U.S. patent application number 09/222357 was filed with the patent office on 2001-08-02 for targeting of peg antibody conjugates to islet cells.
Invention is credited to JACOBS, HARVEY, KIM, SUNG WAN, MENARD, VIRGINIE.
Application Number | 20010010820 09/222357 |
Document ID | / |
Family ID | 21709190 |
Filed Date | 2001-08-02 |
United States Patent
Application |
20010010820 |
Kind Code |
A1 |
JACOBS, HARVEY ; et
al. |
August 2, 2001 |
TARGETING OF PEG ANTIBODY CONJUGATES TO ISLET CELLS
Abstract
A method for delaying onset of insulin dependent diabetes
mellitus (IDDM) in an individual predisposed to developing the
disease is disclosed. The method comprises administering a
composition comprising an immunologically effective monoclonal
antibody or fragment thereof against glutamic acid decarboxylase
(GAD) coupled to a nonimmunogenic hydrophilic polymer that provides
a hydration shell around the monoclonal antibody for inhibiting
immune recognition thereof. Poly(ethylene glycol) is a preferred
polymer. A method of reducing insulitis in an IDDM patient and a
composition therefor are also described.
Inventors: |
JACOBS, HARVEY; (DALLAS,
PA) ; KIM, SUNG WAN; (SALT LAKE CITY, UT) ;
MENARD, VIRGINIE; (SALT LAKE CITY, UT) |
Correspondence
Address: |
ALAN J HOWARTH
PO BOX 1909
SANDY
UT
84091
US
|
Family ID: |
21709190 |
Appl. No.: |
09/222357 |
Filed: |
December 29, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09222357 |
Dec 29, 1998 |
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08710653 |
Sep 20, 1996 |
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5853723 |
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60004109 |
Sep 21, 1995 |
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Current U.S.
Class: |
424/178.1 ;
424/141.1; 424/180.1 |
Current CPC
Class: |
A61K 39/44 20130101;
A61K 47/60 20170801; A61K 47/6871 20170801; A61P 3/10 20180101;
A61K 38/00 20130101; A61P 3/00 20180101; C07K 16/40 20130101; C07K
2317/55 20130101 |
Class at
Publication: |
424/178.1 ;
424/141.1; 424/180.1 |
International
Class: |
A61K 039/395; A61K
039/40; A61K 039/42; A61K 039/44 |
Claims
We claim:
1. A method for delaying onset of insulin dependent diabetes
mellitus in an individual predisposed to developing the disease
comprising administering to said individual an effective amount of
a composition comprising (a) an immunologically active monoclonal
antibody or fragment thereof against glutamic acid decarboxylase
coupled to (b) a nonimmunogenic hydrophilic polymer that provides a
hydration shell around said monoclonal antibody for inhibiting
immune recognition thereof.
2. The method of claim 1 wherein said polymer is a poly(ethylene
glycol).
3. The method of claim 2 wherein said poly(ethylene glycol) has a
molecular weight in the range of about 200 to 8,000.
4. The method of claim 2 wherein said monoclonal antibody or
fragment thereof is an F(ab') fragment.
5. The method of claim 2 wherein said poly(ethylene glycol) is
methoxy-poly(ethylene glycol).
6. The method of claim 5 wherein said monoclonal antibody or
fragment thereof is an F(ab') fragment.
7. The method of claim 2 wherein said monoclonal antibody or
fragment thereof is covalently coupled to said poly(ethylene
glycol).
8. The method of claim 7 wherein said monoclonal antibody or
fragment thereof is covalently coupled to said poly(ethylene
glycol) with a crosslinker.
9. The method of claim 8 wherein said crosslinker is
sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate).
10. The method of claim 9 wherein said monoclonal antibody or
fragment thereof is an F(ab') fragment.
11. The method of claim 1 wherein said monoclonal antibody or
fragment thereof is against a GAD-65 isomer of glutamic acid
decarboxylase.
12. The method of claim 11 wherein the monoclonal antibody or
fragment thereof is derived from ATCC no. HB184.
13. A method for reducing insulitis in beta cells of an individual
predisposed to developing insulin dependent diabetes mellitus
comprising administering to said individual an effective amount of
a composition comprising (a) an immunologically active monoclonal
antibody or fragment thereof against glutamic acid decarboxylase
coupled to (b) a nonimmunogenic hydrophilic polymer that provides a
hydration shell around said monoclonal antibody for inhibiting
immune recognition thereof.
14. The method of claim 13 wherein said polymer is a poly(ethylene
glycol).
15. The method of claim 14 wherein said poly(ethylene glycol) has a
molecular weight in the range of about 200 to 8,000.
16. The method of claim 14 wherein said monoclonal antibody or
fragment thereof is an F(ab') fragment.
17. The method of claim 14 wherein said poly(ethylene glycol) is
methoxy-poly(ethylene glycol).
18. The method of claim 17 wherein said monoclonal antibody or
fragment thereof is an F(ab') fragment.
19. The method of claim 14 wherein said monoclonal antibody or
fragment thereof is covalently coupled to said poly(ethylene
glycol).
20. The method of claim 19 wherein said monoclonal antibody or
fragment thereof is covalently coupled to said poly(ethylene
glycol) with a crosslinker.
21. The method of claim 20 wherein said crosslinker is
sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate).
22. The method of claim 21 wherein said monoclonal antibody or
fragment thereof is an F(ab') fragment.
23. The method of claim 13 wherein said monoclonal antibody or
fragment thereof is against a GAD-65 isomer of glutamic acid
decarboxylase.
24. The method of claim 23 wherein the monoclonal antibody or
fragment thereof is derived from ATCC no. HB184.
25. A composition for delaying onset of insulin dependent diabetes
mellitus in an individual predisposed to developing the disease
comprising (a) an immunologically active monoclonal antibody or
fragment thereof against glutamic acid decarboxylase coupled to (b)
a nonimmunogenic hydrophilic polymer that provides a hydration
shell around said monoclonal antibody for inhibiting immune
recognition thereof.
26. The composition of claim 25 wherein said polymer is a
poly(ethylene glycol).
27. The composition of claim 26 wherein said poly(ethylene glycol)
has a molecular weight in the range of about 200 to 8,000.
28. The composition of claim 26 wherein said monoclonal antibody or
fragment thereof is an F(ab') fragment.
29. The composition of claim 26 wherein said poly(ethylene glycol)
is methoxy-poly(ethylene glycol).
30. The composition of claim 29 wherein said monoclonal antibody or
fragment thereof is an F(ab') fragment.
31. The composition of claim 26 wherein said monoclonal antibody or
fragment thereof is covalently coupled to said poly(ethylene
glycol).
32. The composition of claim 31 wherein said monoclonal antibody or
fragment thereof is covalently coupled to said poly(ethylene
glycol) with a crosslinker.
33. The composition of claim 32 wherein said crosslinker is
sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate).
34. The composition of claim 33 wherein said monoclonal antibody or
fragment thereof is an F(ab') fragment.
35. The composition of claim 25 wherein said monoclonal antibody or
fragment thereof is against a GAD-65 isomer of glutamic acid
decarboxylase.
36. The composition of claim 35 wherein the monoclonal antibody or
fragment thereof is derived from ATCC no. HB184.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/004,109, filed Sep. 21, 1995.
BACKGROUND OF THE INVENTION
[0002] This invention relates to a method of treating insulin
dependent diabetes mellitus (IDDM). More particularly, the
invention relates to a method of delaying onset of IDDM by
systemically administering a composition comprising a modified
anti-glutamic-acid-decarboxylase antibody to an individual in need
thereof.
[0003] The pancreas is composed of two major tissues, the acini,
which secrete digestive juices into the duodenum, and the islet
cells of Langerhans, which secrete insulin and glucagon into the
blood. Insulin functions in the storage of excess energy sources.
