Immunoreactive peptides

Details Immunoreactive peptides Immunoreactive peptides

1996-11-25

2004-11-16

Ewoldt, G. R. (Department: 1644)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

C424S185100, C424S198100, C424S810000, C435S183000, C514S003100, C514S012200, C514S013800, C514S014800, C514S015800, C514S866000, C530S303000, C530S324000, C530S325000, C530S326000, C530S327000, C530S399000

Reexamination Certificate

active

06818217

ABSTRACT:

The present invention relates generally to molecules such as peptides, polypeptides and proteins which interact immunologically with antibodies or T-cells in subjects having pre-clinical or clinical Insulin-Dependent Diabetes Mellitus (IDDM). These molecules are preferentially immunoreactive to T-cells in subjects having pre-clinical or clinical IDDM and are useful in the development of diagnostic, therapeutic and prophylactic agents for IDDM.
Amino acid sequences are referred to herein by sequence identity numbers (SEQ ID NOs) which are defined at the end of the specification.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers, but not to the exclusion of any other element or integer or group of elements or integers.
Insulin-Dependent Diabetes Mellitus is a serious disease resulting from the destruction of insulin-secreting &bgr;-cells, probably mediated by T cells that recognise &bgr;-cell autoantigens. A major antigen implicated in T-cell mediated &bgr;-cell destruction characteristic of IDDM is glutamic acid decarboxylase (GAD), which occurs in two major isoforms, GAD 65 and GAD 67. These two isoforms have approximately 65% similarity at the amino acid sequence level. Subjects with IDDM or at high-risk of the disease show autoantibody and autoreactive T-cell responses to GAD insulin or both autoantigens. In NOD mice, an animal model for spontaneous IDDM, GAD is a dominant and early target antigen (Tisch et al
Nature
366:72-75, 1993).
Identification of the immunodominant epitope(s) of pathogenic autoantigens involved in &bgr;-cell autoimmunity could lead to improved methods of diagnosis as well as therapeutic strategies to prevent IDDM.
In work leading up to the present invention, the inventors sought to identify immunodominant epitopes in GAD and proinsulin molecules in order to improve upon current diagnostic procedures and to further develop therapeutic and prophylactic compositions and treatment approaches for IDDM.
In accordance with the present invention, peptides were synthesised based on a thirteen amino acid region of high similarity between the sequences of human GAD 65 (amino acid residue numbers 506-518) and human proinsulin (amino acid residue numbers 24-36), which region of similarity also extends to human GAD 67 and mouse proinsulins and mouse GADs (FIG.
1
). The immunoreactivity of these peptides is identified in accordance with the present invention on the basis of interactivity of peripheral blood cells or T-cells obtained from the peripheral blood of subjects with pre-clinical or clinical IDDM, thereby forming the basis for a new range of diagnostic, therapeutic and prophylactic procedures for IDDM.
Accordingly, one aspect of the present invention provides a recombinant or synthetic peptide or chemical equivalents thereof of the formula:
X
1
X
2
X
3
wherein:
X
1
and X
3
may be the same or different and each is an amino acid sequence comprising from 0 to 40 naturally or non-naturally occurring amino acid residues;
X
2
is any amino acid sequence of from 10 to 100 residues derived from, homologous to or contiguous within amino acids 506 to 518 inclusive or derivatives thereof of human GAD65 and/or amino acids 24 to 36 inclusive or derivatives thereof of human proinsulin; and wherein said peptide molecule is capable of reacting with T cells and modifying T-cell function when incubated with cells from subjects having pre-clinical or clinical Insulin-Dependent Diabetes Mellitus (IDDM). Preferred cells include but are not limited to peripheral blood mononuclear cells (PBMCs), anticoagulated whole blood and tissue biopsy cells.
Reference to a “peptide” includes reference to a polypeptide or protein or parts thereof.
In a preferred embodiment X
2
comprises not less than about 10 and not greater than about 50, amino acid residues, more preferably not less than about 10 and not greater than about 30 amino acid residues and even more preferably not less than about 10 and not greater than about 15.
In a particularly preferred embodiment X
2
has either of the following amino acid sequences:
F F Y T P K T R R E A E D  [SEQ ID NO:1];
or
F W Y I P P S L R T L E D  [SEQ ID NO:2].
According to this preferred embodiment, there is provided a recombinant or synthetic peptide or chemical equivalent thereof comprising the sequence:
X
1
X
2
X
3
wherein
X
1
and X
3
may be the same or different and each is an amino acid sequence comprising from 0 to 15 naturally or non-naturally occurring amino acid residues;
X
2
is selected from FFYTPKTRREAED and FWYIPPSLRTLED or a derivative or chemical equivalent thereof and wherein said peptide is capable of reacting with T cells and modifying T-cell function when incubated with cells from subjects with pre-clinical or clinical IDDM and determining reactivity by an appropriate assay. Preferred cells include but are not limited PBMCs, anti-coagulated whole blood or tissue biopsy cells and determining reactivity by an appropriate assay.
The peptides of the present invention may be prepared by recombinant or chemically synthetic means. According to a preferred aspect of the present invention, there is provided a recombinant peptide which is preferentially immunologically reactive with T-cells from individuals with clinical or pre-clinical IDDM, which is prepared by the expression of a host cell transformed with a cassette coding for the peptide sequences of the present invention. The peptide may be fused to another peptide, polypeptide or protein. Alternatively, the peptide may be prepared by chemical synthetic techniques, such as by the Merrifield solid-phase synthesis procedure. The synthetic or recombinant peptide may or may not retain GAD activity or proinsulin activity. Furthermore, although synthetic peptides of the formula given above represent a preferred embodiment, the present invention also extends to biologically pure preparations of the naturally occurring peptides or fragments thereof. By “biologically pure” is meant a preparation comprising at least about 60%, preferably at least about 70%, more preferably at least about 80% and still more preferably at least about 90% or greater as determined by weight, activity or other suitable means.
By “pre-clinical IDDM” as used herein means those subjects who may or may not be first degree relatives of someone with IDDM who have genetic and/or immune markers of pancreatic islet (&bgr;) cell autoimmunity. By “immune markers” is meant amongst other parameters known to those in the art to include circulating antibodies and/or T-cells reactive with islet (&bgr;) cell autoantigens.
By “derivatives” as used herein is taken to include any single or multiple amino acid substitution, deletion and/or addition relative to the naturally occurring amino acid sequence in the native molecule from which the peptide is derived including any single or multiple substitution, deletion and/or addition of other molecules associated with the peptide, including carbohydrate, lipid and/or other proteinacious moieties. Such derivatives, therefore, include glycosylated or non-glycosylated forms or molecules with altered glycosylation patterns.
By the term “reacting with T cells and modifying T-cell function” as used herein is taken to include T-cell activation, T-cell inactivation and/or T-cell death.
The present invention also covers chemical analogues of the subject peptides which include, but is not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives, during peptide synthesis and the use of cross-linkers and other methods which impose conformational constraints on the peptides or their analogues.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH
4
; amidination with

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