Cloned glutamic acid decarboxylase

ES 2 179 077 T3

DESCRIPTION

Cloned glutamic acid decarboxylase.

1. Field of the invention

The present invention relates to the polypeptides of glutamic acid decarboxylase65 (GAD65) and to the methods of using the polypeptides of GAD65 in an autoimmune disease from the points of view of diagnosis and therapy.

two. Description of the background of the technique

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 every 300 to 400 subjects, and epidemiological studies suggest that its incidence is increasing. The disease results from the autoimmune destruction of insulin-producing β cells of the pancreas. More specifically, the pre-onset stage is characterized by "insulitis," in which lymphocytes infiltrate the pancreatic islets and selectively destroy the β-cells. Insulitis can be present for many years before the onset of clans. The presentation of topical hyperglycemia in IDDM appears only after at least 80% of the insulin-producing β cells have been lost. The β cells that remain are destroyed during the first 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, serious complications resulting in dysfunction of the eye, kidney, heart, and other organs are common in IDDM patients receiving insulin therapy. Because of this, it is highly desirable to prolong the latency period and avoid progression (eg. g., by administering immunosuppressive drugs to interfere with the autoimmune process and insulin to achieve better control of the effects of continuous hypoglycemia) between the onset of β-cell destruction and the existing requirement of insulin replacement (ie, that is, when 80% of the β cells will be destroyed). Consequently, a diagnostic test to determine the onset of β-cell destruction will allow the clinician to administer immunosuppressive drugs (Silverstein et al., New England Journal of Medicine, 319: 599-604, 1988) or prophylactic insulin therapy ( Keller et al., Lancet, 341: 977, 1993) to extend this latency period and thus significantly delay the onset of insulin substitution side effects.

Many IDDM patients have sera containing antibodies to a 64kD molecule (Baekkeskov et al., J. Clin. Invest .. 79: 926-934, 1987; Atkinson et al., Lancet, 335: 1357-1360, 1990), in islet cell cytopaasmic molecules (ICA) or islet cell surface molecules (ICSA) (Botazzo et al., Lancet, 1 : 668-672, 1980), or on insulin (Palmer et al., Science, 222: 1137-1139, 1983; (Atkinson et al., Lancet, 335: 1357-1360, 1990) have shown that the presence of antibodies in a 64kD molecule in human serum appears to be the earliest and most reliable indicator that initiates IDDM symptoms that will eventually occur.

Recently, Baekkeskov et al. Demonstrated that the 64kD molecule and glutamic acid decarboxylase (GAD) have several antigaenic epitopes in common and thus can be either identical or very similar molecules. Although this identification is an important discovery, it is known that the use of this information as a diagnostic tool to predict IDDM is quite inconvenient and is limited exclusively to the knowledge of the molecular biology of GAD. Studies by Kaufman et al. (J. Clin. Invest., 89: 283, 1992) demonstrated that the 64kD molecule was GAD₆₅ integral. Consequently, the cloning and subsequent production of large amounts of GAD65 or a GAD molecule that is substantially antigenically identical to the GAD65 molecule or fragments of the GAD65 molecule, which can be easily purified, will allow the development of a kit of diagnostic designed to predict IDDM, in addition to effective therapeutic modalities. The present invention provides means to accomplish these results.

WO92 / 19972 discloses that an antiserum peptide raised against the TQSD IDFLI EEIER LGQDL peptide recognizes three forms of GAD in rat islets.

WO92 / 20811 discloses the clonation, sequence and expression by recombinant means of a human pancreatic islet glutamic acid decarboxylase (GAD), an autoanthagen involved in the development of IDDM.

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EP-A-0519469 discloses that the X-Pro-Glu-Val-Lys-Y-Lys-Z polypeptide is useful for ameliorating autoimmune disease.

Document WO94 / 20127, cited against the present application under Article 54 (3) CPE, refers to the clarification of peptide sequences that are capable of binding to MHC class 1 glycoproteins of a type that are specifically encoded by the HLA-A2.1 allele and that, as a consequence of the union, can produce an immune response. ILEAKQKGYV and AALGFGTDNV are two amino acid motifs from human GAD that have been shown to elicit such a response.

Summary of the invention

Following one aspect of the invention, a polypeptide as claimed in claim 1 is provided.

Following another aspect of the invention, a polypeptide as claimed in claim 8 is provided.

Following a further aspect of the invention, a polypeptide as claimed in claim 9 is provided.

The invention further provides a polypeptide as claimed in claim 10.

In a further aspect of the invention, a polynucleotide sequence as claimed in claim 11 is provided.

The invention further provides a monoclonal antibody as claimed in claim

12.

Still following another aspect of the invention, a hybridoma cell line is provided as claimed in claim 13.

Following a further aspect of the invention, a vector as claimed in claim 14 is provided.

Still following another aspect of the invention, a host cell as claimed in claim 15 is provided.

Following still a further aspect of the invention, a method for detecting autoantibodies to GAD as claimed in claim 16 is provided.

Following another aspect of the invention, a use of the polypeptides of the invention is provided when preparing a composition as claimed in claim 17.

Following another aspect of the invention, a use of the polypeptides of the invention is provided when preparing a composition as claimed in claim 18.

The invention also provides equipment as claimed in claim 20.

The present invention presents the discovery that recombinant DNA technology could be used to produce eukaryotic GAD65 polypeptide and that the GAD65 polypeptide could be used in the diagnosis and therapy of patients with autoimmune disease. Particularly relevant is the use of the eukaryotic GAD65 polypeptide in the diagnosis and therapy of patients suffering from, or at risk of suffering from, GAD-related autoimmune disorders such as insulin-dependent diabetes mellitus (IDDM) or rigid man disease.

The main advantage of the present invention is that it provides the technique with an available source of eukaryotic GAD65 polypeptide corresponding to the purified of natural origin, while avoiding the problems related to the isolation of the naturally occurring eukaryotic GAD65 polypeptide when they separate, if it is from other eukaryotic polypeptides other than GAD65. The absence of other eukaryotic polypeptides other than GAD65 is important because it allows the development of test systems that will only detect antibodies specifically reactive with GAD65 polypeptides.

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Another advantage of providing GAD polypeptide₆₅ Eukaryotic in host cells is that by doing so, it is possible to obtain much larger amounts of polypeptide than are currently practically available from natural sources. As a consequence, it is not only possible to use the polypeptide of the invention to more accurately classify and treat patients for said autoimmune diseases such as IDDM, but it is also currently possible to provide commercially useful quantities of GAD polypeptide for use in diagnostic and diagnostic systems. in pharmaceutical compositions.

Description of the drawings

Figure 1 Cloning strategy to obtain GAD65 and GAD67 specific cDNA probes.

Figure 2 DNA sequence and corresponding amino acid sequence for rat GAD65. Figure 3 DNA sequence and corresponding amino acid sequence for human GAD65. Figure 4 Comparison of the amino acid sequences of rat GAD65 and human GAD65. Figure 5 GAD cDNAs₆₅ and GAD₆₇ they hybridize to RNA of different sizes.

Figure 6 Southern blots hybridized with specific cDNA probes for GAD65 and GAD67. Figure 7 Immunological identification of GAD65 and GAD67.

Figure 8 T-cell multiplier responses from NOD mice to β-cell antigens.

Figure 9 Characterization of the GAD-specific T-lymphocyte response of NOD mice as Th1 cells sensitized by increased clonal size (a) and cell surface markers (b) and IFNy secretion.

Figure 10 Intramolecular diffusion of T lymphocyte autoimmunity within the GAD molecule. Figure 11 Delay of the onset of IDDM after immunization with GAD65.

Detailed description of the invention

The present invention refers to the manipulation of genetic materials by means of recombinant DNA procedures that make possible the production of polypeptides that have part or all of the primary structural conformation for one or more of the epitopes, to bind autoantibodies to glutamic acid decarboxylase65 (GAD65 ) and for polypeptides that bind to MHC receptors to block T lymphocyte recognition. These polypeptides are extremely useful for the immunological detection of autoantibodies reactive with them, since said autoantibodies are prediagnosis and indicators of autoimmune diseases such as insulin-dependent diabetes mellitus and "stiff man" syndrome. These polypeptides can also be used for the purpose of identifying drugs, such as those that alter the function of GAD, and for the production of polyclonal and monoclonal antibodies which, in turn, can be used as a diagnosis to identify GAD65.

The development of specific DNA sequences encoding the eukaryotic GAD65 polypeptide for splicing in DNA vectors can be accomplished using various techniques. For example, alternative procedures that may be employed include (1) isolating a double helix DNA sequence from eukaryotic genetic DNA; (2) the chemistry of a DNA sequence to provide the necessary codons for the polypeptide in question; and (3) the in vitro synthesis of a double helix DNA sequence by reverse transcription of mRNA isolated from a donor eukaryotic cell. In the latter case, a double helix DNA complementary to the mRNA that is generally referred to as cDNA is finally formed.

Manufacture of DNA sequences is often the procedure of choice when the complete sequence of amino acid residues of the desired polypeptide is known. When the complete sequence of the amino acid residues of the desired polypeptide is not known, direct manufacture of the DNA sequences is not possible and the procedure of choice is the formation of cDNA sequences. Among the standard procedures for the isolation of the cDNA sequences in question is the formation of plasmid-bearing cDNA libraries, which come from the reverse transcription of the mRNA that is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, they can even be cloned

ES 2 179 077 T3 rare expression products. In those cases where significant parts of the amino acid sequence of the polypeptide are known, the production of DNA or RNA probe sequences of a labeled single or double helix by duplicating a sequence presumably present in the target cDNA can be used in hybridization procedures. DNA / DNA that is made on cloned copies of the cDNA that has been denatured into a helix shape (Jay et al., Nucleic Acid Research, 11: 2325, 1983).

Hybridization procedures are useful for the detection of recombinant clones using mixed, labeled oligonucleootide synoptic probes in which each is potentially the full complement of a specific DNA sequence in the hybridization sample comprising a heterogeneous mixture of denatured DNA. double helix. For such detection, hybridization is preferably carried out on DNA from a helix or DNA from a double helix. These procedures are particularly useful in the detection of cDNA clones originating from sources in which an extremely low amount of mRNA sequences were present relative to the polypeptide in question. In other words, using stringent hybridization conditions aimed at avoiding non-specific binding, it is possible, for example, to allow autoradiographic observation of a specific cDNA clone by hybridizing the target DNA so that the individual probe in the mixture is its complement. full (Wallace et al., Nucleic Acid Research, 9: 879, 1981).

In addition, the GAD cDNA library can be screened by injecting various cDNAs into oocytes, allowing sufficient time for expression of the cDNA gene products to take place, and testing for the presence of the desired cDNA expression product, for example, using the specific antibody for the GAD65 polypeptide, using functional analysis for the enzymatic activity of GAD65, or by measuring the ability of the expression product to stimulate pathogenic T lymphocytes.

On the other hand, a cDNA library for GAD65 peoptides having at least one epotope using antibodies to GAD65 can be indirectly screened (Chang and Gottlieb, J. Neurosci., 8: 2123, 1988). Such antibodies can be obtained polyclonally or monoclonally and can be used to detect the expression product indicating the presence of the GAD65 cDNA. Antibodies directed to an epotope observed in the first 100 amino acids of the N-terminal part of GAD are preferred.₆₅.

Of the three procedures indicated above to develop specific DNA sequences for use in recombinant procedures, the use of genomic DNA strains is the least frequent. This is especially true when it is desired to obtain the microbial expression of mammalian polypeptides due to the presence of introns.

The present invention provides novel GAD65 polypeptides as defined in the appended claims that are part of the entire primary structural conformation, that is, a continuous sequence of amino acid residues, having at least one epotope for antibodies to GAD65, and polynucleotides. encoding said polypeptides. It is possible to use the polypeptide fragments of the invention better than intact GAD to detect antibodies to GAD. The term "polypeptide", as applied to the GAD polypeptide, encompasses any amino acid sequence that has an epotope for autoantibodies to GAD. Thus, the GAD polypeptide fragments comprised by the invention possess biological activity such as the ability to produce and / or bind antibodies to GAD.

The polypeptides that result from the microbial expression of the DNA sequences of the invention or from other synthetic techniques, such as the solid phase peptide synthesis, can also be characterized by their freedom of association with other eukaryotic polypeptides or other contaminants that result from otherwise they could be related to GAD in their natural cellular environment or in extracellular fluids such as plasma or urine.

Studies by the present inventors unequivocally demonstrate that GAD65 and GAD67 are encoded by different genes and are not produced, for example, by post-transcriptional or post-translational modification of a common genomic sequence. Evidence proving that GAD65 and GAD67 are encoded by different genes comprises: (a) the largest contiguous sequence of exact identity between GAD65 and GAD67 cDNAs is only 17 nucleotides long, (b) the GAD65 and GAD67 cDNAs do not hybridize to each other or between asses and the mRNA under low stringency conditions (2.0 x SSC, 0.01% SDS, 23 ° C), and (c) the GAD cDNAs₆₅ and GAD₆₇ they do not hybridize by crossing with isolated genomic clones encoding GAD67 and GAD65, respectively.

The term "host" includes not only prokaryotes, but also eukaryotes such as yeast,

ES 2 179 077 T3 filamentous fungi, plant and animal cells, as well as insect cells that can replicate and express an intron-free DNA sequence of GAD₆₅ eukaryotic. However, prokaryotes are preferred as host organisms for detection purposes whereas eukaryotic cells, especially insect cells, are preferred for expression.

The term "prokaryotes" includes all bacteria that can be transformed or transfected with the gene for the expression of GAD65. Prokaryotic hosts can comprise 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 encoding GAD65 polypeptides can be used to transform or transfect the host using any of the techniques commonly known to those of skill in the art. Especially preferred is the use of a plasmid or a virus containing the sequence encoding GAD65 for the purposes of prokaryoetic transformation or transfection, respectively. On the other hand, liposomes containing the DNA in question can be used to obtain expression in the host (Zhu et al., Science, 261: 209, 1993).

Procedures for preparing genes fused, operably linked and expressed in bacteria are well known in the art (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). The genetic constructs and procedures described in this manual can be used for the expression of GAD65 in prokaryotic hosts.

In general, expression vectors containing activator sequences that facilitate efficient transcription of the inserted eukaryotic genetic sequence are used in relation to the host. The expression vector typically contains an origin of replication, an activator and a terminator, in addition to specific genes that are capable of providing phenotypic selection of transformed cells. Transformed prokaryotic hosts can be grown in fermenters and cultured according to techniques known in the art to achieve optimal cell growth. The polypeptides of the invention can then be isolated from growth medium, from cell lysates or from cell membrane fractions.

