Peptides derived from glutamic acid decarboxylase

Isolated polypeptides useful in ameliorating GAD-associated autoimmune disease as well as diagnostic and therapeutic methods of using the peptides are disclosed.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to glutamic acid decarboxylase.sub.65 
(GAD.sub.65) polypeptides and methods of using GAD.sub.65 polypeptides 
diagnostically and therapeutically in autoimmune disease. 
2. Description of the Background Art 
Insulin-dependent diabetes mellitus (IDDM; type I diabetes) is one of the 
most common metabolic disorders. In the United States, IDDM affects 
approximately one in 300 to 400 people, and epidemiological studies 
suggest that its incidence is increasing. The disease results from the 
autoimmune destruction of the insulin-producing .beta.-cells of the 
pancreas. More specifically, the preonset stage is characterized by 
"insulitis", in which lymphocytes infiltrate the pancreatic islets and 
selectively destroy the .beta.-cells. Insulitis may be present for many 
years before the onset of clinical symptoms. The typical IDDM presentation 
of hyperglycemia appears only after at least 80% of the insulin-producing 
.beta.-cells are lost. The remaining .beta.-cells are destroyed during the 
next few years. 
Although insulin therapy allows most IDDM patients to lead normal lives, 
this replacement is imperfect and does not completely restore metabolic 
homeostasis. Thus, severe complications which result in dysfunctions of 
the eye, kidney, heart, and other organs are common in IDDM patients 
undergoing insulin therapy. Because of this, it is highly desirable to 
extend the latency period and prevent progression (e.g., through 
administration of immunosuppressant drugs to interfere with the autoimmune 
process and insulin to achieve better control of the effects of sustained 
hypoglycemia) between the start of .beta.-cell destruction and the actual 
requirement of insulin replacement (i.e., when 80% of the .beta.-cells are 
destroyed). Therefore, a diagnostic test which determines the beginning of 
.beta.-cell destruction would allow the clinician to administer 
immunosuppressant drugs (Silverstein, et al., New England Journal of 
Medicine, 319:599-604, 1988) or prophylactic insulin therapy (Keller, et 
al., Lancet, 341:927, 1993) to extend this latency period and thus 
significantly delay the onset of insulin replacement side effects. 
Many IDDM patients have sera which contain antibodies to a 64kD molecule 
(Baekkeskov, et al., J. Clin. Invest., 79:926-934, 1987; Atkinson, et al., 
Lancet, 935:1357-1360, 1990), to islet cell cytoplasmic (ICA) molecules or 
islet cell surface (ICSA) molecules (Bottazzo, et al, Lancet, 1:668-672, 
1980), or to insulin (Palmer, et al., Science, 222:1137-1139, 1983; 
Atkinson, et al., Diabetes, 35:894-898, 1986). Atkinson and coworkers 
(Atkinson, et al., Lancet, 935:1357-1360, 1990) have demonstrated that the 
presence of antibodies to the 64kD molecule in human sera appears to be 
the earliest and most reliable indicator that onset of IDDM symptoms will 
eventually occur. Recently, Baekkeskov and coworkers established that the 
64kD molecule and glutamic acid decarboxylase (GAD) have several antigenic 
epitopes in common and thus they may be identical or very similar 
molecules. Although this identification is an important finding, the use 
of this information as a diagnostic tool for predicting IDDM is quite 
cumbersome and limited unless knowledge of the molecular biology of GAD is 
known. Studies by Kaufman, et al, (J. Clin. Invest., 89:283, 1992) 
established that the 64kD molecule was intact GAD.sub.65. Consequently, 
the cloning and subsequent production of large quantities of GAD.sub.65 or 
a GAD molecule which is antigenically substantially identical to the 
GAD.sub.65 molecule or fragments of the GAD.sub.65 molecule, both of which 
can be easily purified, will allow the development of a diagnostic kit 
designed to predict IDDM as well as effective therapeutic modalities. The 
present invention provides a means for accomplishing these results. 
SUMMARY OF THE INVENTION 
The present invention arose out of the discovery that recombinant DNA 
technology could be used to produce eukaryotic GAD.sub.65 polypeptide and 
that GAD.sub.65 polypeptide could be used in the diagnosis and therapy of 
patients with autoimmune disease. Particularly relevant is the use of 
eukaryotic GAD.sub.65 polypeptide in the diagnosis and therapy of patients 
having, or at risk of having, GAD-associated autoimmune disorders such as 
insulin-dependent diabetes mellitus (IDDM) or stiff man disease. 
A major advantage of the present invention is that it provides the art with 
a ready source of eukaryotic GAD.sub.65 polypeptide corresponding to that 
purified from natural sources, while avoiding the problems associated with 
the isolation of naturally occurring eukaryotic GAD.sub.65 polypeptide 
when separating it from other eukaryotic non-GAD.sub.65 polypeptides. This 
absence of other eukaryotic non-GAD.sub.65 polypeptides is significant in 
that it allows the development of test systems which will only detect 
antibodies specifically reactive with GAD.sub.65 polypeptides. 
Another advantage of providing eukaryotic GAD.sub.65 polypeptide in host 
cells is that by so doing, it is possible to obtain much larger quantities 
of the polypeptide than are currently practicably available from natural 
sources. As a consequence, not only is it possible to use the polypeptide 
of the invention to more accurately classify and treat patients with such 
autoimmune diseases as IDDM, but it is also now possible to provide 
commercially useful quantities of GAD polypeptide for use in diagnostic 
systems and pharmaceutical compositions.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to the manipulation of genetic materials by 
recombinant DNA procedures which make possible the production of 
polypeptides possessing part or all of the primary structural conformation 
for one or more of the epitopes for binding autoantibodies to glutamic 
acid decarboxylase.sub.65 (GAD.sub.65) and for polypeptides that bind to 
MHC receptors to block T-cell recognition. These polypeptides are highly 
useful for the immunological detection of autoantibodies reactive with 
them, since such autoantibodies are pre-diagnostic and indicative of 
autoimmune diseases such as insulin dependent diabetes mellitus and "stiff 
man" syndrome. These polypeptides can also be used for purposes of 
screening drugs, such as those that alter GAD function, and for generation 
of polyclonal and monoclonal antibodies which, in turn, can be used 
diagnostically to detect GAD.sub.65. 
The development of specific DNA sequences encoding eukaryotic GAD.sub.65 
polypeptide for splicing into DNA vectors can be accomplished using a 
variety of techniques. For example, alternative methods which can be 
employed include (1) the isolation of a double stranded DNA sequence from 
the genomic DNA of the eukaryote; (2) the chemical manufacture of a DNA 
sequence to provide the necessary codons for the polypeptide of interest; 
and (3) the in vitro synthesis of a double stranded DNA sequence by 
reverse transcription of mRNA isolated from a eukaryotic donor cell. In 
the latter case, a double stranded DNA complement of MRNA is eventually 
formed which is generally referred to as CDNA. 
The manufacture of DNA sequences is frequently the method of choice when 
the entire sequence of amino acid residues of the desired polypeptide 
product is known. When the entire sequence of amino acid residues of the 
desired polypeptide is not known, the direct manufacture of DNA sequences 
is not possible and the method of choice is the formation of cDNA 
sequences. Among the standard procedures for isolating cDNA sequences of 
interest is the formation of plasmid-carrying cDNA libraries which are 
derived from reverse transcription of mRNA which is abundant in donor 
cells that have a high level of genetic expression. When used in 
combination with polymerase chain reaction technology, even rare 
expression products can be cloned. In those cases where significant 
portions of the amino acid sequence of the polypeptide are known, the 
production of labeled single or double stranded DNA or RNA probe sequences 
duplicating a sequence putatively present in the target cDNA may be 
employed in DNA/DNA hybridization procedures which are carried out on 
cloned copies of the cDNA which have been denatured into a single stranded 
form (Jay, et al., Nucleic Acid Research, 11:2325, 1983). 
Hybridization procedures are useful for the screening of recombinant clones 
by using labeled mixed synthetic oligonucleotide probes wherein each is 
potentially the complete complement of a specific DNA sequence in the 
hybridization sample which includes a heterogeneous mixture of denatured 
double stranded DNA. For such screening, hybridization is preferably 
performed on either single stranded DNA or denatured double stranded DNA. 
These procedures are particularly useful in the detection of cDNA clones 
derived from sources where an extremely low amount of mRNA sequences 
relating to the polypeptide of interest are present. In other words, by 
using stringent hybridization conditions directed toward avoidance of 
non-specific binding, it is possible, for example, to allow the 
autoradiographic visualization of a specific cDNA clone by the 
hybridization of the target DNA to that single probe in the mixture which 
is its complete complement (Wallace, et al., Nucleic Acid Research, 9:879, 
1981). 
In addition, a GAD cDNA library can be screened by injecting the various 
cDNAs into oocytes, allowing sufficient time for expression of the cDNA 
gene products to occur, and testing for the presence of the desired cDNA 
expression product, for example, by using antibody specific for GAD.sub.65 
polypeptide, by using functional assays for GAD.sub.65 enzymatic activity, 
or by measuring the ability of the expression product to stimulate 
pathogenic T-cells. 
Alternatively, a cDNA library can be screened indirectly for GAD.sub.65 
peptides having at least one epitope using antibodies to GAD.sub.65 (Chang 
and Gottlieb, J. Neurosci, 8:2123, 1988). Such antibodies can be either 
polyclonally or monoclonally derived and used to detect expression product 
indicative of the presence of GAD.sub.65 cDNA. Preferred are antibodies 
directed to an epitope found in the first 100 amino acids of the 
N-terminal portion of GAD.sub.65. 
Of the three above-noted methods for developing specific DNA sequences for 
use in recombinant procedures, the use of genomic DNA isolates, is the 
least common. This is especially true when it is desirable to obtain the 
microbial expression of mammalian polypeptides because of the presence of 
introns. 
The present invention provides novel polypeptides of GAD.sub.65 which have 
part or all of the primary structural conformation, that is, a continuous 
sequence of amino acid residues, having at least one epitope for 
antibodies to GAD.sub.65 or at least one determinant for T-cell 
recognition. It is possible to use the polypeptide fragments of the 
invention rather than intact GAD to detect autoantibodies to GAD. The term 
"polypeptide," as applied to GAD polypeptide, includes any sequence of 
amino acids having an epitope for autoantibodies to GAD or binds to a 
T-cell MHC receptor. 
Thus, the polypeptide fragments of GAD encompassed by the invention possess 
a biological activity such as the ability to induce and/or bind 
autoantibodies to GAD, bind to T-cell MHC receptors (especially receptors 
on pathogenic T-cells) and the like. 
The polypeptides resulting from microbial expression of the DNA sequences 
of the invention or from other synthetic techniques, such as solid-phase 
peptide synthesis, can be further characterized by their freedom from 
association with other eukaryotic polypeptides or other contaminants which 
might otherwise be associated with GAD in its natural cellular environment 
or in such extracellular fluids as plasma or urine. 
Studies by the present inventors unequivocally establish that GAD.sub.65 
and GAD.sub.67 are encoded by distinct genes and are not produced, for 
example, by post-transcriptional or post-translational modification of a 
common genomic sequence. Evidence proving that GAD.sub.65 and GAD.sub.67 
are encoded by different genes include: (a) the largest contiguous 
sequence of exact identity between GAD.sub.65 and GAD.sub.67 cDNAs is only 
17 nucleotides in length, (b) cDNAs from GAD.sub.65 and GAD.sub.67 do not 
cross hybridize with each other's or with each other's mRNA under low 
stringency conditions (2.0.times.SSC, 0.01% SDS, 23.degree. C.), and (c) 
GAD.sub.65 and GAD.sub.67 cDNAs do not cross hybridize with isolated 
genomic clones encoding GAD.sub.67 and GAD.sub.65, respectively. 
The term "host" includes not only prokaryotes, but also such eukaryotes as 
yeast, filamentous fungi, plant and animal cells, as well as insect cells 
which can replicate and express an intron-free DNA sequence of eukaryotic 
GAD.sub.65. However, prokaryotes are preferred as the host organism for 
screening purposes while eukaryotic cells, especially insect cells, are 
preferred for expression. 
The term "prokaryotes" includes all bacteria which can be transformed or 
transfected with the gene for the expression of GAD.sub.65. Prokaryotic 
hosts may include gram negative as well as gram positive bacteria such as, 
for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus 
subtilis. 
A recombinant DNA molecule coding for the GAD.sub.65 polypeptides can be 
used to transform or transfect the host using any of the techniques 
commonly known to those of ordinary skill in the art. Especially preferred 
is the use of a plasmid or a virus containing the GAD.sub.65 coding 
sequence for purposes of prokaryotic transformation or transfection, 
respectively. Alternatively, liposomes containing the DNA of interest can 
be used to obtain expression in the host (Zhu, et al., Science, 251:209, 
1993) 
Methods for preparing fused, operably linked genes and expressing them in 
bacteria are well-known in the art (Maniatis, et al., Molecular Cloning: A 
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y., 1989). The genetic constructs and methods described therein can be 
utilized for expression of GAD.sub.65 in prokaryotic hosts. 
In general, expression vectors containing promoter sequences which 
facilitate the efficient transcription of the inserted eukaryotic genetic 
sequence are used in connection with the host. The expression vector 
typically contains an origin of replication, a promoter, and a terminator, 
as well as specific genes which are capable of providing phenotypic 
selection of the transformed cells. The transformed prokaryotic hosts can 
be grown in fermentors and cultured according to techniques known in the 
art to achieve optimal cell growth. The polypeptides of the invention can 
then be isolated from the grown medium, cellular lysates, or cellular 
membrane fractions. 
The isolation and purification of the expressed polypeptides of the 
invention may be by any conventional means such as, for example, 
preparative chromatographic separations and immunological separations such 
as those involving the use of monoclonal or polyclonal antibody. 
By having provided the sequence of amino acid residues of GAD.sub.65, the 
present invention provides for the manufacture of DNA sequences which code 
for the host expression of polypeptide analogs or derivatives of 
GAD.sub.65 which differ from naturally-occurring forms in terms of the 
identity or location of one or more amino acid residues and which share 
some or all of the epitopes of naturally-occurring polypeptide forms. 
The novel DNA sequences of the invention include all sequences useful in 
providing the expression in prokaryotic or eukaryotic host cells of 
polypeptides which have at least a part of the primary structural 
conformation for one or more epitopes capable of reacting with 
autoantibodies to GAD.sub.65 which are comprehended by: (a) the DNA 
sequence as set forth in FIGS. 2 or 3 or their complementary strands; (b) 
DNA sequences which hybridize to DNA sequences defined in (a) or fragments 
thereof; and (c) DNA sequences which, but for the degeneracy of the 
genetic code, would hybridize to DNA sequences defined in (a) and (b) 
above. Specifically comprehended in (b) are genomic DNA sequences which 
encode allelic variant forms of GAD.sub.65. Part (c) specifically 
comprehends the manufacture of DNA sequences which encode GAD.sub.65, 
GAD.sub.65 fragments, and GAD.sub.65 analogs wherein the DNA sequences 
thereof may incorporate codons which facilitate translation of mRNA in 
non-vertebrate hosts. 
Since the cDNA sequence of the invention encodes essentially the entire 
human or rat GAD.sub.65 molecule, it is now a matter of routine to 
prepare, subclone, and express smaller polypeptide fragments of cDNA from 
this or a corresponding cDNA sequence which would encode as few as one 
epitope for autoantibodies to human or rat GAD.sub.65. The presence of 
such an epitope on a cloned polypeptide can then be confirmed using, for 
example, serum from a patient with autoantibodies to GAD.sub.65. An 
example of such a smaller peptide is the first approximately 100 amino 
acids from the N-terminus of GAD.sub.65 (shown in FIG. 3). This amino acid 
sequence is essentially absent from GAD.sub.67. Other examples of specific 
peptides of the invention are shown in Table 7 as well as the approximate 
carboxy-terminal two-thirds of GAD from about amino acid 224 to about 
amino acid 585. Especially preferred in the carboxy-terminal two-thirds of 
GAD is the amino acid segment from about amino acid 224 to about amino 
acid 398. 