For example, insulin stimulates (a) the storage of carbohydrates in
the muscles and in the liver as glycogen, (b) the storage of fat in
adipose tissue, and (c) the uptake and storage of amino acids as
proteins in cells. Insulin's main role, however, is in the
homeostatic control of blood glucose through facilitated glucose
uptake into muscle cells. At a normal blood glucose level of about
100 mg/dl, the rate of insulin secretion is about 25 ng/min/kg.
When glucose levels rise after a meal, glucose stimulates beta cell
receptors to secrete insulin, resulting in a 10-25-fold increase
over the basal rate. Once blood glucose levels decrease, the beta
cells rapidly turn off insulin production and return to basal
levels.
[0004] Insulin dependent diabetes mellitus (Type I, IDDM) results
from diminished secretion of insulin by the beta cells. Diabetes is
a process involving different stages of disease progression:
obligatory genetic predisposition, followed by a precipitating
event--either a genetic mutation or an environmental insult, all
leading to autoimmune destruction of islet/beta cells. It has been
noted that up to several years prior to onset of diabetes, greater
than 70% of pre-clinical IDDM patients exhibit circulating
antibodies directed against islet/beta cell antigens. E.g. I.
Deschamps, Life Table Analysis of the Risk of Type 1
(Insulin-dependent) Diabetes Mellitus in Siblings According to
Islet Cell Antibodies and HLA Markers, 35 Diabetologia 951-57
(1992).
[0005] IDDM is characterized by cellular immunological destruction
of beta cells, resulting in a lack of insulin secretion and leading
to the severe pathogenesis of the disease. E.g., L. C. Harrison et
al., Islet-reactive T Cells Are a Marker of Preclinical
Insulin-dependent Diabetes, 89 J. Clin. Invest. 1161-65 (1992).
Different parameters including genetic predisposition, e.g., I.
Deschamps, 35 Diabetologia 951-57, supra, intervenous glucose
tolerance (IVGT), and the presence of antibodies, T. J. Wilkin et
al., Autoimmune Diabetes and the Germ Theory of Disease, 35
Diabetologia 187-89 (1992), have been shown as causative agents in
the development of diabetes.
[0006] The contribution of antibodies in the development of
diabetes, as an autoimmune disease, has been demonstrated by many
researchers. Several preclinical antibody markers have been
isolated, identified, and correlated with IDDM including antibodies
against insulin, insulin receptors, islet cell surface antigens,
and carboxypeptidase H. Statistical studies, e.g., U. Roll et al.,
Associations of Anti-GAD Antibodies with Islet Cell Antibodies and
Insulin Autoantibodies in First-degree Relatives of Type I Diabetes
Patients, 43 Diabetes 154-60 (1994), show that the pattern of
antibody expression, especially involving insulin antibodies (IAA)
and islet cell antibodies (ICA) is highly correlated to the onset
of diabetes. Patients exhibiting antibodies to both IAA and ICA
have a greater than 90% probability of devloping diabetes within 6
years.
[0007] Of particular importance in the causative nature of
prediabetic antibodies is the identification of a subset of ICA
directed against glutamic acid decarboxylase (GAD). Anti-GAD
antibodies have been shown to be present in about 70% of relatives
demonstrating ICA and IAA antibody titers. E.g., U. Roll et al.,
supra. Of this population, about 66% of the patients still develop
diabetes. Furthermore, studies have demonstrated that the immune
system of young mice can develop tolerance to anti-GAD antibodies
through the continued injection of GAD and not develop IDDM. P. Z.
Zimmet et al., Latent Autoimmune Diabetes Mellitus in Adults
(LADA): the Role of Antibodies to Glutamic Acid Decarboxylase in
Diagnosis and Prediction of Insulin Dependency, 11 Diabetic
Medicine 299-303 (1994). In this process, the enzyme GAD is not
recognized as a foreign protein. Thus, autoimmune recognition does
not occur and there is no destruction of beta cells and
normoglycemia is exhibited.
[0008] The initial step in an automimmune response is the
nonspecific recognition, phagocytosis, and processing of proteins
or other molecules as foreign antigens by antigen-presenting cells
(APC's): Langerhans cells (skin), dendritic cells (spleen, lymph
nodes), and monocytes (blood). After phagocytosis, the partially
degraded antigen fragment is transferred to a cell-surface
glycoprotein known as the major histocompatibility complex class II
(MHC II). B cells also assimilate and process specific antigens.
Antigens bind to antibodies on the B cell surface, become
assimilated, processed, and expressed by MHC II proteins, similar
to APC's. The processed antigen is recognized by helper T cells
through their CD4 T cell receptor. The recognition, interaction,
and proliferation of B cells through helper T cell contact is
essential for a strong immune response, since B cells are the chief
antibody secreting cells. The B memory cells lack soluble antibody,
yet they retain surface-bound specific antigen receptors. These
long-lived cells are primed for response to subsequent antigenic
exposures. D. P. Stites et al., Basic and Clinical Immunology
(1987); E. Harlow & D. Lane, Antibodies: A Laboratory Manual
(1988).
[0009] CD8 cytotoxic T cell receptors recognize both antigenic
epitopes and helper T cell surface receptors. E.g., B. J. Bradley
et al., CD8 T Cells Are Not Required for Islet Destruction Induced
by a CD4 Islet-Specific T-Cell Clone, 41 Diabetes 1603-08 (1992).
It is the infiltration, interaction, fusion, and lysis of the
antigen effected cell with the cytotoxic T cell that leads to cell
destruction. In addition, destruction of antigen affected cells can
occur through a humoral immunity process. Briefly, IgG antibodies
bound to antigens will activate the complement fixing pathway
through their Fc fragment, thereby leading to cell death. D. P.
Stites et al., Basic and Clinical Immunology (1987).
[0010] Circulating antibodies against islet/beta cell antigen
(islet cell antibodies (ICA)) were first observed by G. F. Botazzo
et al., Islet Cell Antibodies in Diabetes Mellitus with Autoimmune
Polyendocrine Deficiency, Lancet 1279-83 (1974). The specific
identification and cellular location of the relevant antigens
remains unknown. Initial studies proposed that the ICA target
(antigen) had the biochemical characteristics of a gangliocide. F.
Dotta et al., Pancreatis Islet Ganglioside Expression in Non-obese
Diabetic Mice; Comparison with C57B1/10 Mice and Changes After
Autoimmune B-cell Desctruction, 130 Endocrinology 37-42 (1992). ICA
can be visualized by immunofluorescence; the serum of a newly
diagnosed IDDM patient is incubated with frozen diabetic pancreas
cells, and then islet ICA can be visualized using fluorescent
labeled second antibodies. E.g., C. J. Greenbaum et al., Improved
Specificity of ICA Assays in the Fourth International Immunology of
Diabetes Serum Exchange Workshop, 41 Diabetes 1570-74 (1992); C. J.
Greenbaum et al., Fifth Inernational Serum Exchange Workshop for
Insulin Autoantibody (IAA) Standardization, 35 Diabetologia 798-800
(1992). This method identifies ICA as a group of antibodies, and
does not distinguish between islet or beta cell specificity. As a
group of antibodies, ICA antibodies have been detected at very low
levels in the average population, M. Landin-Olsson et al.,
Predictive Value of Islet Cell and Insulin Autoantibodies for Type
1 (Insulin-dependent) Diabetes Mellitus in a Population-based Study
of Newly-diagnosed Diabetic and Matched Control Children, 35
Diabetologia 1068-73 (1992); C. Levy-Marshal et al., Islet Cell
Antibodies in Normal French School Children, 35 Diabetologia 577-82
(1992), yet high titers predict a high probability of developing
IDDM. E.g., A. Gottsater et al., Islet Cell Antibodies and Fasting
Plasma C-peptide During the First 10 Years After Diagnosis in
Patients with Diabetes Mellitus Diagnosed in Adult Age, 5 Diab.
Nutr. Metab. 243-48 (1992). Furthermore, association of ICA with a
genetic characteristic of major histocompatibility complex
(MHC:DR3+DR4+) results in an 84% probability of developing IDDM. I.
