each line at the time of the second in vitro
stimulation with peptide. GAD and PI-specific T cells responded to
their respective peptides and did not crossreact with other
peptides containing the class II binding motif. Proliferation of
both GAD T cell lines containing the S-E binding motif was
inhibited by the addition of anti-RT1.B antibody (Ox-3 & Ox-6),
but not anti-RT1.D (Ox-17) antibody (FIG. 2, panel A: GAD.sub.412;
panel B: GAD.sub.520). Proliferation of PI-specific T cells to 20
.mu.g/ml PI peptide could be partially inhibited by antibodies
against RT1.B (54-67%) and RT1.D (22-25%) molecules individually,
and significantly inhibited by the addition of RT1.B and D
antibodies together (>90%) (FIG. 2, panel C). This inhibitory
effect of anti-MHC Class II antibodies followed the same pattern
irrespective of the antigen presenting cell (APC) source used
(i.e., DA(RP) or the RT1.B/D.sup.1 congenic Lewis strain rat). In
addition, PI-specific T cells proliferated in response to a
insulin/proinsulin-containing supernatant from the rat insulinoma
cell line, RINm5F, and this proliferation could be blocked by the
addition of anti-RT1.B antibody. Both GAD-specific and PI-specific
cell proliferation was abrogated by monoclonal antibodies against
CD4, but not CD8, indicating that only CD4 positive T cells
responded to GAD and PI peptides.
[0103] PI-specific T cells were monitored in vitro for cytokine
production in response to PI peptide with or without MHC Class II
blocking antibody. PI-specific T cells secreted marked amounts of
both IL-2 and IL-4 in response to PI peptide, even in the presence
of antibodies to MHC Class II that inhibited proliferation.
Furthermore, PI-specific T cells secreted IFN-.gamma. in response
to PI peptide alone (22.5+0.2 ng/ml) as measured by an ELISA
specific for rat IFN-.gamma. (GIBCO). IFN-.gamma. production in
response to PI peptide in combination with individual class II
antibodies (Ox-3 and Ox-17) averaged 23.8+0.3 ng/ml, and then
dropped to 12.5+0.2 ng/ml in cultures where .sup.3H-Thymidine
incorporation in response to PI peptide was significantly inhibited
by the addition of Ox-3 and Ox-17 together (FIG. 2, C).
[0104] Cell surface antigen expression of the T cell lines was
defined by flow cytometric analysis using a FACScan
(Becton-Dickinson) and is shown in FIG. 3. PI-specific T line cells
were positive for TcR.alpha..beta., were predominantly of the
CD4.sup.+/CD8.sup.- phenotype, and exhibited negligible expression
of CD45RC (<1%). The cell surface phenotype exhibited by cells
specific for GAD.sub.412 and GAD.sub.520 lines was similar.
Depletion of the PI-specific T cell line of the small number of
CD8.sup.+ cells (using magnetic beads; DYNAL, Lake Success, N.Y.)
neither abolished the ability of the remaining CD4.sup.+/CD8.sup.-
cells to respond vigorously to the PI peptide, nor inhibited the
ability of the CD4.sup.+ cells to adoptively transfer insulitis.
Therefore, in DA(RP) rats, it appears that CD4.sup.+ T cells
specific for a peptide fragment of PI can mediate the adoptive
transfer of insulitis, rather than T cells specific for comparable
GAD peptides, even though all peptides result in vigorous,
antigen-specific CD4.sup.+ T cell proliferation.
[0105] In order to assess the persistence of antigen specificity of
GAD- and PI-specific T cell lines in vivo, spleens were removed
from rats injected with either GAD- or PI-specific T cells 18 days
post-transfer. Splenocytes from rats injected with PI-specific
cells exhibited the ability to respond specifically to PI peptide,
but not GAD, as measured by .sup.3H-Thymidine incorporation
(anti-PI T cell line response to PI peptide stimulation index
(S.I.)=41.9 vs. response to GAD=1.9). Similarly, splenocytes from
rats injected with GAD-specific T cells were found to proliferate
markedly to GAD peptide (S.I.=21.6) but not PI peptide (S.I.=1.3).
Moreover, the islets of rats which received PI-specific T cells
exhibited more severe insulitis at day +18 than at day +10
(63.+-.10% involvement of islets). Pancreases from rats injected
with GAD-specific T cells still showed no evidence of insulitis 18
days post-transfer.