The present invention further relates to monoclonal antibodies which are 
specific for the polypeptides of the invention as well as the diagnostic 
and therapeutic use of these monoclonal antibodies. This specificity 
enables the monoclonal antibody, and like monoclonal antibodies with like 
specificity, to be used to bind the polypeptide of the invention when the 
polypeptide, or amino acids comprising the polypeptide, are present in 
specimens or a host, such as a human. 
Numerous techniques can be utilized to produce the monoclonal antibodies of 
the invention without resorting to undue experimentation. To a great 
extent, the products of such monoclonal antibodies is rendered routine 
because of the highly defined nature of the polypeptides of the invention. 
Thus, whether the polypeptides of the invention are used for immunization 
and/or screening, the very limited number of immunogenic determinants on 
the polypeptides greatly simplifies the identification of cell lines 
producing monoclonal antibodies of the invention, for example, by limiting 
the repertoire of clonal expression possible. 
One very useful type of cell line for expression of the monoclonal 
antibodies of the invention is the hybridoma. The general method used for 
production of hybridomas producing monoclonal antibody is well known 
(Kohler and Milstein, Nature, 256:495, 1975). The resulting hybridomas 
were then screened for production of monoclonal antibodies capable of 
binding to the polypeptides of the invention. 
The techniques of sensitization and/or immunization, cell fusion, ascites 
production, selection of mixed hybridomas, or subcloning of monoclonal 
hybridomas are generally well known in the art. Attention is directed to 
Koprowski, et al., U.S. Pat. No. 4,172,124, Koprowski, et al., U.S. Pat. 
No. 4,196,265, or Douillard, J. Y. and Hoffman, T., Basic Facts about 
Hybridomas, in Compendium of Immunology, Vol. II, L. Schwartz, ed. (1981), 
which are herein incorporated by reference. In general, the purified 
peptides can be modified to have a cystine attached at the C-terminus to 
permit unidirectional attachment of the synthetic peptide to an 
immunogenic protein through a connecting bridge, for example, 
maleimidobenzoylated (MB)-keyhole limpet hemocyanin (KLH). Other 
immunogenic conjugates can also be used, for example, albumin, and the 
like. The resulting structure may have several peptide structures linked 
to one molecule of protein. 
Somatic cells derived from a host immunized against the synthetic peptides 
can be obtained by any suitable immunization technique. The host subject 
is immunized by administering the antigen, usually in the form of a 
protein conjugate, as indicated above, by any suitable method, preferably 
by injection, either intraperitoneally, intravenously, subcutaneously, or 
by intra-foot pad. Adjuvants may be included in the immunization protocol. 
The initial immunization with the protein bound antigen can be followed by 
several booster injections given periodically at intervals of several 
weeks. The antibody contained in the plasma of each host can then be 
tested for its reactivity with the immunizing polypeptide of the 
invention. The host having the highest response is usually most desirable 
as the donor of the antibody secreting somatic cells used in the 
production of hybridomas. Alternatively, hyperimmunization can be effected 
by repeatedly injecting additional amounts of peptide-protein conjugate by 
intravenous and/or intraperitoneal route. 
The isolation of hybridomas producing monoclonal antibodies of the 
invention can be accomplished using routine screening techniques which 
permit determination of the elementary reaction pattern of the monoclonal 
antibody of interest. Thus, if a monoclonal antibody being tested binds 
with a polypeptide of the invention, then the antibody being tested and 
the antibody produced by the hybridomas of the invention are equivalent. 
Alternatively, since the invention teaches polypeptides or amino acid 
sequences which are specifically required for binding of the preferred 
monoclonal antibodies of the invention, it is now possible to use these 
peptides for purposes of immunization to produce hybridomas which, in 
turn, produce monoclonal antibodies specific for the polypeptide. This 
approach has the added advantage of decreasing the repertoire of 
monoclonal antibodies generated by limiting the number of antigenic 
determinants presented at immunization by the polypeptide. The monoclonal 
antibodies produced by this method can be screened for specificity using 
standard techniques, for example, by binding polypeptide to a microtiter 
plate and measuring binding of the monoclonal antibody by an ELISA assay. 
It is also possible to determine, without undue experimentation, if a 
monoclonal antibody has the same specificity as a monoclonal antibody of 
the invention by ascertaining whether the former prevents the latter from 
binding the polypeptide of the invention. If the monoclonal antibody being 
tested competes with the monoclonal antibody of the invention, as shown by 
a decrease in binding by the monoclonal antibody of the invention, then it 
is likely that the two monoclonal antibodies bind to the same, or a 
closely related, epitope. 
Still another way to determine whether a monoclonal antibody has the 
specificity of a monoclonal antibody of the invention is to pre-incubate 
the monoclonal antibody of the invention with the polypeptide of the 
invention with which it is normally reactive, and then add the monoclonal 
antibody being tested to determine if the monoclonal antibody being tested 
is inhibited in its ability to bind the antigen. If the monoclonal 
antibody being tested is inhibited then, in all likelihood, it has the 
same, or a closely related, epitopic specificity as the monoclonal 
antibody of the invention. 
The GAD.sub.65 of the invention is particularly suited for use in 
immunoassays in which it can be utilized in liquid phase or bound to a 
solid phase carrier. In addition, GAD.sub.65 used in these assays can be 
detectably labeled in various ways. 
Examples of immunoassays which can utilize the GAD.sub.65 of the invention 
are competitive and non-competitive immunoassays in either a direct or 
indirect format. Examples of such immunoassays are the radioimmunoassay 
(RIA), the sandwich (immunometric assay) and the Western blot assay. 
Detection of antibodies which bind to the GAD.sub.65 of the invention can 
be done utilizing immunoassays which run in either the forward, reverse, 
or simultaneous modes, including immunohistochemical assays on 
physiological samples. The concentration of GAD.sub.65 which is used will 
vary depending on the type of immunoassay and nature of the detectable 
label which is used. However, regardless of the type of immunoassay which 
is used, the concentration of GAD.sub.65 utilized can be readily 
determined by one of ordinary skill in the art using routine 
experimentation. 
The GAD and GAD fragments of the invention can be bound to many different 
carriers and used to detect the presence of antibody specifically reactive 
with the polypeptide. Alternatively, the carrier-bound GAD and GAD 
fragments can be used therapeutically for extracorporeal absorption of 
autoimmune antibodies in patients having, or at risk of having, 
GAD-associated disorders. Examples of well-known carriers include glass, 
polystyrene, polyvinyl chloride, polypropylene, polyethylene, 
polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, 
polyacrylamides, agaroses, and magnetite. The nature of the carrier can be 
either soluble or insoluble for purposes of the invention. Those skilled 
in the art will know of other suitable carriers for binding GAD.sub.65, or 
will be able to ascertain such, using routine experimentation. 
There are many different labels and methods of labeling known to those of 
ordinary skill in the art. Examples of the types of labels which can be 
used in the present invention include enzymes, radioisotopes, colloidal 
metals, fluorescent compounds, chemiluminescent compounds, and 
bioluminescent compounds. 
Alternatively, the polypeptide of the invention which comprises the GAD 
enzymatic domain can be used to detect antibodies to GAD by measuring GAD 
enzymatic activity. For example, GAD.sub.65 and a specimen suspected of 
having antibodies to GAD.sub.65 can be incubated for a period of time and 
under conditions sufficient to allow binding to occur between GAD.sub.65 
and the antibodies. The reaction product is precipitated and then tested 
for GAD enzymatic activity. 
For purposes of the invention, the antibody which binds to GAD.sub.65 of 
the invention may be present in various biological fluids and tissues. Any 
sample containing a detectable amount of antibodies to GAD.sub.65 can be 
used. Normally, a sample is a liquid such as urine, saliva, cerebrospinal 
fluid, blood, serum and the like, or a solid or semi-solid such as tissue, 
feces and the like. 
The materials for use in the assay of the invention are ideally suited for 
the preparation of a kit. Such a kit may comprise a carrier means being 
compartmentalized to receive in close confinement one or more container 
means such as vials, tubes and the like, each of the container means 
comprising one of the separate elements to be used in the method. For 
example, one of the container means may comprise GAD.sub.65 bound to a 
carrier. A second container may comprise soluble, detectably-labeled 
second antibody, in lyophilized form or in solution. 
In addition, the carrier means may also contain a plurality of containers 
each of which comprises different, predetermined amounts of GAD.sub.65. 
These latter containers can then be used to prepare a standard curve into 
which can be interpolated the results obtained from the sample containing 
the unknown amount of autoantibodies to GAD.sub.65. 
In using the kit all the user has to do is add, to a container, a 
premeasured amount of a sample containing a measurable, yet unknown amount 
of autoantibodies to GAD.sub.65 to be detected, a premeasured amount of 
carrier-bound GAD.sub.65 present in the first container, and a premeasured 
amount of the detectably labeled second antibody present in the second 
container. Alternatively, the non-detectably labeled GAD.sub.65 can be 
provided attached to the container to which the sample and the detectably 
labeled second antibody are added. After an appropriate time for 
incubation, an immune complex is formed and is separated from the 
supernatant fluid, and the immune complex or the supernatant fluid are 
detected, as by radioactive counting or addition of an enzyme substrate, 
and color development. 
In an alternative embodiment, a kit comprising the GAD polypeptide of the 
invention can be used to detect the stage of GAD-associated autoimmune 
disease in a patient. As further shown herein, Applicants have discovered 
that certain GAD peptides or fragments are associated with different 
levels of progression in the autoimmune disease and that the level of 
disease process can be ascertained by looking at immune cell proliferative 
response, such as that of the pathogenic T-cell of the patient. 
The term "ameliorate" denotes a lessening of the detrimental effect of the 
autoimmune response in the patient receiving therapy. The term 
"therapeutically effective" means that the amount of GAD.sub.65 
polypeptide used is of sufficient quantity to ameliorate the cause of 
disease due to the autoimmune response. 
The GAD.sub.65 polypeptides, including whole GAD.sub.65, of the invention 
can be used therapeutically in patients having, or at risk of having, an 
autoimmune response associated with GAD.sub.65. Such therapy can be 
accomplished, for example, by the administration of GAD.sub.65 polypeptide 
to induce tolerance to GAD. Such administration can utilize unlabeled as 
well as labeled GAD.sub.65 polypeptide. When unlabeled GAD.sub.65 
polypeptide is utilized advantageously, it would be in a form wherein, for 
example, the GAD.sub.65 polypeptides are in fragments which are too small 
to stimulate an immune response, but large enough to bind, or block, the 
continuance of the autoimmune response. For example, GAD.sub.65 could be 
digested enzymatically into epitope-sized peptides (typically 5-12 amino 
acids in length) and thereby bind to Fab binding portions present in the 
body fluids, or on the surface of immune cells, of the patient with 
autoimmune disease. Alternatively, peptides having at least one 
determinant for binding to T-cell MHC receptor can be similarly produced 
or chemically synthesized. 
Alternatively, the GAD.sub.65 polypeptides of the invention can be 
administered labeled with a therapeutic agent. These agents can be coupled 
either directly or indirectly to the GAD.sub.65 polypeptides of the 
invention. One example of indirect coupling is by use of a spacer moiety. 
These spacer moieties, in turn, can be either insoluble or soluble 
(Diener, et al., Science, 231:148, 1986) and can be selected to enable 
drug release from the GAD.sub.65 polypeptide at the target site. Examples 
of therapeutic agents which can be coupled to the GAD.sub.65 polypeptides 
of the invention for immunotherapy are drugs, radioisotopes, lectins, and 
toxins. 
The drugs with which can be conjugated to the GAD.sub.65 polypeptides of 
the invention include compounds which are classically referred to as drugs 
such as mitomycin C, daunorubicin, and vinblastine. 
In using radioisotopically conjugated GAD.sub.65 polypeptides of the 
invention for immunotherapy, certain isotopes may be more preferable than 
others depending on such factors as leukocyte distribution as well as 
stability and emission. Depending on the autoimmune response, some 
emitters may be preferable to others. In general, .alpha. and .beta. 
particle-emitting radioisotopes are preferred in immunotherapy. Preferred 
are short range, high energy .alpha. emitters such as .sup.212 Bi. 
Examples of radioisotopes which can be bound to the GAD.sub.65 
polypeptides of the invention for therapeutic purposes are .sup.125 I, 
.sup.131 I, .sup.90 Y, .sup.67 Cu, .sup.212 Bi, .sup.211 At, .sup.212 Pb, 
.sup.47 Sc, .sup.109 Pd and .sup.188 Re. 
Lectins are proteins, usually isolated from plant material, which bind to 
specific sugar moieties. Many lectins are also able to agglutinate cells 
and stimulate lymphocytes. However, ricin is a toxic lectin which has been 
used immunotherapeutically. This is accomplished by binding the 
.alpha.-peptide chain of ricin, which is responsible for toxicity, to the 
antibody molecule to enable site specific delivery of the toxic effect. 
Toxins are poisonous substances produced by plants, animals, or 
microorganisms that, in sufficient dose, are often lethal. Diphtheria 
toxin is a substance produced by Corynebacterium diphtheria which can be 
used therapeutically. This toxin consists of an .alpha. and .beta. subunit 
which under proper conditions can be separated. The toxic A component can 
be bound to GAD.sub.65 polypeptide and used for site specific delivery to 
a leukocyte expressing a receptor for GAD.sub.65 polypeptide. 
Other therapeutic agents which can be coupled to the GAD.sub.65 
polypeptides of the invention, as well as ex vivo and in vivo therapeutic 
protocols, are known, or can be easily ascertained, by those of ordinary 
skill in the art. 
The present invention also relates to a polypeptide which can be 
administered therapeutically to ameliorate, or utilized diagnostically to 
identify, the disease process in patients having, or at risk of having, 
this disease. The conventional single-letter code used to represent the 
various amino acids relates as follows: 
TABLE 1 
______________________________________ 
Phe: F Leu: L Ile: I Met: M 
Val: V Ser: S Pro: P Thr: T 
Ala: A Tyr: Y His: H Gln: Q 
Asn: N Lys: K Asp: D Glu: E 
Cys: C Trp: W Arg: R Gly: G 
______________________________________ 
A polypeptide sequence of the invention was identified by comparing the 
amino acid sequences of human GAD.sub.65, human GAD.sub.67, and the P2-C 
protein of the picornavirus, coxsackie virus. The P2-C polynucleotide 
plays a role in the virus membrane bound replication complex. These 
analyses established the presence of an extensive sequence similarity 
between both GAD.sub.65 molecules and the coxsackie virus. A core 
polypeptide of six contiguous amino acid residues of the GAD.sub.65 and 
P2-C polypeptide are identical in amino acid sequence. Indeed, of the 24 
amino acids in the polypeptide, 19 are identical or conserved. In 
addition, there also exists a high charge density and the presence of a 
proline residue which would render this region highly antigenic (see Table 
2). 
TABLE 2 
__________________________________________________________________________ 
COMISON OF AMINO ACID SEQUENCES 
Protein Amino Acid Sequence 
__________________________________________________________________________ 
##STR1## 
__________________________________________________________________________ 
The solid line encloses identical amino acids whereas the dashed line 
encloses amino acid residues with similar charge, polarity, or 
hydrophobicity 
In Table 2, the solid line encloses identical amino acids whereas the 
dashed line encloses amino acid residues with similar charge, polarity, or 
hydrophobicity. 