The isolation and purification of the expressed polypeptides of the invention can 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 antibodies.

Having provided the sequence of amino acid residues of GAD65, the present invention provides for the preparation of DNA sequences encoding host expression analogs or polypeptide derivatives of GAD65 that differ from naturally occurring forms in terms of identity or of location of one or more amino acid residues and that share some or all of the epétopes of the forms that occur in nature.

The new DNA sequences of the invention comprise all the sequences useful to provide the expression in the cells of the prokaryotic or eukaryotic host of polypeptides that have at least a part of the primary structural conformation for one or more epitopes capable of reacting with autoantibodies to GAD65 which are comprised of: (a) the DNA sequence as set forth below in Figures 2 or 3 or their complementary helices; (b) DNA sequences that hybridize to the DNA sequences defined in (a) or their fragments; and (c) DNA sequences that, except for the degeneracy of the genetic code, will hybridize to the DNA sequences defined in (a) and (b) above. Genetic DNA sequences encoding allele variant forms of GAD65 are specifically encompassed in (b). Part (c) specifically comprises the preparation of DNA sequences encoding GAD65, GAD65 fragments and GAD65 analogs in which the DNA sequences of these can incorporate codons that facilitate translation of the mRNA in invertebrate hosts.

As the cDNA sequence of the invention essentially encodes the human or rat GAD65 molecule, it is currently a routine matter to prepare, subclone and express smaller polypeptide fragments of cDNA from these or the corresponding cDNA sequence that I will encode. nothing more than an epitope for human or rat GAD65 autoantibodies. The presence of such an epitope in a cloned polypeptide can be confirmed below using, for example, serum from a patient with autoantibodies to GAD.₆₅. An example of such a smaller peptide is the first 100 amino acids approximately from the N-terminus of GAD65 (shown in Figure 3). This amino acid sequence is essentially absent from GAD67. Other examples of peptides

ES 2 179 077 T3 specific to the invention are those shown in Table 7 in addition to the approximate two thirds carboxy terminal of GAD from approximately amino acid 224 to amino acid 585. Especially preferred in the approximately two thirds carboxy terminal of GAD is segment from about amino acid 224 to amino acid 398.

The present invention also relates to monoclonal antibodies that are specific for the polypeptides of the invention as well as the use for the diagnosis and therapy of these monoclonal antibodies. This specificity allows the monoclonal antibody, and similar monoclonal antibodies with similar specificity, to be used to bind the polypeptide of the invention when the polypeptide, or the amino acids that make up the polypeptide, are present in samples or in a host, such as a man. .

Numerous techniques can be used to produce monoclonal antibodies of the invention without resorting to undue experimentation. In large part, the products of such monoclonal antibodies become routine because of the highly defined nature of the polypeptides of the invention. Thus, if the polypeptides of the invention are used for immunization and / or detection, the very limited number of immunogenic determinants in the polypeptides greatly simplifies the identification of cell lines that produce monoclonal antibodies of the invention, for example, limiting the repertoire of possible clonal expression.

A type of cell line widely used for the expression of the monoclonal antibodies of the invention is the hybridoma. The general procedure used for the production of hybridomas that produce monoclonal antibody is well known (Kohler and Milstein, Nature, 256: 495, 1975). The resulting hybridomas were then identified for the 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., US Patent No. 4,172,124, Koprowski et al., US Patent No. 4,196,265, or Douillard, JY and Hoffman, T., Basic Facts about Hybridomas, in Compendium of Immunology, Vol II, L. Schwartz, ed. (1981), which is incorporated herein by reference. In general, purified peptides can be modified to have a C-terminal bound cysteine ​​to allow unidirectional binding of the synthetic peptide to an immunogenic protein via a connecting bridge, eg, keyhole limpet hemocyanin (KLH) - maleimidobenzoylated (MB). Other immunogenic conjugates can also be used, for example, albumin and the like. The resulting structure can have several peptide structures linked to a protein molecule.

Somatic cells from a host immunized against synthetic peptides can be obtained by any suitable immunization technique. The host is immunized by administration of the antigen, generally in the form of a protein pool, as indicated above, by any suitable procedure, preferably by injection, intraperitoneally, intravenously, subcutaneously, or by internal foot pad. Adjuvants can be included in the immunization protocol.

The initial immunization with the antigen-bound protein may be followed by several booster injections given periodically at intervals of several weeks. The reactivity of the antibody contained in the plasma of each host with the immunizing peptide of the invention can then be tested. The host with the highest response is generally the most desirable donor of the somatic cells that secrete the antibody used in the production of hybridomas. On the other hand, hyperimmunization can be effected by repeatedly injecting additional amounts of the peptide-protein combination intravenously and / or intraperitoneally.

The isolation of hybridomas producing monoclonal antibodies of the invention can be performed using routine detection techniques that allow the determination of the elemental reaction pattern of the monoclonal antibody in question. 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.

On the other hand, as the invention teaches the polypeptide or amino acid sequences that are specifically needed for the binding of the preferred monoclonal antibodies of the invention, it is currently possible to use these peptides for immunization purposes to produce hybridomas which, in turn, produce monoclonal specific antibodies for the polypeptide. This approach has the added advantage

ES 2 179 077 T3 to decrease the repertoire of monoclonal antibodies generated by limiting the number of antigenic determinants presented in immunization by the polypeptide. The specificity of monoclonal antibodies produced by this procedure can be detected using standard techniques, for example, by binding a polypeptide to a microtiter plate and measuring the binding of the monoclonal antibody by ELISA analysis.

It is also possible to determine, without undue experimentation, whether a monoclonal antibody has the same specificity as a monoclonal antibody of the invention by ascertaining whether the former prevents the last of the binding of the polypeptide of the invention. If the monoclonal antibody under test competes with the monoclonal antibody of the invention, as demonstrated by a decrease in binding by the monoclonal antibody of the invention, then it is likely that the two monoclonal antibodies will bind to the same or a closely related epitope.

Yet another way to determine if 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 reacts normally, and then add the monoclonal antibody that is tested to determine whether the monoclonal antibody being tested inhibits its ability to bind to the antigen. If the monoclonal antibody under test is inhibited then in all likelihood it has the same, or closely related, epotope specificity as the monoclonal antibody of the invention.

The GAD65 of the invention is particularly suitable for use in immunoanaolysis where it can be used in the liquid phase or bound to a carrier in the solid phase. In addition, the GAD65 used in these analyzes can be detectably labeled in a number of ways.

Examples of immunoassays that can use the GAD65 of the invention are competitive and noncompetitive immunoassays in direct or indirect format. Examples of such immunoanolysis are radioimmunoanalysis (RIA), sandwich (immunometry) and Western blot assay. Detection of antibodies that bind to the GAD65 of the invention can be done using immunoanolysis that is performed in forward, reverse, or simultaneous modes, comprising immunohistochemistry analyzes on physiological samples. The concentration of GAD65 used varied depending on the type of immunoassay and the nature of the detectable label used. However, regardless of the type of immunoassay used, the concentration of GAD65 used can easily be determined by someone of ordinary skill in this field 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. Furthermore, carrier-bound GAD and GAD fragments can be used therapeutically by extracorporeal absorption of autoimmune antibodies in patients who have, or are at risk for, GAD-related disorders. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nyloon, amyloses, natural and modified celluloses, polyacrylamides, agarose, and magnetite. The nature of the carrier can be soluble or insoluble for the purposes of the invention. Other suitable carriers are known to those of skill in the art for binding GAD65, or would be able to discover such carriers, using routine experimentation.

There are many different markers and labeling procedures known to those of ordinary skill in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisootopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds.

On the other hand, the polypeptide of the invention comprising the enzymatic field of GAD can be used to detect antibodies to GAD by measuring the enzymatic activity of GAD. For example, GAD65 and a sample suspected of having antibodies to GAD65 can be incubated for a period of time and under conditions sufficient to allow binding between GAD65 and the antibodies to take place. The reaction product is precipitated and then the enzyme activity of GAD is tested.

For the purposes of the invention, the GAD65-binding antibody of the invention may be present in various biological fluids and tissues. Any sample that contains a detectable amount of antibodies to GAD65 can be used. Normally, the sample is a liquid such as urine, saliva,

ES 2 179 077 T3 cerebrospinal fluid, blood, serum and the like, and a solid or semi-solid such as tissue, feces and the like.

The materials for use in the analysis of the invention are theoretically suitable for the preparation of a kit. Said equipment may comprise a carrier that is compartmentalized to admit in a compact case one or more containers such as vials, tubes or the like, each container comprising one of the separate elements to be used in this procedure. For example, one of the containers may contain GAD65 attached to a carrier. A second container may comprise a detectably labeled, soluble second antibody, in lyophilized form or in solution.

In addition, the carrier may also contain a variety of containers each comprising different, predetermined amounts of GAD65. These latter containers can then be used to prepare a pattern curve into which the results obtained from the sample containing the unknown amount of autoantibodies to GAD65 can be interpolated.

When using the equipment, what every user has to do is add, to a container, a previously measured quantity of a sample containing a measurable, yet unknown quantity of GAD65 autoantibodies to be detected, a previously measured quantity of GAD65 bound to the carrier present in the first container and a pre-measured amount of the second detectably labeled antibody present in the second container. Alternatively, the undetectable labeled GAD65 can be provided bound to the container to which the sample and the detectably labeled second antibody are added. After an appropriate incubation time, an immune complex is formed and separated from the supernatant fluid and the immune complex or supernatant fluid is detected, by radioactive counting or addition of an enzyme substrate and color development.

In another embodiment, a kit comprising the GAD polypeptide of the invention can be used to detect the GAD-related stage of autoimmune disease in a patient. As further demonstrated herein, Applicants have discovered that certain GAD peptides or fragments are related to different levels of progression in autoimmune disease and that the level of the pathological process can be determined by examining the multiplying response of the immune cell, such as such as that of the patient's pathogenic T lymphocyte.

The term "improve" indicates a decrease in the detrimental effect of the autoimmune response in the patient receiving therapy. The term "therapeutically effective" means that the amount of GAD65 polypeptide used is an amount sufficient to ameliorate the cause or disease due to the autoimmune response.

The GAD65 polypeptides, including all of the GAD65, of the invention can be used therapeutically in patients who have a GAD65-related autoimmune response. Such therapy can be accomplished, for example, by administering GAD65 polypeptide to produce tolerance to GAD. Such administration can use unlabeled as well as labeled GAD65 polypeptide. When unlabeled GAD65 polypeptide is suitably used, it should be in a form in which, for example, the GAD65 polypeptides are in fragments that are too small to stimulate an immune response, but large enough to bind, or block, the immune response. continuation of the autoimmune response. For example, GAD65 should be enzymatically digested into epitope-sized peptides (typically 5 to 12 amino acids in length) and bind for that reason to Fab-binding portions present in body fluids, or on the surface of immune cells. of the patient with autoimmune disease. On the other hand, peoptides having at least one determinant to bind to the MHC receptor of the T cell can be produced or chemically synthesized in a similar way.

Furthermore, the GAD65 polypeptides of the invention can be administered labeled with a therapeutic agent. These agents can be directly or indirectly coupled to the GAD65 polypeptides of the invention. An example of indirect coupling is by use of a spacer group. These spacer groups, in turn, can be insoluble or soluble (Diener et al., Science, 231: 148,

1986) and can be selected to allow release of the GAD65 polypeptide drug at the target site. Examples of therapeutic agents that can be coupled to the GAD65 polypeptides of the invention for immunotherapy are drugs, radioisotopes, lectins, and toxins.

The drugs with which the GAD65 polypeptides of the invention can be combined comprise compounds classically referred to as drugs such as mitomycin C, daunorubicin and vimblastin.

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When using GAD65 polypeptides of the invention for immunotherapy, certain isotopes may be more preferable than others depending on factors such as distribution, as well as leukocyte stability and emission. Depending on the autoimmune response, some emitters may be preferable to others. In general, α and β particle-emitting radioisootopes are preferred in immunotherapy. Narrow-band, high-energy emitters such as²¹²Bi. Examples of radioisootopes that can bind to the GAD65 polypeptides of the invention for therapeutic purposes are¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd and ¹⁸⁸Re.

Lectins are proteones, generally isolated from plants, that bind to specific sugar groups. Many lectins are also capable of agglutinating cells and stimulating lymphocytes. However, castor is a toxic lectin that has been used immunotherapeutically. This is done by binding the castor α-peptide chain, which is responsible for toxicity, to the antibody molecule to allow the specific site to be rid of the toxic effect.

Toxins are poisonous substances produced by plants, animals or microorganisms that, in sufficient doses, are often lethal. Diphtheria toxin is a substance produced by Corynebacterium diphtheria that can be used therapeutically. This toxin consists of α and β subunits that can be separated under appropriate conditions. The toxic component A can be bound to the GAD65 polypeptide and used for site-specific delivery for a lymphocyte expressing a GAD65 polypeptide receptor.

Other therapeutic agents that can be coupled to the GAD65 polypeptides of the invention, as well as ex vivo and in vivo therapeutic protocols, are known, or can be readily discovered by those skilled in the art.

The present invention also relates to a polypeptide that can be administered therapeutically to improve, or be used as a diagnostic tool to identify, the pathological process in patients suffering from, or at risk of suffering from, this disease. The conventional code of a single letter used to represent the different amino acids is established as follows:

TABLE 1

<td>Phe:</td><td>F</td><td>Leu: L</td><td>lle: I</td><td>Met: M</td>

<td>Val:</td><td>V</td><td>Ser: S</td><td>Pro: P</td><td>Thr: T</td>

<td>To:</td><td>TO</td><td>Tyr: And</td><td>His: H</td><td>Gln: Q</td>

<td>Asn:</td><td>N</td><td>Lys: K</td><td>Asp: D</td><td>Glu: E</td>

<td>Cys:</td><td>C</td><td>Trp: W</td><td>Arg: R</td><td>Gly: G</td>

A polypeptide sequence of the invention was identified by comparing the amino acid sequences of human GAD65, human GAD67, and the P2-C proteon of picornavirus and coxsackie virus. The P2-C polypeptide plays a role in the membrane-bound replication complex of the virus. These analyzes demonstrated the presence of extensive sequence similarity between the GAD65 molecules and the coxsackie virus. The amino acid sequences in a six contiguous amino acid residue polypeptide nucleus of GAD65 and in the P2-C polypeptide are identical. Indeed, of the 24 amino acids of the polypeptide, 19 are either identical or conserved. In addition, there is also a high charge density and the presence of a proline residue that would make this area highly antigenic (see Table 2).

