Reduction of protein immunogenicity

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Claimed is therapeutically active fused protein with reduced immunogenicity. Protein consists of two proteins derived from human proteins connected through the fusion region. Connective region, which covers or surrounds fusion region within the limits from 1 to 25 amino acid residues, contains modification, which removes T-cell epitope, in norm absent in humans. Claimed is application of fused protein for obtaining pharmaceutical composition for tumour treatment. Claimed is nucleic acid coding fused protein. Method of reduction of fused protein immunogenicity by introduction of substitutes of corresponding amino acids is described. Application of the invention allows reducing ability of connective epitope of therapeutically active fused protein to bind with molecules of the main complex of hystocompatibility (MHC) of class II, which finally reduces interaction of epitope with receptors of T-cells and can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.

EFFECT: reduction of interaction of epitope with receptors of T-cells, which can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.

23 cl, 12 ex

 

The text descriptions are given in facsimile form.

1. Therapeutically active protein with reduced immunogenicity, consisting of two proteins, derived from human, connected to each other through the mail merge pane, and connector pane in the range from 1 to 25 amino acid residues surrounding or overlying the mail merge pane, contains a modification that removes the T-cell epitope, normally absent in the human body selected from the group consisting of:

(i) site of N-linked glycosylation, which is an Asn-X-Ser/Thr, where X is an amino acid,

(ii) the amino acid sequence Ala-Thr-Ala-Thr instead of Leu-Ser-Leu-Ser if the component merge presents IgG or

(iii) mutation of Thr, Ala or Pro instead of Leu, Val, IIe, Met, Phe, Tyr or Trp.

2. Fused protein according to claim 1, characterized in that X represents Gly.

3. Fused protein according to claim 1, characterized in that the connecting region includes a spacer or linker.

4. Fused protein according to claim 1, characterized in that one component of the fused protein is an albumin.

5. Fused protein according to claim 1, characterized in that one component of the fused protein has the activity of a cytokine or hormone.

6. Fused protein according to claim 1, characterized in that one component of the fused protein is an IgG or fragment.

7. Fused protein according to claim 1, characterized in that one component of the fused protein is an IgG or fragment, and the other component is a protein having the activity of a cytokine or hormone.

8. Fused protein according to claim 7, characterized in that the end of the specified IgG or its fragment is connected to the N-end of the other component.

9. Fused protein according to claim 7, characterized in that the fragment of IgG is an Fc molecule.

10. Fused protein according to claim 7, wherein the IgG or fragment contains the amino acid sequence of two isotypes of antibodies.

11. Protein of claim 10, wherein the IgG or fragment contains the amino acid sequence IgG1 and IgG2.

12. Fused protein according to claim 11, wherein the IgG or fragm the t represents the isotype IgG2, modified in the hinge region to IgG1.

13. Protein of claim 8, representing huKS-IL2, including the connecting region in the sequence IgG instead of the sequence Leu-Ser-Leu-Ser sequence Ala-Thr-Ala-Thr.

14. Protein of claim 8, representing Fc-IL12-IL2, including the connecting region in the sequence IgG instead of the sequence Leu-Ser-Leu-Ser sequence Ala-Thr-Ala-Thr.

15. Protein of claim 8, representing Fc-IL12-IL2, including the first positions in the IL2 molecule glycosylation site Asn-Gly.

16. Protein of claim 8, representing the Fc-EPO, including the connecting region in the sequence IgG instead of the sequence Leu-Ser-Leu-Ser sequence Ala-Thr-Ala-Thr.

17. Protein according to clause 16, which represents an Fc-EPO, in which IgG is an IgG2, containing the hinge region of IgG1.

18. The use of therapeutically active fused protein as defined in any of PP-15, to obtain a pharmaceutical composition for the treatment of tumors.

19. Nucleic acid encoding a protein as defined in any one of claims 1 to 17.

20. The method of reducing the immunogenicity of fused protein described in claim 1, by removing the T-cell epitope, normally absent in the body, including the modification of the connecting region surrounding or overlapping is her area of fusion in the range from 1 to 25 amino acid residues, method selected from the group consisting of:

(i) the introduction of N-linked glycosylation, which is an Asn-X-Ser/Thr, where X is an amino acid,

(ii) replacement of the amino acid sequence Leu-Ser-Leu-Ser in the amino acid sequence Ala-Thr-Ala-Thr if the component merge is an IgG, or

(iii) replacement of the amino acids Leu, Val, Ile, Met, Phe, Tyr or Trp at Thr, Ala or Pro;

21. The method according to claim 20, characterized in that X represents Gly.

22. The method according to claim 20, characterized in that one component of the merger is an IgG or fragment.

23. The method according to claim 20, characterized in that one component of the merger is an IgG or fragment, and the other component merge is a cytokine.



 

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