The discovery of this common polypeptide region supports an etiologic role 
for "molecular mimicry" in the precipitation of diabetes. Thus, where a 
patient genetically susceptible to IDDM is infected by a coxsackie virus, 
the immune response to the similar GAD sequence in the patient's 
.beta.-cells. The immunological response is maintained by the 
antigenically similar GAD polypeptides resulting in the eventual 
destruction of the .beta.-cells and the subsequent presentation of IDDM. 
At present, it is believed that the destruction of pancreatic .beta.-cells 
in IDDM is mediated by a cellular autoimmune response. As described 
herein, a polypeptide of the invention can ameliorate the autoimmune 
response to GAD. Because of the complexity of autoimmune disease, it is 
possible to envision numerous possible therapeutic modalities which would 
allow the polypeptides of the invention to be used to ameliorate such 
diseases. In one embodiment, it appears that the polypeptides of the 
invention can be utilized to block recognition by a specific T cell 
receptor (TCR) or an MHC receptor presenting an autoimmune antigen on the 
surface of an antigen presenting cell (APC). The inhibition of such 
recognition might occur, for example, by providing the patient with the 
polypeptide of the invention which, in turn, can displace the autoimmune 
antigen being presented in the antigen-cleft of the MHC receptor. However, 
although not wanting to be bound to a particular theory, it is believed 
that the polypeptides of the invention probably act to induce or restore a 
tolerogenic state by direct interaction with the appropriate TCR on the 
surface of a GAD specific pathogenic T-cell. This latter therapeutic 
approach of direct interaction with the TCR is supported by the examples 
and suggests that suppression of the autoimmune response can be achieved 
through induction of high-zone tolerance by use of high concentrations of 
polypeptide, preferably soluble. Another possible mechanism is that the 
polypeptide of the invention may play a role in anergizing pathogenic T 
cells by binding to the T cell MHC receptor, thereby preventing the 
appropriate costimulatory signal. 
Alternatively, the polypeptides of the invention could be used to stimulate 
a T-suppressor cell population in order to restore self-recognition and, 
thereby, ameliorate the autoimmune disease. Stimulation of T-suppressor 
cell populations could be achieved, for example, by use of a bi-specific 
antibody having one variable region specific for an epitope present on the 
autoimmune antigen residing in the cleft of the MHCII receptor and, a 
second variable region specific for an epitope present on the CD8.sup.+ 
receptor. The production of antibody specific for the polypeptide of the 
invention is a matter of routine to those of skill in the art, as is the 
preparation of bi-specific antibodies having specificity for 2 or more 
epitopes. 
Polypeptide analogs of the present invention may be designed which will 
compete for recognition of self-antigens at the level of antigen 
presentation or induce anergy in T cells, due to a lack of a costimulatory 
signal. Since MHC molecules contain a single peptide binding site, it is 
possible to design polypeptides which will bind with high affinity to 
disease-associated MHC molecules, but will not activate disease-causing 
T-helper cells. Such polypeptides act as antagonists for self-antigen 
recognition. In the present invention, support for this mechanism is found 
in the examples, especially Example 7. Precedent for such an approach 
arises from observation that a mouse lysozyme polypeptide, itself 
non-immunogenic, can compete for MHC binding with an immunogenic 
polypeptide a hen-egg white lysozyme and thereby reduce T cell activation 
by that polypeptide (Adorini, et al., Nature, 334:623-625, 1988) as well 
as studies using T-cell receptor peptides to block formation of complex 
between T-cells, autoantigen and MHC (Howell, et al., Science, 246:668, 
1989). Similarly, such a therapeutic approach for screening effective 
polypeptide analogs has been utilized in such autoimmune diseases as 
experimental autoimmune encephalomyelitis (EAE) (Wraith, et al., Cell, 
59:248, 1989; Urban, et al., Cell, 59:257, 1989). 
The single-letter symbols used to represent the amino acid residues in the 
polypeptides of the present invention are those symbols commonly used in 
the art. The peptides of the invention include not only the natural amino 
acid sequences, but also peptides which are analogs, chemical derivatives, 
or salts thereof. The term "analog" or "conservative variation" refers to 
any polypeptide having a substantially identical amino acid sequence to a 
polypeptide provided herein and in which one or more amino acids have been 
substituted with chemically similar amino acids. For example, one polar 
amino acid, such as glycine or serine, may be substituted for another 
polar amino acid; or one acidic amino acid, such as aspartic acid may be 
substituted for another acidic amino acid, such as glutamic acid; or a 
basic amino acid, such as lysine, arginine, or histidine may be 
substituted for another basic amino acid; or a non-polar amino acid such 
as alanine, leucine, or isoleucine may be substituted for another 
non-polar amino acid. 
The term "analog" or "conservative variation" also means any polypeptide 
which has one or more amino acids deleted from or added to a polypeptide 
of the present invention, but which still retains a substantial amino acid 
sequence homology to such peptide. A substantial sequence homology is any 
homology greater than 70%, preferably at least about 80%, and more 
preferably at least about 90%. The term "fragment" also means any shorter 
version of the polypeptides identified herein having at least 6 amino acid 
residues, wherein the fragment possesses biological activity, or is a 
fragment capable of inhibiting the stimulation of T-cells by a stimulating 
polypeptide fragment or substantially full-length molecule. 
The term "chemical derivative" means any polypeptide derived from a 
polypeptide of the present invention and in which one or more amino acids 
have been chemically derivatized by reaction of the functional side groups 
of amino acid residues present in the polypeptide. Thus, a "chemical 
derivative" is a polypeptide that is derived from the sequences or 
polypeptides identified herein by one or more chemical steps. Such 
derivatized molecules include, for example, those molecules in which free 
amino groups have been derivatized to form amine hydrochlorides, P-toluene 
sulfoamides, benzoxycarboamides, T-butyloxycarboamides, thiourethane-type 
derivatives, trifluoroacetylamides, chloroaceamides, or formamides. Free 
carboxyl groups may be derivatized to form salts, methyl and ethyl esters 
or other types of esters or hydrazides. Free hydroxyl groups may be 
derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen 
of histidine may be derivatized to form N-imbenzylhistidine. Also included 
as chemical derivatives are those polypeptides which contain one or more 
naturally occurring amino acids derivatives of the 20 standard amino 
acids. For example, 4-hydroxyproline may be substituted for proline; 
5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be 
substituted for histidine; homoserine may be substituted for serine, and 
ornithine may be substituted for lysine. 
It should be understood that the present invention is not limited to the 
illustrative polypeptides depicted in Table 2 and Table 9, instead, a 
polypeptide falling within the scope of this invention may extend outside 
of, or comprise less than, the region between amino acid 28 and amino acid 
50 of coxsackie virus P2-C, or between amino acid 250 and amino acid 273 
of GAD.sub.65, or between amino acid 258 and amino acid 281 of GAD.sub.67, 
as well as the region between amino acid 78 and amino acid 97, or between 
amino acid 247 and amino acid 266, or between amino acid 335 and amino 
acid 356, or between amino acid 479 and amino acid 498, or between amino 
acid 509 and amino acid 528, or between amino acid 524 and amino acid 543, 
or between amino acid 539 and amino acid 556, or between amino acid 564 
and amino acid 583 of GAD.sub.65, as long as a substantial part of a given 
polypeptide is characterized by an amino acid sequence from that region, 
or segments or combinations thereof, and the polypeptide demonstrates the 
desired immunological or biological activity against autoimmune disease. 
In addition, polypeptides according to this invention include those having 
amino acid sequences which are longer or shorter in length than those of 
the polypeptides illustrated in Table 2 and Table 9, or which comprise 
segments or combinations thereof, as long as such polypeptides consist 
substantially of the region between the amino acids illustrated in Table 2 
and Table 9 and demonstrate immunological or biological activity. All 
polypeptides of the invention should not stimulate or enhance the 
autoimmune disease. 
Accordingly, it should be understood that the specific selection of any one 
polypeptide within the polypeptides of the invention does not involve 
undue experimentation. Such a selection may be carried out by taking a 
number of polypeptides and testing them for their immunological and 
biological activity in ameliorating the autoimmune disease or for 
detecting antibody. The NOD mouse represents an excellent and well 
characterized model for screening polypeptides of the invention capable of 
ameliorating or preventing diabetes. Example 7 illustrates an acceptable 
procedure for routine screening of candidate polypeptides with biologic 
activity. 
The polypeptides according to the present invention may be prepared by 
recombinant techniques or by conventional synthesis using known 
polypeptide synthetic methods, including synthesis on a solid support. An 
example of a suitable solid phase synthetic technique is that described by 
Merriweather (J.Am.Chem.Soc., 85:2149, 1963). Other polypeptide synthetic 
techniques may be found, for example, in Bodanszky, et al., Peptide 
Synthesis, John Wiley & Sons, 2d ed., 1976, as well as other references 
known to those skilled in the art. A summary of polypeptide synthesis 
techniques can be found in Stewart, et al., Solid Phase Peptide Synthesis, 
Pierce Chemical Company, Inc., Rockford, Ill., 1984. The synthesis of 
polypeptides by solution methods may also be used, for example, as 
described in The Proteins, Vol. II, 3d ed., Neurath, et al., eds., 105, 
Academic Press, New York, N.Y., 1976. Appropriate protective groups for 
use in such synthesis can be found in the above references as well as in 
J. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, 
N.Y., 1973. 
The polypeptides of the invention may also be prepared in an appropriate 
host transformed with DNA sequences that code for the desired polypeptide. 
For example, a polypeptide may be prepared by the fermentation of 
appropriate hosts that have been transformed with and which express a DNA 
sequence encoding the polypeptide. Alternatively, a DNA sequence encoding 
several of the polypeptides of this invention may be linked together and 
those sequences may then be used to transform an appropriate host to 
permit the expression of polypeptides involved in the autoimmune disease. 
The dosage ranges for the administration of the GAD polypeptides of the 
invention are those large enough to produce the desired effect in which 
the symptoms or cellular destruction of the autoimmune response are 
ameliorated. The dosage should not be so large as to cause adverse side 
effects, such as unwanted cross-reactions, anaphylactic reactions, and the 
like. Generally, the dosage will vary with the age, condition, sex, and 
extent of the disease in the patient and can be determined by one of skill 
in the art. The dosage can be adjusted by the individual physician in the 
event of any counterindications. Dosage can vary from about 0.1 mg/m.sup.2 
to about 2000 mg/m.sup.2, preferably about 0.1 mg/m.sup.2 to about 500 
mg/m.sup.2 /dose, in one or more dose administrations daily, for one or 
several days. 
The GAD polypeptides of the invention can be administered parenterally by 
injection or by gradual perfusion over time. The GAD polypeptides of the 
invention can be administered intravenously, intraperitoneally, 
intramuscularly, subcutaneously, intracavity, transdermally, intranasally, 
or enterally. 
Preparations for parenteral administration include sterile aqueous or 
non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous 
solvents are propylene glycol, polyethylene glycol, vegetable oils such as 
olive oil, and injectable organic esters such as ethyl oleate. Aqueous 
carriers include water, alcoholic/aqueous solutions, emulsions or 
suspensions, including saline and buffered media. Parenteral vehicles 
include sodium chloride solution, Ringer's dextrose, dextrose and sodium 
chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include 
fluid and nutrient replenishers, electrolyte replenishers (such as those 
based on Ringer's dextrose), and the like. Preservatives and other 
additives may also be present such as, for example, antimicrobials, 
anti-oxidants, chelating agents, and inert gases and the like. 
The invention also relates to a method for preparing a medicament or 
pharmaceutical composition comprising the GAD.sub.65 polypeptides of the 
invention, the medicament being used for therapy of autoimmune response to 
GAD.sub.65. 
The above disclosure generally describes the present invention. A more 
complete understanding can be obtained by reference to the following 
specific examples which are provided herein for purposes of illustration 
only and are not intended to limit the scope of the invention. 
EXAMPLE 1 
Cloning and Expression of GAD.sub.65 
A. Recombinant DNA Procedures 
In order to obtain cDNA probes specific for GAD.sub.65 and GAD.sub.67, 
total RNA was extracted from adult rat brain by guanidine 
isothiocyanate-cesium gradient using the method of Chirgwin, et al. 
(Biochemistry, 18:5294, 1979). Poly (A) RNA was purified on oligo dT 
cellulose, using the protocol by Bethesda Research Laboratories (BRL). 
First strand synthesis was performed by using MMLV-reverse transcriptase 
(BRL), with conditions suggested, except that poly d(N.sub.6)-mers 
(Pharmacia) were used as primers. This cDNA-RNA mixture was heat 
inactivated at 65.degree. C. for 15 min and stored at -20.degree. C. For 
PCR, 1/50 of the sample was added to the 100 .mu.l reaction. Degenerate 
oligonucleotides were synthesized (Applied Biosystems) to encode the 
underlined common amino acid sequences of feline (from cDNA) (Kobayashi, 
et al., J. Neurosci., 7:2768, 1987) and rat (from peptides) (Chang and 
Gottlieb, J. Neurosci., 8:2123, 1988) GAD (FIG. 1). The 5'-end sequence of 
each degenerate oligonucleotide contained one strand of the DNA sequence 
recognized by either Sstl and HindIII (5' oligo) or Sstl and Sstll (3'-end 
oligo). These primers were used for selective amplification by polymerase 
chain reaction of the generated cDNA template as described by Gould, et 
al. (Proc. NatI. Acad. Sci.,USA, 86:1934, 1989). PCR products were 
subcloned into Hindlll/Sstl double digested Bluescript SK vector 
(Stratagene), transformed into DH5 (BRL), and plated by standard methods 
(Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring 
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). 
Colony hybridization was done with an 5'-.sup.32 P end labeled 
oligonucleotide specific to feline GAD.sub.67 (Kobayashi, et al., J. 
Neurosci., 7:2768, 1987). End labeling of oligonucleotide, hybridization 
conditions, and washing conditions were done as described (Wallace, et 
al., in Guide to Molecular Cloning Techniques; Berger, et al., Eds. in 
Methods of Enzymology; Abelson, et al., Eds. Academic Press, Inc., San 
Diego, 432-442, 1987), except that the nitrocellulose filters were washed 
at 50.degree. C. for 15 min. Colonies which were positive and negative in 
the hybridization were individually picked and grown overnight in Terrific 
Broth (Tartof, et al., Focus, 9:12, 1987). DNA was isolated using a 
boiling method (Maniatis, et al., Molecular Cloning: A Laboratory Manual, 
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989) and 
templates were denatured by 0.2N NaOH and purified by Sephacryl S400 spun 
columns (Pharmacia). Sequencing of denatured double stranded template was 
by the chain-termination method (Sanger, et al., Proc. Natl. Acad. 
Sci.,USA, 74:5463, 1977) using the T7-sequencing kit (Pharmacia). 
As shown in FIG. 1, PCR-generated rat GAD.sub.65 and GAD.sub.67 cDNAs were 
used as probes to screen a lambda ZAP (Stratagene) rat hippocampus library 
provided by S. Heinemann (Salk Institute) by standard techniques 
(Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring 
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). A 2400 nucleotide 
GAD.sub.65 cDNA (the largest clone) was isolated and subcloned by 
"zapping" as described by Stratagene. When a rat GAD.sub.67 cDNA was 
obtained which was smaller than a 3.2 kb rat GAD.sub.67 cDNA clone already 
on hand, the larger cDNA was sequenced. Exo III deletions (Henikoff, Gene, 
25:351, 1984) were made in both directions for GAD.sub.65 and GAD.sub.67 
and templates were prepared and sequenced as described above. Anchored PCR 
(Frohman, et al., Proc.Natl.Acad.Sci.,USA, 85:8998, 1988) was done to 
clone the remaining 5'-ends of GAD.sub.65 and GAD.sub.67 mRNAs which were 
not represented in the original cDNA clones isolated in the library 
screening. Sequencing of these clones revealed that neither GAD.sub.65 nor 
GAD.sub.67 mRNAs contained any further initiation codons (AUGs) in frame 
with the previously designated initiation codons of the original cDNA 
clones. 
