Production of MHC II/CII complexes
OwnerNadine Schneider, Bingze Xu +3inventors
InventorsNadine Schneider, Bingze Xu, Sylvia Cienciala, Rikard Holmdahl, Harald Burkhardt
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Abstract
The present invention relates to in situ glycosylated MHC II/CII peptide complexes, i.e., complexes naturally glycosylated during recombinant protein expression in the host cell. The invention further relates to methods of producing glycosylated MHC II/CII peptide complexes in mammalian cells. Furthermore the invention relates to the use of such post-translationally modified, preferably glycosylated MHC/CII complexed for use in treating rheumatoid arthritis, preferably in humans.
Claims18
A composition comprising recombinant MHC II/CII peptide complexes comprising
- (a) an extracellular region of an MHC class II alpha chain comprising at least one alpha 1 domain;
- (b) an extracellular region of an MHC class II beta chain comprising at least one beta 1 domain;
- and
- (c) a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain by a linker peptide;
- wherein the CII peptide comprises the amino acid sequence selected from the group consisting of AGFKGEQGPKG (SEQ ID NO:
- 1) and AGFKGEQGPXG (SEQ ID NO:
- 2), wherein X is not lysine, and
- the CII peptide is post-translationally modified, wherein the first lysine residue of the CII peptide is O-glycosylated hydroxylysine (Hyl);
- and
- wherein the at least one alpha 1 domain is from DRA*0101 and the at least one beta 1 domain is from a HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402 and DRB1*1303.
A method for producing a MHC II/CII peptide complex comprising a post-translationally modified CII peptide comprising
- (a) transfecting a mammalian cell with
- (i) a polynucleotide encoding an extracellular region of the MHC II alpha chain comprising at least an one alpha 1 domain;
- (ii) a polynucleotide encoding an extracellular region of the MHC II beta chain comprising at least one beta 1 domain;
- and
- (iii) a polynucleotide encoding a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain by a linker peptide, wherein the CII peptide comprises the amino acid sequence selected from the group consisting of AGFKGEQGPKG (SEQ ID NO:
- 1) and AGFKGEQGPXG (SEQ ID NO:
- 2), wherein X is not lysine;
- (b) cultivating the mammalian cell under conditions suitable to produce the MHC II/CII peptide complex;
- and
- (c) harvesting a cell supernatant and optionally cells comprising the MHC II/CII peptide complex comprising a post-translationally modified CII peptide, wherein the first lysine residue of the CII peptide is O-glycosylated hydroxylysine (Hyl) and wherein the at least one alpha 1 domain is from DRA*0101 and the at least one beta 1 domain is from a HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402, and DRB1*1303;
- optionally further comprising a step of analysing the post-translational modification of the CII peptide of the MHC II/CII peptide complex.
A recombinant MHC II/CII peptide complex comprising a post-translationally modified CII peptide, wherein the first lysine residue of the CII peptide is O-glycosylated hydroxylysine (Hyl), obtained by a method for producing a MHC II/CII peptide complex comprising a post-translationally modified CII peptide comprising
- (a) transfecting a mammalian cell with
- (i) a polynucleotide encoding an extracellular region of the MHC II alpha chain comprising at least one alpha 1 domain;
- (ii) a polynucleotide encoding an extracellular region of the MHC II beta chain comprising at least one beta 1 domain;
- and
- (iii) a polynucleotide encoding a collagen II peptide (CII peptide) fused to the N-terminus of the MHC class II alpha chain or the MHC class II beta chain by a linker peptide, wherein the CII peptide comprises the amino acid sequence selected from the group consisting of AGFKGEQGPKG (SEQ ID NO:
- 1) and AGFKGEQGPXG (SEQ ID NO:
- 2), wherein X is not lysine;
- (b) cultivating the mammalian cell under conditions suitable to produce the MHC II/CII peptide complex;
- and
- (c) harvesting a cell supernatant and optionally cells comprising the MHC II/CII peptide complex comprising a post-translationally modified CII peptide, wherein the first lysine residue of the CII peptide is O-glycosylated (Hyl) and wherein the at least one alpha 1 domain is from DRA*0101 and the at least one beta 1 domain is from a HLA-DR allele selected from the group consisting of DRB1*0401, DRB1*0404, DRB1*0405, DRB1*0408, DRB1*0409, DRB1*0101, DRB1*0102, DRB1*1001, DRB1*1402, and DRB1*1303.
Dependencies are inferred from the claim text.