[0106] Of particular interest is the finding that the pathogenic T
cell epitope identified in proinsulin spans the endogenous cleavage
site between the B-chain and C-peptide of insulin. Under normal
enzymatic processing of proinsulin to the insulin B-chain and
C-peptide, the exact area which contains the two overlapping
binding motifs (as shown in Table I) is cleaved so that both motifs
are destroyed (see FIG. 1). The aforementioned area of proinsulin
would exist intact in significant quantities only in islet
.beta.-cells or in their vicinity where proinsulin is processed in
an alternative manner. These results demonstrate that pathogenic T
cell epitopes can be located in portions of molecules which are
subsequently degraded during normal enzymatic processing. Since
proinsulin is found in highest concentrations in the .beta.-cells
of pancreatic islets, it is possible that this molecule, and not
its individual degradation products (i.e., insulin and C-peptide)
may serve as an autoantigen in the pathogenesis of Type I diabetes.
Almost 99% of proinsulin is destined to become insulin via a
regulated-release pathway from the .beta.-cell granule, however,
residual proinsulin travels in secretory vesicles along a
constitutive release pathway (see Sizonenko, S. et al. (1991)
Biochem. J. 278:621-625; Sizonenko, S. et al. (1993) Diabetes
42:933-936; Hutton, J. C. et al. (1989) Diabetologia 32:271-281;
Halban, P. A. et al. (1991) Diabetologia 34:767-778). Of interest
relative to the clinical onset of Type I diabetes is the finding
that circulating proinsulin levels can be more than two times
greater in recently diagnosed diabetics than in nondiabetics
(Heaton, D. et al. (1988) Diabetologia 31:182-184; Heding, L. et
al. (1981) Acta. Med. Scand Suppl. 656:509).
[0107] In this example, it was possible to use both GAD and PI
peptides to successfully generate peptide-specific T cell lines,
the majority of which were CD4.sup.+ and MHC Class II-restricted in
recognition of their respective peptides in vitro. Both PI-specific
and GAD-specific T cells were found to persist at least three weeks
in the spleen following transfer and retain their antigen
specificity. However, adoptive transfer experiments revealed that
only PI-specific T cells were capable of mediating insulitis in
vivo. Although splenocyte responses to GAD occur prior to
detectable insulitis and suggest a role for GAD in the pathogenesis
of autoimmune insulitis in NOD mice (see Kaufman, D. L. et al.
(1993) Nature 366:69-72; Tisch, R. et al. (1993) Nature 366:72-75),
the present studies found that rat T cells specific for selected
GAD peptides do not produce islet lesions. The present study has
identified another potentially significant autoantigen in addition
to GAD by demonstrating that proinsulin-reactive T-cells are
sufficient to yield insulitis. It has been shown that insulitis
precedes diabetes by 2-3 weeks in BB rats (see Colle, E. (1990)
Clin. Immunol. Immunopathol. 57:1-9). Given that circulating
insulin levels in humans and mice with Type I diabetes are not
significantly reduced until >95% of the islets are destroyed
(Katz, J. D. et al. (1993) Cell 74:1089-1100), ongoing studies are
monitoring insulitis and blood glucose levels for extended periods
in rats given PI-specific T cells to determine if overt diabetes
ensues as islets are increasingly involved by inflammatory
cells.
EXAMPLE 2
Proinsulin Peptide-Specific T Cell Clones Induce Diabetes in BB
Rats
[0108] In experiments similar to those described in Example 1,
(Lewis.times.WF)F1, BB(DP) and BB(DR) rats were immunized for T
cell line production to determine if the proinsulin peptide was
also immunogenic in rats with RT1.sup.u haplotype. As in the
RT1.sup.1 rats described in Example 1, all RT1.sup.u and
RT1.sup.1/u rats responded. Both the F1 hybrid, and BB(DR) rats
produced a vigorous, antigen specific, predominantly CD4+ cell line
against the proinsulin peptide. The BB(DP) responded in a specific
manner. Anti-PI cells then were adoptively transferred to naive
animals and the development of insulitis and full-blown diabetes in
the animals was evaluated. Negative controls for the nonspecific,
diabetogenic nature of the anti-PI cells included: BB(DR) rats
given 30 million cells of a syngeneic cell line specific for myelin
basic protein (which did not develop diabetes or EAE) and rats
given similar number of T cells specific for GAD (which developed
neither insulitis nor diabetes).
[0109] The results of these studies are summarized below in Table
II.
