Detoxified TNF and method of preparing

An immunogenic analogue of a human TNFα protein, wherein said analogue comprises

• a) two or three complete TNFα monomers joined end-to-end by a peptide linker, wherein at least one peptide linker includes at least one MHC Class II binding amino acid sequence, or
• b) two or three complete TNFα monomers joined end-to-end by an inert peptide linker, wherein at least one of the monomers include at least one foreign MHC Class II binding amino acid sequence or wherein at least one foreign MHC Class II binding amino acids sequence is fused to the N- or C-terminal monomer, optionally via an inert linker, or
• c) a human TNFα monomer or an analogue defined in a or b, wherein has been inserted or in-substituted at least one foreign MHC Class II binding amino acid sequence into flexible loop 3, or
• d) a human TNFα monomer or an analogue defined in a or b, wherein has been introduced at least one disulfide bridge that stabilises the TNFα monomer 3D structure, or
• e) a human TNFα monomer or an analogue defined in a or b, wherein any one of amino acids 1, 2, 3, 4, 5, 6, 7, 8, and 9 in the amino terminus have been deleted, or
• f) a human TNFα monomer or an analogue defined in a or b, wherein is inserted or in-substituted at least one foreign MHC Class II binding amino acid sequence into loop 1 in an intron position, or
• g) a human TNFα monomer or an analogue defined in a or b, wherein at least one foreign MHC Class II binding amino acid sequence is introduced as part of an artificial stalk region in the N-terminus of human TNFα, or
• h) a human TNFα monomer or an analogue defined in a or b, wherein at least one foreign MHC Class II binding amino acid sequence is introduced so as to stabilize the monomer structure by increasing the hydrophobicity of the trimeric interaction interface, or
• i) a human TNFα monomer or an analogue defined in a or b, wherein at least one foreign MHC Class II binding amino acid sequence flanked by glycine residues is inserted or in-substituted in the TNFα amino acid sequence, or
• j) a human TNFα monomer or an analogue defined in a or b, wherein at least one foreign MHC Class II binding amino acid sequence is inserted or in-substituted in the D-E loop, or
• k) a human TNFα monomer or an analogue defined in a or b, wherein at least one foreign MHC Class II binding amino acid sequence is inserted or in-substituted between two identical subsequences of human TNFα, or
• l) a human TNFα monomer or an analogue defined in a or b, wherein at least one salt bridge in human TNFα has been strengthened or substituted with a disulphide bridge, or
• m) a human TNFα monomer or an analogue defined in a or b, wherein solubility or stability towards proteolysis is enhanced by introducing mutations that mimic murine TNFα crystalline structure,
• wherein potential toxicity is reduced or abolished by introduction of at least one point mutation selected from the group consisting of Y87S, D143N or A145R, the amino acid numbering setting out from the N-terminal valine in human TNFα.