TABLE 2

Comparison of amino acid sequences

<td>Protein</td><td>Amino acid sequence</td>

<td>GAD₆₇ human GAD₆₅ Human Coxsackie Virus PC-2</td><td>₂₅₈S IMAARYKYFPEVKTKGMAAVPK L_{281 250}a mmiarfkmfpevkekgmaalpr l₂₇₃25FIEWLKVKKILPEVKEKHEF-LSRL50</td>

The lone continuum comprises the idótic amino acids while the hyphen comprises the amino acid residues with similar charge, polarity, or hydrophobicity.

ES 2 179 077 T3

In Table 2, the continuous line comprises the idáentico amino acids while the guioan comprises the amino acid residues with similar charge, polarity or hydrophobicity.

The discovery of this polypeptide common zone supports an etiological role for "molecular mimicry" in the onset of diabetes. Thus, when a patient genetically susceptible to IDDM is infected by a coxsackie virus, the immune response to the GAD sequence is similar in the patient's β-cells. The immunological response is maintained by antigenically similar GAD polypeptides that result from the consequent destruction of β-cells and the subsequent presentation of IDDM.

Currently, it is believed that the destruction of pancreatic β cells in IDDM is mediated by an autoimmune cellular response. As described herein, a polypeptide of the invention can enhance the immune response to GAD. Due to the complexity of autoimmune disease, it is possible to envision numerous possible therapeutic modalities that will allow the polypeptides of the invention to be used to improve said diseases. In one embodiment, it appears that the polypeptides of the invention can be used to block recognition by a specific T-lymphocyte receptor (TCR) or an MHC receptor presenting an autoimmune antigen on the surface of a cell-presenting antigen ( APC). Inhibition of such recognition can occur, for example, by providing the patient with the polypeptide of the invention which, in turn, can displace the autoimmune antigen that is presented in the MHC receptor antigen cleft. However, while not wishing to be bound by any particular theory, it is believed that the polypeptides of the invention likely act to produce or reestablish a tolerogenic state by direct interaction with the appropriate TCR on the surface of a pathogenic T cell specific to GAD. This latest therapeutic approach of direct interaction with TCR is supported by the examples and suggests that suppression of the autoimmune response can be achieved by eliciting upper zone tolerance by using large concentrations of polypeptide, preferably soluble. Another possible mechanism is that the polypeptide of the invention may play a role by anergizing pathogenic T lymphocytes by binding to the MHC receptor of T lymphocytes, thereby avoiding adequate synergistic signaling.

On the other hand, the polypeptides of the invention can be used to stimulate a population of suppressor T lymphocytes to restore self-recognition and, for this reason, improve autoimmune disease. Stimulation of suppressor T lymphocyte populations can be achieved, for example, by using a bispecific antibody that has a variable specific zone for an epitope present on the autoimmune antigen that resides in the MHCII receptor cleft, and a second specific zone variable for an epitope present on the CD8 receptor⁺. The production of specific antibody for the polypeptide of the invention is a routine matter for those skilled in the art, since it is the preparation of bispecific antibodies that have specificity for 2 or more epatopes.

Polypeptide analogs of the present invention can be designed that will compete for recognition of autoanthagens at the antigen presentation level or will produce anergy in T lymphocytes, due to a lack of synergistic signal. Since MHC molecules contain a unique peptide binding site, it is possible to design polypeptides that will bind with high affinity to disease-related MHC molecules, but will not activate disease-causing helper T cells. Said polypeptides act as antagonists in the recognition of autoanthagen. In the present invention, support for this mechanism is found in the examples, especially in the Example

7. The precedent for such an approach arises from the observation that a non-immunogenic rat lysozyme polypeptide can compete for MHC binding with an immunogenic hen egg white lysozyme polypeptide and thereby reduce the activation of the T lymphocyte by means of this polypeptide (Adorini et al., Nature, 334: 623 to 625, 1988) in addition to studies using T lymphocyte receptor peptides to block complex formation between T lymphocytes, autoanthagen and MHC (Howell et al., Science, 246: 668, 1989). Similarly, such an approach to detect effective polypeptide analogues has been used in such autoimmune diseases as experimental autoimmune encephalomyelitis (EAE) (Wraith et al., Cell, 59: 248, 1989; Urban et al., Cell, 59: 257, 1989) .

The one-letter symbols used to represent amino acid residues in the polypeptide of the present invention are the symbols commonly used in the art. The peptides of the invention comprise not only natural amino acid sequences, but also peptides that are analogous, chemical derivatives and their salts. The term "analog" or "conservative variant" refers to any polypeptide having an amino acid sequence substantially identical to a polypeptide provided herein and in which one or more amino acids have been substituted for chemically similar amino acids. For example, a polar amino acid, such as glycine or serine, can be substituted for

ES 2 179 077 T3 tuir by another polar amino acid; or an acidic amino acid, such as aspeartic acid, can be substituted for another acidic amino acid, such as glutaemic acid; or a basic amino acid, such as lysine, arginine or histidine can be substituted for another basic amino acid; or a non-polar amino acid such as alanine, leucine or isoleucine can be substituted for another non-polar amino acid.

The term "analogous" or "moderate variation" also means any polypeptide that has one or more amino acids deleted from or added to a polypeptide of the present invention, but that still retain substantial amino acid sequence homology for said peptide. Substantial sequence homology is any homology greater than 70%, preferably at least about 80%, and most preferably at least about 90%. The term "fragment" also means any shorter version of the polypeptides identified herein that have at least 6 amino acid residues, in which the fragment possesses biological activity, or is a fragment capable of inhibiting the stimulation of T lymphocytes by a stimulating polypeptide fragment or substantially the entire length of the molecule.

The term "chemically derived" means any polypeptide derived from a polypeptide of the present invention and in which one or more amino acids have been chemically derived by reaction of the secondary functional groups of the amino acid residues present in the polypeptide. Thus, a "chemical derivative" is a polypeptide that is derived from the sequences or polypeptides identified herein in one or more chemical steps. Said derived molecules comprise, for example, those molecules in which derivatives of free amino groups have been obtained to form amine chlorides, p-toluene sulfonamides, benzoxycarboamides, T-butyloxycarboamides, thiourethane derivatives, trifluoroacetylamides, chloroacetamides or formamides. Derivatives of free carboxyl groups can be obtained to form salts, methyl and ethyl esters or other types of esters or hydrazides. Derivatives of free hydroxyl groups can be obtained to form O-acyl or O-alkyl derivatives. A nitrogen imidazole derivative of histidine can be obtained to form N-im-benzylhistidine. Also included as chemical derivatives are polypeptides containing one or more naturally occurring amino acids derived from the standard 20 amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine and ornithine can be substituted for lysine.

It should be understood that the present invention is not limited to the illustrative polypeptides described in Table 2 and Table 9, instead, a polypeptide within the scope of the invention may extend beyond, or comprise less than, the area between amino acid 28 and amino acid 50 of coxsackie virus P2-C, or between amino acid 250 and amino acid 273 of GAD65, or between amino acid 258 and amino acid 281 of GAD67, in addition to the zone 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 528 amino acid, or between 524 amino acid and 543 amino acid, or between 539 amino acid and 556 amino acid, or between 564 amino acid and 583 amino acid of GAD65, as long as a substantial part of a given polypeptide is characterized by an amino acid sequence from this region, or its segments or combinations, and the polypeptide demonstrates the desired immunological or biological activity against autoimmune disease. Furthermore, the polypeptides according to the invention comprise those that have amino acid sequences that are shorter in length than those of the polypeptides illustrated in Table 2 and Table 9, or that comprise their segments or combinations, while said polypeptides are substantially composed of the zone 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 autoimmune disease.

Thus, it should be understood that the specific selection of any polypeptide within the polypeptides of the invention does not involve undue experimentation. Said selection can be carried out by taking a number of polypeptides and testing their immunological and biological activity in the improvement of autoimmune disease or in the detection of antibodies. NOD raton represents an excellent and well-characterized model for detecting polypeptides of the invention capable of improving or preventing diabetes. Example 7 illustrates an acceptable procedure for the routine detection of candidate polypeptides for biological activity.

The polypeptides according to the present invention can be prepared by recombinant techniques or by conventional synthesis using known polypeptide synthesis procedures, including synthesis on solid support. An example of a suitable solid phase synthesis technique is that described by Merriweather (J. Amónico. Chem. Soc., 85: 2149, 1963). Other polypeptide synthesis techniques can be found, for example, in Bodanszky et al., Peptide Synthesis, John Wiley &amp; Sons, 2-ed., 1976, in addition to other references known to those of skill in the art. A summary of

ES 2 179 077 T3 techniques for polypeptide synthesis in Stewart et al., Solid Phase Peptide Synthesis, Pierce Chemical Company, Inc., Rockford, Ill., 1984. The synthesis of polypeptides by solution procedures can also be used, for example, as described in The Proteins, Vol. II, 3-ed., Neurath et al., eds., Academic Press, New York, NY, 1976. Suitable protecting groups for use in such syntheses can be found in the above references as well as in J. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, NY, 1973.

The polypeptides of the invention can also be prepared in an appropriate host transformed with DNA sequences encoding the desired polypeptide. For example, a polypeptide can be prepared by fermentation of appropriate hosts that have been transformed with a DNA sequence encoding and expressing the polypeptide. Furthermore, a DNA sequence encoding various polypeptides of the invention can be coupled and those sequences can then be used to transform an appropriate host to allow expression of polypeptides involved in autoimmune disease.

The dosage ranges for the administration of the polypeptides of the invention are wide enough to produce the desired effect in which the symptoms or cellular destruction of the autoimmune response are improved. The dose should not be so large as to produce adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, and the like. Generally, the dose will vary with the age, disease, sex, and extent of disease in the patient and can be determined by one of skill in the art. The doctor can adjust the dose in case of any contraindication. The dose can range from approximately 0.1 mg / m²/ dose at approximately 2000 mg / m²/ dose, preferably from about 0.1 mg / m² / dose at approximately 500 mg / m²/ dose, in one or more dose administrations per day, for one or more days.

The GAD polypeptides of the invention can be administered parenterally by injection or by gradual intravenous infusion over time. The GAD polypeptides of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitary, transcutaneously, intranasally or parenterally.

Preparations for parenteral administration comprise sterile aqueous or nonaqueous 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 excipients comprise water, aqueous / alcoholic solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles comprise sodium chloride solution, Ringer's dextrose, sodium dextrose and chloride, lacteal Ringer's solution, or treated oils. Intravenous vehicles comprise filling fluids and nutrients, electrolyte fillers (such as those based on Ringer's dextrose), and the like. In addition, preservatives and other additives may be present such as, for example, antimicrobials, antioxidants, chelators, inert gases, and the like.

The invention also relates to a process for preparing a drug or pharmaceutical composition comprising the GAD65 polypeptides of the invention, the drug being used for the therapy of the autoimmune response to GAD65.

The foregoing discussion generally describes the present invention. A more complete understanding can be obtained by referring 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

GAD cloning and expression₆₅

A. Recombinant DNA Procedures

To obtain specific cDNA probes for GAD65 and GAD67, whole RNA was extracted from adult rat brain by cesium guanidine isothiocyanate gradient using the method of Chirgwin et al. (Biochemistry, 18: 5294, 1979). Poly (A) RNA was purified on oligo dT cellulose, using the Bethesda Research Laboratories (BRL) protocol. First helix synthesis was carried out using MMLV reverse transcriptase (BRL), under the suggested conditions, unless d (N6) polymers were used as primers (Pharmacia). This cDNA-RNA mixture was heat inactivated at 65^°C for 15 minutes and kept at -20^°C. For PCR, 1/50 of the sample was added at 100 μ! reaction. Degenerate oligonucleetides (Applied Biosystems) were synthesized to encode

ES 2 179 077 T3 the underlined feline common amino acid sequences (from cDNA) (Kobayashi et al., J. Neurosci., 7: 2768, 1987) and rat (from peptides) (Chang and Gottlieb, J Neurosci., 8: 2123, 1988) GAD (Figure 1). The 5 'ending sequence of each degenerate oligonucleootide contained a helix of the DNA sequence recognized by SstI and HindIII (5' oligo) or SstI and SstII (3 'ending oligo). These primers were used for selective polymerase chain reaction amplification of the generated cDNA template as described by Gould et al. (Proc. Natl. Acad. Sci., USA, 86: 1934, 1989). The PCR products were subcloned into double HindIII / SstI, the Bluescript SK vector (Stratagene) was digested, transformed into DH5 (BRL) and plated by standard procedures (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).

The colony hybridization was done with a 5'- terminal labeled oligonucleootide.³²GAD specific P₆₇ feline (Kobayashi et al., J. Neurosci., 7: 2768, 1987). The oligonucleotide terminal labeling, 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. Academis Press , Inc., San Diego, 432-442, 1987), except that the nitrocellulose filters were washed at 50^°C for 15 min. Colonies that were hybridized positive and negative were selected one by one and grew overnight in Terrific Broth (Tartof et al., Focus, 9:12,

1987). DNA was isolated using a boiling procedure (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989) and templates were denatured with 0.2N NaOH and purified on columns of Sephacryl S400 wool (Pharmacia). Obtaining the sequence of the denatured double helix template was done by the chain termination procedure (Sanger et al., Proc. Natl. Acad. Sci., USA, 74: 5463, 1977) using the T7 sequencing kit (Pharmacia).

As shown in Figure 1, PCR-produced rat GAD65 and GAD67 cDNAs were used as tests to identify a lambda ZAP (Stratagene) provided by the S. Heinemann rat hippocampal library (Salk Institute) using standard techniques (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). A 2400 nucleotide GAD65 cDNA (the largest clone) was isolated and subcloned by "zapping" as described by Stratagene. When a GAD cDNA was already available₆₇ which was smaller than a 3.2 kb rat GAD67 cDNA clone, the sequence of the largest cDNA was obtained. Deletions of Exo III (Henikoff, Gene, 28: 351, 1984) for GAD₆₅ and GAD₆₇ they were run in both directions and templates were prepared and sequences were obtained as described above. Anchored PCR (Frohman et al., Proc. Natl. Acad. Sci., USA, 85: 8998, 1988) was prepared to clone the 5 'terminals of the GAD65 and GAD67 mRNAs that were not represented in the original cDNA clones isolated in ID from the library. Obtaining the sequence of these clones revealed that neither the GAD65 nor the GAD67 mRNAs contained any additional initiation codons (AUGs) in the framework with the previously designed initiation codons of the original cDNA clones.

