Patent EP-1403280-B1 (EP)
Inventor: WESTRITSCHNIG KERSTIN
IPC: C07K14/415
Filed: 2002-09-27
Granted: 2005-08-31
Abstract: A mutated polypeptide (I) derived from the pollen allergen Phl p 7 is chosen from polypeptides comprising 1-15 amino acid deletions, substitutions and/or additions in a fully defined Phl p 7 sequence (S1) of 78 amino acids as given in the specification and polypeptides comprising a fragment of (S1) and having reduced IgE binding activity compared to wild-type Phl p 7, is new. A mutated polypeptide (I) derived from the pollen allergen Phl p 7 is chosen from: (a) polypeptides comprising an amino acid sequence in which in respect to the amino acid sequence of a fully defined Phl p 7 sequence (S1) of 78 amino acids as given in the specification, where 1-15 amino acid residues are deleted, substituted and/or added; (b) polypeptides comprising a fragment of (a), where the fragment has a length of at least 15, preferably at least 10 amino acids and at least 90% or 80% of the amino acid residues of the fragment are identical to corresponding residues of (S1); and (c) polypeptide comprising a fragment of (S1), where the fragment has a length of at least 15 amino acids, preferably at least 10 amino acids, where (I) has reduced IgE binding activity compared to wild-type Phl p 7 or comprising amino acid sequence (S2) chosen from 3 (mutant 1.6, mutant 2A and mutant 4) fully defined sequences of 78 amino acids as given in the specification; or (d) mutated polypeptide derived from a 2-EF hand pollen allergen, where in respect to the wild-type sequence of 2-EF hand pollen allergen amino acids position having substituted or deleted which corresponds to the amino acid residue which are substituted or deleted in respect to (S1) or (S2). Ala-Asp-Asp-Met-Glu-Arg-Ile-Phe-Lys-Arg-Phe-Asp-Thr-Asn-Gly-Asp- Gly-Lys-Ile-Ser-Leu-Ser-Glu-Leu-Thr-Asp-Ala-Leu-Arg-Thr-Leu-Gly- Ser-Thr-Ser-Ala (peptide 1) Ser-Ala-Asp-Glu-Val-Gln-Arg-Met-Met-Ala-Glu-Ile-Asp-Thr-Asp-Gly- Asp-Gly-Phe-Ile-Asp-Phe-Asn-Glu-Phe-Ile-Ser-Phe-Cys-Asn-Ala-Asn- Pro-Gly-Leu-Met-Lys-Asp-Val-Ala-Lys-Val-Phe (peptide 2) Independent claims are also included for: (1) polynucleotide (II) encoding (I); (2) vector or plasmid (III) containing (II); (3) host cell (IV) transformed or transfected with (III); (4) pharmaceutical composition (V) comprising (I) and a carrier or diluent; and (5) pharmaceutical kit (VI) comprising (I) or (V). ACTIVITY : Antiallergic. No biological data given. MECHANISM OF ACTION : Vaccine. Rabbits were immunized with Phl p7 wild-type, the peptides or the Phl p 7 wild-type, the peptides or the Phl p 7 molecule and Phl p 7 mutant as well as with keyhole limpet hemocyanin (KLH)-conjugated peptides/proteins using Freund's adjuvant. Eight rabbits were immunized with a peptide-KLH conjugate, the mutant-KLH conjugate, Phl p 7-KLH conjugate, unconjugated peptides, mutants and Phl p 7 wild-type, respectively, (200 Microg/injection) using Freund's complete and incomplete adjuvants. Serum samples were obtained in four week intervals. Sera were stored at -20 [deg]C until analysis. Reactivity of peptide-induced IgG antibodies and Phl p 7-mutant-induced IgG antibodies to recombinant Phl p 7 wild-type and a cross reactive allergen was studied by dot blot experiments. Phl p 7 wild-type as well as the corresponding immunogen (peptide 1, peptide2, Mut-4) were dotted onto nitrocellulose-strips (1 Microg/dot). Strips were exposed to different dilutions of rabbit antiserum (1:500, 1:1000, 1:2000). Similarly, recombinant Aln g 4, a Phl p 7-cross-reactive calcium-biding allergen from alder, was dotted onto nitrocellulose and strips were exposed to 1:1000 diluted rabbit antiserum. Bound rabbit antibodies were detected with a 1:1000 diluted 1> 2> 5>l-labeled donkey anti-rabbit antiserum. The peptides coupled to KLH induced IgG anti-Phl p 7 antibody responses as well as the Phl p 7-derived mutant (M4, coupled and uncoupled). Similarly, rabbit antisera raised against M4, M4-KLH and the coupled peptides recognized the Phl p 7-cross-reactive allergen, Aln g 4. The ability of peptide or mutant-induced rabbit IgG to inhibit the binding of allergic patient's IgE to complete Phl p 7 was investigated by enzyme linked immunosorbent assay (ELISA) competition assay. Strongest inhibition of IgE binding was observed.