Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Patent
1998-06-23
2000-07-25
Carlson, Karen Cochrane
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
435197, 4353201, 4352523, 4352542, 435348, 435325, 536232, A61K 3846, C12N 918, C12N 1500, C12N 120, C07H 2104
Patent
active
060933968
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a genetically-modified recombinant form of the 65 kDa human glutamic acid decarboxylase (hGAD65) that retains structural integrity for immuno-reactivity characteristic of the unmodified form, but that is without (or with considerably reduced) enzyme activity. The novel modified GAD is advantageously used in pharmaceutical compositions for treating autoimmune diseases.
BACKGROUND OF THE INVENTION
The inhibitory neurotransmitter .gamma.-aminobutyric acid (GABA), derived from L-glutamic acid by GAD is present in brain as well as several tissues outside the central nervous system. Biological functions of GAD and GABA extend beyond regulation of neurotransmission to include effects on the immune system as well as modulation of cell proliferation, protein synthesis and metabolism.
GAD has recently been associated with autoimmune insulin-dependent diabetes mellitus (IDDM) (Boekkeskov et al, Nature 347:151 (1990), incorporated by reference) because of the increased incidence of IDDM in patients with stiff-man syndrome, a disease associated with autoantibodies against GAD. Antibodies from diabetic sera have also been shown to bind GAD. At least two different GAD, i.e. GAD 65 and 67 have been identified and sequenced in human beings. hGAD65 and hGAD67 have several epitopes in common. However, different epitopes are involved in different reactions and contexts.
Binding of the coenzyme pyridoxal-5'-phospate (PLP) to GAD was first reported by Roberts & Frankel in 1951 (J. Biol. Chem. 188:789, fully incorporated in the description by reference), and formation of a Schiff base during GAD-mediated decarboxylation was subsequently proposed by W T Jenkins 1961, Fed. Proc. 20:978 (fully incorporated in the description by reference) (i.e. referenced in Roberts & Simonsen 1963, Biochem. Pharmacol. 12:113-134, fully incorporated in the description by reference). Accordingly, PLP has been identified as a critical co-factor in decarboxylation by GAD.
Cloning of GAD is disclosed in WO92/05446 and WO92/20811 (both incorporated by reference).
Location of the PLP binding site within the amino acid sequence of hGAD65 was enabled by publication of the cDNA sequence of this enzyme (P.N.A.S. 88:8337, fully incorporated in the description by reference). The site was found because of the homology centred around the amino acid lysine at a/a #396 in hGAD65--to the sequence: X-His-Lys-X that was previously published as the PLP binding site in crystallised E.coli GAD (Biochemistry 9:226-233; Biochemistry 13:670-676, both of which are fully incorporated in the description by reference).
This region in hGAD65 has the following amino acid sequence (SEQ ID NOS:9 and 10):
______________________________________ Thr Trp Asn Pro His Lys* Met Met Gly Val
T W N P H K* M M G V
.cndot. .cndot. .cndot.
#391 #396 #400
______________________________________
*selected herein for mutation to change K to Arg (R)
This homology in this region has subsequently been confirmed by Lernmark (unpublished) using computer homology matches to ca. 10 other PLP-binding (non-GAD) proteins.
The approach of the present invention is a novel GAD without enzyme activity by mutating a single codon in the PLP binding site in the cDNA sequence for hGAD65. This results in the substitution of a single amino acid that is incapable of supporting wild-type enzyme activity.
As the lysine at #396 has been identified as critical for enzyme activity (via formation of a Schiff base during GAD-mediated decarboxylation), the present invention is directed to substitute an amino acid incapable of Schiff base formation at position #396 as a subtle means of achieving this effect.
In addition, a "conservative amino acid substitution" (i.e. replacement of lysine by an amino acid of comparable size and hydrophobicity) is intended to minimise any structural or conformational changes to the hGAD65 protein, and thereby avoid alterations to either antigenic etitopes or T cell determinants originally present in recombinant human GAD (rhGAD65).
SUMMA
Essen-Moller Anders
Falorni Alberto
Lernmark Ake
Robertson John
Carlson Karen Cochrane
Diamyd Therapeutics AB
Srivastava Devesh
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