Deschamps, 35 Diabetologia 951-57, supra.
[0011] Recent research has identified antibodies directed against a
beta cell antigen known as glutamic acid decarboxylase (GAD). E.g.,
M. R. Christie et al., Binding of Antibodies in Sera from Type 1
Diabetic Patients to Glutamate Decarboxylase from Rat
Tissues--Evidence for Antigenic and Non-antigenic Forms of the
Enzyme, 35 Diabetologia 380-84 (1992); M. R. Christie, et al.,
Antibodies to GAD and Tryptic Fragments of Islet 64K Antigen as
Distinct Markers for Development of IDDM--Studies with Identical
Twins, 41 Diabetes 782-87 (1992). GAD is an enzyme associated with
the generation of the neurotransmitter, GABA. H. J. DeAizpurua
& L. C. Harrison, Glutamic Acid Decarboxylase in Insulin
Dependent Diabetes Mellitus, 8 Diabetes/Metabolism Reviews 133-47
(1992). GAD has a molecular weight of about 64,000, and has been
shown to exist in different isomeric forms, such as GAD-65 and
GAD-67. Circulating antibodies directed against a GAD-67 epitope
have also been discovered in Stiffman Syndrome. E. Bjork et al.,
GAD Autoantibodies in IDDM, Stiff-Man Syndrome, and Autoimmune
Polyendocrine Syndrome Type I Recognize Different Epitopes, 43
Diabetes 161-65 (1994).
[0012] In the case of IDDM, anti-GAD antibodies are becoming
recognized as significant predictors for the development of
diabetes; 60-70% of the patients who present anti-GAD antibodies
will develop diabetes. E.g., C. H. Thivolet et al., Glutamic Acid
Decarboxylase (GAD) Autoantibodies are Additional Predictive
Markers of Type 1 (Insulin-dependent) Diabetes Mellitus in High
Risk Individuals, 35 Diabetologia 570-76 (1992). Like ICA, the
presence of anti-GAD antibodies is associated with genetic MHC
characteristics, thereby increasing the probability of IDDM. S. W.
Serjeantson et al., Antibodies to Glutamic Acid Decarboxylase Are
Associated with HLA-DR Genotypes in both Australians and Asians
with Type 1 (Insulin-dependent) Diabetes Mellitus, 35 Diabetologia
996-1001 (1992). Others have shown that the presence of anti-GAD
antibodies correlates with T cell activation and the development of
insulitis. R. Tisch et al., Immune Response to Glutamic Acid
Decarboxylase Correlates with Insulitis in Non-obese Diabetic Mice,
366 Nature 72-75 (1993). In yet another pertinent study, GAD was
injected into young (prediabetic) NOD mice and GAD was found to
block the development of T cell reactivity toward beta cell
antigens, thereby preventing insulitis and diabetes. D. L. Kaufman
et al., Spontaneous Loss of T-Cell Tolerance to Glutamic Acid
Decarboxylase in Murine Insulin-dependent Diabetes, 344 Nature
69-72 (1993).
[0013] Other types of antibodies have been found in the serum of
prediabetic and diabetic patients. The antibodies with the greatest
signficance for diagnosis are the ICA antibodies, but many other
types have been studied, although their importance has yet to be
determined. The finding of insulin autoantibodies in the serum of
prediabetic patients, prior to insulin therapy, correlates with the
rate of progression of diabetes. As with the ICA antibodies,
international workshops have resulted in standardization of IAA
titration. C. J. Greenbaum et al., 35 Diabetologia 798-800, supra.
On the other hand, it is possible to induce diabetes in NOD mice by
transfection of specific genes related to the insulin receptor.
E.g., M. A. Lipes et al., Progression to Diabetes in Non-obese
Diabetic (NOD) Mice with Transgenic T Cell Receptors, 259 Science
1165-69 (1993). These antibodies have been reported in some
studies, but their role and importance have not been clarified.
E.g., G. S. Eisenbarth et al., Pathogenesis of Insulin-dependent
(Type I) Diabetes Mellitus, in C. R. Kohn & G. Weiz eds.,
Joslin's Diabetes Mellitus 216-39 (13th ed., 1994). Some studies
have investigated the role of islet cells surface antibodies, but
the difficulty in measuring these antibodies and the lack of real
evidence of their presence in prediabetic pateints make this a
difficult tool to estimate the onset of diabetes. E.g, M. Vives et
al., Reevaluation of Autoantibodies to Islet Cell Membrane in
IDDM-Failure to Detect Islet Cell Surface Antibodies Using Human
Islet Cells as Substrate, 41 Diabetes 1624-31 (1992). Finally,
antibodies to carboxypeptidase H (CPH) have been found in 30% of
prediabetic patients who will develop diabetes. G. S. Eisenbarth et
al., in Joslin's Diabetes Mellitus 216-39, supra. These statistics
and current knowledge of this CPH antibody allow the evaluation of
these autoantibodies as markers compared to anti-GAD and ICA
antibodies.
[0014] The only currently approved treatment for IDDM is the daily
administration of exogenous insulin. E.g., R. B. Tattersall &
E. Gale, Patient Self-monitoring of Blood Glucose and Refinements
of Coventional Insulin Treatment, 70 Am. J. Med. 177-80 (1981).
Other experimental protocols include computer controlled infusion
pumps to deliver insulin in relation to blood glucose, e.g., F. J.
Fogt et al., Development and Evaluation of a Glucose Analyzer for a
Glucose-Controlled Insulin Infusion System (Biostator), 24 Clin.
Chem. 1366-81 (1978), bio-feedback mechanisms with concanavalin
A-glycoylated insulin, e.g. S. W. Kim et al., A self-regulating
Insulin Delivery System, Excerpta Medica 25-32 (1990), and insulin
delivery devices utilizing glucose oxidase, K. Ishihara et al.,
Glucose Induced Permeation Through a Complex Membrane Consisting of
Immobilized Glucose Oxidase and a Poly(amine), 16 Polymer J. 625-42
(1984). In these treatments, exogenous insulin must be administered
since all beta cell function has been previously destroyed. Another
approach to treating IDDM is to transplant functioning islet/beta
cells into the diabetic patient. E.g., E. Reich et al., Prevention
of Diabetes in NOD Mice by Injection of Autoreactive T-lymphocytes,
38 Diabetes 1647-51 (1989). Theoretically, this approach will
provide euglycemia for the patient. However, the islet cells are
usually from a different species, and transplant-related rejection
is often observed. E.g., R. P. Lanza et al., Xenotransplantation of
Canine, Bovine and Porcine Islets in Diabetic Rats without
Immunosuppresion, 88 Proc. Nat'l Acad. Sci. USA 11100-03
(1991).
[0015] A different approach is to prevent or delay the development
of diabetes. In some studies, immunosuppressant therapy was
initiated after IDDM markers (genetic, ICA, IAA, GAD) were
detected. E.g., P. J. Bingley et al., Can We Really Predict IDDM?,
42 Diabetes 213-20 (1993). Immunosuppressives, such as
corticosteroids, azathioprine, and cyclosporin A have been used to
treat diabetes. E.g., A. Lernmark, Immune Intervention Yes, but for
What Reason, for Whom, When and How?, 35 Diabetologia 1096-98
(1992). If administered prior to total beta cell destruction, these
agents specifically blocked cellular immune activity. The beta
cells were protected from further destruction, allowing
regeneration of viable cells, and transient return to euglycemia.
E.g., H. Kolb et al., Immunomodulatory Drugs in Type I Diabetes, in
G. S. Eisenbarth ed., Immunotherapy of Diabetes and Selected
Autoimmune Diseases 111-23 (1989). However, the side effects of
these drugs, especially in diabetic children, outweighed their
usefulness. A. Lernmark, 35 Diabetologia 1096-98, supra.