EXAMPLE 2 
Characterization of Cloned GAD.sub.65 
A. Northern Blot Hybridization 
Two PCR-derived cDNA probes were hybridized to Northern blots containing 
rat brain RNA in order to determine whether the GAD.sub.67 and GAD.sub.65 
cDNAs were derived from two different mRNAs. RNA was extracted as 
described in Example 1. Poly (A) RNA was separated by electrophoresis in 
formaldehyde and transferred onto Biotrans (ICN) membranes, and 
hybridization was performed as described by Well, et al. (J. Neurosci., 
16:311, 1986), except that 100 .mu./ml of poly (A) was added. Probes were 
labeled to approximately 10.sup.9 dpm/.mu.g by the oligolabeling procedure 
of Feinberg and Vogelstein (Anal.Biochem., 132:6, 1983). Identical results 
were subsequently obtained with full-length clones of GAD.sub.65 and 
GAD.sub.67 cDNAs. 
As shown in FIG. 5, lanes 1 and 2 contain 1 .mu.g of poly (A) selected RNA 
extracted from rat cerebellum. Lane 1 was hybridized to a cDNA probe for 
the rat cognate of feline GAD.sub.67 (Kobayashi, et al., J. Neurosci., 
7:2768, 1987) and lane 2 with a cDNA probe for the rat peptide sequence 
(which corresponds to GAD.sub.65). 
The cDNA probe for the rat peptide sequence hybridized to a 5.7 kb RNA, 
while the cDNA probe for the rat cognate of feline GAD.sub.67 cDNA, 
hybridized to a 3.7 kb RNA. This demonstrates that GAD.sub.65 and 
GAD.sub.67 are not derived from the same mRNA. 
B. Genomic Hybridization of GAD.sub.67 and GAD.sub.65 
In order to investigate the possibility that GAD.sub.67 and GAD.sub.65 
arise from separate genes, cDNAs of both GAD.sub.67 and GAD.sub.65 were 
hybridized to DNA blots containing genomic DNA. 
For Southern blots, genomic DNA was extracted from rat liver as described 
(Kaiser, et al., in DNA Cloning, vol. I, A Practical Approach, D. M. 
Glover ed., IRL Press, Oxford, pp. 38-40, 1985). DNA (10 .mu.g/sample) was 
digested to completion with EcoRI and HindIII using conditions recommended 
by the suppliers (BRL, Gaithersburg, Md.). DNA fragments were separated by 
electrophoresis at 1.5 v/cm for 16 hrs in 0.8% agarose. The DNA was then 
transferred to Zeta-Probe membranes (Bio-Rad), hybridized, and washed, as 
described by Gatti, et al. (Biotechniques, 2:148, 1984), except that 5 
.mu.g/ml Carnation dried milk was substituted for Denhardt's solution. 
Probes for Southern blots were labeled as described in Example 1, above. 
As shown in FIG. 6, genomic DNA digested with HindIII and EcoRI are in 
lanes 1 and 3 and lanes 2 and 4, respectively. GAD.sub.67 cDNA was 
hybridized to lanes 1 and 2, whereas GAD.sub.65 cDNA was hybridized to 
lanes 3 and 4. Numbers along the side of the gel are the DNA fragment 
sizes in kilobases. 
This data shows that the two cDNAs hybridize to genomic fragments of 
different sizes. In addition, the greatest contiguous stretch of identical 
nucleotide sequence of GAD.sub.65 and GAD.sub.67 cDNAs is only 17 
nucleotide bases in length. Thus, GAD.sub.67 and GAD.sub.65 are encoded by 
two distinct genes. 
C. Enzymatic Comparison of GAD.sub.67 and GAD.sub.65 
Studies were done comparing the effect of PLP on the activity of GAD.sub.67 
and GAD.sub.65. In so doing, both cDNAs were subcloned into vectors that 
allowed their expression in bacteria (Studier, et al., J. Mol.Biol., 
189:113, 1986). Overexpression of "fusionless" GAD.sub.65 and GAD.sub.67 
was accomplished by subcloning GAD.sub.65 cDNA into the Ncol site of 
pET-8c and GAD.sub.67 cDNA into the Nhel site of pET-5c vectors (Studier, 
et al., J. Mol.Biol., 189:113, 1986). 
To obtain compatible sticky ends for correct in-frame subcloning of both 
cDNAs, selective amplification was performed by PCR using conditions 
suggested by United States Biochemical (USB), with 200 .mu.M dNTPs and 1.5 
mM MgCl.sub.2 in the mixture and annealing at 55.degree. C. with 20 cycles 
to decrease infidelity of AmpliTAQ (USB). Primers specific for GAD.sub.65 
and GAD.sub.67 contained one DNA strand of the Ncol and Spel recognition 
sites, respectively. Since there is a Nhel restriction site within the 
coding region of GAD.sub.67, Spel (which is compatible with Nhel) was 
used. 
PCR products were subcloned into their respective pET vectors, transformed 
into DH5 and plated as described (Maniatis, et al., Molecular Cloning: A 
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y., 1989). Colonies were picked and grown overnight in LB broth with 50 
.mu.g/ml ampicillin. Subclones with correct orientation were transformed 
into BL21(DE3) strain (Studier, et al., J. MoI. Biol., 189:113, 1986) for 
overexpression. As a negative control, the pET-8C vector with no insert 
was transformed and subsequently induced. Single colonies were picked, 
grown, induced by 1 mM isopropyl-B-D-thiogalacto-pyranoside (IPTG), and 
analyzed on SDS-PAGE gels as described (Sambrook, et al., Molecular 
Cloning a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold 
Spring Harbor, 17.15-17.16, 1989). 
To measure GAD activity, we induced 10 ml cultures of bacteria at 
OD.sub.600 -0.5 with 1 mM IPTG. Two hours after induction, bacteria was 
spun down and resuspended and sonicated in 1 ml of homogenizing buffer (1 
mM phenylmethylsulfonyl fluoride (PMSF), 1 mM 2-aminoethylisothiouronium 
bromide (AET), and 60 mM potassium phosphate, pH 7.1). After sonication, 
cell debris was removed by centrifugation and protein concentration was 
measured (Bradford, Anal.Biochem., 72:248, 1986) in the supernatant 
(supernatant was stored in aliquots at -70.degree. C). Brain homogenates 
were prepared as described (Legay, et al., J. Neurochem., 46:1478, 1986). 
GAD activity was measured as described (Krieger, et al., J. Neurochem., 
33:299, 1984) with 0.2 mM PLP present or absent and 20 .mu.l of brain 
homogenate or bacterial lysate in the incubation mixture. Production of 
.sup.14 CO.sub.2 in bacterial lysates was linear with respect to time of 
incubation and protein concentration. 
TABLE 3 
______________________________________ 
GAD Specific Activity.sup.a 
Fold Increase 
Source - PLP + PLP in Induction 
______________________________________ 
BL21 (DE3) + pET-8c 
12 .+-. 0.4 
9 .+-. 1 -- 
BL21 (DE3) + pET-GAD.sub.65 
115 .+-. 3 
773 .+-. 61 
6.7 
BL21 (DE3) + pET-GAD.sub.67 
160 .+-. 2 
389 .+-. 8 
2.4 
Rat Brain 131 .+-. 5 
216 .+-. 2 
1.6 
______________________________________ 
.sup.a cpms of .sup.14 CO.sub.2 /.mu.gprotein/hr of triplicates .+-. 
S.E.M. 
As shown in Table 3, bacterial lysates containing GAD.sub.65 or GAD.sub.67 
catalyze the conversion of 1-.sup.14 C!-glutamate to GABA and .sup.14 
CO.sub.2. 
PLP stimulates the enzymatic activity of GAD.sub.65 more than GAD.sub.67. 
This greater stimulation probably reflects the faster cycling of 
GAD.sub.65 through the inactivation cycle proposed by Martin and coworkers 
(Martin, Cell.Mol.Neurobiol., 7:237, 1987). This faster cycling suggests 
that GAD.sub.65 contributes more to the pool of apo-GAD that exists in 
vivo (Miller, et al., Brain Res. Bull., 5(Suppl.2):89, 1980). Thus, in 
vivo, PLP appears to regulate GAD.sub.65 activity more than GAD.sub.67 
activity. 
GAD.sub.65 activity in bacterial lysates is similar to the five-fold PLP 
stimulation of GAD activity found in synaptosomes prepared from rat 
substantia nigra (Miller, et al., J.Neurochem., 33:533, 1979). Because 
both GADs are more dependent upon added PLP in bacteria than is the GAD 
activity in crude rat brain homogenates, the endogenous PLP concentration 
of bacteria lysates may be less than rat brain homogenates. 
D. Immunological Identification of GAD.sub.65 and GAD.sub.67 
Rat brain homogenates and bacterial lysates were extracted as described 
above. Equal volumes of loading buffer were added to each sample as 
described (Harlow, et al., Antibodies, A Laboratory Manual, Cold Spring 
Harbor Laboratory, Cold Spring Harbor, N.Y., 1988). Proteins were 
separated by electrophoresis in a 10% acrylamide gel in SDS and 
electrophoretically transferred to nitrocellulose (Harlow, et al., 
Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold 
Spring Harbor, N.Y., 1988). The unreacted sites were blocked with a 
phosphate buffered saline (PBS) solution containing 2% bovine serum 
albumin (fraction V), 1% gelatin, and 1% Triton-X-100 at 42.degree. C. for 
one hr. After washing, the nitrocellulose filter was then cut into three 
sections and incubated with the following primary antibodies: lanes 1 to 4 
with a 1/2000 dilution of the antiserum of Oertel, et al. (Neuroscience, 
6:2689, 1981), which recognizes both GAD.sub.67 and GAD.sub.65 ; lanes 5-8 
with a 1/2000 dilution of K-2 antiserum, which recognizes only GAD.sub.67 
; lanes 9-12 with a 1/2000 dilution of GAD-6 monoclonal antibody, which is 
specific for GAD.sub.65 (Chang, et al., J. Neurosci., 8:2123, 1988). All 
filters were extensively washed and appropriate secondary antibodies were 
incubated and washed. Bound antibodies were detected with .sup.125 
I-labeled protein A and autoradiography. Each lane contained the 
following: lanes 1, 5, and 9 are BL21 (DE3)+pET-GAD.sub.67 ; lanes 2, 6, 
and 10 are BL21(DE3)+pET-GAD.sub.65 ; lanes 3, 7, and 11 are rat brain 
homogenate; and lanes 4, 8, and 12 are BL21(DE3)+pET-8c. 
The immunoblots of bacterially produced GAD.sub.65 and GAD.sub.67 
demonstrated that GAD.sub.65 indeed corresponds to the smaller GAD in 
brain extracts, and GAD.sub.67 to the larger form (FIG. 7). Previous work 
has demonstrated the correspondence of GAD.sub.67 to the larger GAD for 
feline GAD.sub.67, and for mouse GAD.sub.67 (Katarova, et al., 
Eur.J.Neurosci., 2:190, 1990; 235, 1987). The mobilities of bacterially 
produced GAD.sub.65 and GAD.sub.67 (as detected with the antiserum of 
Oertel, et al. (Neuroscience, 6:2689, 1981) are identical to the 
immunoreactive doublet seen in rat brain homogenate. 
The smaller molecular weight and larger molecular weight forms of GAD in 
rat brain are thus identical in antigenicity and size to the products of 
GAD.sub.65 and GAD.sub.67 cDNAs, respectively. Consequently, the two GADs 
in rat brain are GAD.sub.65 and GAD.sub.67. From these data it can also be 
concluded that the molecular identity of the reported PLP-dependent and 
PLP-independent GADs by Tapia (Bayon, et al., J.Neurochem., 29:519, 1977) 
are GAD.sub.65 and GAD.sub.67, respectively. Martin and coworkers (Spink, 
et al., Brain Res., 421:235, 1987) have reported the existence of four 
kinetically different forms of rat brain GAD. However, immunoblotting 
experiments (with the antisera used here) of these forms have not been 
reported. 
E. Distribution of GAD.sub.65 and GAD.sub.67 in RNAs in Brain Tissue 
Experiments were done to determine the distribution of GAD.sub.65 and 
GAD.sub.67 in RNAs in cerebellum using in situ hybridization. 
Transcripts of, respectively, 3.2 kb and 2.3 kb from GAD.sub.65 and 
GAD.sub.67 cDNAs, were radiolabeled with .sup.35 S according to 
Wuenschell, et al. (Proc.Natl.Acad.Sci.,USA, 83:6193, 1986) procedure. 
Hydrolyzed fragments of 200 bp were hybridized to coronal sections of a 
rat cerebellum. Animals were anesthetized under halothane and decapitated. 
The brain was rapidly frozen in dry ice and coronal frozen sections (12 
.mu.m) were fixed for 30 min in freshly prepared 4% formaldehyde in 
phosphate-buffered saline (PBS; 130 mM NaCl, 10 mM Na phosphate, pH 7.0). 
The tissue was dehydrated through graded ethanol solutions and stored at 
-70.degree. C. 
In order to increase tissue permeability, the sections were submitted to 
the following pretreatments: rehydration through graded ethanol solutions 
(5 min each in 95%, 85%, 70%, 50%, and 30% ethanol); PBS (5 min); 0.02N 
HCl (10 min); PBS (5 min); 0.01% Triton N-101 in PBS (1 min); PBS 
(2.times.5 min); 1 .mu.g/ml proteinase K (7.5 min); and glycine (to 
inhibit proteinase K) in PBS (3.times.5 min). Proteinase K was digested 
for 30 min at 37.degree. C. before use. Sections were then incubated at 
37.degree. C. in 50% formamide, 750 mM NaCl, 25 mM EDTA, 0.2% SDS, 0.02% 
BSA, 0.002% Ficoll, 0.02% polyvinylpyrrolidone, 250 .mu.g/ml yeast tRNA, 
250 .mu.g/ml poly A, and 25 mM PPES (pH 6.8). 
For the hybridization, 100 mM DTT, 10% dextran sulfate, and sense or 
antisense .sup.35 S-RNA were added to the prehybridization solution. An 
aliquot (50 .mu.l) of the hybridization solution containing about 3 ng 
(10.sup.6 cpm) of probe (sense or antisense) was added onto the slides. 
Each slide was coverslipped and incubated for 16 hrs at 50.degree. C., 
following which the siliconized coverslips were removed by brief washing 
in 4.times.SSC (1.times.SSC-150 mM NaCl, 60 mM Na citrate, pH 7.0). 
Sections were then treated with ribonuclease A (50 .mu.g/ml in 0.5M NaCl, 
10 mM Na thiosulfate, 1 mM EDTA, 10 mM TrisHCL, pH 8.0) for 20 min at 
37.degree. C. and rinsed for 2 hrs at room temperature in 2.times.SSC, 10 
mM Na thiosulfate, for 30 min at 55.degree. C. Sections were dehydrated in 
ethanol, delipidated in xylene, coated with Kodak NTB2 emulsion and 
exposed for 10 days at 4.degree. C. The emulsion was developed with Kodak 
D19, and the tissue counterstained with cresyl violet. 
Autoradiographic grains were detected using reflected polarized light and 
grain numbers, densities, nd cell areas were determined with an Analytic 
Imaging Concepts image analyzer system. Due to the low background level, 
the criteria for defining a cell "labeled" was based on the presence of 
more than 5 clustered grains. The GAD labeled cells were found scattered 
throughout the brain, enabling the measurement of the number of grains 
over individual cells. The boundary of the cell and the area covered by a 
grain allowed the calculation of the number of grains per cell. This 
analysis was done at a high magnification (800.times.), using both 
reflected polarized light and transmitted light to simultaneously 
visualize the stained cell and the superimposed grains. Numbers are means 
.+-.S.E.M. of "n" cells. 
