Endos mutant enzyme

Patent metadata

Field	Value
Publication number	US-11001819-B2
Application number	US-201615745357-A
Country	US
Kind code	B2
Filing date	Jul 15, 2016
Priority date	Jul 16, 2015
Publication date	May 11, 2021
Grant date	May 11, 2021

Abstract

The present invention provides an EndoS mutant enzyme having an amino acid sequence of EndoS D233Q and further having a particular additional mutation and exhibiting a reduced hydrolysis activity, in comparison with the activity of EndoS D233Q, to an N-linked sugar chain (N297-linked sugar chain) linked to Asn at position 297 in IgG and a gene encoding the same.

First claim

The invention claimed is:

1. An EndoS mutant enzyme comprising the amino acid sequence of amino acid numbers 37 to 995 of the amino acid sequence of SEQ ID NO: 2 with a mutation only at an amino acid position selected from the group consisting of: amino acid 122, amino acid 184, amino acid 279, amino acid 282, amino acid 303, amino acid 348, amino acid 350, amino acid 402, amino acid 405, amino acid 406, and a combination thereof, wherein said EndoS mutant enzyme exhibits a reduced hydrolysis activity in comparison with the hydrolysis activity of an EndoS D233Q mutant enzyme, the activity being an activity on an N-linked sugar chain (N297 linked sugar chain) linked to Asn at position 297 in IgG.

2. The EndoS mutant enzyme according to claim 1 , wherein the mutation is at 1 to 4 amino acid positions selected from the group consisting of: 122, 184, 279, 282, 303, 348, 350, 402, 405, and 406.

3. The EndoS mutant enzyme according to claim 1 , wherein the mutation is a mutation at one amino acid position selected from the group consisting of 303, 350, and 405.

4. The EndoS mutant enzyme according to claim 1 , wherein the mutation is at least one selected from the group consisting of:
a mutation at 122 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro, Ala, Glu, Asp, Leu, Ile, Met, or Phe;
a mutation at 184 to the amino acids: Gly, Cys, Thr, Ser, Val, Ala, Gln, or Asn;
a mutation at 279 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro, Ala, or Gln;
a mutation at 282 to the amino acids: Arg, Lys, or His;
a mutation at 303 to the amino acids: Met, Pro, or Leu;
a mutation at 348 to the amino acids: His or Trp;
a mutation at 350 to the amino acids: Lys, Arg, His, Tyr, Gln, Asn, Asp, Gly, Cys, Thr, Ser, Val, Pro, or Ala;
a mutation at 402 to the amino acids: Phe or Trp;
a mutation at 405 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro, or Ala; and
a mutation at 406 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro, Ala, Glu, Asp, Gln, or
Asn.

5. The EndoS mutant enzyme according to claim 4 , wherein the mutation is at least one selected from the group consisting of:
a mutation at H122 to Ala (H122A) or Phe (H122F);
a mutation at P184 to Gln (P184Q);
a mutation at D279 to Ser (D279S) or Gln (D2790);
a mutation at Y282 to Arg (Y282R);
a mutation at 0303 to Leu (0303L);
a mutation at Y348 to His (Y348H);
a mutation at E350 to Ala (E350A), Asn (E350N), Asp (E350D), or Gln (E3500);
a mutation at Y402 to Phe (Y402F) or Trp (Y402W);
a mutation at D405 to Ala (D405A); and
a mutation at R406 to Gln (R4060).

6. The EndoS mutant enzyme according to claim 5 , comprising at least one mutation selected from the group consisting of 0303L, E350A, E35N, E350D, E350Q, and D405A.

7. The EndoS mutant enzyme according to claim 6 , wherein the mutation is 0303L, E350A, E350N, E350D, E350Q, D405A, H122A/0303L, H122F/0303L, P184Q/0303L, D279Q/0303L, D279S/0303L, Y282R/0303L, 0303L/Y348H, 0303L/E350A, 0303L/E350N, 0303L/E350D, 0303L/E3500, 0303L/Y402F, 0303L/Y402W, 0303L/D405A, 0303L/R4060, H122A/E350A, H122F/E350A, P1840/E350A, D279Q/E350A, D279S/E350A, Y282R/E350A, Y348HZE350A, E350A/Y402F, E350A/Y402W, E350A/D405A, E350A/R4060, H122A/E350N, H122F/E350N, P1840/E350N, D2790/E350N, D279S/E350N, Y282R/E350N, Y348H/E350N, E350N/Y402F, E350N/Y402W, E350N/D405A, E350N/R4060, H122A/E350D, H122F/E350D, P184Q/E350D, D2790/E350D, D279S/E350D, Y282R/E350D, Y348H/E350D, E350D/Y402F, E350D/Y402W, E350D/D405A, E350D/R4060, H122A/E350Q, H122F/E350Q, P1840/E350Q, D279Q/E350Q, D279S/E3500, Y282R/E350Q, E3500/Y348H, E3500/Y402F, E3500/Y402W, E3500/D405A, E350Q/R4060, H122A/D405A, H122F/D405A, P1840/D405A, D279Q/D405A, D279S/D405A, Y282R/D405A, Y348H/D40 5A, or D405A/R4060.

8. The EndoS mutant enzyme according to claim 7 , wherein the mutation is 0303L, E350A, E350N, E350D, E350Q, D405A, 0303L/E350A, 0303L/E350N, 0303L/E350D, 0303L/E35010, 0303L/D405A, E350A/D405A, E350N/D405A, E350D/D405A, or E3500/D405A.

9. The EndoS mutant enzyme according to claim 4 , wherein the mutation is H122A, H122F, P1840, D2790, D279S, Y282R, Y348H, Y402F, Y402W, or R4060.

10. A polynucleotide encoding the EndoS mutant enzyme according to claim 1 .

11. A vector comprising a nucleotide complementary to the polynucleotide according to claim 10 .

12. A host cell transformed with the vector according to claim 11 .

13. A method of production of a molecule comprising a sugar-remodeled Fc region, comprising reacting a molecule comprising an Fc region of IgG having a core GlcNAc optionally with fucose addition as an N297-linked sugar chain with a sugar donor having a structure comprising oxazolinated GlcNAc in the presence of the EndoS mutant enzyme according to claim 1 to produce a molecule comprising an Fc region with a N297-linked sugar chain having a structure in which a sugar chain possessed by the sugar donor is transferred to the core GlcNAc of the N297-linked sugar chain.

14. The method of production according to claim 13 , wherein the molecule comprising an Fc region is an IgG monoclonal antibody.

Assignees

• Daiichi Sankyo Co Ltd

Inventors

• KAWAGUCHI Yoshirou
• Nakamura Kensuke
• Sekiguchi Yukiko
• Iwamoto Mitsuhiro

Classifications

• C12N15/02

  Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion {(monoclonal antibodies C07K16/00; apparatus for cell fusion C12M)} · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N9/2402 Primary

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• C07K16/32

  against translation products of oncogenes · CPC title