[0016] From an immunotherapeutic approach, overt early stage
diabetes has been treated by blocking the activating receptors on T
cells with monoclonal antibodies. In one such study,
anti-lymphocyte serum (ALS) and antibodies directed against CD4 and
Cd8 T cell receptors were administered to diabetic mice. T. Maki et
al., Long-term Abrogation of Autoimmune Diabetes in Non-obese
Diabetic Mice by Immunotherapy with Anti-lymphocyte Serum, 89 Proc.
Nat'l Acad. Sci. USA 3434-38 (1992). In this study, ALS or anti-DC4
and CD8 treatment given within 14 days after disease onset resulted
in remission rates (euglycemia) of 75% and 65%, respectively.
Euglycemia occurred within 30 days after treatment and lasted for
about 200 days. Several significant points about the autoimmunity
of diabetes were observed. The lymphocytic antibodies were
responsible for termination of the immune response, thereby
allowing islet recovery. Also, if antibody treatment was initiated
prior to 14 days after disease onset, there were sufficient numbers
of viable islets that could regenerate to resume normal glycemic
conditions. Unfortunately, this procedure may lead to severely
immune-compromised patients and may not be useful for human
applications. Similarly, other studies demonstrate that the
injection of anti-CD3 into NOD mice can prevent diabetes.
[0017] In view of the foregoing, it will be appreciated that
providing a method of delaying the onset of IDDM in an individual
predisposed to this disease would be a significant advancement in
the art.
BRIEF SUMMARY OF THE INVENTION
[0018] It is an object of the present invention to provide a method
for treating insulin dependent diabetes mellitus (IDDM).
[0019] It is also an object of the invention to provide a method
for delaying the onset of IDDM in a person predisposed to
developing the disease.
[0020] It is another object of the invention to provide a method
for treating IDDM and delaying the onset thereof by administering a
modified anti-GAD antibody to a person predisposed to developing
the disease.
[0021] It is still another object of the invention to provide a
composition for administering to a person predisposed to developing
IDDM for delaying the onset of IDDM.
[0022] These and other objects can be achieved by providing a
method for delaying onset of insulin dependent diabetes mellitus in
an individual predisposed to developing the disease comprising
administering to the individual an effective amount of a
composition comprising (a) an immunologically active monoclonal
antibody or fragment thereof against glutamic acid decarboxylase
coupled to (b) a nonimmunogenic hydrophilic polymer that provides a
hydration shell around the monoclonal antibody or fragment thereof
for inhibiting immune recognition thereof.
[0023] Preferably, the polymer is a poly(ethylene glycol), and more
preferably has a molecular weight in the range of about 200 to
8,000, although higher molecular weight polymers, branched
polymers, star molecules, and PEG block copolymers are also within
the scope of the invention. Methoxy-PEG is a particularly preferred
polymer. It is also preferred that the monoclonal antibody or
fragment thereof is an F(ab') fragment. Preferably, the monoclonal
antibody or fragment thereof is against the GAD-65 isomer of
glutamic acid decarboxylase. In one illustrative preferred
embodiment, the monoclonal antibody and polymer are covalently
coupled together with a crosslinker.
[0024] A method for reducing insulitis in beta cells of an
individual predisposed to developing insulin dependent diabetes
mellitus comprises administering to the individual an effective
amount of a composition comprising (a) an immunologically active s
monoclonal antibody or fragment thereof against glutamic acid
decarboxylase coupled to (b) a nonimmunogenic hydrophilic polymer
that provides a hydration shell around the monoclonal antibody or
fragment thereof for inhibiting immune recognition thereof.
[0025] A composition for delaying onset of insulin dependent
diabetes mellitus in an individual predisposed to developing the
disease comprises (a) an immunologically active monoclonal antibody
or fragment thereof against glutamic acid decarboxylase coupled to
(b) a nonimmunogenic hydrophilic polymer that provides a hydration
shell around the monoclonal antibody or fragment thereof for
inhibiting immune recognition thereof.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0026] FIG. 1 shows the reactions for coupling methoxy-PEG-amine to
an F(ab') fragment with a heterobifunctional crosslinker.
[0027] FIG. 2 shows the reactions for coupling diamino-PEG to an
F(ab') fragment with a heterobifunctional crosslinker and labeling
the PEG moiety with label R, such as Bolton-Hunter reagent or
fluorescein isothiocyanate.
[0028] FIG. 3 shows a plot of diabetic incidence as a function of
time for NOD mice injected weekly with 0.3 mg anti-GAD Mab
(.circle-solid.); 0.1 mg anti-GAD Mab (O); or not injected
(.quadrature.).
[0029] FIG. 4 shows a histogram of the percentage of islets
exhibiting a relative islet insulitis score for NOD mice injected
weekly with 0.3 mg anti-GAD Mab (hatched); 0.1 mg anti-GAD Mab
(shaded); or not injected (unshaded).
[0030] FIG. 5 shows a histogram of in vitro splenocyte activation
by anti-GAD Mab (unshaded) and polyclonal mouse IgG (shaded) at
various Mab concentrations.
DETAILED DESCRIPTION
[0031] Before the present method for treating IDDM, and in
particular delaying the onset thereof, is disclosed and described,
it is to be understood that this invention is not limited to the
particular configurations, process steps, and materials disclosed
herein as such configurations, process steps, and materials may
vary somewhat. It is also to be understood that the terminology
employed herein is used for the purpose of describing particular
embodiments only and is not intended to be limiting since the scope
of the present invention will be limited only by the appended
claims and equivalents thereof.
[0032] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to a composition comprising "an
antibody" includes reference to two or more of such antibodies, and
reference to "a polymer" includes reference to one or more of such
polymers.
[0033] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0034] As used herein, "effective amount" means an amount of the
presently claimed composition to provide the selected effect and
performance at a reasonable benefit/risk ratio attending any
medical treatment. The guidance provided herein is sufficient to
permit a person skilled in the art to determine appropriate dosages
without undue experimentation.
[0035] As used herein, "administering" and similar terms mean
delivering the composition to the individual being treated such
that the composition is capable of being circulated systemically to
the parts of the body where the antibody portion of the composition
can bind its cognate antigen, e.g. islet cells. Thus, the
composition is preferably administered to the individual by
systemic administration, typically by subcutaneous, intramuscular,
or intravenous administration, or intraperitoneal administration.
Injectables for such use can be prepared in conventional forms,
either as a liquid solution or suspension or in a solid form
suitable for preparation as a solution or suspension in a liquid
prior to injection, or as an emulsion. Suitable excipients include,
for example, water, saline, glycerol, ethanol, and the like; and if
desired, minor amounts of auxiliary substances such as wetting or
emulsifying agents, buffers, and the like can be added.
[0036] As used herein, "delaying onset" of IDDM and similar terms
mean delaying the date wherein an individual predisposed to
developing IDDM exhibits clinical symptoms of the disease, and
include delaying IDDM to the extent that the disease does not fully
develop.
[0037] An used herein, "poly(ethylene glycol)," "PEG," and similar
terms mean poly(ethylene glycol) and various derivatives thereof,
such as methoxy-PEG-amine, diamine-PEG, and the like. Preferred
poly(ethylene glycols) include polymers of a molecular weight of
about 200 to 8,000, but higher molecular weight polymers are also
within the scope of the invention. PEG includes linear and branched
polymers, star molecules, and PEG block copolymers formed by the
coupling of at least two different PEG polymers to form a higher
molecular weight polymer.
[0038] In the present invention, anti-GAD monoclonal antibodies
(Mab) are modified to maintain binding to their cognate antigens
while further preventing recognition by other aspects of the immune
system. In an illustrative embodiment, the anti-GAD antibody is
modified by digestion with a protease and chemical reduction with a
reducing agent to yield F(ab') fragments, which are then conjugated
with various poly(ethylene glycol) polymers (PEG). The F(ab')
fragment retains the antigen-specific Fab binding fragment, while
the immune and complement activating Fc fragment is removed. In
addition, the poly(ethylene glycol) moiety provides an increased
hydration sphere and dynamic mobility that prevents protein and
cellular interaction. Thus, the present anti-GAD-F (ab') -PEG
composition simultaneously binds GAD and prevents or inhibits
further recognition by the immune system.