TABLE 4 
______________________________________ 
GRAINS/CELL 
CELL TYPE 
GAD.sub.67 mRNA 
GAD.sub.66 mRNA 
GAD.sub.67 :GAD.sub.65 
______________________________________ 
Purkinje 172 .+-. 34 (87).sup.a 
43 .+-. 2 (70) 
4.0 
Golgi II 96 .+-. 8 (80) 
64 .+-. 9 (65) 
1.5 
Basket 61 .+-. 12 (102) 
16 .+-. 1 (57) 
3.8 
Stellate 55 .+-. 15 (65) 
18 .+-. 3 (37) 
3.1 
______________________________________ 
.sup.a .+-. S.E.M. (n) 
In all neuronal types GAD.sub.67 mRNA levels are greater. The observations 
with in-situ hybridization are consistent with previous findings (Nitsch, 
J.Neurochem., 34:822, 1980; Denner, et al., J.Neurochem., 44:957, 1985; 
Itoh, et al., Neurochem. Res. 6:1283, 1981) that the ratio of PLP 
dependent to independent GAD activities in the cerebellum is one of the 
lowest in brain regions tested. In addition, as shown in Table 3, the 
order of amounts for GAD.sub.67 mRNA is Purkinje&gt;Golgi II&gt;Basket&gt;Stellate 
cells; in contrast, for GAD.sub.65 mRNA, this order is Golgi 
II&gt;Purkinje&gt;Basket&gt;Stellate cells. 
The expression of GAD.sub.65 and GAD.sub.67 mRNAs thus differs among 
classes of neurons. The contribution of each to total GAD activity in turn 
affects how GABA production is regulated. For example, the substantia 
nigra contains one of the highest ratios of PLP-dependent to 
PLP-independent GAD activities (Nitsch, J. Neurochem., 34:822, 1980). 
Increasing GABA concentration in the substantia nigra by local injection 
of inhibitors of GABA catabolism is especially effective in reducing 
seizure susceptibility (Gale, Fed. Proc., 44:2414, 1985). Experimental 
animals undergoing seizures induced by PLP-antagonists may therefore be 
unable to inhibit seizure propagation because of inhibition of GAD.sub.65 
particularly in nerve terminals within the substantia nigra. 
F. Subcellular Location of GAD.sub.65 and GAD.sub.67 
The distribution of GAD.sub.65 and GAD.sub.67 was evaluated in the S.sub.2 
and synaptosome subcellular fractions. S.sub.2 is a high speed supernatant 
consisting of the cytosol of all cells in the brain, while the 
synaptosomal fraction consists primarily of nerve endings (Gray, et al., 
J. Anat., Lond, 96:79, 1962). For these studies, whole rat brain 
fractionation was performed as described by Booth and Clark (Booth, et 
al., Biochem. J., 176:365, 1978). Protein concentrations were determined 
by Schaffner and Weissman (Schaffner, et al., Anal Biochem. 56:502, 1973). 
Samples were prepared as described (Kaiser, et al., DNA Cloning, Vol I, A 
Practical Approach, D. M. Glover ed. (IRL Press, Oxford, 1985, pp. 38-40), 
and immunoblotting was done as described above using GAD-6 monoclonal 
antibody and K-2 antiserum. Equal amounts of protein (16 .mu.g) were added 
to each lane. Autoradiography showed a linear response of increasing 
amount of .sup.125 I-protein A bound to antibody with protein 
concentrations of 1, 3, 10, 30, 100 .mu.gs with both K-2 antiserum and 
GAD-6 monoclonal antibody (data not shown). 
The results showed that GAD.sub.67 was present in equal amounts in both 
fractions. Since the S.sub.2 fraction contains the cytosolic proteins of 
glial (as well as other non-neuronal) and neuronal cells, the 
concentration of GAD.sub.67 must be greater in neuronal cell bodies than 
in nerve endings. In contrast, the concentration of GAD.sub.65 was greater 
in synaptosomes than in S.sub.2. These subcellular fractionation 
experiments suggest that, in contrast to GAD.sub.65, a much greater 
fraction of GAD.sub.67 is present in cell bodies of neurons than in nerve 
terminals. Thus, subcellular fractionation, like immunohistochemistry, 
shows that GAD.sub.65 and GAD.sub.67 have different subcellular 
distributions. 
In vivo experiments utilizing inhibitors of GABA synthesis and degradation 
have suggested that the GABA pool in neuronal cell bodies is different 
from that in the nerve terminals (ladarola, et al., Mol. Cell. Biochem., 
39:305, 1981). GABA produced by GAD.sub.67 may be involved more in 
cellular metabolism (for example, in the GABA shunt) and in 
dendrodendritic synapses. The dendrites of granule cells in the olfactory 
bulb, which form dendrodendritic synapses with mitral dendrites (Shepard, 
Physiol. Rev., 52:864, 1972) and probably release GABA (McLennan, Brain 
Res., 29:177-184, 1971), label intensely with K-2 antiserum. While not 
shown here, it has also been found greater GAD.sub.67 than GAD.sub.65 mRNA 
levels (2-3 fold) in the olfactory bulb. This distribution is consistent 
with the reported finding that most GAD activity in the olfactory bulb is 
present in S.sub.2 and P.sub.1 (crude nuclear pellet) and not in 
synaptosomes (Quinn, et al., J. Neurochem., 35:583, 1980). 
The differing subcellular distributions of GAD.sub.65 and GAD.sub.67 could 
result from cytoskeletal anchoring or from some unknown protein targeting 
mechanism. Some cytoskeletal proteins have distributions that resemble 
GAD.sub.65 and GAD.sub.67. For example, in cultured sympathetic neurons 
Peng, et al. (J Cell. Biol., 102:252, 1986), demonstrate that 84% of tau 
is in axons while 100% of MAP-2 is in cell bodies and dendrites. In 
addition, 43kd protein, a cytoskeletal protein, is thought to anchor the 
acetylcholine receptor to the underlying membrane cytoskeleton (Flucher, 
et al., Neuron, 3:163, 1989). 
EXAMPLE 3 
Detection of GAD Autoantibodies in Clinical Specimens 
A. Materials and Methods 
1. Patient Specimens. 
Sera from four groups of individuals were selected from a previous study by 
Atkinson and co-workers (Atkinson, et al., Lancet, 335:1357-1360, 1990). 
These groups consisted of: Group (1), 1 new onset IDD patients diagnosed 
according to the established National Diabetes Data Group (NDDG) criteria 
(Gleichman, et al., Diabetes, 36:578-584, 1987) that had been referred to 
the University of Florida, Diabetes Clinics; Group (2), 5 randomly 
selected islet cell cytoplasmic antibody (ICA) negative non-diabetic 
controls without any known family history of autoimmune disease; Group 
(3), 13 individuals whose sera had been collected 3 to 66 months prior to 
their documented clinical onsets of IDD; Group (4), non-diabetic controls 
and relatives, and those who were studied prior to their onsets of IDD; 
and Group (5), 3 patients at risk for IDDM, but where onset has not yet 
occurred. This latter group had been ascertained through ongoing 
prospective ICA screening studies of more than 5000 first degree relative 
of IDD probands, and 8200 individuals from the general population (of 
which 4813 were school children). 
2. Islet Cell Autoantibodies. 
ICA were assayed by indirect immunofluorescence on blood group O cryocut 
pancreatic (Atkinson, et al., Lancet, 335:1357-1360, 1990). All results 
were interpreted on coded samples, with control negative and positive sera 
in each batch. The degrees of ICA positivity were analyzed with the 
guidelines established by the Immunology Diabetes Workshop (IDW) for the 
standardization of ICA (Gleichman, et al., Diabetes, 36:578-584, 1987). 
All positive sera were titered by end point dilution, and the Juvenile 
Diabetes Foundation (JDF) units were determined by reference to a standard 
serum previously calibrated to the international JDF standard of 80 units. 
In the studies reported here, a positive ICA result was defined by 
replicate titers of 10 JDF units or greater. 
3. HLA DR Typing. 
HLA DR typing was performed as adapted from the method described by Van 
Rood and Van Leuwen (Nature, 262:795-797, 1976), using DR trays (One Lamda 
Laboratories, Los Angeles, Calif.). 
4. Human Islet Cells. 
Human pancreatic islets were isolated from cadaveric pancreases and 
maintained in vitro as previously described (Ricordi, et al., Diabetes, 
37:413-420, 1988). The islet cells were metabolically labeled with .sup.35 
S methionine (Amersham, Arlington Heights, Ill.) in vitro (95% air/5% 
CO.sub.2). 
5. Islet Cell Extractions and Immunoprecipitations. 
Islet cells were extracted as previously described by Atkinson, et al. 
(Lancet, 335:1357-1360, 1990) with the following modifications. For 
immunoprecipitation studies, the islet cell lysates were precleared twice 
by incubation (2 h, 4.degree. C.) with either control, IDD serum (100 
.mu.l), or GAD-6 (Chang, et al., J.Neuro, 8:2123-2130, 1988) (1 .mu.l in 
99 .mu.l of Tris buffer (Atkinson, et al., Lancet, 335:1357-1360, 1990) 
for every 1000 islets. Immune complexes were then absorbed (1 h 4.degree. 
C.) with an excess of protein A Sepharose CL-4B (Pharmacia, N.J.). Aliquot 
volumes representing 1000 islet cells containing unbound (precleared) 
lysate were then incubated (12 h, 4.degree. C.) with either IDD or control 
sera (25 .mu.l), or GAD-6 (Chang, et al., J. Neuro, 8:2123-2130, 1988) (1 
.mu.l in 25 .mu.l Tris buffer). Following another incubation with protein 
A Sepharose CL-4B (1 h, 4.degree. C.), the complexes were then washed 5 
times with 50 mM Tris HCL (pH 7.4) with 0.1% SDS, 1.0% Triton X-114, and 2 
mM EDTA, and then washed again one time in double distilled water. The 
protein A Sepharose CL-4B was then boiled in Laemmli sample buffer 
(Laemmli, Nature, 227:680-685, 1970), and the samples were subjected to 
SDS-PAGE and fluororadiography (Kodak, X-omat AR5) using Enhance (New 
England Nuclear). Alternatively, the autoradiographs were analyzed by a 
BETAGEN (Boston, Mass.) analyzer. Both 64 KA positive and negative sera 
were used in each assay, to serve as interassay controls. All 
fluororadiographs were analyzed and rated as positive or negative after 
comparison with the known interassay controls. Positive serum samples were 
designated as 1 when a sample resulted in immunoprecipitation of a low 
intensity 64,000 M.sub.r band, 2 if a moderate intensity band was observed 
and 3 if the intensity of the immunoprecipitated protein was high. A 
similar rating procedure was employed for the intensity of bands 
corresponding to immunoprecipitated .sup.35 S-GAD.sub.65 and .sup.35 
S-GAD.sub.67. 
6. Immunoprecipitations. 
Immunoprecipitation of bacterial lysates containing .sup.35 -GAD.sub.65 or 
.sup.35 S-GAD.sub.67, and GAD from human brain homogenate, was completed 
as described above in immunoprecipitation studies of human islet cell 
extractions. 
7. GAD Assays. 
Human brain homogenates were incubated with patient sera as described above 
in human islet cells. After absorption and washes, the protein A agarose 
slurry was aliquoted into three equal volumes and GAD activity was 
measured as described (Krieger, et al., Neurochem. 33:299, 1984). Briefly, 
Protein A agarose beads were incubated with (1-.sup.14 C)-glutamate 
(Amersham) in a designated incubation mixture (Krieger, et al., J. 
Neurochem. 33:299, 1984) and production of .sup.14 CO.sub.2 was 
quantitated by a liquid scintillation counter. 
8. Production of .sup.35 S-GAD.sub.65 and .sup.35 S-GAD.sub.67. 
Rat GAD.sub.65 and GAD.sub.67 cDNAs were subcloned into a bacterial 
expression system as previously described. Labeling of .sup.35 S-GADs was 
completed by pulsing IPTG induced bacterium (growing in Minimal Media) for 
15 minutes with TRAN .sup.35 S-label (ICN). Cultures were then spun down 
and resuspended and sonicated in 1 ml of homogenizing buffer (1 mM 
phenylmethylsulfonyl fluoride (PMSF), 1 mM 2-aminoethylisothiouronium 
Bromide (AET) and 60 mM potassium phosphate, pH 7.1). After sonication, 
cell debris was removed by centrifugation and protein concentration was 
measured (Bradford, Anal.Biochem., 72:248, 1986) in the supernatant 
(supernatant was stored in aliquots at -70.degree. C.). 
B. Immunoreactivity of IDDM Specimens 
Sera from patients with IDDM were tested for the ability to precipitate GAD 
from human brain homogenates. 
TABLE 5 
______________________________________ 
SERA FROM IDDM PATIENTS IMMUNOPRECIPITATE 
GAD ACTIVITY 
Pre-IDDM GAD Activity.sup.44 
Patient 
IDDM Period.sup.11 
64K.sup.22 
JDF.sup.33 
cpm's 
______________________________________ 
DA .sup. *.sup.5 
&gt;24 3 164 13,762 
DC * &gt;1 3 20 1,719 
RS + 5 3 40 588 
NL + 0 2 80 440 
DM * &gt;1 2 10 184 
C - na 0 0 280 
C - na 0 0 285 
C - na 0 0 325 
C - na 0 0 275 
C - na 0 0 270 
______________________________________ 
.sup.1 Expressed as months 
.sup.2 64K titers as described in Experimental Methods 
.sup.3 The islet cell antibody test as expressed in Juvenile Diabetes 
Foundation (JDF) units 
.sup.4 Not adjusted for background 
.sup.5 At risk for diabetes (also, failed glucose test) 
na -- Not applicable 
As shown in Table 5, the sera of four (out of five) at risk for IDDM or 
IDDM patients bound significantly greater amounts of enzymatically active 
GAD of human brain extracts than sera from control patients. In addition, 
sera from one of the patients was drawn in a pre-IDDM period, thus 
autoantibodies to GAD are present prior to the onset of IDDM symptoms (see 
C below). 
Further experiments (results not presented) showed that the sera of two at 
risk IDDM patients (DA, DC) immunoprecipitated recombinantly produced 
.sup.35 S-GAD.sub.65 whereas recombinantly produced as .sup.35 
S-GAD.sub.67 was only recognized by sera of patient DA (and to a lesser 
degree than .sup.35 S-GAD.sub.65). 
Additional studies using patient DA sera showed the presence of antibodies 
which recognize specific polypeptides produced in human pancreatic islet 
cells. Electrophoretic analysis of the bound polypeptides demonstrated the 
presence of autoantibodies to a 64kD component, as previously shown by 
others in human IDDM (Baekkeskov, et al., Nature, 298:167-169, 1982) and 
in animal models (Baekkeskov, et al., Science, 224:1348-1350, 1984; 
Atkinson, et al., Diabetes, 37:1587-1590, 1988). Prior absorption of these 
sera with GAD-6 monoclonal, which recognized GAD.sub.65 but not 
GAD.sub.67, or with bacterially produced GAD.sub.65, abolished the ability 
of the sera to recognize the 64kD pancreatic polypeptide. The epitopes 
recognized by autoantibodies to the 64kD autoantigen are thus present in 
GAD.sub.65, indicating that the 64kD autoantigen is indeed GAD.sub.65. In 
order to investigate the predictive value of GAD.sub.65, sera drawn from 
patients prior to onset of clinical manifestation of IDDM were tested for 
autoantibodies to GAD.sub.65. 