Example 2

Characterization of the cloned GAD65

A. Northern blot hybridization

Two PCR-derived cDNA probes were hybridized by Northern blots containing rat brain RNA to determine whether the GAD67 and GAD65 cDNAs were derived from two different mRNAs. RNA was extracted as described in Example 1. Poly (A) RNA was separated by formaldehyde electrophoresis, transferred to Biotrans membranes (ICN), and hybridization was performed as described by Well et al. (J. Neurosci., 16 : 311, 1986), except that 100 µ / ml of poly (A) was added. The probes were labeled at approximately 10⁹ dpm / 'g by the oliglabeling procedure of Feinberg and Vogelstein (Anal. Biochem., 132: 6, 1983). Identical results were subsequently obtained with full length clones of GAD65 and GAD67 cDNAs.

C omo shown in Figure 5, lanes 1 and 2 contain 1 .mu.g of poly (A) RNA from the selected extraódo rat cerebellum. Lane 1 was hybridized in a cDNA probe for the rat afon to feline GAD67 (Kobayashi et al., J. Neurosci., 7: 2768, 1987) and lane 2 with a cDNA probe for the rat peptide sequence (corresponding to GAD65).

The cDNA probe for the rat peptide sequence hybridized to 5.7 kb RNA, while the rat cDNA probe related to feline GAD67 DNA hybridized to 3.7 kb RNA.

ES 2 179 077 T3

This shows that GAD65 and GAD67 do not come from the same mRNA.

B. Genomic hybridization of GAD₆₇ and GAD₆₅

To investigate the possibility that GAD67 and GAD65 originate from separate genes, GAD65 and GAD67 cDNAs were hybridized to DNA blots containing genetic DNA.

For Southern blots, genetic DNA was extracted from rat liver as described (Kaiser et al., In DNA Cloning, vol. I, A Practical Approach, DM Glover ed., IRL Press, Oxford, pp. 38-40, 1985 ). DNA (10 µ / sample) was fully digested with EcoRI and HindIII using the conditions recommended by the suppliers (BRL, Gaithersburg, MD). The DNA fragments were separated by electrophoresis at 1.5v / 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 μ / ml Carnation milk powder they were replaced by Denhardt's solution. Probes were labeled for Southern blotting as described in Example 1, above.

As shown in Figure 6, the HindIII and EcoRI digested genoemic DNA are in bands 1 and 3 and in bands 2 and 4 respectively. The GAD67 cDNA hybridized in lanes 1 and 2, while the GAD65 cDNA hybridized in lanes 3 and 4. The numbers along the sides of the gel are the dimensions of the DNA fragment in kilobases.

These data demonstrate that the two cDNAs hybridize to gene fragments of different dimensions. Furthermore, the largest contiguous stretch of identical nucleotide sequence of the GAD65 and GAD67 cDNAs is only 17 nucleotide bases in length. So, GAD67 and GAD65 are encoded by two different genes.

C. Enzyme comparison between GAD₆₇ and GAD₆₅

Studies were done comparing the effect of PLP on the activity of GAD67 and GAD65. Acting in this way, both cDNAs were subcloned into vectors that allowed their expression in bacteria (Studier et al., J. Mol. Biol., 189: 113, 1986). The overexpression of GAD₆₅ and GAD₆₇ "Fuseless" is performed by subcloning GAD cDNA₆₅ at the NcoI site of pET-8c and the GAD cDNA₆₇ at the NheI site of the pET-5c vectors (Studier et al., J. Mol. Biol., 189: 113, 1986).

To obtain compatible adhesive ends for a correct subcloning in the correct structure of both cDNAs, selective PCR amplification will be carried out using the conditions suggested by United States Biochemical (USB), with 200 μΜ dNTPs and MgCl.₂ 1.5mM in the mix and tempering at 55 C with 20 cycles to decrease the infidelity of AmpliTAQ (USB). The primers specific for GAD65 and GAD67 contained a DNA haelix from the NcoI and SpeI recognition sites, respectively. As there is a NheI restriction site in the GAD67 coding region, SpeI (which is compatible with NheI) 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, NY, 1989). Colonies were picked and grown overnight in LB broth with 50 µg / ml ampicillin. Correctly oriented subclones were transformed into strain BL21 (DE3) (Studier et al., J. Mol. Biol., 189: 113, 1986) for overexpression. As a negative control, the pET-8C vector without any insertion was transformed and subsequently stimulated. Individual colonies were selected, grown, stimulated by 1mM isopropyl-BD-thiogalactopyranoside (IPTG) and analyzed on SDS-PAGE gels as described (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 17.1517.16, 1989).

To measure GAD activity, 10 ml of bacteria cultures were produced at OD600-0.5 with 1mM IPTG. Two hours after production, they were turned over, resuspended, and sonicated in 1 ml of homogenizing buffer (1mM phenylmethylsulfonyl fluoride (PMSF), 1mM aminoethylisothiouronium bromide (AET), and 60 mM of potassium phosphate, pH 7.1). After ultrasound, cell debris was removed by centrifugation and protein concentration was measured (Bradford, Anal. Biochem., 72: 248, 1986) in the supernatant (the supernatant was stored in aliquots at -70 ° C). Brain homogenates were prepared as described (Legay et al., J. Neurochem., 33: 299, 1984) with or without 0.2mM PLP and 20μ of brain homogenate or bacterial lysate.

ES 2 179 077 T3 in the incubation mix. The production of¹⁴CO2 in bacterial lysates was linear in relation to incubation time and protein concentration.

TABLE 3

<td rowspan="2">Source</td><td colspan="2">GAD specific activity^to</td><td rowspan="2">Duplicates in production</td>

<td>- PLP</td><td>+ PLP</td>

<td>BL21 (DE3) + pET-8c</td><td> 12 ± 0,4</td><td> 9±1</td><td>_—</td>

<td>BL21 (DE3) + pET-GAD65</td><td> 115 ± 3</td><td> 773 ± 61</td><td> 6,7</td>

<td>BL21 (DE3) + pET-GAD67</td><td> 160 ± 2</td><td> 389 ± 8</td><td> 2,4</td>

<td>Rat brain</td><td> 131 ± 5</td><td> 216 ± 2</td><td> 1,6</td>

^to cpms of ¹⁴CO₂^ g protein / hr of triplicates ± SEM

As shown in Table 3, bacterial lysates containing GAD65 or GAD67 catalyze the conversion of [1-C] -glutamate to GABA and ¹⁴CO2.

PLP stimulates the enzyme activity of GAD65 more than that of GAD67. This increased stimulation probably reflects the more rapid cyclization of GAD65 by the inactivation cycle proposed by Martin et al. (Martin, Cell. Mol. Neurobiol., 7: 237, 1987). This faster cycling suggests that GAD65 contributes more to the apo-GAD association than exists in vivo (Miller et al. Brain Results. Bull., 5 (Suppl 2): ​​89, 1980). Thus, in vivo, PLP appears to regulate GAD activity.₆₅more than the activity of GAD67.

GAD65 activity in bacterial lysates is similar to a five-fold PLP stimulation of the activity found in synaptosomes prepared from rat substance nigra (Miller et al., J. Neurochem., 33: 533, 1979) Because both GADs are more dependent on added PLP in bacteria than is GAD activity in crude rat brain homogenates, the endogenous PLP concentration of bacterial lysates may be lower to those of rat brain homogenates.

D. Immunological identification of GAD₆₅ and GAD₆₇

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, NY,

1988). Proteins were separated by 10% acrylamide gel electrophoresis in SDS and electrophoretically transferred to nitrocellulose (Harlow et al., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988). Unreacted sites were blocked with phosphate buffered saline (PBS) containing 2% bovine serum albumin (fraction V), 1% gelatin, and 1% Triton-X-100 at 42 ° C for one hr. After washing, the nitrocellulose filter is then cut into three sections and incubated with the following primary antibodies: bands 1 to 4 at a 1/2000 dilution of Oertel et al. Antiserum (Neuroscience, 6: 2689, 1981), that recognizes GAD₆₇and GAD65; Lanes 5 to 8 at a 1/2000 dilution of K-2 antiserum, which recognizes only GAD67; Lanes 9 to 12 with a 1/2000 dilution of monoclonal antibody GAD-6, which is specific for GAD65 (Chang et al., J. Neurosci., 8: 2123, 1988). All filters were washed extensively and appropriate secondary antibodies were incubated and washed. Bound antibodies were detected with protein A labeled with¹²⁵I and autoradiography. Each band contained the following: bands 1, 5 and 9 are BL21 (DE3) + pETGAD67; Lanes 2, 6 and 10 are BL21 (DE3) + pET-GAD65; Lanes 3, 7 and 11 are rat brain homogenates and Lanes 4, 8 and 12 are BL21 (DE3) + pET-8c.

Immunoblots of GAD65 and GAD67 produced by bacteria demonstrated that GAD65 actually corresponds to the smallest GAD in brain extracts and to GAD67 in the largest form (Figure 7). Previous work has shown the correspondence of GAD67 to the major GAD for GAD₆₇ feline and for GAD₆₇ mouse (Karatova et al., Eur. J. Neurosci., 2: 190, 1990; 235, 1987). The mobilities of GAD65 and GAD67 produced by bacteria as detected with the antiserum of Oertel et al. (Neuroscience, 6: 2689, 1981) are identical to the immunoreactive doublet observed in the rat brain homogenate.

ES 2 179 077 T3

The lower molecular weight and higher molecular weight forms of GAD in the rat brain are thus identical in antigenicity and size to the products of the GAD cDNAs.₆₅ and GAD67, respectively. Consequently, the two GADs in the rat brain are GAD65 and GAD67. From these data it can also be concluded that the molecular identity of the GADs determined to be PLP-dependent and PLP-independent by Tapia (Bayon et al., J. Neurosci., 29: 519, 1977) are GAD₆₅ and GAD₆₇ respectively. Martin et al. (Spink et al., Brain Res., 421: 235, 1987) have reported the existence of four kinetically different forms of GAD in the rat brain. However, immunoblot experiments (with the antiserum used here) in these ways have not been published.

E. Distribution of GAD₆₅ and GAD₆₇ in RNAs in brain tissue

Experiments were done to determine the distribution of GAD65 and GAD67 in RNAs in the cerebellum using in situ hybridization.

The 3.2 kb and 2.3 kb transcripts of GAD65 and GAD67 cDNAs, respectively, were radiolabeled with ³⁵S according to the procedure of Wuenschell et al. (Proc. Natl. Acad. Sci., USA, 83: 6193, 1986). The 200 bp hydrolyzed fragments hybridized in frontal sections of the rat cerebellum. Animals were anesthetized with halothane and decapitated. The brain was snap frozen on dry ice and frozen frontal sections (12 μm) were fixed for 30 min in 4% formaldehyde freshly prepared in phosphate buffered saline (PBS; 130 mM ClNA, 10 mM Na phosphate, pH 7 , 0). The tissue was dehydrated using graduated ethanol solutions and stored at -70 ° C.

To increase the permeability of the tissue, the sections were subjected to the following pretreatments: rehydration using graduated 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 x 5 min); 1 μg / m! proteinase K (7.5 min) and glycine (to inhibit proteinase K) in PBS (3 x 5 min). Proteinase K was digested for 30 min at 37 ° C before use. Sections were then incubated at 37^°C in 50% formamide, 750 mM NaCl, 25 mM EDTA, 0.2% SDS, 0.02% BSA, 0.002% Ficoll, 0.02% polyvinylpyrrolidone, 250 µg / m! yeast tRNA, 250 μg / m! of poly A and 25 mM PPES (pH 6.8).

For hybridization, 100 mM DTT, 10% dextran sulfate and ³⁵S-RNA sense and antisense. An aliquot part (50 μl) of the hybridization solution containing approximately 3 ng (10⁶ cpm) of probe (sense or antisense). Each slide was covered with the coverslip and incubated for 16 hrs at 50^°C, after which the silico slides were removed by brief washing in 4 x SSC (1 x SSC-150 mM NaCl, 60 mM Na citrate, pH 7.0).

The sections were then treated with ribonuclease A (50 μg / ml in 0.5M NaCl, 10mM Na thiosulfate, 1mM EDTA, 10mM TrisHCL, pH 8.0) for 20 min at 37^°C and rinsed for 2 hrs at room temperature in 2 x SSC, 10mM Na thiosulfate, for 30 min at 55^°C. The sections were dehydrated in ethanol, degreased in xylene, covered with Kodak NTB2 emulsion and exposed for 10 days at 4 ° C. The emulsion was developed with Kodak D19 and the tissue was contrast stained with cresyl violet.

Autoradiographic grains were detected using reflected polarized light and grain numbers, densities, and nd cell surfaces were determined with an Analytic Imaging Concepts image analyzer system. Due to the low background level, the criteria to define a "tagged" cellula were based on the presence of more than 5 clustered grains. GAD-labeled cells were found dispersed throughout the brain, allowing the measurement of a number of grains in individual cells. The boundary of the cell and the surface covered by a grain allowed the calculation of the number of grains per cell. This analysis was done at high magnification (800x), using reflected polarized light and transmitted light to simultaneously observe the stained cell and the overlapping grains. The numbers are means ± SEM of cells "n".

ES 2 179 077 T3

TABLE 4

<td colspan="4">Grains / Cell</td>

<td>Cell type</td><td>GAD67 mRNA</td><td>GAD65 mRNA</td><td>GAD67: GAD65</td>

<td>Purkinje</td><td> 172 ± 34 (87)^to</td><td> 43 ± 2 (70)</td><td> 4,0</td>

<td>Golgi II</td><td> 96 ± 8 (80)</td><td> 64 ± 9 (65)</td><td> 1,5</td>

<td>Basket case</td><td> 61 ± 12 (102)</td><td> 16 ± 1 (57)</td><td> 3,8</td>

<td>Starry</td><td> 55 ± 15 (65)</td><td> 18±3(37)</td><td> 3,1</td>

^to ± SEM (n)

In the GAD67 mRNA of all neuronal types the concentrations are higher. 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) in which the ratio of PLP-dependent to PLP-independent GAD activities in the cerebellum is one of the lowest in the brain regions tested. Furthermore, as shown in Table 4, the order of quantities for the GAD67 mRNA is Purjinke&gt; Golgi II&gt; basket&gt; stellate cells; conversely, for the GAD65 mRNA, this order is Golgi II&gt; Purjinke&gt; basket&gt; stellate cells.