[0039] Modern immunological techniques have made biosynthesis,
isolation, and purification of an antigen-specific Mab a common
laboratory procedure, E. Harlow & D. Lane, Antibodies: A
Laboratory Manual (1988), hereby incorporated by reference. Animals
can be immunized with a specific antigen to generate high plasma
titers of a selected antigen. Concurrently, primed B cells are
produced that synthesize the specific antibody. Primed B cells
(plasma cells) are then fused with a myeloma cell line to produce
large quantities of the antibody. Through cell screening
procedures, these hybridoma cells can be selected for the specific
Mab desired and will produce the specific Mab either in cell
culture or after injection into a host animal.
[0040] As is well known in the art, antibodies of the IgG class are
divalent molecules. The so-called Fab portion binds with the
antigen, and the Fc portion can bind with complement proteins or
with other immunity-related cells, such as T cells and macrophages,
thereby eliciting another antigenic-like component. However, an
intact IgG molecule can be reproducibly digested by papain to yield
single Fab fragments or with pepsin to obtain F(ab').sub.2
fragments, both of which lack the Fc domain. Digestion with pepsin
cleaves the F(ab').sub.2 fragment below the disulfide hinge region.
Thus, chemically reducing the disulfide bonds between the hinge
region of the F(ab').sub.2 fragment yields two F(ab') fragments,
each with free, reactive sulfhydryl groups. Since the antigen
binding region is associated with the V (variable) region of the
F(ab) domain, F(ab') fragments maintain the same antigen binding
properties as the intact IgG molecule. Such F(ab') fragments are
advantageous because of the availability of free sulfhydryl groups
for subsequent formation of covalent chemical linkages with
fluorescent labels, anticancer drugs, polymers, and the like.
[0041] Furthermore, Mab's and fragments thereof have been used in
targeted drug delivery. P. Tyler & B. P. Ram, Monoclonal
Antibodies, Immunoconjugates and Lipsomes as Targeted Therapeutic
Systems, in P. Tyle & B. P. Ram eds., Targeted Therapeutic
Systems 3-24 (1990). Antibodies administered to experimental
animals and humans have been targeted to cell surface antigens
presented on the tumor cells. The Mab's can destroy tumor cells
through inhibition of these surface receptors involved in metabolic
activity, or through signaling events. Increased therapeutic
activity of drugs against tumors has been achieved through coupling
cytotoxic drugs with Mab's, thus directing the agent directly to
the tumor mass. S. Ramakrishnan, Current Status of
Antibody-toxinconjugates for Tumor Therapy, in P. Tyle & B. P.
Ram eds., Targeted Therapeutic Systems 189-215 (1990). In addition,
liposomes bound with Mab F(ab') fragments have also been used in
antitumor treatment. The liposomes act as a drug reservoir, which
can be deposited and accumulated in the vicinity of the tumor mass
through the targeting or homing properties of the Mab/F(ab')
fragment. Therefore, Mab's can be administered to an individual
such that the Mab's bind to and accumulate on a selected target
antigen.
[0042] Mab's against the specific beta cell antigen, GAD, can be
prepared according to methods well known in the art. G. Kohler
& C. Milstein, Continuous Cultures of Fused Cells Secreting
Antibody of Pre-defined Specificity, 256 Nature 495-97 (1975);
Wunderlich et al., 17 Eur. J. Cancer Clin. Oncol. 719 (1981);
Schlom et al., 77 Proc. Nat'l Acad. Sci. USA 6841 (1980); E. Harlow
& D. Lane, Antibodies: A Laboratory Manual (1988), hereby
incorporated by reference. A hybridoma cell line generating
anti-GAD-IgG is also available from the ATCC (accession no.
HB184).
[0043] The Mab generated against GAD is digested to obtain the
F(ab') fragment, according to methods well known in the art. J.
Rousseaux et al., Optimal Conditions for the Preparation of
Proteolytic Fragments from Monoclonal IgG of Different Rat IgG
Subclasses, 121 Meth. Enzymol. 663-69 (1986); S. I. Wie et al.,
Characterization of the Proteolytic Fragments of Bovine Colostral
IgG.sup.1, 121 J. Immunol. 98-104 (1978), hereby incorporated by
reference. To further increase the immune reactivity of the F(ab')
fragment, poly(ethylene glycol) (PEG) is conjugated to the F(ab')
molecule. PEG is a linear or branched, neutral polyether, available
in a broad range of molecular weights, and is soluble in water and
most organic solvents. PEG is effective at excluding other polymers
or peptides from its presence when in water, primarily through its
high dynamic chain mobility and hydrophilic nature, thus creating a
water shell or hydration sphere when attached to other proteins or
polymer surfaces. PEG is nontoxic, non-immunogenic, and approved by
the Food and Drug Administration for internal consumption.
[0044] PEG conjugated polyers, proteins, or enzymes have
demonstrated bioactivity, non-antigenic properties, and decreased
clearance rates when administered in animals. F. M. Veronese et
al., Preparation and Properties of Monomethoxypoly(ethylene
glycol)-modified Enzymes for Therapeutic Applications, in J. M.
Harris ed., Poly(Ethylene Glycol) Chemistry--Biotechnical and
Biomedical Applications 127-36 (1992). This is due to the exclusion
properties of PEG in preventing recognition by the immune system.
In addition, PEG has been widely used in surface modification
procedures to decrease protein adsorption and improve blood
compatibility. S. W. Kim et al., Nonthrombogenic Bioactiev
Surfaces, 516 Ann. N.Y. Acad. Sci. 116-30 (1987); H. Jacobs et al.,
Surface Modification for Improved Blood Compatibilty, 12 Artif.
Organs 500-01 (1988); K. D. Park et al., Synthesis and
Characterization of SPUU-PEO-Heparin Graft Copolymers, 29 J. Poly.
Sci, Part A 1725-31 (1991). Hydrophobic polymer surfaces, such as
polyurethanes and polystyrene were modified by the grafing of PEG
(MW 3,400) and employed as nonthrombogenic surfaces. In these
studies, surface properties (contact angle) were more consistent
with hydrophilic surfaces, due to the hydrating effect of PEG. More
importantly, protein (albumin and other plasma proteins) adsorption
was greatly reduced, resulting from the high chain motility,
hydration sphere, and protein exclusion properties of PEG.
[0045] PEG MW 3,400 was determined as an optimal size in surface
immobiliztion studies, K. D. Park et al., Blood Compatibility of
SPUU-PEO-Heparin Graft Copolymers, 26 J. Biomed. Mat. Res. 739-45
(1992), while PEG MW 5,000 was most beneficial in decreasing
protein antigenicity, F. M. Veronese et al., in J. M. Harris ed.,
Poly(Ethylene Glycol) Chemistry--Biotechnical and Biomedical
Applications 127-36, supra.
EXAMPLE 1
Purification of Antibodies from Cell Culture
[0046] A hybridoma cell line producing anti-GAD-IgG (IgG.sub.1),
ATCC no. HB184, was stored frozen (-20.degree. C.). An aliquot of
frozen cells was thawed and suspended in a tissue culture flask
containing 90% Dulbecco's modified Eagle medium with 4.5 g/l
glucose and 10% fetal bovine serum (Hyclone, Logan, Utah). The
medium was renewed every 2-3 days by dilution with fresh medium to
maintain a cell density between 10.sup.5 and 10.sup.6 cells/ml. The
colony was maintained at 37.degree. C. under 6.5% CO.sub.2
atmosphere.