TABLE 6 
__________________________________________________________________________ 
IDDM PATIENTS ANALYZED FOR AUTOANTIBODIES PRIOR TO 
THE ONSET OF DISEASE 
Age Pre-IDD 
Patient 
Sex HLA 
Onset.sup.11 
Peroid.sup.22 
JDF 
64KA.sup.33 
GAD.sup.33.sub.65 
GAD.sup.33.sub.67 
__________________________________________________________________________ 
TA M 3,2 
17 11 20 2 0 1 
CA F 4,5 
38 4 0 1 1 0 
RA M 2,1 
5 34 0 2 1 0 
TB M 2,4 
11 66 40 1 1 0 
AB M N.D. 
23 6 160 
3 3 2 
VC F 4,6 
15 3 40 1 0 1 
JD M 6,1 
34 25 10 3 1 1 
DR F 3,4 
14 42 320 
2 1 0 
JG M 3,3 
12 8 40 1 0 0 
BR M 3,3 
5 9 0 0 1 1 
KR F 4,X 
34 14 10 3 2 0 
JT F 4,6 
7 10 N.D. 
1 1 1 
__________________________________________________________________________ 
.sup.1 Age of IDDM donset expressed as months 
.sup.2 The time interval between sera drawn and IDDM onset expressed as 
months 
.sup.3 1 = lowest; 2 = medium; and 3 = highest band intensities 
N.D. -- not determined 
As shown in Table 6, 9 out of 12 specimens (75%) were immunoreactive with 
.sup.35 S-GAD.sub.65. In addition, two patients (JA and VC) were 
immunoreactive to GAD.sub.67, but not GAD.sub.65 under these conditions. 
Therefore, in combination, autoantibodies to GAD.sub.65 and GAD.sub.67 
were present in 11 out of 12 (91%) of these patients sera. This finding 
suggests that although autoantibodies to GAD.sub.65 are more common than 
autoantibodies to GAD.sub.67, the use of both recombinant GADs (GAD.sub.65 
and GAD.sub.67) in an assay would allow for greater predictability of 
IDDM. Previous tests of these sera (Atkinson, et al., Langet, 
335:1357-1360, 1990) demonstrated that 11 out of 12, or 92%, immunoreacted 
with the .sup.35 S-64 kD molecule from human pancreatic islet cells. The 
serum which contained detectable autoantibodies to the 64kD molecule and 
not GAD.sub.65 was a serum which contained the lowest titer (or "1") for 
the 64kD molecule. Thus, the false negative obtained was due to a lack of 
sensitivity in this assay. Furthermore, this assay predicted IDDM in one 
patient (BR) who was negative for 64K. 
These results show that the 64kD molecule identified in .beta.-cells of 
human pancreas is identical in size and antigenicity to rat GAD.sub.65. 
Furthermore, sera drawn from patients prior to IDDM onset contain 
autoantibodies to GAD.sub.65. Consequently, the GAD.sub.65 recombinant 
molecule is of great utility as a diagnostic tool for predicting IDDM. The 
ability of a physician to diagnose IDDM prior to actual symptoms may 
result in a greater extension of time before insulin therapy is needed. 
The sensitivity of such immunoassays will improve with the use of a 
recombinant GAD.sub.65 of human origin which represents the GAD form 
present in .beta.-cells of the pancreas. 
EXAMPLE 4 
Immune Proliferative Response to Polypeptide 
Polypeptides were synthesized using an automatic instrument (Applied 
Biosystems) and standard conditions. These polypeptides were then tested 
to compare their relative ability to stimulate proliferation of splenic 
lymphocytes and islet infiltrating T lymphocytes (IITLs). In this study, 
polypeptides derived from the GAD.sub.65 core sequence and from the 
homologous region of polio virus were compared. Appropriate cells were 
cultured for 5 days with the respective polypeptide in the presence of 
5.times.10.sup.4 irradiated spleen cells. .sup.3 H-thymidine was added 
during the last 16 hours of culture. 
TABLE 7 
______________________________________ 
.sup.3 H-THYMIDINE 
INCORPORATION (cpm) 
BY LYMPHOID 
AMINO ACID CELL POPULATION 
ANTIGEN SEQUENCE IITLs.sup.a 
SPLEEN.sup.b 
______________________________________ 
None -- 1,100 6,500 
Poliovirus 
MKSMCPQAQLKVKYL 900 22,500 
GAD.sub.65 
ARFKMFPEVKEKGMAA 
9,500 23,300 
______________________________________ 
.sup.a islet infiltrating T lymphocytes (3 .times. 10.sup.4 cells/well) 
.sup.b 1 .times. 10.sup.5 cells/well 
In these studies, there was no significant difference in the proliferative 
activity of cultures of spleen lymphocytes exposed to either the polio or 
the GAD.sub.65 polypeptides. However, both polypeptides stimulated a T 
cell response which was higher than that found in the media control. The 
lack of difference in proliferation in the spleen cell population may be 
due to a lower frequency of GAD polypeptide specific T cells. 
The IITL population, when evaluated in the same manner, showed a marked 
difference in cell proliferation. In this system, the response to the 
GAD.sub.65 polypeptide was 9-fold greater than that of either the culture 
media or the polio polypeptide. This data strongly suggests that the 
GAD.sub.65 is an important antigen for T cell responses in the IITL 
population. This data suggests that molecular mimicry plays a role in the 
pathogenesis of diabetes. 
EXAMPLE 5 
GAD Induces Proliferation of Spleen Cells of Nod Mice 
Proliferative T-cell responses to .beta.-cell antigens (.beta.CA) develop 
spontaneously in the nonobese diabetic (NOD) mouse model in a defined 
chronological order. The NOD mouse experimental model is considered the 
most analogous in vivo system available for studying IDDM in humans. This 
example describes studies on the antigen-induced blastogenesis of spleen 
cells from newborn to 5 month old female NOD mice when exposed to GAD and 
other peptides. 
The .beta.CAs tested included one of the two forms of GAD (Kaufman, et al., 
Science, 232:1138-1140, 1986; Erlander, et al., Neuron, 7:91-100, 1991; 
Kaufman, et al., Trends in Pharm. Sci. (in press)), (GAD.sub.65, 
previously known as the 64K autoantigen (Baekkeskov, et al., Nature, 298: 
167-169, 1981; Baekkeskov, et al., Nature, 347:151-156, 1990), 
carboxypeptidase H (CPH) (Castano, et al., J. Clin. Endoctrinol Metab., 
73:1197-1201, 1991), insulin (Palmer, Predicting IDDM, Diabetes Reviews, 
1:104-115, 1993) and a peptide of hsp which has been shown to be the 
immunodominant determinant recognized by NOD T-cells (Elias, Proc. Natl. 
Acad. Sci., 88:3088-3091, 1991). GAD in particular, is a good candidate 
for the initial target antigen in IDDM since autoantibodies to GAD arise 
early in the natural history of the disease (Baekkeskov, supra; Atkinson, 
et al., Lancet, 335:1357-1360, 1990; Kaufman, et al., J. Clin. Invest., 
89:283-292,1992). Furthermore, unlike the ubiquitous hsp, GAD is expressed 
primarily in .beta.-cells and the immunologically privileged central 
nervous system (CNS) and gonads. As control antigens, irrelevant prototype 
foreign and self antigens including hen eggwhite lysozyme (HEL), human 
serum albumin (HSA), E. coli. .beta.-galactosidase (.beta.-gal) and murine 
myelin basic protein (MBP) were used. 
NOD (Taconic farms) and BALB/c mice (Jackson Laboratories) were kept under 
specific pathogen free conditions. The mice were sacrificed at the ages 
indicated and the spleen cells were tested directly ex vivo for their 
proliferative recall response to antigen. Single cell suspensions of 
spleen cells were plated at 1.times.10.sup.6 cells per well in 96 well 
microtiter plates in 200 .mu.l serum free HL-1 medium (Ventrex) that was 
supplemented with 2 mM glutamine with or without 10 .mu.g/ml antigen (or 7 
.mu.M peptide) in triplicate cultures. During the last 16 h of the 72 h 
culture period, 1 .mu.Ci.sup.3 H!-thymidine was added per well. 
Incorporation of label was measured by liquid scintillation counting. 
Both human GAD.sub.65 (Bu, et al., Proc. Natl. Acad. Sci., 89:2115-2119, 
1992) and E. coil .beta.-gal (control) were purified from recombinant 
bacteria on the basis of a hexahistidine tag which allows their rapid 
affinity purification by metal affinity chromatography (Hochuli, et al., 
Bio/Technology, 6:1321-1325, 1988). Bovine CPH was the generous gift of L. 
Fricker (Albert Einstein Col. Med.) and human insulin was purchased from 
Eli Lilly. 
As illustrated in FIG. 8, while proliferative T-cell responses were not 
detected at any time point to the control antigens, a response to GAD 
arose at 4 weeks of age in NOD mice, concurrent with the onset of 
insulitis in the colony. The blastogenesis induced by GAD increased during 
the next four weeks and then declined to background levels by week 16. At 
6 weeks of age, near the peak of anti-GAD reactivity, T-cell responses to 
hsp appeared and increased until week 15 and then diminished as well (FIG. 
8). In all NOD mice tested, hsp reactivity was preceded by an anti-GAD 
response, suggesting that the former reactivity developed as a secondary 
event during the autoimmune process. Similarly, while no response was 
detected to CPH at 4 weeks of age, a strong anti-CPH response was observed 
by week 8. In some mice, a weak response to insulin was observed at 12 
weeks, which became more prevalent at 15 weeks of age (FIG. 8 and Table 
8). None of the antigens induced proliferation in T-cells from age-matched 
control BALB/c or (NOD.times.BALB/c) F.sub.1 mice, both of which do not 
develop insulitis or IDDM. T-cell reactivity subsequently arises to other 
.beta.CAs, consistent with the inter-molecular diversification of the 
autoimmune response. Thus, the autoimmune response to GAD was the first to 
occur among the autoantigens tested. In view of this, tolerization to GAD 
should prevent the spread of autoimmunity to other .beta.CAs and 
insulitis. If this were not the case then tolerization to GAD should have 
no effect on the response to these other antigens. 
Blastogenesis provides an approximation of the relative clonal sizes of 
antigen-specific CD4+ T-cells (Corradin, et al., J. Immunol., 
119:1048-1053, 1977). The data in FIG. 8 shows that GAD reactive T-cells 
"spontaneously" undergo clonal expansion concurrent with the onset of 
insulitis. These findings are consistent with an endogenous priming event. 
EXAMPLE 6 
Induction of Tolerance with GAD 
This example describes a study which shows that induced tolerance to GAD 
can ameliorate IDDM. 
1. In these experiments female NOD mice were intravenously injected at 3 
weeks of age with 50 .mu.g GAD, .beta.-galactosidase, mycobacterial hsp65 
(m-hsp) or 0.1 .mu.g of the immunodominant hsp peptide (hsp-p), in PBS. At 
12 weeks of age, mice were examined for insulitis and autoantigen reactive 
T-cells. At this age both indications are established in untreated NOD 
mice. Pancreatic tissue sections were stained by immunoperoxidase 
techniques for insulin and were counterstained with hematoxylin. Insulitis 
was scored in a blinded manner by examining 54 to 87 islets on 5 
interrupted tissue sections from each pancreas. Proliferative splenic 
T-cell responses induced by various antigens were performed as described 
above in Example 4. Data in Table 8 are expressed as the average .sup.3 
H!-thymidine label (cpm) incorporated in triplicate cultures. 
TABLE 8A 
__________________________________________________________________________ 
GAD Induced Tolerance 
Spleen Cell Proliferation (SI .+-. SEM).sup.b 
Insulitis GAD Peptides hsp 
Treatment 
Score.sup.a 
N .beta.-Gal 
GAD #17 #34 #35 Peptide 
CPH 
__________________________________________________________________________ 
Uninjected 
2.4 .+-. 0.2 
5 1.0 .+-. 0.2 
9.5 .+-. 2.1 
4.8 .+-. 0.4 
6.0 .+-. 0.1 
2.9 .+-. 0.2 
6.7 .+-. 1.0 
ND.sup.c 
.beta.-Gal. 
2.6 .+-. 0.6 
5 1.1 .+-. 0.1 
15.4 .+-. 1.8 
5.1 .+-. 0.6 
5.1 .+-. 0.6 
4.0 .+-. 0.2 
6.6 .+-. 0.5 
11.5 .+-. 0.9 
GAD 0.1 .+-. 0.1 
8 1.1 .+-. 0.03 
1.6 .+-. 0.3 
1.0 .+-. 0.05 
1.2 .+-. 0.1 
1.0 .+-. 0.1 
1.2 .+-. 0.1 
1.1 .+-. 0.02 
hsp-p 
1.7 .+-. 0.4 
5 1.1 .+-. 0.05 
5.8 .+-. 0.2 
4.5 .+-. 0.1 
4.1 .+-. 0.3 
4.2 .+-. 0.1 
1.1 .+-. 0.04 
4.4 .+-. 0.2 
m-hsp 
1.8 .+-. 0.5 
5 1.0 .+-. 0.1 
4.2 .+-. 0.1 
3.9 .+-. 0.1 
3.9 .+-. 0.1 
3.4 .+-. 0.2 
1.0 .+-. 0.03 
4.3 .+-. 0.2 
__________________________________________________________________________ 
.sup.a Severity of mononuclear cell infiltration was defined 
histologically (0 = no lymphocytic infiltration; 1 = &lt;25%; 2 = 25-50%; 3 
50-75%; 4 = &gt;75%) (Qin, et al., Immunol., 150:2072-2080, 1993). Score is 
mean .+-. SE. 
.sup.b Significant responses noted by solid underline, borderline 
responses noted by double underline. 
.sup.c Not determined. 
TABLE 8B 
__________________________________________________________________________ 
GAD Induced Tolerance 
Spleen Cell Proliferation (SI .+-. SEM).sup.b 
Insulitis GAD Peptides hsp 
Treatment 
Score.sup.a 
N .beta.-Gal 
GAD #17 #34 #35 Peptide 
CPH 
__________________________________________________________________________ 
11 peptide 
.apprxeq.2.5 
1.0 .+-. 0.1 
21.1 .+-. 2.2 
13.7 .+-. 1.5 
11.4 .+-. 1.5 
11.3 .+-. 0.7 
13.3 .+-. 0.9 
ND 
34/35 peptides 
0.7 .+-. 0.4 
1.0 .+-. 0.2 
1.9 .+-. 1.1 
2.2 .+-. 1.2 
1.1 .+-. 0.3 
1.0 .+-. 0.1 
1.8 .+-. 1.1 
ND 
(+IFA) 
IFA alone 
.apprxeq.2.5 
1.0 .+-. 0.1 
8.1 .+-. 0.5 
5.0 .+-. 0.5 
4.8 .+-. 0.5 
4.8 .+-. 0.4 
6.6 .+-. 0.6 
ND 
(+IFA) 
__________________________________________________________________________ 
.sup.a Severity of mononuclear cell infiltration was defined 
histologically (0 = no lymphocytic infiltration; 1 = &lt;25%; 2 = 25-50%; 3 
50-75%; 4 = &gt;75%) (Qin, et al., Immunol., 150:2072-2080, 1993). Score is 
mean .+-. SE. 
.sup.b Significant responses noted by solid underline, borderline 
responses noted by double underline. 
.sup.c Not determined. 
TABLE 8C 
__________________________________________________________________________ 
GAD Induced Tolerance 
Spleen Cell Proliferation (SI .+-. SEM).sup.b 
Insulitis GAD Peptides hsp 
Treatment 
Score.sup.a 
N .beta.-Gal 
GAD #17 #34 #35 Peptide 
CPH 
__________________________________________________________________________ 
11 peptide 
.apprxeq.(?) 