The expression of the GAD65 and GAD67 mRNAs thus differs between classes of neurons. The contribution of each to total GAD activity in turn affects how GABA production is regulated. For example, substantia nigra contains one of the highest ratios of PLP-dependent to PLP-independent GAD activities (Nitsch, J. Neurochem., 34: 822, 1980). Increasing the concentration of GABA in the substantia nigra by local injection of GABA catabolism inhibitors is especially effective in reducing susceptibility to attack (Gale, Fed. Proc., 44: 2414, 1985). Experimental animals suffering seizures produced by PLP antagonists may therefore be unable to inhibit the spread of the seizure due to inhibition of GAD65 particularly at nerve endings within the substantia nigra.

F. Subcellular status of GAD₆₅ and GAD₆₇

The distribution of GAD65 and GAD67 was evaluated in the subcellular fractions of S2 and of the synaptosome. S2 is a high-speed supernatant that is made up of the cytosol of all cells in the brain, while the synaptosomal fraction is mainly made up of nerve endings (Gray et al.,

J. Anat., Lond. 96:79, 1962). For these studies, whole rat brain division was performed as described by Booth and Clark (Booth et al., Biocchem. J., 176: 365, 1978). Protein concentrations were determined according to Schaffner and Weissman (Schafner et al., Anal. Biochem. 56: 502, 1973). Samples were prepared as described (Kaiser et al., DNA Cloning, Vol. I, A practical Approach,

DM 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 µg) were added to each band. Autoradiography showed a linear response of increasing amount of¹²⁵I-protein A bound at antibody protein concentrations of 1, 3, 10, 30 and 100μgs with K-2 antiserum and GAD-6 monoclonal antibody (data not shown).

The results showed that GAD67 was present in equal amounts in both fractions. As the S2 fraction contains the cytisiole proteins of the glial (as well as other non-neuronal) and neurons, the concentration of GAD67 must be higher in the bodies of the neurons than in the nerve endings. On the contrary, the concentration of GAD65 was higher in synaptosomes than in S2. These subcellular division experiments suggest that, in contrast to GAD65, a much higher fraction of GAD67 is present in neuron bodies than in nerve terminals. Thus, subcellular division, like immunohistochemistry, shows that GAD65 and GAD67 have different subcellular distributions.

In vivo experiments using GABA synthesis and degradation inhibitors have suggested that the GABA group in the bodies of neurons is different from those of nerve terminals (Iadarola et al., Mol. Cell. Biochem., 39: 305, nineteen eighty one). The GABA produced by GAD₆₇ it may be more involved in cellular metabolism (for example, in the GABA derivation) and in the dendrodendritic synapse. The

ES 2 179 077 T3 dendrites of graonule cells in the olfactory bulb, which form dendrodendrotic synapses with mitral dendrites (Shepard, Physiol. Rev., 52: 864, 1972) and probably release GABA (Mc Lennan, Brain Results .. 29 : 177-184, 1971), marked intensely with K-2 antiserum. Although not shown herein, higher concentrations of GAD67 than GAD65 mRNA (2 to 3 times) have been observed in the olfactory bulb. This distribution is consistent with the published finding that most of the GAD activity in the olfactory bulb was present in S2 and P1 (crude nuclear agglomerate) and not in synaptosomes (Quinn et al., J. Neurochem., 35: 583, 1980).

The different subcellular distributions of GAD65 and GAD67 could come from cytoskeletal anchoring or from some unknown mechanisms targeting proteones. Some cytoskeletal proteones have distributions reminiscent of GAD65 and GAD67. For example, in cultured sympathetic neurons Peng et al., (J. Cell. Biol., 102: 252, 1986), demonstrated that 84% of tau is in axons while 100% of MAP-2 is in cell bodies. and dendrites. In addition, the 43kd proteon, cytoskeleetic proteon, is believed to anchor the acetylcholine receptor to the underlying membrane cytoskeleton (Flucher et al., Neuron, 3: 163, 1989).

Example 3

Detection of GAD autoantibodies in clonal samples

A. Materials and procedures

1. Patient samples. Sera from four individual groups were selected from a previous study by Atkinson et al. (Atkinson et al., Lancet, 335: 1357-1360, 1990). These groups consist of: Group (1), 1 new group of patients at the beginning of IDD diagnosed according to criteria established by the National Diabetes Data Group (NDDG) (Gleichman et al., Diabetes, 36: 578 to 584, 1987) who They were referred to the University of Florida, Diabetes Clones; Group (2), 5 randomly selected islet cell cytoplasmic antibody (ICA) negative non-diabetic controls with no known family history of autoimmune disease; Group (3), 13 individuals whose sera have been collected 3-66 months before their documented clonal onset of IDD; Group (4), non-diabetic and related controls, and those that were studied before the onset of IDD; and Group (5), 3 patients at risk of IDDM, but in whom it has not yet started. This last group has been investigated through studies of probable detection of continuous AHF of more than 5000 first grades related to IDD probands and 8200 individuals from the general population (of which 4813 were schoolchildren).

two. Islet cell autoantibodies. ICA were analyzed by indirect immunoinfluorescence in cryocautery pancreotic blood group 0 (Atkinson et al., Lancet, 335: 1357-1360, 1990). All results were interpreted on coded samples with positive and negative control sera in each lot. The degrees of positivity of ICAs were analyzed with the guidelines established by the Immunology Diabetes Working Group (IDW) for the standardization of ICAs (Gleichman et al., Diabetes, 36: 578 to 584, 1984). All positive sera were assessed by end-point dilution and the units of the Juvenile Diabetes Foundation (JDF) were determined by reference to a standard serum previously calibrated to the international JDF standard of 80 units. In the studies published herein, a positive ICA result was defined by replicate values ​​of 10 JDF units or higher.

3. HLA DR characterization. The DR characterization of HLA was carried out by adaptation of the procedure described by Van Rood and Van Leuwen (Nature, 262: 795 to 797, 1976), using DR plates (One Lamda Laboratories, Los A^or Angeles, CA).

Four. Coellulae of the human islet. Human pancreatic islets were isolated from cadover pancreases and maintained in vitro as previously described (Ricordi et al., Diabetes, 37: 413-420, 1988). The islet cells were metabolically labeled with methionine³⁵S (Amersham, Arlington Heights, IL) in vitro (95% air / 5% CO2).

5. Islet cell extractions and immunoprecipitations. Cellulae were removed from the islets as previously described by Atkinson et al. (Lancet, 335: 1357-1360, 1990) with the following modifications. For immunoprecipitation studies, islet cell lysates were previously purified by incubation twice (2h, 4 ° C) with control IDD serum (100 µl) or GAD-6 (Chang et al., J. Neuro, 8: 2123-2130, 1988) (1 µl in 99 µl) Tris buffer (Atkinson et al., Lancet, 335: 1357-1360, 1990) per 1000 islets. Immune complexes were then absorbed (1h, 4^°C)

ES 2 179 077 T3 with excess protein A Sepharose CL-4B (Pharmacia, NJ). Aliquot volumes representing 1000 islet cells containing unbound (prepurified) lysates were then incubated (12 h, 4^°C) with IDD or control sera (25 µl), or GAD-6 (Chang et al., J. Neuro, 8: 2123 to 2130, 1988) (1 µl in 25 µl Tris buffer). After another incubation with protein A Sepharose CL-4B (1h, 4 ° C), the complexes were then washed 5 times with Tris HCL (pH 7.4) with 0.1% SDS, Triton X-114 al 1 % and 2mM EDTA and then washed again in redistilled water. Protein A Sepharose CL-4B was then boiled in Laemmli sample buffer (Laemmli, Nature, 227: 680 to 685, 1970), and the samples were subjected to SDS-PAGE and fluororaradiography (Kodak, X-omat AR5) using Enhance (New England Nuclear). On the other hand, autoradiographs were analyzed using a BETAGEN analyzer (Boston MA). 64KA positive and negative sera were used in each run to serve as inter-run controls. All fluororadiographs were analyzed and classified as positive and negative after comparison with known inter-assay controls. Positive serum samples were designated 1 when the sample produced low intensity immunoprecipitation in the 64,000 MT band, 2 if a moderate intensity band was observed and 3 if the intensity of the immunoprecipitated protein was high. A similar classification procedure was used for the intensity of the bands corresponding to ³⁵S-GAD65 and ³⁵S-GAD67 immunoprecipitated.

6. Immunoprecipitations. Immunoprecipitation of bacterial lysates containing³⁵S-GAD65 and ³⁵S-GAD67, and GAD from human brain homogenates, was completed as described above in immunoprecipitation studies of cell extractions from the human isote.

7. GAD analysis. Human brain homogenates were incubated with patient sera as described above for human islet cells. After absorption and washings, the agarose suspension in protein A was divided into three equal volumes and GAD activity was measured as described (Krieger et al., Neurochem. 33: 299, 1984). Briefly, protein A agarose drops were incubated with (1-¹⁴C) -glutamate (Amersham) in an incubation mixture for this purpose (Krieger et al., Neurochem. 33: 299, 1984) and the production of ¹⁴CO using a liquid scintillation counter.

8. Production of ³⁵S-GAD65 and ³⁵S-GAD67. Rat GAD65 and GAD67 cDNAs were subcloned into a bacterial expression system as described above. The marking of the³⁵S-GADs by pulsing the produced IPTG bacteria (growth in minimal medium) for 15 minutes with TRAN ³⁵S-marker (ICN). The cultures were then turned, resuspended, and sonicated in 1 ml of homogenization buffer (1mM phenylmethylsulfonyl fluoride (PMSF), 1mM 2-aminoethylisothiouronium bromide (AET), and 60mM potassium phosphate). , pH 7.1). After ultrasound, cell debris was separated by centrifugation and protein concentration was measured (Bradford, Anal. Biochem., 72: 248, 1986) in the supernatant (the supernatant was stored in aliquots at -70 ° C).

B. Immunoreactivity of IDDM samples

Human brain homogenates were tested for the ability to precipitate GAD from human brain homogenates.

TABLE 5

Immunoprecipitated GAD Activity Sera from IDDM Patients

<td>Patient</td><td>IDDM</td><td>Period Pre-IDDM¹</td><td>64K²</td><td>JDF³</td><td>GAD activity⁴ cpm's</td>

<td>GIVES</td><td> *5</td><td> &gt;24</td><td> 3</td><td> 164</td><td> 13.762</td>

<td>DC</td><td> *</td><td> &gt;1</td><td> 3</td><td> 20</td><td> 1.719</td>

<td>RS</td><td> +</td><td> 5</td><td> 3</td><td> 40</td><td> 588</td>

<td>NL</td><td> +</td><td> 0</td><td> 2</td><td> 80</td><td> 440</td>

<td>DM</td><td> *</td><td> &gt;1</td><td> 2</td><td> 10</td><td> 184</td>

<td>C</td><td> -</td><td>na</td><td> 0</td><td> 0</td><td> 280</td>

<td>C</td><td> -</td><td>na</td><td> 0</td><td> 0</td><td> 285</td>

<td>C</td><td> -</td><td>na</td><td> 0</td><td> 0</td><td> 325</td>

<td>C</td><td> -</td><td>na</td><td> 0</td><td> 0</td><td> 275</td>

<td>C</td><td> -</td><td>na</td><td> 0</td><td> 0</td><td> 270</td>

ES 2 179 077 T3 ¹ Expressed in months ² 64K titrations are as described in the Experimental Procedures ³ Islet cell antibody test expressed in units of the Juvenile Diabetes Foundation (JDF) ⁴ Not adjusted for background ⁵ At risk of diabetes (additionally, negative glucose test) na - not applicable

As shown in Table 5, sera from four (out of five) IDDM or IDDM-risk patients bound significantly higher amounts of enzyme-active GAD from human brain extracts than from control patient sera. In addition, the sera from one of the patients were collected in a pre-IDDM period, thus GAD antibodies are present before the onset of IDDM symptoms (see below).

Additional experiments (results not shown) demonstrated that sera from two patients at risk of IDDM (DA, DC) produced ³⁵Recombinantly immunoprecipitated S-GAD65 while ³⁵Recombinantly produced S-GAD67 was only recognized by sera from patient DA (and to a lesser extent than ³⁵S-GAD67).

Additional studies using serum from patient DA demonstrated the presence of antibodies that recognize specific polypeptides produced in the cells of the human pancreatic islet. Electrophoretic analyzes of the bound polypeptides demonstrated the presence of antibodies in a 64 kD component, as previously demonstrated 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). Before absorption of these sera with monoclonal GAD-6, which recognized GAD65 but not GAD67, or with GAD65 produced with bacteria, the ability of the sera to recognize the 64kD pancreatic polypeptide was suppressed. The epitopes recognized by autoantibodies to the 64kD autoantigens are thus present in GAD65, indicating that the 64kD autoantigens are actually GAD65. To investigate the predictive value of GAD65, sera drawn from patients prior to the onset of clinical manifestation of IDDM were tested for antibodies to GAD65.