[0047] The ability of the cell suspension to secrete anti-GAD-IgG
was continually monitored using an enzyme linked immunosorbent
assay (ELISA). In this assay, GAD (Sigma Chemical Co., St. Louis,
Mo.) was dissolved in a coating buffer (50 mM sodium phosphate, 1.2
M NaCl, pH 7.5) and the solution (0.5 mg/ml) was transferred to
wells of a microplate (50 .mu.l/well) and incubated for 1 hour at
37.degree. C. The wells were washed with 200 .mu.l of blocking
buffer (10 mM Tris, 1 mM EDTA, 150 mM NaCl, 0.02% Tween 20, 1%
bovine serum albumin, pH 7.3) and then incubated in 250 .mu.l of
blocking buffer for 1 hour at 37.degree. C. After incubation, the
blocking buffer was removed and the microplate was dried. Duplicate
dilutions (50 .mu.l) of samples containing anti-GAD (IgG, F(ab'),
or F(ab')-PEG) (serum or dilutions of chromatographic fractions)
were placed in the wells and incubated for 2 hours at 37.degree.
C., followed by 3 washes with washing buffer (blocking buffer
without BSA). Rabbit anti-IgG.sub.1 mouse immunoglobulin conjugated
with alkaline phosphatase, at a concentration of {fraction (1/200)}
in blocking buffer, was added to all wells and incubated for 2
hours at 37.degree. C., followed by 3 washes with washing buffer.
The wells were then incubated with p-nitrophenol phosphate
chromogen at 1 mg/ml in 1 M diethylethanolamine buffer for 40
minutes at room temperature. The optical density at 405 nm was then
determined using a microplate autoreader (EL311, Bio-Tek
Instruments). These values were compared to standard curves
prepared with known anti-GAD concentrations to extrapolate the
anti-GAD antibody concentration.
[0048] The IgG-containing cell culture medium was isolated by
centrifuging the cell suspension for 10 minutes at 1400 g and
discarding the cell pellet. The supernatant containing anti-GAD-IgG
was then concentrated 10 to 15 times against a sodium phosphate
buffer with a concentrator (Centriprep, MWCO=100,000).
[0049] The concentrated medium was then filtered through a 0.2
.mu.m filter (Sterile Acrodisc, Gelman Sciences) and loaded onto a
DEAE-cellulose column, previously equilibrated with 25 mM sodium
phosphate pH 7.3. The bound proteins were eluted with PBS buffer at
pH 7.33 (40 mM dibasic sodium phosphate, 10 mM monobasic sodium
phosphate, 1.5 M NaCl) at a flow rate of 1 ml/minute. After each
run, the column was rinsed with 2 volumes of 0.1 M HCl, followed by
2 volumes of 0.1 M NaOH, and reequilibrated with 25 mM sodium
phosphate, pH 7.3. Typically, antibodies were eluted in the
non-retained fraction. The purity of the fraction was characterized
by electrophoresis (Phastgel 8/25, Pharmacia) under native and
denaturing conditions. The activity of the anti-GAD-IgG fraction
was estimated by ELISA as described above. This procedure typically
resulted in a yield of about 70-80% of the initial anti-GAD-IgG
concentration.
EXAMPLE 2
Purification of Antibodies from Ascites Fluid
[0050] Ascites is an intraperitoneal fluid extracted from mice with
peritoneal tumors. Ascites fluid results from hybridoma cells being
injected intraperitoneally into mice for growth of the hybridoma
cells and Mab production. The hybridoma cells grow to high
densities in the peritoneal cavity and continually secrete
antibodies specific to the hybridoma cell line. Typically, antibody
production by this method yields approximately 3 ml of ascites
fluid per mouse, containing between 5 and 10 mg/ml of IgG,
representing between 90-98% of the crude antibody titer.
[0051] Retired Breeders BALB/c mice were purchased from Jackson
Laboratory. The mice were primed with pristane, which acts as an
intraperitoneal irritant to recruit nutrients, monocytes, and
lymphoid cells into the peritoneum, thereby creating a favorable
growth environment for the hybridoma cells. The mice were then
injected with hybridoma cells (ATCC no. HB184) based on the
following timetable. Day 0 is the end of the isolation period, upon
which the mice were each injected intraperitoneally with 0.5 ml of
pristane. On day 3, each mouse was again injected with pristane as
before. On day 10, each mouse was injected intraperitonally with
0.5 ml of hybridoma cell suspension (ATCC no. HB184) containing
10.sup.6 cells per ml. Between days 20 and 25, the mice were
euthanized by carbon dioxide asphyxiation, and the ascites fluid
was extracted from the intraperitoneal cavity. After about 25 days,
the animals were euthanized with CO.sub.2 gas, and the ascites
fluid was extracted from the peritoneal cavity. The fluid was
centrifuged and the pellet containing the red cells and the fibrin
clot was discarded. The ascites fluid was then stored at
-20.degree. C. before purification.
[0052] The anti-GAD-IgG from the ascites fluid was purified by
precipitation with saturated ammonium sulfate (SAS), followed by
anion exchange chromatography and protein A-SEPHAROSE
chromatography. The ascites fluid from 10 mice was slowly added to
an equal volue of SAS at 4.degree. C. under agitation. The solution
was centrifuged and the supernatant was separated from the
precipitate, both being retained. The pellets were redissolved in
PBS and precipitated again with an equal volume of SAS, the
supernatant was reprecipitated with 0.5 volume of SAS, and then the
two mixtures were incubated overnight at 4.degree. C. After
incubation, the two solutions were centrifuged, the supernatant of
each was discarded, and the pellets were redissolved in a volume of
PBS corresponding to 0.25 of the initial volume of ascites fluid.
The solutions were dialyzed overnight at 4.degree. C., filtered
through a 0.2 .mu.m filter, and eluted on a DEAE-cellulose column,
as described in Example 1.
[0053] Depending on the purity, a second chromatographic
purification on a protein A-SEPHAROSE column may be necessary. The
Mab-containing fraction was diluted with an equal volume of binding
buffer (1.5 M glycine, 3 M NaCl, pH 8.9). The bound protein was
eluted from the protein A column with 0.1 M sodium citrate, pH 6.0,
and subsequently with 0.1 M sodium citrate, pH 3.0. After each
purification, the column was rinsed with two volumes of 6 M
guanidine. Typically, the anti-GAD antibody emerged as a sharp peak
eluted in 0.1 M sodium citrate, pH 6.0. The yield of the
anti-GAD-IgG from ascites fluid was typically 30-40%.
[0054] It was anticipated that the antibodies isolated during the
previous procedures were a mixture of anti-GAD and indigenous mouse
antibodies. As a final purification step, a GAD-immobilized
affinity column was prepared that binds only anti-GAD-IgG.
CDI-activated SEPHAROSE beads (Pierce Chemical Co.) were covalently
bound to GAD through .epsilon.-amino groups of lysine residues. The
beads were prepared and the GAD coupled to the beads according to
the manufacturer's specifications. The Mab preparation was
dissolved in 0.1 M borate buffer (pH 8.5) and added to the
CDI-activated bead slurry (about 2 mg GAD/ml of beads) and gently
mixed overnight at 4.degree. C. The beads were filtered and washed
with 2 M Tris (pH 8.0) to block remaining CDI groups. The Mab
solution in PBS was then bound to the beads packed in a column. The
column was washed with 10 volumes of PBS to remove unbound IgG.
Anti-GAD-IgG was displaced and eluted by an ionic strength
gradient. The purified fractions were pooled, dialyzed,
lyophilized, and stored at -20.degree. C.
EXAMPLE 3
Preparation of Anti-GAD-F(ab') Fragments
[0055] The anti-GAD-IgG prepared according to the procedures of
either Example 1 or Example 2 was enzymatically digested and then
chemically reduced to obtain F(ab') fragments, which could then be
coupled to PEG. The rationale behind this procedure is to obtain an
antibody fragment capable of binding to the GAD antigen yet which
lacks the Fc domain, and is conjugated with PEG to further decrease
protein and cellular interactions.
