Modified human chorionic gonadotropin (-hCG) proteins and their medical use

The present invention relates to modified human chorionic gonadotropin (-hCG) proteins and their medical use as immunological contragestatives. The modification causes a reduction in the cross-reactivity of the modified -hCG protein with luteinizing hormone (LH) as defined by the ability of both proteins to react with the same antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically a modified -hCG protein in which an epitope which cross-reacts with LH is modified; more particularly an epitope deletion mutant which has lost epitope (▪) cross-reacting with -LH but still retains -hCG specific epitope ().

FIG. 2 comprises FIGS. 2A-2D, and shows the fluorescence of cell transfected with -hCG mutant 6 determined by Facscan:

2A-control antibody;

2B-stained by anti-C terminal epitope;

2C-stained by anti-hCG-specific

1 epitope;

2D-stained with antibody to LH cross-reacting

3 epitope showing loss of staining;

FIG. 3 shows the relative spatial distribution of the -hCG epitope clusters;

FIG. 4 shows the results of the binding of Mabs to different mutants; and,

FIG. 5 shows the amino acid substitutions made in -hCG.

1. A modified -hCG protein, the protein amino acid sequence being modified by recombinant means by one or more amino acid substitutions selected from the group consisting of 20 (Lys) to Asn, 21 (Glu) to Arg and 22 (Gly) to Glu; 24 (Pro) to His; 25 (Val) to Tyr; 68 (Arg) to Glu; 74 (Arg) to Ser; 75 (Gly) to His; 79 (Val) to His; and 71 (Gly) to Arg and 74 (Arg) to Ser; so as to reduce the cross-reactivity of the modified -hCG protein with LH as defined by the ability of both proteins to react with the same antibody, wherein the modified -hCG protein retains one or more conformational epitopes specific to the native -hCG.

2. The modified -hCG protein according to claim 1 being modified by at least amino acid substitution 68 (Arg) to Glu.

3. The modified -hCG protein according to claim 1 being modified by at least amino acid substitution 74 (Arg) to Ser.

4. The modified -hCG protein according to claim 1 being modified by at least amino acid substitution 20 (Lys) to Asn, 21 (Glu) to Arg and 22 (Gly) to Glu.

5. The modified -hCG protein according to claim 1 being modified by at least amino acid substitution 24 (Pro) to His.

6. The modified -hCG protein according to claim 1 being modified by at least amino acid substitution 25 (Val) to Tyr.

7. The modified -hCG protein according to claim 1 being modified by at least amino acid substitution 71 (Gly) to Arg, and 74 (Arg) to Ser.

8. The modified -hCG protein according to claim 1 being modified by at least amino acid substitution 75 (Gly) to His.

9. The modified -hCG protein according to claim 1 being modified by at least amino acid substitution 79 (Val) to His.

10. The modified -hCG protein according to claim 1 wherein the protein amino acid sequence is modified by point mutation.

11. The modified -hCG protein according to claim 1 wherein the modified -hCG protein is chemically linked by a chemical linkage to an immunogenic substance.

12. The modified -HCG protein according to claim 11 wherein the chemical linkage is by co-expression as a fusion protein.

13. An isolated and purified nucleic acid sequence encoding a modified -hCG protein according to claim 1.

14. The isolated and purified nucleic acid according to claim 13 further encoding a fusion protein comprising an immunogenic carrier protein fused to the modified -hCG protein.

15. An expression vector comprising a nucleic acid according to claim 13.

16. An expression vector comprising a nucleic acid according to claim 14.

17. A mammalian host cell comprising a vector according to claim 15.

18. A mammalian host cell comprising a vector according to claim 16.

19. A mammalian host cell comprising nucleic acid according to claim 13.

20. A mammalian host cell comprising nucleic acid according to claim 14.

21. A microbial host cell comprising a vector according to claim 15.

22. A microbial host cell comprising a vector according to claim 16.

23. A microbial host cell comprising nucleic acid according to claim 13.

24. A microbial host cell comprising nucleic acid according to claim 14.

25. A composition comprising a modified -hCG protein according to claim 1 and a pharmaceutically acceptable carrier.

26. A composition comprising a nucleic acid according to claim 13 and a pharmaceutically acceptable carrier.

27. A composition comprising a nucleic acid according to claim 14 and a pharmaceutically acceptable carrier.

28. A composition comprising an expression vector according to claim 15 and a pharmaceutically acceptable carrier.

29. A composition comprising an expression vector according to claim 16 and a pharmaceutically acceptable carrier.

30. A contragestative composition, comprising a modified -hCG protein according to claim 1 and a pharmaceutically acceptable carrier.

31. A modified -hCG protein having a contragestative function in a female mammal, wherein the modified -hCG protein has an amino acid sequence that is modified by recombinant means by one or more amino acid substitutions selected from the group consisting of 20 (Lys) to Asn, 21 (Glu) to Arg and 22 (Gly) to Glu; 24 (Pro) to His; 25 (Val) to Tyr; 68 (Arg) to Glu; 74 (Arg) to Ser; 75 (Gly) to His; 79 (Val) to His; and 71 (Gly) to Arg and 74 (Arg) to Ser; so as to reduce the cross-reactivity of the modified -hCG protein with LH as defined by the ability of both proteins to react with the same antibody, wherein the modified -hCG protein retains one or more conformational epitopes specific to the native -hCG.

32. A modified -hCG protein according to claim 31 being modified by at least an amino acid substitution 68 (Arg) to Glu.

33. A modified -hCG protein according to claim 31 being modified by at least an amino acid substitution 74 (Arg) to Ser.