TABLE 6

IDDM patients tested for antibodies before disease onset

<td>Patient</td><td>Sex</td><td>HLA</td><td>Starting age¹</td><td>Period Pre-IDD²</td><td>JDF</td><td>64K³</td><td>GAD³65</td><td>gad3₇</td>

<td>TA</td><td>M</td><td> 3,2</td><td> 17</td><td> 11</td><td> 20</td><td> 2</td><td> 0</td><td> 1</td>

<td>AC</td><td>F</td><td> 4,5</td><td> 38</td><td> 4</td><td> 0</td><td> 1</td><td> 1</td><td>or</td>

<td>RA</td><td>M</td><td> 2,1</td><td> 5</td><td> 34</td><td> 0</td><td> 2</td><td> 1</td><td>or</td>

<td>TB</td><td>M</td><td> 2,4</td><td> 11</td><td> 66</td><td> 40</td><td> 1</td><td> 1</td><td>or</td>

<td>AB</td><td>M</td><td>ND</td><td> 23</td><td> 6</td><td> 160</td><td> 3</td><td> 3</td><td> 2</td>

<td>VC</td><td>F</td><td> 4,6</td><td> 15</td><td> 3</td><td> 40</td><td> 1</td><td> 0</td><td> 1</td>

<td>JD</td><td>M</td><td> 6,1</td><td> 34</td><td> 25</td><td> 10</td><td> 3</td><td> 1</td><td> 1</td>

<td>DR</td><td>F</td><td> 3,4</td><td> 14</td><td> 42</td><td> 320</td><td> 2</td><td> 1</td><td> 0</td>

<td>JG</td><td>M</td><td> 3,3</td><td> 12</td><td> 8</td><td> 40</td><td> 1</td><td> 0</td><td> 0</td>

<td>BR</td><td>M</td><td> 3,3</td><td> 5</td><td> 9</td><td> 0</td><td> 0</td><td> 1</td><td> 1</td>

<td>KR</td><td>F</td><td>4, X</td><td> 34</td><td> 14</td><td> 10</td><td> 3</td><td> 2</td><td> 0</td>

<td>JT</td><td>F</td><td> 4,6</td><td> 7</td><td> 10</td><td>ND</td><td> 1</td><td> 1</td><td> 1</td>

¹ IDDM start age expressed in months

ES 2 179 077 T3 ² Time interval between serum extraction and the onset of IDDM expressed in months ³ Band intensities 1 = lower; 2 = mean; and 3 = higher

ND - not determined

As shown in Table 6, 9 out of 12 samples (75%) were immunoreactive with ³⁵SGAD65 Furthermore, two patients (JA and VC) were immunoreactive to GAD67, but not to GAD65 under these conditions. Therefore, in combination, GAD65 and GAD67 autoantibodies were present in 11 out of 12 (91%) of these patient sera. This discovery suggests that although GAD65 antibodies are more common than GAD67 antibodies, the use of both recombinant GADs (GAD65 and GAD67) in an analysis will allow greater predictability of IDDM. Previous tests of these sera (Atkinson et al., Lancet, 335: 1357-1360, 1990) showed that 11 out of 12, or 92%, immunoreacted with the molecule. ³⁵S-64kD from human pancreatic islet cells. The serum containing detectable antibodies to the 64kD molecule and not GAD65 was a serum containing the lowest value (or "1") for the 64kD molecule. Thus, the false negative obtained was due to a lack of sensitivity in this analysis. Furthermore, this analysis predicted IDDM in one patient (BR) who was negative for 64K.

These results demonstrate that the 64kD molecule identified in the β-cells of the human pancreas is identical in size and antigenicity to rat GAD65. Furthermore, sera collected from patients before the start of IDDM contain GAD65 autoantibodies. Therefore, the recombinant molecule is very useful as a diagnostic tool for predicting IDDM. The ability of a physician to diagnose IDDM before actual symptoms can result in a longer time before insulin therapy is needed. The sensitivity of said immunoassays will improve with the use of a recombinant GAD65 of human origin that represents the GAD form present in the β cells of the pancreas.

Example 4

Multiplying immune response to polypeptide

Polypeptides were synthesized using an automated instrument (Applied Biosystems) and standard conditions. These polypeptides were then tested to compare their relative ability to stimulate the multiplication of splenic lymphocytes and islet infiltrator T lymphocytes (IITLs). In this study, polypeptides derived from the nucleus sequence of GAD65 and the homogenous region of the polio virus were compared. Appropriate cells were cultured for 5 days with the respective polypeptide in the presence of 5 x 10⁴ irradiated spleen cells. It was added³H-thymidine during the last 16 hours of culture.

TABLE 7

<td>Antigen</td><td>Amino acid sequence</td><td colspan="2">Incorporation of ³H-thymidine (cpm) by lymphoid cell population</td>

<td></td><td></td><td>IITLs^to</td><td>Spleen^b</td>

<td>None</td><td> —</td><td> 1.100</td><td> 6.500</td>

<td>Poliovirus</td><td>MKSMCPQAQLKVKYL</td><td> 900</td><td> 22.500</td>

<td>GAD65</td><td>ARFKMFPEVKEKGMAA</td><td> 9.500</td><td> 23.300</td>

^to Islet infiltrating T lymphocytes (3 x 10⁴ cells / well) ^b 1 x 10⁵ cells / well

In these studies, there was no significant difference between the multiplying activity of spleen lymphocyte cultures exposed to poliovirus or GAD65 polypeptides. However, both polypeptides stimulated a T cell response that was greater than that seen in a medium control. The absence of difference in the multiplication of the spleen cell population may be due to a lower frequency of GAD polypeptide-specific T lymphocytes.

ES 2 179 077 T3

When the IITL population was calculated in the same way, it showed a marked difference in cell multiplication. In this system, the response to the GAD65 polypeptide was 9 times greater than that of the culture medium or that of the poliovirus poliopeptide. These data strongly suggest that GAD65 is an important antigen for T cell responses in the IITL population. These data suggest that molecular mimicry plays a role in the pathogenesis of diabetes.

Example 5

GAD causes the multiplication of spleen cells in NOD mice

The multiplier T cell responses to β-cell antigens (eCA) develop spontaneously in the non-obese diabetic mouse (NOD) model in a defined chronological order. The NOD mouse experimental model is considered the most widely available in vivo system for studying IDDM in man. This example describes antigen-induced blastogenesis studies of spleen cells from female NOD mice born up to 5 months of age when exposed to GAD and other peptides.

The eCAs tested comprise one of two forms of GAD (Kaufman et al., Science, 232: 1138-1140, 1946; Enlander et al. Neuron, 7:91-100, 1991; Kaufman et al., Trends in Pharm. Sci. (in press)), GAD65 previously known as the 64K autoanthogen (Baekkeskov et al., Nature, 298: 167-169, 1981); (Baekkeskov et al., Nature, 347: 151-156, 1990), carboxypeptidase H (CPH) (Castano et al., J. Clin. Endocrinol. Metab., 73: 1197-1201, 1991), insulin (Palmer, IDDM Prediction, Diabetes Reviews, 1: 104-115, 1993), and an hsp peptide that has been shown to be the determining immunodominant recognized by NOD T cells. (Elias, Proc. Nat. Acad. Sci., 88: 3088-3091,

1991). GAD in particular is a good candidate for the initial target antigen in IDDM, since autoantibodies to GAD appear early in the natural course 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 β cells, the immunologically privileged central nervous system (CNS), and the goonads. As control antigens, a foreign irrelevant prototype and autoanthogens comprising hen egg lizosime (HEL), human serum albumin (HSA), E. coli β-galactosidase were used. (β-gal) and the basic murine myelin protein (MBP).

NOD (Tatonic farms) and BALB / c (Jackson Laboratories) mice were kept under specific pathogen-free conditions. Mice were sacrificed at the indicated ages and spleen cells were tested directly ex vivo for their response to the antigen multiplier call. Coell suspensions isolated from spleen cells were plated at 1 x 10⁶ cells per well in 96-well microtiter plates in 200 µl of serum-free HL-1 medium (Ventrex) supplemented with 2mM glutamine with or without 10 µg / ml of antigen (or 7 µM peptide) in triplicate cultures. During the last 16 h of the 72 h culture period, 1μθϊ of [³H] -thymidine per well. The incorporation of the marker was measured by liquid scintillation counting.

GAD was purified₆₅ human (Bu et al., Proc. Natl. Acad. Sci., 89: 2115 to 2119, 1992) and E. coli β-gal (control) from recombinant bacteria based on a hexahistidine marker that allows its rapid affinity purification by metal affinity chromatography (Hochuli et al., Bio / Technology, 6: 1321-1325, 1988). Bovine CPH was a generous gift from L. Fricker (Albert Einstein Col. Med.) And human insulin was purchased from Eli Lilly.

As illustrated in Figure 8, while multiplier T lymphocyte responses were not detected at any time for the control antigens, at 4 weeks of age a response appeared in NOD mice, coinciding with the onset of insulitis. in the colony. GAD-induced blastogenesis increased over the next four weeks and then declined to background levels at week 16. At 6 weeks of age, near the peak of anti-GAD reactivity, T lymphocyte responses to hsp appeared and increased until week 15 and then decreased as well (Figure 8). In all NOD mice tested, the hsp reactivity was preceded by an anti-GAD response, suggesting that the above reactivity developed as a secondary event during the autoimmune process. Likewise, while no response to CPH was detected at 4 weeks of age, a strong anti-CPH response was observed at week 8. In some mice, a weak response was observed at 12 weeks, which became severe. predominant at 15 weeks of age (Figure 8 and Table 8). None of the antigens caused multiplication in the T lymphocytes of age-matched control BALB / co (NOD x BALB / c) F1 mice, which did not develop insulitis or IDDM. The reactivity of T lymphocytes appears later in other eCAs, consistent with the diversity23

ES 2 179 077 T3 intermolecular efficacy of the autoimmune response. Thus, the autoimmune response to GAD was the first to occur among the autoanthagens tested. In view of this, tolerance to GAD would prevent the spread of autoimmunity to other eCAs and to insulitis. If this is not the case then tolerance to GAD should have no effect on respect for 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 Figure 8 demonstrate that GAD-reactive T cells "spontaneously" undergo clonal expansion coinciding with the onset of insulitis. These findings are consistent with the endogenous sensitization event.

Example 6

Provocation of tolerance with GAD

This example describes a study demonstrating that induced GAD tolerance can improve IDDM.

1. In these experiments, female NOD mice were injected intravenously at 3 weeks of age with 50 μg of GAD, β-galactosidase, mycobacterial hsp-65 (m-hsp) or 0.1 μg of the immunodominant hsp peptide (hsp-p) , on PBS. At 12 weeks of age, the mice are examined for insulitis and autoantigen reactive T lymphocytes. At this age, both indications are verified in untreated NOD mice. Pancreatic tissue sections were stained by immunoperoxidase techniques for insulin and hematoxylin contrast stains were made. Insulitis is scored blindly by examining 54 to 87 islets in 5 discontinuous tissue sections from each pancreas. Spleen multiplier T cell responses elicited by various antigens were performed, as described above in Example

Four. The data in Table 8 are expressed as a marker of [³Mean H] -thymidine (cpm) incorporated in triplicate cultures.

TABLE 8A

Tolerance caused by GAD

<td>Punctuation Treatment of insulitis^to N</td><td>Spleen cell multiplication (SI ± SEM)^b</td><td></td>

<td>β-Gal</td><td>GAD Peptides from GAD Peptide</td><td>CPH</td>

<td></td><td>n ° 17 n ° 34 n ° 35 hsp</td><td></td>

Without injecting β-Gal. GAD hsp-p m-hsp

2,4 ± 0,2 2,6 ± 0,6 0,1 ± 0,1

1.7 ± 0,4

1.8 ± 0,5

1,0 ± 0,2 1,1 ± 0,1 1,1 ± 0,03 1,1 ± 0,05 1,1 ± 0,1

9.5 ± 2,1 15,4 ± 1,8

1.6 ± 0,3 5,8 ± 0,2 4,2 ± 0,1

4.8 ± 0,4 5,1 ± 0,6 1,0 ± 0,05 4,5 ± 0,1

3.9 ± 0,1

6,0 ± 0,1 2,9 ± 0,2

5.1 ± 0,6 4,0 ± 0,2

1.2 ± 0,1 1,0 ± 0,1 4,1 ± 0,3 4,2 ± 0,1 3,9 ± 0,1 3,4 ± 0,2

6,7 ± 1,0 6,6 ± 0,5 1,2 ± 0,1 1,1 ± 0,04 1,0 ± 0,03

ND^c

11,5 ± 0,9 1,1 ± 0,02 44 ± 0,2 4,3 ± 0,2 ^to The severity of mononuclear cell infiltration was histologically defined (0 = no lymphocytic infiltration; 1 &lt;25%; 2 = 25-50%; 3 = 50-75%;

&gt; 75%) (Qin et al., Immunol., 150: 2072-2080, 1993). The score is the mean ± SE.

^b Important answers indicated in normal underlines, intermediate answers indicated in double underlines.

^c Not determined

ES 2 179 077 T3

TABLE 8B

Tolerance caused by GAD

<td>Treats- I lie</td><td colspan="2">Insulitis score^to N</td><td colspan="5">Spleen cell multiplication (SI ± SEM)^b</td>

<td rowspan="2"></td><td colspan="2" rowspan="2">β-Gal</td><td rowspan="2">GAD</td><td colspan="2">GAD peptides</td><td rowspan="2">Peptide hsp</td><td rowspan="2">CPH</td>

<td>n^°17</td><td>n^°3. 4 n^°35</td>

<td>Peptide 11</td><td> « 2,5</td><td> 1,0 ± 0,1</td><td> 21,1± 2,2</td><td> 13,7 ± 1,5</td><td> 11,4 ± 1,5 11,3 ± 0,7</td><td> 13,3 ± 0,9</td><td>ND^c</td>

<td>Peptides 34/35</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>(+ IFA)</td><td> 0,7 ± 0,4</td><td> 1,0 ± 0,2</td><td> 1,9 ± 1,1</td><td> 2,2 ± 1,2</td><td> 1,1 ± 0,3 1,0 ± 0,1</td><td> 1,8 ± 1,1</td><td>ND</td>

<td>IFA only (+ IFA)</td><td> « 2,5</td><td> 1,0 ± 0,1</td><td> 8,1 ± 0,5</td><td> 5,0± 0,5</td><td> 4,8 ± 0,5 4,8 ± 0,4</td><td> 6,6 ± 0,6</td><td>ND</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

^to The severity of mononuclear cell infiltration was histologically defined (0 = no lymphocytic infiltration; 1 &lt;25%; 2 = 25 - 50%; 3 = 50 - 75%;

&gt; 75%) (Qin et al., Immunol., 150: 2072-2080, 1993). The score is the mean ± SE.

^b Important answers indicated in normal underlines, intermediate answers indicated in double underlines.

^c Not determined.