[0056] Digestion of intact IgG to yield F(ab').sub.2 domains is a
common laboratory procedure. Anti-GAD-IgG was dissolved in PBS at a
concentration of 1-2 mg/ml, and then 1 ml of 100 mM sodium citrate
(pH 4.2) was added. The pH of the solution was maintained at pH 4.2
with the addition of 0.1 M acetic acid. Pepsin (1 mg/ml),
corresponding to a ratio of 6 mg pepsin per 33 mg anti-GAD-IgG, was
added to the Mab solution and incubated for 12 hours at 37.degree.
C. A person of ordinary skill in the art can modify the incubation
time and ratio of pepsin to antibody, if necessary, to obtain an
optimal yield of F(ab').sub.2 fragments. The digestion reaction was
terminated by adjusting the pH of the solution to pH 6.5 with 0.1 M
carbonate buffer (pH 9.5). The reaction mixture was then
immediately loaded onto a size exclusion chromatography column
(HILOAD 16/60, Pharmacia).
[0057] Additional purification of the F(ab').sub.2 fragments was
performed on a protein A column. The Fc fragments and intact IgG
molecules bind to protein A, whereas the F(ab').sub.2 fragments do
not. The F(ab').sub.2 peak was identified by UV spectroscopy at 280
nM, and the F(ab').sub.2 fractions were collected, lyophilized, and
stored at -20.degree. C.
[0058] The disulfide bonds joining the two heavy chain fragments of
the F(ab').sub.2 were reduced with dithiothreitol (DTT) to yield
two F(ab') fragments per F(ab').sub.2 fragment, each with free,
reactive sulfhydryl groups. An aliquot of F(ab').sub.2 fragments
(0.67 ml at 3 mg/ml) was added to 0.23 ml of buffer (100 mM sodium
acetate, 88 mM NaCl, pH 5.5) and 0.1 ml of 200 mM DTT, and then was
gently mixed at room temperature for 90 minutes.
[0059] Separation of unreacted F(ab').sub.2 fragments and reagents
was perfomed on a PB10 GPC column using 100 mM sodium acetate, 88
mM NaCl, pH 5.5, as the elution buffer. Chromatography was
performed in a nitrogen atmosphere (in a glove box) with all
reagents and buffers thoroughly degassed and flushed with nitrogen.
The elution volume containing the F(ab') fragments was determined
by UV spectroscopy at 280 nm.
EXAMPLE 4
Activation of Methoxy-PEG-amine
[0060] As discussed previously, poly(ethylene glycols) (PEGS) with
molecular weights between about 2,000 and 8,000 have been used to
prevent plasma protein adhesion on blood contacting surfaces and to
decrease the antigenicity of foreign immunogenic proteins and
enzymes. Therefore, PEGs of various molecular weights are coupled
to the F(ab') fragments through the sulfhydryl groups thereof.
These anti-GAD-F(ab')-PEG compositions maintain ability to bind to
islet/beta cells while the PEG moiety masks the remainder of the
F(ab') molecule from eliciting additional immunological events.
[0061] PEG coupling to F(ab') fragments can use a
heterobifunctional crosslinking agent, such as sulfosuccinimidyl
4-(N-maleimidomethyl)cycloh- exane-1-carboxylate) (sulfo-SMCC;
Pierce Chemical Co.). This agent is first coupled to an amine group
of methoxy-PEG-amine
(CH.sub.3--[O--CH.sub.2--CH.sub.2].sub.n-NH.sub.2) and then is
linked to the F(ab') fragment by the sulfydryl group thereof (FIG.
1).
[0062] Methoxy-PEG-amine, with a molecular weight ranging from
about 200 to 8,000 (2.times.10.sup.-4 moles) is dissolved in 0.1
sodium phosphate, pH 9.0. A 0.5 molar equivalent of sulfo-SMCC
(0.044 mg, 1.times.10.sup.-4 moles) is added and the reaction
proceeds for 24 hours at 4.degree. C.
[0063] A cationic exchange column is used to separate the reaction
mixture into substituted and unsubstituted SMCC-PEG derivatives.
The reaction mixture containing 100 mg of PEG derivatives is
applied to a MONO S chromatography column (Pharmacia) connected in
line with a fast protein liquid chromatography (FPLC; Pharmacia)
system. A linear gradient of NaCl is used to elute the PEG
derivatives, wherein the substituted and unsubstituted derivatives
elute at different ionic strengths. The eluate is monitored with a
UV detector set at the maximum absorbance of the maleimide group.
The peak corresponding to the pure substituted SMCC-PEG is
collected, dialyzed against distilled water, lyophilized, and
stored at -20.degree. C.
EXAMPLE 5
Activation of Diamino-PEG
[0064] For cell staining and whole body perfusion (pharmacokinetic)
evaluations, it is useful to label anti-GAD-F(ab')-PEG with, for
example, a radioactive or fluorescent label. In vivo therapeutic
applications of the anti-GAD-F(ab')-PEG generally do not require
such labels. Current methods of labeling antibodies involve forming
conjugates through amine groups (fluorescent or .sup.125I labels)
or through oxidation of tyrosine residues (.sup.125I label). These
labeling methods can interfere with antibody binding through
reaction with the active site of the antibody. Therefore, this
example shows coupling of the label to the PEG moiety. The labeled
PEG moiety is later coupled to the F(ab') fragment. This procedure
assures that labeled and unlabeled compositions have similar
affinities for the antigen.
[0065] The procedure of Example 4 is followed with the exception
that diamino-PEG (H.sub.2N--[O--CH.sub.2--CH.sub.2].sub.n-NH.sub.2)
is used instead of methoxy-PEG-amine. The aim is to modify one
amine group of the diamino-PEG while leaving the other amine group
available for reaction with a label (FIG. 2). The reaction of
diamino-PEG with sulfo-SMCC results in a mixture of di-, mono-, and
un-substituted SMCC-PEG derivatives. This mixture is separated
according to the procedure of Example 4. Elution with low ionic
strength buffer results in elution of the disubstituted SMCC-PEG
derivatives (both amine groups coupled, no ionic binding).
Monosubstituted and unsubstituted PEGs elute at higher ionic
strengths. The pure monosubstituted SMCC-PEG is collected, dialyzed
against distilled water, lyophilized, and stored at -20.degree.
C.
EXAMPLE 6
.sup.125I Labeling of Activated PEG
[0066] In this example, the monosubstituted SMCC-PEG-amine of
Example 5 is labeled by iodination. The SMCC-PEG-amine (0.5 g) is
dissolved in 0.1 M sodium borate (pH 8.5) to a final concentration
of 0.1 mg/ml and transferred to an ice bath. About 1 mCi of
.sup.125I-labeled Bolton-Hunter reagent is added to a 1.5 ml tube
at 0.degree. C., and the solvent is evaporated with nitrogen gas.
The SMCC-PEG-amine solution is added to the dried Bolton-Hunter
reagent and permitted to react for 15 minutes on ice. An equal
volume of "stop" solution (0.5 M ethanolamine, 10% glycerol, 0.1%
xylene cyanol in 0.1 M sodium phosphate, pH 8.5) is added and
incubated for 5 minutes at room temperature. Purification will be
accomplished by dialysis (MWCO 3,500) against distilled water. The
purified product is then lyophilized and stored at -20.degree.
C.
EXAMPLE 7
Fluorescein Labeling of Activated PEG
[0067] In this example, the monosubstituted SMCC-PEG-amine of
Example 5 is labeled with fluorescein isothiocyanate (FITC).
SMCC-PEG-amine (2 mg/ml) is dissolved in 0.1 M sodium carbonate (pH
9.0). FITC is dissolved in dimethyl sulfoxide at a concentration of
1 mg/ml. FITC is slowly added to the SMCC-PEG-amine solution with
stirring until a concentration of 0.05 ml/mg of SMCC-PEG-amine is
reached, and then the reaction is permitted to proceed in the dark
for 8 hours at 4.degree. C. A stop solution (50 mM ammonium
chloride, 0.1% cylene cyanol, and 5% glycerol) is addd an incubated
for 2 hours at 4.degree. C. The resulting SMCC-PEG-FITC is purified
and isolated by dialysis in the dark at 4.degree. C. against
distilled water. The final compound is then lyophilized and stored
at -20.degree. C.
