Novel hyaluronic acid hydrolase variants with improved stability and pharmaceutical compositions containing the same

Abstract

The present invention provides a novel PH20 mutant or section thereof, which is a human hyaluronidase, specifically wild-type PH20, and preferably has improved stability, enzyme activity, and expression rate compared to mature wild-type PH20. [Solution] The present invention provides a novel PH20 mutant or section thereof of human hyaluronidase, a hyaluronic acid hydrolase, in which the enzymatic activity and thermal stability have been improved. In the mutant having a specific amino acid sequence, one or more amino acid residues are substituted, and additional N-terminal and / or C-terminal amino acid residues are selectively deleted.

Claims

1. A PH20 mutant or a section thereof, characterized in that a mutant having the amino acid sequence of SEQ ID NO: 3 includes substitution, insertion, or deletion of one or more amino acid residues, and has a higher aggregation temperature compared to wild-type PH20.

2. The PH20 mutant or section thereof according to claim 1, characterized in that the mutant having the amino acid sequence of SEQ ID NO: 3 includes substitution of amino acid residues at one or more positions selected from the group consisting of R39, D65-L68, N82, T84, I102-I105, T132-Y134, N166, L179-T182, T185-K187, V241-K244, N266-Q269, P271, V272, K290-P292, Q311-K314, G340-N363, L441, S442, D451-D453, D461, V463 and D461-V463.

3. The substitutions of the aforementioned amino acid residues are R39K, D65A, E66A, P67A, L68A, N82A, T84N, I102A, D103A, S104A, S104N, I105A, I105Q, T132A, T132S, F133A, Y134A, N166A, N166K, L179A, L179S, L179I, L179F, S180T, S180A, L181A, L181M, T182A, T185A, E186A, E186D, K187A, V241A, E242A, I243A, K244A, N266A, T267A, Q268A, Q268D, Q26 8I, Q268N, Q269A, P271A, V272A, K290A, I291A, I291G, I291L, P292A, P292D , Q311A, V312A, L313A, L313P, L313M, K314A, G340Q, S341H, S341D, S341T, W 342I, W342D, W342H, W342L, E343V, E343S, E343Y, E343Q, N344F, N344I, T34 5E, T345K, T345S, R346M, R346F, R346L, R346T, R346S, R346A, T347Q, T347E, T347V, T347W, T347H, T347S, K348Q, K348F, K348D, K348T, K348E, K348M, E3 49L, E349W, E349A, S350Q, S350I, S350D, S350T, S350E, S350N, Q352E, Q352 G, Q352Y, Q352W, Q352T, A353E, A353Y, A353H, A353K, I354E, I354Q, I354S, I354V, I354A, I354N, I354T, I354R, I354W, I354L, K355Q, K355H, K355D, E35 6M, E356F, E356I, E356L, E356Q, E356V, E356D, Y357W, Y357F, M358V, M358R , M358Y, M358L, D359K, D359V, D359Y, D359Q, D359T, D359S, D359E, T360Y, T 360R, T360L, T360D, T360S, T361M, T361E, T361H, T361L, T361D, T361I, L36 2A, N363M, N363E, L441A, S442A, D451A, D451S, T452A, T452D, T452H, T452K,A PH20 mutant or section thereof according to claim 2, characterized in that it is one or more selected from the group consisting of T452G, T452P, T452M, T452F, D453A, D461R, D461A, G462A, V463Y, and V463A.

4. Furthermore, the PH20 mutant or section thereof according to any one of claims 1 to 3, characterized in that some amino acid residues at the C-terminus and / or N-terminus are deleted.

5. The PH20 mutant or section thereof according to claim 4, characterized in that some amino acid residues are deleted by being cleaved before an amino acid residue selected from the group consisting of M1 to P42 at the N-terminus.

6. The PH20 mutant or section thereof according to claim 5, characterized in that some amino acid residues are deleted by cleavage before the N-terminal L36, N37, F38, R39, A40, P41, or P42 residue.

7. The PH20 mutant or section thereof according to claim 4, characterized in that it is cleaved after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus, resulting in the deletion of some amino acid residues.

8. The PH20 mutant or section thereof according to claim 7, characterized in that it is cleaved after an amino acid residue selected from the group consisting of V455 to S490 at the C-terminus, resulting in the deletion of some amino acid residues.

9. The PH20 mutant or section thereof according to claim 7, characterized in that it is cleaved after the C-terminal residues V455, D456, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488, or S490, resulting in the deletion of some amino acid residues.

10. The PH20 mutant or section thereof according to claim 4, characterized in that some amino acid residues are deleted by cleavage before the N-terminal F38 residue and after the C-terminal F468 residue.

11. A PH20 mutant or section thereof according to any one of claims 1 to 10, characterized by being selected from the group consisting of amino acid sequences of SEQ ID NOs: 163 to 316.

12. A cancer treatment composition comprising a PH20 mutant or a section thereof according to any one of claims 1 to 11.

13. The cancer treatment composition according to claim 12, characterized in that it is used in combination therapy with other anticancer drugs.

14. The cancer treatment composition according to claim 13, characterized in that the other anticancer agent is an immunosuppressant.

15. The cancer treatment composition according to claim 14, characterized in that the immunosuppressant is an immune checkpoint inhibitor.

16. A nucleic acid encoding a PH20 variant or a section thereof according to any one of claims 1 to 11.

17. A recombinant expression vector comprising the nucleic acid described in claim 16.

18. A host cell transformed with the recombinant expression vector according to claim 17.

19. A method for producing a PH20 mutant or a section thereof, comprising the step of culturing the host cells described in claim 18.

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