TABLE 8C

Tolerance caused by GAD

<td>Treats- I lie</td><td>Insulitis score^to</td><td>N</td><td colspan="5">Spleen cell multiplication (SI ± SEM)^b</td>

<td></td><td></td><td>β-Gal</td><td colspan="3">GAD GAD peptides</td><td>Peptide</td><td>CPH</td>

<td></td><td></td><td></td><td>n^°17</td><td>n^°34</td><td>n^°35</td><td>hsp</td><td></td>

<td>Peptide 11</td><td> ©?)</td><td> 1,4 ± 0,4</td><td> 12,1± 3,5 6,9 ± 0,4</td><td> 6,4 ± 1,0</td><td> 6,8 ± 0,3</td><td> 8,7 ± 0,9</td><td>ND^c</td>

<td>Peptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>HEL</td><td> (?)</td><td> 1,2 ± 0,2</td><td> 10,7 ± 3,5 6,4 ± 1,4</td><td> 5,9 ± 1,6</td><td> 6,9 ± 1,9</td><td> 8,0 ± 1,8</td><td>ND</td>

<td>Peptides 34/35</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td rowspan="2">(+ IFA)</td><td rowspan="2"> (?)</td><td> 1,0 ± 0,1</td><td> 4,4 ± 2,6 1,8 ± 1,1</td><td> 1,0 ± 0,1</td><td> 1,1 ± 0,2</td><td rowspan="2"> 2,7 ± 1,3</td><td>ND</td>

<td></td><td></td><td></td><td></td><td></td>

^to The severity of mononuclear cell infiltration was histologically defined (0 = no lymphocytic infiltration; 1 &lt;= 25%; 2 = 25 - 50%; 3 = 50 -75%;

&gt; 75%) (Qin et al., Immunol., 150: 2072-2080, 1993). The score is the mean ± SE.

^b Important answers indicated in normal underlines, intermediate answers indicated in double underlines.

^c Not determined.

Seventy-five percent of the GAD-treated mice, but none of the controls, showed no T cell reactivity with GAD (indicating complete tolerance) or with other eCAs. These mice were also totally free of insulitis (score (0,0). If there were another population of effector T lymphocytes in the islets, specific for an unknown eCA, which preceded the response

ES 2 179 077 T3 anti-GAD, cytokine release by this population should have stimulated T cell responses to eCAs and insulitis (Sarvetnick et al., Nature, 346: 844, 1990; Heath et al., Nature, 359: 547, 1992). Twenty-five percent of the treated mice did not fully tolerate GAD, as demonstrated by weak residual reactivity to GAD (SI of approximately 3), and had very limited periinsulitis. In contrast, while tolerance to both hsp antigens was complete, these treatments reduced, but did not prevent, the development of T cell responses to other eCAs or insulitis. Thus, while inactivation of GAD-reactive T cells prevented β-cell autoimmunity, tolerance to hsp only partially reduced it, as would be expected if a secondary element were separated from the amplifying stream.

In ongoing experiments examining the effects of GAD tolerance on the incidence of diabetes, all GAD-treated mice (n = 17, currently 37 weeks old) have normal glucose levels, while 70% of mice that received control antigens developed hyperglycemia for 19 weeks (n = 20). At 30 weeks of age, five GAD-treated mice were sacrificed. All were free of detectable eCA-reactive T cells. Of these five animals, four mice were totally free of insulitis and a few mice had very limited periinsulitis. These data demonstrate that inactivation of GAD-reactive T lymphocytes prevents the development of insulitis and diabetes in the long term.

two. In a second series of experiments, newborn female NOD mice were injected intraperitoneally with IFA with peptide 11, a mixture of peptides 34 and 35 with IFA, or with IFA alone, and at 12 weeks of age, insulitis and Self-antigenic reactive T lymphocytes in mice as in Example 6.1. Multiplying splenic T cell responses elicited by various antigens were performed as in Example 4, and the data is expressed in Table 8B as a marker of [³H] -thymidine medium (cpm) incorporated in triplicate cultures.

The data in Table 8B demonstrate that tolerance to control peptide 11 does not prevent the autoantibody response to GAD or to peptides 17, 34 or 35 to GAD. There was no response to the hsp peptide prevented by tolerance to peptide 11. IFA alone it was somewhat effective in suppressing the immune response. On the contrary, tolerance to the mixture of peptides 34 and 35 suppressed the autoimmune response of the multiplication of spleen cells in all the species tested: e-gal, GAD, peptides 17, 34 and 35 of GAD and peptide of hsp. . Furthermore, tolerance to peptides 34 and 35 of GAD greatly reduces insulitis but does not completely prevent it as GAD65 does in its entirety.

3. In a third series of experiments, female NOD mice were injected intravenously at three weeks of age with peptide 11 (control), a mixture of 34 and 35 peptides plus IFA, or with HEL peptides and at 12 weeks of age. The mice were examined for insulitis and autoantigen reactive T lymphocytes as in Example 6.1. Multiplying splenic T cell responses elicited by various antigens were performed according to Example 4, and the data is expressed in Table 8C as a marker of [³H] -thymidine medium (cpm) incorporated in triplicate cultures.

The data in Table 8C demonstrate that tolerance to control peptide 11 does not prevent the autoantibody response to GAD or to GAD peptides 17, 34, or 35 or hsp although the response was not as great as in Example 6.2. There was no response to the hsp peptide prevented by immunization with peptide 11. In contrast, immunization with the mixture of peptides 34 and 35 plus IFA suppressed the autoimmune response of spleen cell multiplication in all species tested: e-gal, GAD, peptides 17, 34 and 35 of GAD and peptide. from hsp.

Example 7

Characterization of GAD-reactive T lymphocytes

This example describes studies on GAD-reactive T lymphocytes for additional properties that distinguish activated / memory from remaining / single lymphocytes.

In a series of experiments, measurements of interferon a (IFNa) were made by ELISA in culture supernatants (CSN) of spleen cells of 6 to 9-week-old mice, after testing with GAD or the HEL control antigens. and MBP. Additionally, the frequency of the specific antigen, the IFNy-producing cells, was determined by the ELISA blot technique (T. Taguchi et al., J. Immunol., 145: 68-77, 1990). The antigen-induced frequency of spot-forming cells (CFS) between 10³ Spleen cells are depicted in Figure 9 (a). Values ​​are mean + SEM of 5 individual female NOD mice, each tested in triplicate cultures

ES 2 179 077 T3 with or without antigens. The results of a single experiment are shown. AND^and These are representative of 3 separate experiments.

Performing these experiments, freshly isolated spleen cells were cultured with or without antigen as described in Example 4. CSNs were collected after 48 h and the IFNy concentration was determined by ELISA (Macy et al., FASEB J., 3003 a 3009, 1988). The IFNy-specific monoclonal antibody (mAb) R4-6A2 (Pharmingen) was used as a scavenger reagent and biotinized XMG 1.2 mAb (Pharmington, also specific for IFNy) was used together with streptavidin alkaline phosphatase (Zymed) and p-nitrophenol for detection. of bound lymphokine Recombinant murine IFNy (Pharmingen) was used as standard. ELISA blot analysis for specific antigen detection of IFNy-producing cells were performed as described (Taguchi et al., J. Immunol., 145: 68-77, 1990). After 24 h of spleen cell preactivation culture with or 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 separated and the IFNy spots were observed using XMG 1.2-biotin together with bromochloroindolyl-terazolium nitroblue phosphate substrate (Sigma). The spots were counted visually and the frequency of the antigenspecific cells was determined from the difference between the number of spots observed with and without antigen.

As demonstrated in Figure 9 (a), when freshly isolated T lymphocytes from 6 to 9 week old NOD mice were stimulated with GAD or control antigens, high concentrations of IFNy were detected only in cultures containing GAD, suggesting that GAD-specific T cells had been pre-activated in vivo, since only pre-activated T cells (Th1) produce IFNy 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, age-matched BALB / c mouse T cells do not respond to GAD or control antigens by IFNy production (data not shown).

Results of ELISA blot analysis to directly measure the frequency of GAD-specific T cells demonstrated that while in 6 to 9 week old NOD mice, T cells reactive to control antigens constituted about 1 in 10⁵ cells in the spleen, the frequency of GAD-reactive T lymphocytes was approximately two orders of magnitude higher, ranging from 90 to 291 cells per 10⁵ cells (Figure 9 (a)), confirming the data obtained by multiplication tests (Figure 8) that these cells have expanded clonally in vivo.

In another series of experiments, GAD-specific T lymphocytes were characterized by the expression of the cell surface marker L-selectin, since murine T lymphocytes convert from a phenotype to L-selectin activation.⁺ (L-sel⁺) up to L-selectin^- (L-sel^-) (Bradley et al., J. Immunol., 148: 324-331, 1992).

To perform these studies, mixed spleen cells from 3 to 4 age-matched mice were spread over plates coated with goat Ig-anti-razoen (Zymed) to separate adherent macrophages as well as B lymphocytes. CD8 + cells with mAb 58.6-72 (ATCC) and were removed by distributing them on plates coated with goat-anti-mouse Ig (Zymed). The non-adherent CD4 + cell fraction was labeled with anti-L-selectin mAb MEL-14 (ATCC) and spread on plates coated with goat-anti-rat Ig. Adherent fractions (CD4 + L-sel⁺) and non-adherent (CD4 + L-sel^-). The purity of the cell fraction was assessed by FACS analysis; cells that were&gt; 90% CD4 + and&gt; 95% were enriched for the L-sel phenotype^- or L-sel⁺. Purified cell fractions were tested for reactivity with GAD by seeding at 2 × 10⁵ cells per well in 96-well microtiter plates with or without antigen. Undissociated spleen cells from 3-week-old irradiated (3000 rd) NOD mice were added at 5 × 10⁵ cells per well as a source of cell donor antigens. The triplicate culture supernatants were taken and their IFNy content was determined by ELISA.

The results of this study demonstrated that at 2 to 3 weeks of age, GAD-reactive T cells could not be detected in the L-sel population.⁺ nor in the L-sel^-, consistent with a low frequency of reactive antigen precursors at that time. However, at 6 weeks of age, GAD (but not control antigens) produced high concentrations of IFNy in the L-sel subpopulation.(but not in the L-sel⁺) of CD4 + cells (Figure 9 (b)).

The increase in clonal size of GAD-reactive T lymphocytes, their production of IFNy and their L-sel phenotype^- provided three independent lines of evidence that the response of potentially pathogenic Th1-type cells (Ando et al., Cell Immunol., 124: 132-143, 1989) is sensitive27

ES 2 179 077 T3 spontaneously lized for GAD in vivo initially in the development of NOD.

Example 8

Characterization of GAD-specific T lymphocyte

Determining identification

The good specificity of the T cell response will be represented graphically using a series of 38 peptides (numbered successively from the N-terminal) whose complete GAD sequence was 20 to 23 amino acids long and measured (aa) (Bu et al., Proc. Natl. Acad. Sci., 89: 2115-2119, 1992) with overlaps of 5aa (Figure 10).

Spleen cell multiplier responses were tested in 4 (Figure 10a), 5 (Figure 10b), and 7 (Figure 10c) week-old mice (will continue to be described in Example 4) for GAD peptides. Peptides were present in cultures at 7 µM and the marker was added during the last 16 hours of a 5 day culture. The peptides were synthesized using the biochemical pattern Fmoc and purified by reverse phase HPLC (Advanced Chemtech). The sequence of the stimulatory peptides is presented below in Table 9.

TABLE 9

<td>Peptide number</td><td>GAD area</td><td>Amino Acid Sequence</td>

<td> 6</td><td>78a97</td><td>KPCSCSKVDVNYAFLHATDL</td>

<td> 17</td><td>247 to 266</td><td>NMYAMMIARFKMFPEVKEKG</td>

<td> 23</td><td>335 to 356</td><td>TAGTTVYGAFDPLLAVADICKK</td>

<td> 32</td><td>479 to 498</td><td>EYLYNIIKNREGYEMVFDGK</td>

<td> 34</td><td>509 to 528</td><td>IPPSLRYLEDNEERMSRLSK</td>

<td> 35</td><td>524 to 543</td><td>SRLSKVAPVIKARMMEYGTT</td>

<td> 36</td><td>539 to 558</td><td>EYGTTMVSYQPLGDKVNFER</td>

<td> 38</td><td>566 to 585</td><td>ATHQDIDFLIEEIERLGQDL</td>

Murine and human GAD65s are 95% identical in the amount of amino acids (555/585) and 98% are conserved, with most of the differences located near their N-terminus. The underlined amino acids in the above stimulatory peptide sequences are conservatively substituted in the GAD65 of the mutant. In separate experiments the murine forms of the key peptides (# 17 and # 34) were tested and produced similar results.

Still shown in Figure 10, the peptides that elicited stimulation indices&gt; 3 are indicated in black bars. These peptides do not multiply in T lymphocytes from NOD mice &lt;3 to&gt; 16 weeks of age, or control (BALB / cx NOD) F1 mice (data not shown). These data are represented as mean SI ± standard error calculated from 3 to 6 individual mice tested twice in each age group. Characteristic results for blastogaenia produced by the peptide in individual mice are shown in Table 6. The first detectable response, at 4 weeks of age, was confined to the carboxy terminal zone of GAD, and involved two adjacent peptides (aa 509 to 528 and 524 to 543, n^° 34 yn^° 35, respectively, Figure 10a). At 5 weeks of age, responses to an additional determinant (aa 247 to 266, peptide n^°17, which contains a region of sequence similarity to coxsackie virus (Kaufman et al., J. Clin. Invest., 89: 283-292, 1992) (Figure 10b). Over the next two weeks, responses to peptide n increased.^° 17 (aa 247 to 266) and spread T cell autoimmunity to two additional peptides at the carboxy terminal (aa 479 to 498 and 539 to 558, n^° 32 yn^° 36, respectively, Figure 10c). Subsequently, the reactivity to the GAD peptides will decrease (data not shown), the loss of response of the whole protein being analogous (Figure 8). It is not clear why the initial response of eCAs T lymphocytes disappears in NOD mice. Possible explanations include: a) immune regulatory mechanisms; b) response depletion due to continuous stimulation by endogenous antigen; and c) production of anergy in specific T lymphocytes due to their recognition of autoanthagen in "non-professional" antigen donating cells such as the β-cells themselves (Markmann et al., Nature, 336: 476-479, 1988).

ES 2 179 077 T3

The gradual diversification of the repertoire of sensitized autoreactive T lymphocytes observed in this naturally occurring autoimmune disease is analogous to the newly observed changes in T lymphocyte recognition in experimentally produced autoimmunity in CNSs where autoimmunity is spread intra- and intermolecularly between CNS proteins (Lehmann et al., Nature, 358: 155-157, 1992; Perry et al., J. Immunol., 33: 7-15, 1991; Watanabe et al., Nature, 305: 150-153, 1983; Liebert et al., J. Neuroimmunol., 17: 103-118, 1988). Lymphokine secretion by the first wave of specific autoantigenic T cells in the target organ appears to result in upregulation of antigen presentation and creates a microenvironment that favors sensitization 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). As hsp-reactive CD4 + T cells are capable of producing IDDM (Elias et al., Proc. Nat. Acad. Sci., 87: 1576-1580, 1990; Elias, Proc. Nat. Acad. Sci., 88: 3088 to 3091, 1991), their enhancement in the activated T lymphocyte pool, along with reactive T lymphocytes to other eCAs, probably reflects an amplifying torrent that ultimately leads to β-cell destruction.

