Subtilisin carlsberg proteins with reduced immunogenicity
OwnerFiona Hardinginventor
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Abstract
The present invention provides methods for the identification of CD4+ T-cell epitopes in subtilisin Carlsberg proteins. The present invention also provides for the production of altered peptides which, when incorporated into a wild-type subtilisin Carlsberg protein produce an altered immunogenic response, preferably a low immunogenic response in humans. In particular, the present invention provides means, including methods and compositions suitable for reducing the immunogenicity of ALCALASE® enzyme.
Claims21
A method for identifying at least one T-cell epitope of a microbial subtilisin, wherein said subtilisin comprises subtilisin Carlsberg, comprising the steps of:
- (i) obtaining from a from a single human blood source, a solution of dendritic cells and a solution of naïve CD4+ and/or CD8+ T-cells;
- (ii) differentiating said dendritic cells to produce a solution of differentiated dendritic cells;
- (iii) combining said solution of differentiated dendritic cells and said naïve CD4+ and/or CD8+ T-cells with peptide fragments of said subtilisin Carlsberg;
- and
- (iv) measuring proliferation of said T-cells in said step (iii).
A method of reducing the immunogenicity of a microbial subtilisin Carlsberg, comprising the steps of:
- (a) identifying at least one T-cell epitope in said protein by
- (i) contacting an adherent monocyte-derived dendritic cell that has been differentiated by exposure to at least one cytokine in vitro, with at least one peptide comprising said T-cell epitope;
- and
- (ii) contacting said dendritic cell and said peptide with a naïve T-cell, wherein said naïve T-cell has been obtained from the same source as said adherent monocyte-derived dendritic cell, and whereby said T-cell proliferates in response to said peptide;
- and
- (b) modifying said subtilisin Carlsberg to neutralize said T-cell epitope to produce a variant protein, such that said variant protein induces less than or substantially equal to the baseline proliferation of said naïve T-cells.
A modified subtilisin Carlsberg, wherein said subtilisin Carlsberg comprises at least one alteration in at least one epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:90, SEQ ID NO:15, SEQ ID NO:30, and SEQ ID NO:40.
Dependencies are inferred from the claim text.