1.4 .+-. 0.4 
12.1 .+-. 3.5 
6.9 .+-. 0.4 
6.4 .+-. 1.0 
6.8 .+-. 0.3 
8.7 .+-. 0.9 
ND 
HEL peptide 
(?) 1.2 .+-. 0.2 
10.7 .+-. 3.5 
6.4 .+-. 1.4 
5.9 .+-. 1.6 
6.9 .+-. 1.9 
8.0 .+-. 1.8 
ND 
34/35 peptides 
(?) 1.0 .+-. 0.1 
4.4 .+-. 2.6 
1.8 .+-. 1.1 
1.0 .+-. 0.1 
1.1 .+-. 0.2 
2.7 .+-. 1.3 
ND 
(+IFA) 
__________________________________________________________________________ 
.sup.a Severity of mononuclear cell infiltration was defined 
histologically (0 = no lymphocytic infiltration; 1 = &lt;25%; 2 = 25-50%; 3 
50-75%; 4 = &gt;75%) (Qin, et al., Immunol., 150:2072-2080, 1993). Score is 
mean .+-. SE. 
.sup.b Significant responses noted by solid underline, borderline 
responses noted by double underline. 
.sup.c Not determined. 
Seventy five percent of the GAD treated mice, but none of the controls, 
showed no T-cell reactivity to GAD (indicating complete tolerization) or 
to other .beta.CAs. These mice were also completely free of insulitis 
(score 0.0). If there were another effector T cell population in the 
islets, specific for an unknown .beta.CA, that preceded the anti-GAD 
response, the release of cytokines by this population should have promoted 
T-cell responses to .beta.CAs and insulitis (Sarvetnick, et al., Nature, 
346:844, 1990; Heath, et al., Nature, 359:547, 1992). Twenty five percent 
of the GAD-treated mice were not completely tolerized to GAD, as evidenced 
by a weak residual GAD reactivity (SI of about 3) and displayed very 
limited peri-insulitis. In contrast, while tolerization to both of the hsp 
antigens was complete, these treatments reduced, but did not prevent, the 
development of T cell responses to other .beta.CAs or insulitis. Thus, 
while the inactivation of GAD-reactive T cells prevented .beta. cell 
autoimmunity, hsp tolerization only partially reduced it, as would be 
expected if a secondary element was removed from the amplifactory cascade. 
In ongoing experiments examining the effects of GAD tolerization on 
diabetes incidence, all of the GAD treated mice (n=17, presently 37 weeks 
old) have normal glucose levels, while 70% of the mice receiving control 
antigens developed hyperglycemia by 19 weeks of age (n=20). Five GAD 
treated mice were sacrificed at 30 weeks of age. All were free of 
detectable .beta.CA reactive T cells. Of these five animals, four mice 
were completely free of insulitis and one mouse displayed very limited 
peri-insulitis. These data show that inactivation of GAD reactive T-cells 
prevents the long term development of insulitis and diabetes. 
2. In a second set of experiments neonatal female NOD mice were injected 
intraperitoneally IFA with peptide 11, a mixture of peptides 34 and 35 
plus IFA, or with IFA alone and at 12 weeks of age the mice were examined 
for insulitis and autoantigen reactive T-cells as in Example 6.1. 
Proliferative splenic T-cell responses induced by the various antigens 
were performed as in Example 4, and data in Table 8B are expressed as the 
average .sup.3 H!-thymidine label (cpm) incorporated in triplicate 
cultures. 
The data in Table 8B show that tolerization with control peptide 11 did not 
prevent auto antibody response to GAD or to GAD peptides 17, 34 or 35. Nor 
was response to hsp peptide prevented by tolerization with peptide 11. IFA 
alone was somewhat effective at suppressing immune response. By contrast, 
tolerization with the mixture of peptides 34 and 35 suppressed the 
autoimmune response of spleen cell proliferation to all species tested: 
.beta.-gal, GAD, GAD peptides 17, 34 and 35, and hsp peptide. In addition, 
tolerization to GAD peptides 34 and 35 greatly reduces insulitis but does 
not completely prevent it as whole GAD65 does. 
3. In a third set of experiments female NOD mice were intravenously 
injected at three weeks of age with peptide 11 (control), a mixture of 
peptides 34 and 35 plus IFA, or with HEL peptides and at 12 weeks of age 
the mice were examined for insulitis and autoantigen reactive T-cells as 
in Example 6.1. Proliferative splenic T-cell responses induced by the 
various antigens were performed as in Example 4, and data in Table 8C are 
expressed as the average .sup.3 H!-thymidine label (cpm) incorporated in 
triplicate cultures. 
The data in Table 8C show that tolerization with control peptide 11 did not 
prevent auto antibody response to GAD, to GAD peptides 17, 34 or 35 or to 
hsp although the response was not as great as in Example 6.2. Nor was 
response to hsp peptide prevented by immunization with peptide 11. By 
contrast, immunization with the mixture of peptides 34 and 35 plus IFA 
suppressed the autoimmune response of spleen cell proliferation to all 
species tested: .beta.-gal, GAD, GAD peptides 17, 34 and 35, and hsp 
peptide. 
EXAMPLE 7 
Characterization of GAD-Reactive T-Cells 
This example describes studies on GAD-Reactive T-Cells for additional 
properties that distinguish activated/memory from resting/naive 
lymphocytes. 
In one series of experiments .gamma. interferon (IFN.gamma.) was measured 
by ELISA in culture supernatants (CSN) of spleen cells of 6-9 week old 
mice after challenge with GAD or control antigens HEL and MBP. 
Additionally, the frequency of antigen specific, IFN.gamma.-producing 
cells was determined by an ELISA spot technique (T. Taguchi, et al., J. 
Immunol., 145:68-77, 1990). Frequency of antigen-induced, spot forming 
cells (SFC) among 10.sup.3 spleen cells is represented in FIG. 9(a). 
Values are the mean+SEM from 5 individual female NOD mice, each tested in 
triplicate cultures with or without antigen. Results from a single 
experiment are shown. These are representative of 3 separate experiments. 
In performing these experiments, freshly isolated spleen cells were 
cultured with or without antigen as described in Example 4. CSN were taken 
after 48 h and the concentration of IFN.gamma. was determined by ELISA 
(Macy, et al., FASEB J., 3003-3009, 1988). IFN.gamma. specific monoclonal 
antibody (mAb) R4-6A2 (Pharmingen) was used as the capturing reagent and 
biotinylated mAb XMG 1.2 (Pharmingen, also specific for IFN.gamma.) was 
used in conjunction with streptavidin-alkaline phosphatase (Zymed) and 
p-nitrophenol for detection of bound lymphokine. Recombinant murine 
IFN.gamma. (Pharmingen) was used as a standard. ELISA spot assays for the 
detection of antigen-specific, IFN.gamma.-producing cells were performed 
as described (Taguchi, et al., J. Immunol., 145:68-77, 1990). After a 24 h 
pre-activation culture of spleen cells with our without antigen, cells 
were transferred by serial dilution to 96 well microtiter plates 
(Millipore) that had been pre-coated with mAb R4-6A2. After 24 h, the 
cells were removed and IFN.gamma. spots were visualized using XMG 
1.2-biotin in conjunction with nitroblue terazolium-bromochloroindolyl 
phosphate substrate (Sigma). Spots were counted visually and the frequency 
of antigen specific cells was determined from the difference between the 
number of spots seen with and without antigen. 
As shown in FIG. 9(a), when freshly isolated T-cells from 6-9 week old NOD 
mice were challenged with GAD or control antigens, high concentrations of 
IFN.gamma. were detected only in cultures containing GAD, suggesting that 
the GAD specific T-cells had been pre-activated in vivo, since only 
pre-activated T-cells (Th1) produce IFN.gamma. within 48 hours after 
antigen recognition (Ehlers, et al., J. Exp. Med., 173:25-36 1991; Croft, 
et al., J. Exp. Med., 176:1431-1437, 1992). In contrast, T-cells from age 
matched BALB/c mice did not respond to GAD or to control antigens by 
IFN.gamma. production (data not shown). 
Results of the ELISA spot assay to measure directly the frequency of 
GAD-specific T-cells showed that while in 6-9 week old NOD mice, T-cells 
reactive to control antigens constituted approximately 1 in 10.sup.5 cells 
in the spleen, the frequency of GAD-reactive T-cells was about two orders 
of magnitude higher, ranging from 90-291 cells per 10.sup.5 cells (FIG. 
9(a), confirming the data obtained by proliferation assays (FIG. 8) that 
these cells had been clonally expanded in vivo. 
In another series of experiments, GAD specific T-cells were characterized 
for expression of the cell surface marker L-selectin, since murine T-cells 
convert from an L-selectin.sup.+ (L-sel.sup.+) to an L-selectin.sup.- 
(L-sel.sup.-) phenotype upon activation (Bradley, et al., J. Immunol., 
148:324-331, 1992). 
To perform these studies, pooled spleen cells from 3 to 4 age matched mice 
were panned on plates coated with goat-anti-mouse Ig (Zymed) to remove 
adherent macrophages as well as B cells. Next, CD8+ cells were coated with 
mAb 58.6-72 (ATCC) and removed by panning over plates coated with 
goat-anti-rat Ig (Zymed). The non-adherent CD4+ cell fraction was labeled 
with anti-L-selectin mAb MEL-14 (ATCC) and panned on goat-anti-rat Ig 
coated plates. Both the adherent (CD4+ L-sel.sup.+) and non-adherent 
(CD4+,L-sel.sup.-) fractions were sampled. Purity of the cell fraction was 
assessed by FACS analysis; cells were &gt;90% CD4+ and &gt;95% enriched for the 
L-sel.sup.- or L-sel.sup.+ phenotype. The purified cell fractions were 
tested for GAD reactivity by seeding them at 2.times.10.sup.5 cells per 
well in 96 well microtiter plates with or without antigen. Irradiated 
(3000 rad), unseparated spleen cells of 3 week old NOD mice were added at 
5.times.10.sup.5 cells per well as a source of antigen presenting cells. 
Supernatants of triplicate cultures were taken 48 h later and their 
IFN.gamma. content was determined by ELISA. 
The results of this study showed that by 2-3 weeks of age, GAD reactive 
T-cells could not be detected in either the L-sel.sup.+ or the 
L-sel.sup.- population, consistent with a low frequency of antigen 
reactive precursors at this time point. However, by 6 weeks of age high 
levels of IFN.gamma. were induced by GAD (but not by control antigens) in 
the L-sel.sup.- (but not the L-sel.sup.+) subpopulation of CD4+ cells 
(FIG. 9(b)). 
The increase in clonal size of GAD reactive T-cells, their production of 
IFN.gamma. and their L-sel.sup.- phenotype provide three independent lines 
of evidence that a potentially pathogenic (Ando, et al. Cell Immunol., 
124:132-143, 1989) Th1 type T-cell response is spontaneously primed to GAD 
in vivo early in NOD development. 
EXAMPLE 8 
Characterization of GAD Specific T-Cell Determinant Recognition 
The fine specificity of the anti-GAD T-cell response was mapped using a set 
of 38 peptides (numbered successively from the N-terminus) that were 20-23 
amino acids (aa) long and span the entire GAD.sub.65 (Bu, et al., Proc. 
Natl. Acad. Sci., 89:2115-2119, 1992) sequence with 5 aa overlaps (FIG. 
10). 
Spleen cells were tested from 4 (FIG. 10a), 5 (FIG. 10b) and 7 (FIG. 10c) 
week old NOD mice for proliferative responses (as described in Example 4) 
to the GAD peptides. Peptides were present in cultures at 7 .mu.M and the 
label was added during the last 16 hours of a 5-day culture. The peptides 
were synthesized using standard Fmoc chemistry and purified by reverse 
phase HPLC (Advanced Chemtech). The sequence of stimulatory peptides are 
shown below in Table 9. 
TABLE 9 
______________________________________ 
Peptide GAD Amino Acid 
Number Region Sequence 
______________________________________ 
6 78-97 KPCSCSKVDVNYAFLHATDL 
17 247-266 NMYAMMIARFKMFPEVKEKG 
23 335-356 TAGTTVYGAFDPLLAVADICKK 
32 479-498 EYLYNIIKNREGYEMVFDGK 
34 509-528 IPPSLRYLEDNEERMSRLSK 
35 524-543 SRLSKVAPVIKARMMEYGTT 
36 539-558 EYGTTMVSYQPLGDKVNFFR 
38 566-585 ATHQDIDFLIEEIERLGQDL 
______________________________________ 
Murine and human GAD.sub.65 are 95% identical at the amino acid level 
(555/585) and are 98% conserved, with most of the differences localized 
near their N-termini. The underlined amino acid in the stimulatory peptide 
sequences above are conservatively substituted in murine GAD.sub.65. In 
separate experiments, the murine form of key peptides (#17 and #34) were 
tested and produced similar results. 
As shown in FIG. 10, peptides that triggered stimulation indices &gt;3 are 
indicated as black bars. These peptides did not induce proliferation in 
T-cells from NOD mice &lt;3 or &gt;16 weeks in age, or from control 
(BALB/c.times.NOD)F1 mice (data not shown). The data are represented as 
the mean SI.+-.standard error calculated from 3-6 individual mice tested 
twice in each age group. Characteristic results for peptide induced 
blastogenesis in individual mice are shown in Table 6. The first 
detectable response, at 4 weeks of age, was confined to the 
carboxy-terminal region of GAD, and involved two adjacent peptides (aa 
509-528 and 524-543, peptides #34 and #35, respectively, FIG. 10a). At 5 
weeks of age, responses to an additional determinant (aa 247-266, peptide 
#17, which contains a region of sequence similarity with Coxsackievirus 
(Kaufman, et al., J. Clin. Invest., 89:283-292, 1992) (FIG. 10b) were 
regularly recorded. During the next two weeks, responses to peptide #17 
(aa 247-266) increased and T-cell autoimmunity spread to two additional 
peptides at the carboxy terminus (aa 479-498 and 539-558; peptides #32 and 
#36 respectively, FIG. 10c). Subsequently, reactivity to the GAD peptides 
declined (data not shown), paralleling the loss of response to the whole 
protein (FIG. 8). It is unclear why the initial T-cell response to 
.beta.CAs fades in NOD mice. Possible explanations include: a) immune 
regulatory mechanisms; b) exhaustion of the response due to the continuous 
stimulation by the endogenous antigen; and c) induction of anergy in 
specific T-cells owing to their recognition of the autoantigen on "non 
professional" antigen presenting cells such as the .beta. cells themselves 
(Markmann, et al., Nature, 336:476-479, 1988). 
The gradual diversification of the primed autoreactive T-cell repertoire 
that was observed in this naturally occurring autoimmune disease parallels 
the shifts in T-cell recognition recently observed in experimentally 
induced autoimmunity to the CNS where autoreactivity spreads both intra- 
and intermolecularly among CNS proteins (Lehmannn, et al., Nature, 
359:155-157, 1992; Perry, et al., J. Neuroimmunol., 33:7-15, 1991; 
Watanabe, et al.,Nature, 305:150-153, 1983; Liebert, et al., J. 
Neuroimmunol. ,17:103-118, 1988). Apparently, lymphokine secretion by the 
first wave of autoantigen specific T-cells in the target organ results in 
up-regulation of antigen presentation and creates a microenvironment that 
favors priming of additional autoreactive T-cells (Lehmann, et al., 
Immunol. Today, 14:203-208, 1993; Sarvetnick, et al., Nature, 346:844-847, 
1990; Heath, et al., Nature, 359:547-549, 1992). Since hsp reactive CD4+ 
T-cells are capable of inducing IDDM (Elias, et al., Proc. Natl. Acad. 
Sci., 87:1576-1580, 1990; Elias, et al., Proc. Natl. Acad. Sci., 
88:3088-3091, 1991), their recruitment into the activated T-cell pool, 
along with T-cells reactive to other .beta.CAs, probably reflects an 
amplificatory cascade that eventually leads to .beta. cell destruction. 
In summary, the data above establish GAD as a critical target antigen in 
the pathogenesis of IDDM in NOD mice. The results show that T-cell 
responses to .beta.CAs diversify both intramolecularly and 
intermolecularly as the disease progresses, consistent with a dynamic 
autoimmune repertoire (Lehmann, et al., Immunol. Today, 14:203-208, 1993). 