EXAMPLE 8
Coupling of Anti-GAD-F(ab') to Activated PEG
[0068] In this example, a PEG intermediate prepared according to
the procedure of Examples 4, 6, or 7 is coupled to anti-GAD-F(ab')
prepared according to the procedure of Example 3. All of the PEG
intermediates described have maleimide groups for coupling to the
sulfhydryl moieties of F(ab') fragments. The purified
anti-GAD-F(ab') fragments are added to solutions of the selected
PEG intermediate in a molar ratio of 3 PEG intermediates to 2
F(ab') fragments in 100 mM sodium acetate, 88 mM NaCl, pH 5.5. The
reaction proceeds in a nitrogen atmosphere for 24 hours at
4.degree. C.
[0069] Purification of the anti-GAD-F(ab')-PEG is accomplished by
equilibrium dialysis at 4.degree. C. Appropriate precautions are
taken for FITC-labeled (dark) and radioactively-labeled
compositions. The reactions products are placed in cellulose
acetate dialysis bags (MWCO<14,000) and dialyzed against PBS for
48 hours with frequent changes of buffer. The higher molecular
weight anti-GAD-F(ab')-PEG is retained in the bag, while unreacted
PEG intermediates are removed.
[0070] The protein content of the purified composition is
determined by standard protein assay, e.g., M. Bradford, A Rapid
and Sensitive Method for the Quantitation of Microgram Quantities
of Protein Utilizing the Principle of Protein Dye Binding, 72 Anal.
Biochem. 248-54 (1976). Labeled compositions are assayed by, for
example, fluorescence for FITC-labeled compositions or gamma
counting for .sup.125-labeled compositions.
EXAMPLE 9
In Vivo Treatment of NOD Mice with Anti-GAD Mab
[0071] The non-obese diabetic (NOD) mouse is an excellent model to
study Type I diabetes in relation to humans. H. Ikegami et al.,
Immunogenetics and Immunopathogenesis of the NOD Mouse, in G. S.
Eisenbarth ed., Immunotherapy of Diabetes and Selected Autoimmune
Diseases 24-31 (1980); S. Makino et al., Breeding of a Non-obese,
Diabetic Strain of Mice, 12 Exp. Anim. 1-15 (1980). NOD mice
spontaneously develop diabetes within 16 weeks of birth and
demonstrate cell pathology and organ system failure similar to
humans. Generally, overt diabetes begins at about 12 weeks of age
and is observed in nearly 90% of females and 20% of males within 16
weeks. The animals display preclinical antibody titers, insulitis
within 4-6 weeks, and cell infiltration (macrophage, T cell, and B
cell) prior to beta cell destruction, as observed in humans. They
develop clinical symptoms such as polyuria, glycosuria, and loss of
weight, as well as hyperglycemia, increased hemoglobin A1
(glycosylated hemoglobin), and deficit of intrinsic insulin
production. Extensive studies on gene manipulation, immunotherapy,
and lymphocyte inhibition have established the similarity of IDDM
between NOD mice and humans. Therefore, the NOD mouse is an animal
model viewed by one skilled in the art as being reasonably
predictive of utility of a treatment for IDDM in humans.
[0072] In this example, a group of NOD mice was administered
anti-GAD Mab prepared by the procedure of Example 2 by weekly
intraperitoneal injection with either 0.1 or 0.3 mg of the Mab
beginning at 7 weeks of age. Controls were not injected. The
animals were weighed and their blood glucose levels determined
weekly. Blood glucose levels were determined by cutting the tail
and collection a blood sample (about 150 .mu.l) in a capillary
tube. A portion of the blood was placed on commercial glucose test
strips and the blood glucose levels determined. Prior to
experimental evaluation, hyperglycemic animals received 0.3-0.5 IU
of 50:50 regular/NPH insulin twice daily. Animals were considered
to be diabetic with a blood glucose level of >200 mg/dl. FIG. 3
shows that the mice injected with anti-GAD Mab exhibited a
substantial reduction in disease incidence, and those animals that
did develop diabetes did so at a substantially later date than
controls. These results show that administration of anti-GAD
antibodies can both reduce disease incidence and delay the onset of
IDDM.
EXAMPLE 10
[0073] In this example, the procedure of Example 9 is followed
except that the NOD mice are injected with anti-GAD-F(ab')-PEG
prepared according to the procedure of Example 8.
EXAMPLE 11
[0074] In this example, euglycemic NOD mice were injected with
anti-GAD Mab at doses of 0.3 mg and 0.1 mg (5 to 6 mice/group)
according to the procedure of Example 9. The mice were sacrificed
at 32 weeks of age and control (uninjected) mice were sacrificed at
about 20 weeks of age, suffering from hyperglycemia. The pancreata
were removed, fixed with 10% buffered formalin, embedded in
parafin, sectioned at 5 .mu.m, and stained with hematoxylin and
eosin. The severity of insulitis was assessed using the following
scoring system: (0) normal islet; (1) mononuclear infiltration in
less than 25% of the islet; (2) 25 to 50% of the islets
infiltrated; (3) over 50% infiltrated; (4) extensive intra-islet
infiltration with obvious beta cell damage. The mean score for each
pancreas was calculated by dividing the total score by the number
of islets scored (FIG. 4).
[0075] These pancreas histology studies show that the anti-GAD Mab
treated mice have a lower level of insulitis as compared to the
control group by 16 weeks.
EXAMPLE 12
[0076] In this example, the procedure of Example 11 is repeated
with the exception that anti-GAD-F(ab')-PEG prepared according to
the procedure of Example 8.
EXAMPLE 13
[0077] In this example, 8-week-old NOD mice were sacrificed and
their spleens were removed aseptically and placed in ice-cold RPMI
1640 medium. Splenocytes were extracted by disrupting the spleens
in a solution containing 0.15 M Tris, 0.83% NH.sub.4Cl, and then
passing the cells through a fine wire mesh. Splenocytes
(5.times.10.sup.6 cells/well) were cultured for 4 days in different
concentrations of anti-GAD Mab (0 to 10 .mu.g/well) or polyclonal
mouse IgG (0 to 10 .mu.g/well; Zymed). The extent of cell
proliferation in each cell was assayed using a colorimetric
technique using microtetrazolium (MTT). Thus, MTT was dissolved in
sterile PBS, pH 7.3, at a concentration of 5 mg/ml and then filter
sterilized. This solution (25 .mu.l) was added to each well.
Incubation was continued for 4 hours, after which 100 .mu.l of a
solubilizing solution (20% (w/v) SDS, 50% dimethylformamide, 50%
H.sub.2O, adjusted to pH 4.7 by addition of acetic acid and HCl)
was added to each well, and incubation was continued for 4
additional hours. The absorbance of each well was then read at 570
nm. MTT reacts with an enzyme in metabolically active cells such
that it is converted to a product that absorbs at 570 nm. Thus, the
absorbance measured in each well is proportional to the number of
viable cells per well.
[0078] FIG. 5 shows the results of these experiments demonstrating
the effects of the anti-GAD Mab on splenocyte cells. The incidence
of splenocyte activation by anti-GAD Mab demonstrated a direct
effect of anti-GAD Mab on lymphocyte activation. This indicates
that a specific regulation of the idiotypic network by anti-GAD Mab
can induce the regulation of the immune system and prevent or
inhibit diabetes in NOD mice. This activation appears to be
concentration dependent in that there was little or no effect
observed at low concentrations (0-1 .mu.g/well), but there was
significant stimulation of cell proliferation at higher
concentrations (10 .mu.g/well) of anti-GAD Mab. There was no
significant proliferation observed in the polyclonal mouse IgG
treated control cells under the same conditions.
EXAMPLE 14
[0079] In this example, the procedure of Example 13 is followed
with the exception that anti-GAD-F(ab')-PEG prepared according to
the procedure of Example 8 is substituted for anti-GAD Mab.
* * * * *