The modified polypeptide with increased half-life, its receipt and use of

 

The invention relates to biotechnology, can be used in medical practice for a polypeptide, which is excreted through the kidneys and does not contain in its original form Fc-region of IgG. The original polypeptide modified to include rescue receptornegative epitope of the Fc-region of IgG and through this to increase circulatory half-life. The modified polypeptide contains the amino acid sequence of Ig domain or Ig-like, which is not CH2domain, and amino acid sequence of rescue receptornegative epitope of the Fc-region of IgG, located within the Ig domain or Ig-like domain. Amino acid sequence of the modified polypeptide at least 70% identical to that of the original polypeptide. Disclosed is a method of obtaining the specified polypeptide with DNA (design vector) that encodes the specified polypeptide. The resulting polypeptide can be used to ensure mediated LFA-1 disorders in mammals, including man. The invention allows to increase the half-life of the polypeptide, thereby increasing the efficiency of treatment. 8 C. and 17 C.p. f-crystals, 4 Il., 3 table.

1. The modified polypeptide having an increased length of time half-life in vivo and characterized in that it is obtained on the basis of interest polypeptide that does not contain the Fc-region of IgG and excreted through the kidneys, and the amino acid sequence of interest polypeptide is modified so that it contains a) the amino acid sequence of Ig domain or Ig-like domain, which is not CH2domain, and b) amino acid sequence of rescue receptornegative epitope of the Fc-region of IgG, which is located within the Ig domain or Ig-like domain, where the amino acid sequence of the modified polypeptide at least 70% identical to the amino acid sequence of interest polypeptide and its biological activity is retained.

2. The modified polypeptide under item 1, characterized in that the Ig-domain or Ig-like domain contains the domain of CH1.

3. The modified polypeptide under item 1, characterized in that the rescue receptornegative epitope derives from one or two loops Fc-region and is located in the Ig-domain or Ig-like domain.

4. Modificar thedomain Fc-region, the modified polypeptide further includes CH1-, CH3or VH-area and the specified epitope is located in CH1-, CH3or Vn-the field or in several fields such Ig-domain or Ig-like domain.

5. The modified polypeptide under item 1, characterized in that the rescue receptornegative epitope derives from CH2domain Fc-region and is located in the Ig-domain or Ig-like domain.

6. The modified polypeptide under item 1, characterized in that interest, the polypeptide is a Fab, (Fab’)2, dentition, Fv fragment, single-chain Fv fragment, or a receptor.

7. The modified polypeptide under item 1, characterized in that interest, the polypeptide is an antagonist of LFA-1.

8. The modified polypeptide under item 7, characterized in that interest, the polypeptide is a Fab or (Fab’)2antibodies against LFA-1.

9. The modified polypeptide under item 8, characterized in that interest, the polypeptide is an anti-CD18 Fab or anti-CD18(Fab’)2.

10. The modified polypeptide under item 9, characterized in that interest, the polypeptide is a human or humanized.

yuushi epitope contains a sequence PKNSSMISNTR (SEQ ID NO:3).

12. The modified polypeptide according to p. 11, characterized in that the rescue receptornegative the epitope further comprises a sequence HQSLGTQ (SEQ ID NO:11).

13. The modified polypeptide according to p. 11, characterized in that the rescue receptornegative the epitope further comprises a sequence HQNLSDGK (SEQ ID NO:1).

14. The modified polypeptide according to p. 11, characterized in that the rescue receptornegative the epitope further comprises a sequence HQNISDGK (SEQ ID NO:2).

15. The modified polypeptide according to p. 11, characterized in that the rescue receptornegative the epitope further comprises a sequence VISSHLGQ (SEQ ID NO:31).

16. The modified polypeptide under item 1, characterized in that the rescue receptornegative epitope has the sequence HQNLSDGK (SEQ ID NO:1), HQNISDGK (SEQ ID NO:2), HQSLGTQ (SEQ ID NO:11) or VISSHLGQ (SEQ ID NO:31) and the sequence PKNSSMISNTP (SEQ ID NO:3).

17. The modified polypeptide under item 16, characterized in that the rescue receptornegative epitope fused with interest the polypeptide.

18. A DNA molecule encoding a modified polypeptide according to any one of paragraphs.1-16.

19. A replicable vector containing the DNA molecule under item 18.

20. The method of obtaining mazany modified polypeptide nucleic acid, in culture medium and removing the specified modified polypeptide from the culture medium, cells or from the culture medium and cells.

21. A method of obtaining a modified polypeptide according to any one of paragraphs.1-15, including changing interest of the polypeptide in such a way that it contains rescue receptornegative epitope of the Fc-region of IgG has an increased half-life in vivo.

22. The method according to p. 21, characterized in that the phase change is performed using site-directed, cassette or PCR mutagenesis.

23. The method of obtaining modified polypeptide under item 1, including (a) identification of the sequence and conformation rescue receptornegative epitope of the Fc region of the IgG molecule, where the specified epitope derives from CH2domain Fc-region; b) a change in the sequence specified interest polypeptide, which is excreted through the kidneys and does not contain the Fc-region of IgG, to include the sequence and conformation of the identified binding epitope; (C) validation of the modified polypeptide from step (b) for longer half-life in vivo than the interest polypeptide; (g) if ismaningerstrasse interest of the polypeptide to further include the sequence and conformation of the identified binding epitope and check for the longer half-life in vivo as long until I get a longer half-life in vivo.

24. The method of treatment mediated by LFA-1 violations, characterized in that the mammal in need of treatment is administered an effective amount of a modified polypeptide according to any one of paragraphs.1-15.

25. The method of treatment mediated by LFA-1 disorders characterized by the fact that the patient in need of treatment is administered an effective amount of a modified polypeptide according to any one of paragraphs.1-15.

 

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