However, interference with the early autoreactive T-cell population can 
prevent the recruitment of additional autoantigens into the primed 
repertoire thereby halting a cascade of autoimmune responses that 
eventually leads to .beta. cell destruction. As a similar autoimmune 
progression is also likely to occur during the development of human IDDM 
(Palmer, J. P., Predicting IDDM, Diabetes Reviews, 1:14-115, 1993; 
Atkinson, et al., Lancet, 339:458-459, 1992), these findings suggest that 
peptide-based immunotherapeutic agents would be useful in predicting and 
ameliorating human IDDM. 
EXAMPLE 9 
Autoantibody Reactivity with GAD Fragments 
This example describes a study which examined the variability in 
recognition of epitopes in human GAD.sub.65 polypeptides by IDDM 
autoantibodies in sera of human patients. 
Portions of human GAD.sub.65 cDNA were amplified by the polymerase chain 
reaction (PCR; Saiki, et al., Science, 239:487, 1988) to produce DNA 
segments encoding three polypeptide segments: amino acid residues 1-224 
(segment A); 224-398 (segment B); and 398-585 (segment C). Each construct 
also contained a T.sub.7 promoter, a consensus sequence for the initiation 
of translation and an initiating methionine codon (Korak, M., J. Cell 
Biol., 108:229, 1989). Each PCR product was then trascribed in vitro with 
T.sub.7 RNA polymerase and translated in vitro in a rabbit reticulocyte 
cell-free system in the presence of .sup.35 S-methionine, using conditions 
recommended by the supplier (Amersham Corp., Arlington Heights, Ill.). 
Each test serum (30 .mu.l) was incubated with the resulting .sup.35 S 
labeled-polypeptides. The bound peptides were isolated with PAS and 
analyzed by SDS-PAGE in 12% polyaacrylamide and autoradiography. 
TABLE 10 
______________________________________ 
IDDM PATIENT SERA REACTIVITY WITH GAD SEGMENTS 
SEGMENT 
PATIENT A B C 
______________________________________ 
Control (N = 7) 
- - - 
052 - + + 
723 - - - 
705 - + + 
UC2 - + + 
N.L. - - - 
L.I. - - - 
T.L. - - - 
P.T. - + - 
J.D. - - - 
B.Y. - + + 
M.C. - - - 
R.S. - - - 
K.O. - - - 
T.B. - - - 
S.M. - - - 
A.W. - + - 
J.B. - + + 
J.A. - - - 
P.C. - + + 
L.R. - - - 
J.M. - + - 
G.A. - - - 
______________________________________ 
As shown in Table 10, none of the specimens had detectable levels of 
antibodies to the amino terminal third (segment A) of GAD whereas 9 
patients (41%) had antibodies reactive with the middle third (segment B) 
and 6 patients (27%) had antibodies to the carboxyl-terminal third 
(segment C) of GAD. 
EXAMPLE 10 
Prediction of Incipient IDDM by GAD Epitope Recognition Pattern 
The increasing likelihood of an IDDM interventive therapy and the (recently 
acknowledged) benefits of managed glucose homeostasis in preventing IDDM 
associated complications makes the early detection of .beta. cell 
autoimmunity before clinical IDDM onset and in NIDDM patients (10% of whom 
eventually convert to IDDM) a crucial goal. Autoanitbodies to GAD may 
provide the earliest and most reliable marker of impending IDDM among the 
molecularly defined IDDM associated autoantigens. To determine whether GAD 
peptides will bind to IDDM associated autoantibodies the following study 
was conducted. 
A set of peptides (20-23 amino acids in length, with 5 aa overlaps) that 
span the human GAD65 molecule were synthesized to determine whether sera 
from most individuals at risk, pre-IDDM and with IDDM (in contrast to 
healthy controls) do in fact produce antibodies that differentially 
recognize GAD65 linear epitopes distributed throughout the molecule. 
Patient sera and most control sera were those used in a previous study 
(Kaufman, et al., J. Clin. Investigation, supra) All samples were coded 
and tested in a blind manner. Peptides were synthesized using an automatic 
instrument (Applied Biosystems, Foster City, Calif.) and standard 
conditions. Peptides were dissolved in 60 mM sodium bicarbonate buffer (pH 
9.6) at 20 .mu.g/ml and 100 .mu.l of each was added to duplicated wells of 
a 96 well Nunc-lmmuno Plate. Peptides were allowed to bind at 4.degree. C. 
overnight. The plates were then washed three times with PBS+0.1% Tween 20 
(wash buffer), after which the plates were pre-absorbed with 3% BSA in 
sodium bicarbonate buffer for 0.5 hours at 37.degree. C., or at room 
temperature overnight. The plates were then washed 5 times with the above 
wash buffer. 100 .mu.l of serum at a 1/300 dilution in PBS + 0.1% Tween 20 
and 1% BSA was added to each well and antibodies were allowed to bind for 
1 hour at 37.degree. C. The plates were washed 5 times with wash buffer. 
100 .mu.l of a 1/600 dilution of HRP-goat anti-human IgG (BRL, 
Gaithersberg, Md.) was added to each well and allowed to bind for 1 hour 
at 37.degree. C. The plates were then washed 7 times and 100 .mu.l of 
substrate buffer was added to each well for 30 minutes at room 
temperature. The color development was measured at 410 nm using an ELISA 
plate reader (ICN, Biomedicals, Costa Mesa, Calif.). Positive sera were 
defined as: OD.sub.410 of the sample/negative control.gtoreq.3.0. 
The data shown in Table 11 establish that a number of GAD peptides were 
recognized by patients previously shown to be 64K positive, but not by 
control sera. Each patient showed a different pattern of GAD epitope 
recognition. Peptides 20, 21 and 25, were each recognized by 6/8 patients, 
and none of the controls--with the exception of peptide 25 which was 
recognized by 1 out of 13 controls. Based on immunoreactivity to 2 of 
these peptides (#20 and 21) 7/8 (88%) of the patients (and none of the 
controls) could be identified as possessing GAD autoantibodies. Peptides 
3, 6, 22, 25 and 37 were each recognized by only 25-37% of the patients 
(and none of the control sera), but taken together, 75% of the patients 
recognized at least one of these. Peptides 5, 9 and 24 were often positive 
for immunoreactivity by both control and patient sera. 
This level of sensitivity is comparable to the best currently available 
assays using whole GAD65 purified from brain or recombinant organisms. 
Besides avoiding laborious antigen purification, peptide based 
autoantibody screening, together with PCR based HLA typing, may reveal 
epitope recognition patterns associated with progression or lack of 
progression to IDDM and its associated complications. Individuals 
determined to be at high risk could then consider therapeutic 
intervention. 
It should also be noted that the GAD peptides recognized by autoantibodies 
were different from those recognized by NOD GAD reactive T cells in 
Example 6. 
3 TABLE 11 
- EPITOPE RECOGNIATION OF HUMAN GAD65 PEPTIDES 
PEPTIDE 
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 2 
8 29 30 31 32 33 34 35 36 37 38 
Controls 
A.P. X X X 
P.T. X X 
5748 X b X 
5620 X b X b b 
4380 X b X b 
S.M. X X b X X b 
FA8 X 
FA12 X b 
1688 b X 
FA9 X X 
H.B b X 
FA2 X X 
FA11 X X 
2 1 
IDDM 
At Risk 
052 b X X b b b b X X X b X b b 
825 X X X b 
356 X X X X X 
L.I. b X X b b X b 
Pre-IDDM 
J.A. X X X 
723 X X X X X X X X X 
P.T. 
(1) X X b X b X b X b 
(2) b X b b b X X X X b b 
J.B. 
(1) b b b X 
(2) b X b b b b b X b b 
R.S. 
(1) X X b b X X 
(2) X X b b b b b X X 
B.Y. 
(1) X X X 
(2) X X 
J.B. 
(1) b b b X 
(2) b X b b b b b X b b 
At Onset 
705 X X X X X X X X X X 
S.H. b X X X X X X b 
291 b X X b X X X X X X b b 
048 X X X X X X X X X X X X 
5 1 2 2 3 2 1 7 7 2 8 1 4 1 1 1 1 2 1 
X = positive for immunoreactivity as defined by OD/background 3.0 
b = borderline response (OD/background ?2.5-2.9). 
EXAMPLE 11 
GAD Immunization Protects NOD Mice from IDDM 
The availability of cDNAs encoding GAD65 allows the testing of this 
molecule in new interventive therapies designed to interfere with 
GAD-specific T cells. Tests were conducted to examine the ability of GAD65 
immunization to protect NOD mice at 8 weeks of age, a time at which T cell 
responses to a number of b cell antigens and insulitis is already well 
established. If GAD immunotherapy was effective at this stage, it would 
hold promise for treatment in humans in which the autoimmune process has 
already been established. 
Methods 
Antigens 
An IPTG inducible T7 expression vector was used to express both human GAD65 
and E. coil b galactosidase (.beta.-gal). In IPTG induced recombinant E. 
coil, GAD and .beta.-gal constitute about 10-20% of the total bacterial 
protein. However, almost all of the GAD was in inclusion bodies, which 
could be isolated and extensively washed to obtain material that is about 
80% GAD. We then did affinity purifications of GAD and .beta.-gal on the 
basis of a hexa-histidine "tag" which was attached to GAD during the 
subcloning process. These extra histidine residues allow the rapid 
affinity purification (Novagen) of GAD by metal affinity chromatography 
(Hochuli, et al., Bio Technology, 6:1321-1325, 1988). The inclusion body 
material is solubilized in 6M guanidine hydrochloride (GHCL), 10 mm 
.beta.-mercaptoethanol and 1% triton X-100. After binding to the column, 
the column was extensively washed with GHCL and 8M urea in phosphate 
buffers. Only the central peak GAD fraction was utilized for subsequent 
studies. Human GAD65 shares 96% amino acid sequence identity with murine 
GAD65, with most of the amino acid differences being conservative 
substitutions. 
The GAD preparation appeared to be free of immunologically detectable 
contaminants. It also appeared to be free of bacterial contaminants on 
overloaded silver stained gels. Analysis by a national reference 
laboratory found &lt;0.06ng LPS/ug GAD. Human GAD65 did not induce T cell 
proliferation in &lt;4 or &gt;16 week old NOD or control BALB/c or (NOD/BALB/c) 
F1 spleen cells. The results using synthetic GAD peptides (FIG. 10) 
precisely parallel the data using whole recombinant GAD (FIG. 8). Other 
antigens described herein elsewhere that are not involved in IDDM (such as 
the beta galactosidase) did not induce NOD T cell responses. After 
immunizing mice with GAD, we were unable to detect cross reactive T cell 
responses in recall experiments with other proteins that were purified 
from recombinant E. coil by the same metal affinity chromatography 
procedure. Amino acid sequence analysis of GAD and .beta.-gal each gave a 
single expected amino acid N-terminal sequence. If there had been 
appreciable endotoxins, heat shock proteins, or other contaminants present 
in the GAD preparation, spleen, PBMC (Atkinson, et al.), and T cell 
proliferation responses that were not disease specific would have been 
expected. 
Breeder mice were purchased from Taconic Farms and housed under specific 
pathogen-free conditions. Only female NOD mice were used in this study. 
The average age of IDDM onset in unrelated females in the colony was 22 
weeks. Insulitis is generally observed beginning at 4 weeks of age. T cell 
responses to GAD, HSP, CPH were found by 6 weeks in age. The incidence of 
IDDM in female mice is 70-90% by one year of age. 
Immunizations 
At 8 weeks of age, 25 .mu.g GAD or control .beta.-gal. was injected 
intraperitoneally (ip) in 100 .mu.l of incomplete Freunds adjuvant (IFA). 
Because there may be a requirement for continual antigen presentation 
(Ramsdell, et al., Science, 257:1130-1133, 1992) mice were treated again 
every 6 weeks. Urine glucose levels were monitored twice weekly. After 
observing above normal glucose in urea, blood glucose levels were 
monitored twice weekly. Two consecutive blood glucose level readings of 
300 mg/ml was considered as IDDM onset, after which the mice were 
sacrificed and spleen cells were tested as described above in Example 6 
for evidence of spleen cell proliferation. 
Immunization of 8 week old NOD mice produced a clear delay in the onset of 
IDDM compared to control .beta.-gal immunized mice (FIG. 11). While two of 
the GAD immunized mice (open circles) developed IDDM at about the normal 
age of onset (20 weeks), the other 8 GAD immunized mice showed no signs of 
hyperglycemia until 36 weeks in age. Four of the GAD treated mice 
developed IDDM between 37 and 40 weeks in age. Four of the GAD treated 
mice currently remain disease free (at 52 weeks of age). In contrast, the 
majority of .beta.-gal injected mice (closed circles) had hyperglycemia by 
22 weeks of age and 6/10 developed IDDM by 27 weeks in age. At 52 weeks of 
age, 2 of the .beta.-gal treated mice remain disease free. This experiment 
shows that GAD immunization significantly delayed (&lt;0.02) or prevented 
diabetes of NOD mice in which .beta. cell autoimmunity has already 
significantly progressed. 
.beta. cell autoimmunity is already well established at 8 weeks of age, and 
it is likely to also be in individuals determined to be at risk for IDDM 
on the basis of circulating autoantibodies. Although the mechanism of this 
protection is not clear, periodic injections of GAD have a profound 
moderating effect on the induction of disease. 
TABLE 11 
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AMINO ACID SEQUENCES FOR GAD65 
______________________________________ 
1 MASPGSGFWSFGSEDGSGDS 
2 GSGDSENPGTARAWCQVAQKFTG 
3 QKFTGGIGIGNKLCALLYGD 
4 LLYGDAEKPAESGGSQPPRA 
5 QPPRAAARKAACACDQKPCSC 
6 KPCSCSKVDVNYAFLHATDL 
7 HATDLLDGERPTLAFLQ 
8 LAFLQDVMNILLQYVVKSFDRS 
9 SFDRSTKVIDFHYPNELLQE 
10 ELLQEYNWELADQPQNLEEILM 
11 EEILMHCQTTLKYAIKTGHP 
12 KYGHPRYFNQLSTGLDMVGL 
13 DMVGLAADWLTSTANTNMFT 
14 TNMFTYEIAPVFVLLEYVTL 
15 EYVTLKKMREIIGWPGGSGD 
16 GGSGDGIFSPGGAISNMYAM 
17 NMYAMMIARFKMFPEVKEKG 
18 PEVKEKGMAALPRLIAFTSE 
19 AFTSEHSHFSLKKGAAALGI 
20 AALGIGTDSVILIKCDERGK 
21 DERGKMIPSDLERRILEAKQ 
22 LEAKQKGFVPFLVSATAGTT 
23 TAGTTVYGAFDPLLAVADICKK 
24 DICKKYKIWMHVDAAWGGGLLMS 
25 GLLMSRKHKWKLSGVERANS 
26 ERANSVTWNPHKMMGVPLQC 
27 VPLQCSALLVREEGLMQNCNQ 
28 QNCNQMHASYLFQQDKHYDL 
29 KHYDLSYDTGDKALQCGRHV 
30 CGRHVDVFKLWLMWRAKGTTG 
31 KGTTGFEAWDKCLELAEYLYN 
32 EYLYNIIKNREGYEMVFDGK 
33 VFDGKPQHTNVCFWYIPPSL 
34 IPPSLRTLEDNEERMSRLSK 
35 SRLSKVAPVIKARMMEYGTT 
36 EYGTTTMVSYQPLGDKVNFFR 
37 VNFFRMVISNPAATHQDIDF 
38 ATHQDIDFLIEEIERLGQDL 
______________________________________ 
The invention now being fully described, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
without departing from the scope of the invention.