In summary, the above data test GAD as a critical target antigen in the pathogenesis of IDDM in NOD mice. The results demonstrate that T cell responses diversify intra- 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 may prevent the enhancement of additional autoantigens in the sensitized repertoire, thereby disrupting a cascade of autoimmune responses that ultimately lead to β cell destruction. According to a similar autoimmune progression it is also likely to occur during the development of IDDM (Palmer, JP, Predicting IDDM, Diabetes Reviews, 1: 14-115, 1993; Atkinson et al., Lancet, 339: 458-459, 1992), these Findings suggest that peptide-based immunotherapeutic agents will be useful in predicting and enhancing human IDDM.

Example 9

GAD fragment autoantibody reactivity

This example describes a study examining variability in the recognition of epitopes on human GAD65 polypeptides by IDDM antibodies in human patient sera.

Human GAD65 cDNA portions were augmented by polymerase chain reaction (PCR; Saiki et al., Science, 239: 487, 1988) to produce DNA segments encoding three polypeptide segments: amino acid residues 1 to 224 (segment TO); 224 to 398 (segment B); and 398 to 595 (segment C). Each construct also contains a T7 activator, an agreement sequence for the initiation of translation, and a methionine initiator codon (Korak, M., J. Cell Biol., 108: 229,

1989). Each PCR product was then transcribed in vitro with T7 RNA polymerase and translated in vitro in a rabbit reticulocyte cell-free system in the presence of³⁵S-methionine, using conditions recommended by the supplier (Amersham Corp., Arlington Heights, Il). Each test serum (30 μl) was incubated with the produced polypeptides labeled with³⁵S. PAS-linked peptides were isolated and analyzed by SDS-PAGE in 12% polyacrylamide and autoradiography.

ES 2 179 077 T3

TABLE 10

Reactivity of IDDM Patient Sera with GAD Segments

<td rowspan="2">Patient</td><td colspan="3">Segment</td>

<td>TO</td><td>B</td><td>C</td>

<td>Control (N = 7)</td><td> -</td><td> -</td><td>_-</td>

<td> 052</td><td> -</td><td> +</td><td> +</td>

<td> 723</td><td> -</td><td> -</td><td> -</td>

<td> 705</td><td> -</td><td> +</td><td> +</td>

<td>UC2</td><td> -</td><td> +</td><td> +</td>

<td>NL</td><td> -</td><td> -</td><td> -</td>

<td>LI</td><td> -</td><td> -</td><td> -</td>

<td>TL</td><td> -</td><td> -</td><td> -</td>

<td>PT</td><td> -</td><td> +</td><td> -</td>

<td>JD</td><td> -</td><td> -</td><td> -</td>

<td>BY</td><td> -</td><td> +</td><td> +</td>

<td>MC</td><td> -</td><td> -</td><td> -</td>

<td>RS</td><td> -</td><td> -</td><td> -</td>

<td>KO</td><td> -</td><td> -</td><td> -</td>

<td>TB</td><td> -</td><td> -</td><td> -</td>

<td>YE</td><td> -</td><td> -</td><td> -</td>

<td>AW</td><td> -</td><td> +</td><td> -</td>

<td>JB</td><td> -</td><td> +</td><td> +</td>

<td>HA</td><td> -</td><td> -</td><td> -</td>

<td>Pc</td><td> -</td><td> +</td><td> +</td>

<td>LR</td><td> -</td><td> -</td><td> -</td>

<td>JM</td><td> -</td><td> +</td><td> -</td>

<td>GA</td><td> -</td><td> -</td><td> -</td>

Seguín is shown in Table 10, no sample has detectable concentrations of antibodies in the amino terminal third (segment A) of GAD while 9 patients (41%) presented reactive antibodies with the middle third (segment B) and 6 patients (27 %) presented antibodies in the third of the carboxy terminal (segment C) of GAD.

Example 10

Early IDDM Prediction Using GAD

Epotope recognition model

The increasing likelihood of IDDM surgical therapy and the (recently recognized) benefits of treated glucose homeostasis in the prevention of IDDM-related complications make early detection of cell autiimmunity a crucial target and before of the clinical onset of IDDM and in NIDDM patients (10% of which eventually become IDDM). Autoantibodies to GAD can provide the earliest and most reliable marker of impending IDDM among the molecularly defined IDDM related to autoantigens. The following study was conducted to determine whether GAD peptides bind to IDDM-related autoantibodies.

A series of peptides (20 to 23 amino acids in length, with 5 aa overlapping) that bridge the human GAD65 molecule were synthesized to determine whether sera from most at-risk individuals, prior to IDDM and with IDDM (compared to health controls) does produce antibodies that differentially recognize the linear epitopes of GAD65 distributed throughout the molecule.

ES 2 179 077 T3

Patient sera and most of the control sera were those used in the previous study (Kaufman et al., J. Clin. Investigation, supra). All samples were coded and blind tested. The peptides were synthesized using an automaotic instrument (Applied Biosystems, Foster City, CA) and standard conditions. The peptides were dissolved in 60 mM sodium bicarbonate buffer (pH 9.6) at 20 µg / ml and 100 µl of each were added to duplicate wells of a 96-well Nunc-Immuno plate. Peptides were allowed to join 4^°C all night. 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^°C or at room temperature overnight. The plates were then washed 5 times with the wash buffer above. 100 μl of serum at a 1/300 dilution in PBS + 0.1% Tween 20 and 1% BSA was added to each well and the antibodies were allowed to bind for 1 hour at 37^°C. Plates were washed 5 times with wash buffer. 100 μl of a 1/600 dilution of goat anti-human IgG HRP (BRL, Gaithersberg, MD) was added to each well and allowed to bind for 1 hour at 37^°C. The plates were then washed 7 times and 100 µl of substrate buffer was added to each well for 30 minutes at room temperature. Color development was measured at 410 nm using an ELISA plate reader (ICN, Biomedicals, Costa Mesa, CA). Positive sera were defined according to: OD₄₁₀ sample / negative control&gt; 3.0. The data shown in Table 11 shows that a number of GAD peptides were recognized by patients previously shown to be 64K positive, but not by control serum. Each patient showed a different pattern of GAD epitope recognition. Peptides 20, 21 and 25 were recognized each by 6 to 8 patients, and none of the controls except for peptide 25, which was recognized by 1 out of 13 controls. Based on the immunoreactivity to 2 of these peptides (n^°s. 20 and 21) from 7 to 8 (88%) of the patients (and none of the controls) could be identified as having GAD autoantibodies. Peptides 3, 6, 22, 25 and 37 were each recognized by only 25 to 37% of the patients (and none of the control sera), but considered together, 75% of the patients recognized at least one of these. . Peptides 5, 9 and 24 were positive for immunoreactivity many times in control and patient sera.

This level of sensitivity is comparable to the best analyzes currently available using all GAD65 purified from brain or recombinant organisms. In addition to avoiding laborious antigen purification, peptide-based antibody detection, together with PCR-based HLA typing, can reveal epitope recognition patterns related to the evolution or lack of evolution of IDDM and its related complications. Therapeutic intervention of individuals defined as being at high risk could be considered below.

It should also be noted that the GAD peptides recognized by autoantibodies were different from those recognized by the GAD-reactive T cells of NOD in Example 6.

ES 2 179 077 T3

<img file="ES2179077T3_D0001.tif" />

ES 2 179 077 T3 eeexicq

<img file="ES2179077T3_D0002.tif" />

ES 2 179 077 T3

Example 11

GAD immunization protects NOD mice from IDDM

The availability of cDNAs encoding GAD65 allows this molecule to be tested in new surgical therapies designed to interfere with GAD-specific T lymphocytes. Tests were conducted to examine the immunization ability of GAD65 to protect NOD mice at 8 weeks of age, by which time the T lymphocyte responds to a number of B cell antigens, and insulitis is well established. If GAD immunotherapy were effective at this stage, it would hold hope for treatment in humans in which the autoimmune procedure has already been demonstrated. Procedures

Autogenous

An IPTG-elicited T7 expression vector will be used to express human GAD65 and E. coli b galactosidase (β-gal). In recombinant E. coli caused by IPTG, GAD and β-gal constitute 10-20% of the total bacterial protein. However, almost all of the GAD was in inclusioan bodies, which can be isolated and washed extensively to obtain material that is approximately 80% GAD. Affinity purifications of GAD and β-gal were then done on the basis of a hexa-histidine "terminal" that bound to GAD during the subclonation process. These additional histidine residues allow rapid affinity purification (Novagen) of GAD by metal affinity chromatography (Hochuli et al., Bio Technology, 6: 1321-1325, 1988). Inclusion of body material is solubilized in 6M guanidine chloride (GHCL), 10mM β-mercaptoethanol, and 1% Triton X-100. After fixation to the column, the column was washed extensively with GHCL and 8M urea in phosphate buffers. Only the GAD fraction of the central peak will be used for further studies. Human GAD65 shares 96% amino acid sequence identity with murine GAD65, with most amino acid differences being conservative substitutions.

The GAD preparation is presented free of immunologically detectable contaminants. In addition, it is presented free of bacterial contaminants in overloaded silver gels. Analyzes by a national reference laboratory gave &lt;0.06ng LPS / 'g GAD. The GAD₆₅ Human does not cause T lymphocyte multiplication in NOD &lt;4 or&gt; 16 weeks of age or in control F1 BALB / c (NOD / BALB / c) spleen cells. The results using syntatic GAD peptides (Figure 10) are exactly analogous to the data using total recombinant GAD (Figure 8). Other antigens described elsewhere in this specification that are not involved in IDDM (such as β-galactosidase) do not elicit T cell responses in NOD. After immunizing the mice with GAD, we were unable to detect cross-responses of reactive T lymphocytes in calling experiments with other proteans that were purified from recombinant E. coli by the same metal affinity chromatography procedure. GAD and β-gal amino acid sequence analyzes each gave a unique expected N-terminal amino acid sequence. If endotoxins had been seen, one would have expected dermal shock proteans or other contaminants present in the preparation of GAD, spleen, PBMC (Atkinson et al.), And T cell multiplication responses that were not disease specific.

Hatchery mice were purchased from Tatonic Farms and housed under specific pathogen-free conditions. Only female NOD mice were used in this study. The average age at onset of IDDM in unrelated females in the colony was 22 weeks. Insulitis is seen to generally begin at 4 weeks of age. The response of T lymphocytes to GAD, HSP, CPH was observed at the age of 6 weeks. The incidence of IDDM in female mice is 70 to 90% for one year of age.

Immunizations

At 8 weeks of age, 25 μg of GAD or β-gal was injected intraperitoneally (ip). in 100 μ! of incomplete Freund's adjuvant (IFA). Because it may be a requirement for continuous antigen presentation (Ramsdell et al., Science, 257: 1130-1133, 1992) mice were treated again every 6 weeks. Urine glucose concentrations were monitored twice a week. After observing previous normal glucose in urea, blood glucose concentrations were monitored twice a week. Two consecutive blood glucose concentration readings of 300 mg / ml were considered as the start of IDDM, after which the mice were sacrificed and spleen cells tested as described above in Example 6 to test multiplication. cells of the spleen.

ES 2 179 077 T3

Immunization of the 8 week old NOD mice produced a clear delay in the onset of IDDM compared to the control β-gal immunized mice (Fig. 11). While two of the GAD-immunized mice (open circles) developed IDDM at the normal age of onset (20 weeks), the other 8 GAD-immunized mice did not show signs of hyperglycemia until the age of 36 weeks. Four of the GAD-treated mice developed IDDM between 37 and 40 weeks of age. Four of the GAD-treated mice are currently disease-free (at 52 weeks of age). In contrast, the majority of mice injected with β-gal (black circles) had hyperglycemia at 22 weeks of age and between 6 and 10 developed IDDM during 27 weeks of age. At the age of 52 weeks, 2 of the β-gal treated mice remain disease free. This experiment demonstrates that immunization with GAD significantly delays (&lt;0.02) or prevents diabetes in NOD mice in which β-cell autoimmunity has already progressed significantly.

Β-cell autoimmunity is already well demonstrated at 8 weeks of age, and it will probably also be in individuals defined to be at risk for IDDM based on circulating antibodies. Although the mechanism of this protection is not clear, periodic GAD injections have a profound moderating effect on disease initiation.

TABLE 12

Amino Acid Sequences for GAD65

MASPGSGFWSFGSEDGSGDS

GSGDSENPGTARAWCQVAQKFTG

QKFTGGIGIGNKLCALLYGD

LLYGDAEKPAESGGSQPPRA

QPPRAAARKAACACDQKPCSC

KPCSCSKVDVNYAFLHATDL

HATDLLPACDGERPTLAFLQ

LAFLQDVMNILLQYVVKSFDRS

SFDRSTKVIDFHYPNELLQE

ELLQEYNWELADQPQNLEEILM

EEILMHCQTTLKYAIDTGHP

KYGHPRYFNQLSTGLDMVGL

DMVGLAADWLTSTANTNMFT

TNMFTYEIAPVFVLLEYVTL

EYVTLKKMREIIGWPGGSGD

GGSGDGIFSPGGAISNMYAM

NMYAMMIARFKMFPEVKEKG

PEVKEKGMAALPRLIAFTSE

AFTSEHSHFSLKKGAAALGI

AALGIGTDSVILIKCDERGK

DERGKMIPSDLERRILEAKQ

LEAKQKGFVPFLVSATAGTT

TAGTTVYGAFDPLLAVADICKK

DICKKYKIWMHVDAAWGGGLLMS

GLLMSRKHKWKLSGVERANS

ERANSVTWNPHKMMGVPLQC

VPLQCSALLVREEGLMQNCNW

QNCNQMHASYLFQQDKHYDL

KHYDLSYDTGDKALQCGRHV

CGRHVDVFKLWLMWRAKGTTG

ES 2 179 077 T3

TABLE 12 (continued)

KGTTGFEAHVDKCLELAEYLYN

EYLYNIIKNREGYEMVFDGK

VFDGKPQHTNVCFWYIPPSL

IPPSLRTLEDNEERMSRLSK

SRLSKVAPVIKARMMEYGTT

EYGTTTMVSYQPLGDKVNFFR

VNFFRMVISNPAATHQDIDF

ATHQDIDFLIEEIERLGQDL

In the invention which has been described in its entirety, 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.