5TABLE II Insulitis RT-1 after anti-PT Diabetes after Rat Strain
MHC type cell transfer after anti-PI cells Lewis A.sup.1, B.sup.1,
D.sup.1 0/10 0/10 (Lew x WF)F1 A.sup.1/u, B.sup.1/u, D.sup.1/u 4/4
0/4 DA(RP) A.sup.u, B.sup.1, D.sup.1 18/18* 0/18 BB(DR) A.sup.u,
B.sup.u, D.sup.u 14/14 14/14 BB(DP) A.sup.u, B.sup.u, D.sup.u 4/4
4/4 (DA(RP)xBB(DR))F.sub.1 A.sup.u/u, B.sup.u/1, D.sup.u/1 2/10
0/10 *in the (Lew x WF)F1 animals, insulitis involved under 20% of
the islets ten days following transfer; in the DA(RP) over 40% of
the islets were involved by insulitis ten days following transfer.
# All adoptive transfers employed between 20 to 30 million
activated anti-PI cells injected intravenously.
[0110] Injection of the (Lewis.times.WF)F1 anti-PI cell line into
naive rats of the same F1 type produced a mild insulitis in some
animals; but none became diabetic. However, adoptive transfer of
the BB(DR) anti-PI cell line (and subsequently other anti-PI T cell
lines derived similarly from BB(DR) rats) resulted in the
development of overt insulin dependent diabetes mellitus in both
BB(DR) or prediabetic BB(DP) rat. For both the BB(DP) and BB(DR)
rats, cell transfer led to diabetes in only those getting anti-PI
cells; litter mates given no cells, or infused with anti-GAD cells,
remained normal for 30 days or more following the time of
injections. Thus, this example demonstrates that the PI-specific T
cells are sufficient to induce diabetes when transferred into
genetically appropriate recipient animals.
EXAMPLE 3
Proinsulin Peptide-Reactive T Cells are Present in the Circulation
of Type I Diabetic Humans
[0111] In this example, peripheral blood from Type I diabetic and
non-diabetic control individuals was studied to determine whether T
cells reactive against human proinsulin peptide are present in
diabetic individuals. Peripheral blood was obtained from Type I
diabetics (n=9) and non-diabetic, age-matched controls (n=8).
Lymphocytes were obtained by standard density centrifugation and
cultured in the presence of human proinsulin peptide (10 .mu.g/ml)
or tetanus toxoid peptide (10 .mu.g/ml). The amino acid sequences
of the peptides were as follows:
6 Human proinsulin .sup. 47G F F Y T P K T R R E A E D L Q V
G.sup.64 (SEQ ID NO: 4) .sup. Tetanus toxoid .sup.828L M Q Y I K A
N S K F I G I T E L.sup.840 (SEQ ID NO: 23)
[0112] To generate antigen-specific clones, 2.times.10.sup.5
cells/well were cultured in 96-well plates, whereas to generate
antigen-specific lines, 5.times.10.sup.6 cells/well were cultured
in 24-well plates, both for 10-14 days in medium containing
autologous serum. The later 7-14 day culture period included
supplementation with medium containing interleukin-2. At 14 days
after the initiation of cell cultures, an antigen-specific
proliferation assay was performed where T cell lines and clones
were cultured with or without the appropriate peptide and
irradiated, autologous leukocytes for 4.5 days, the last 12 hr
including a pulse with .sup.3H-thymidine. These data are summarized
below in Table III.
7TABLE III # PI PI. Slim. Subject Sex Clones Index Max. CPM TT
Stim. Index CONTROLS JR F 0 5.3 1,039 58.7 KC F 0 12.8 4,264 13.9
AG F 0 1.4 393 38.1 RR M 3 11.8 4,093 21.6 MF M 0 2.8 597 3.8 CH M
0 1.0 470 8.3 KW M 7 1.3 2,190 20.5 MR M 0 4.3 928 155.8 DIABETICS
MW F 4 40.0 5,685 1.4 BL F 3 3.5 1,918 8.3 SJ F 3 5.5 712 2.0 GG M
12 2.4 539 3.3 TP M 11 16.9 4,864 19.1 JW M 4 4.8 1,508 16.7 WD M
16 17.6 21,336 4.8 JS M 2 1.4 246 12.5 TE M 3 5.8 1,733 8.3
[0113] As determined by .sup.3H-thymidine uptake as a measure of
antigen-specific proliferation, proinsulin-reactive T cell lines (8
or 9) and clones (9 of 9) (100%) were detected in Type I diabetic
patients, whereas only 2 of 8 (25%) non-diabetic controls exhibited
proinsulin-reactive T cells from peripheral blood. All subjects
were found to have tetanus toxoid-reactive T cells as expected for
a recall antigen response. Thus, this example demonstrates that
proinsulin-reactive T cell lines and clones can be generated from
the peripheral blood of human Type I diabetic patients, whereas
such lines and clones are not readily generated from the peripheral
blood of non-diabetic controls.
[0114] Equivalents
[0115] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
* * * * *