Process for the modification of a glycoprotein using a glycosyltransferase that is or is derived from A β(1,4)-N-acetylgalactosaminyltransferase

Patent metadata

Field	Value
Publication number	US-9988661-B2
Application number	US-201615318248-A
Country	US
Kind code	B2
Filing date	Apr 25, 2016
Priority date	Apr 23, 2015
Publication date	Jun 5, 2018
Grant date	Jun 5, 2018

Abstract

The present invention relates to a process for the enzymatic modification of a glycoprotein. The process comprises the step of contacting a glycoprotein comprising a glycan comprising a terminal GlcNAc-moiety, in the presence of glycosyltransferase that is, or is derived from, a β-(1,4)-N-acetylgalactosaminyltransferase, with a non-natural sugar-derivative nucleotide. The non-natural sugar-derivative nucleotide is according to formula (3): wherein A is selected from the group consisting of —N 3 , —C(O)R 3 , —(CH 2 ) i C≡C—R 4 , —SH, —SC(O)R 8 , —SC(O)OR8, —SC(S)OR8, —F, —CI, —Br —I, —OS(O) 2 R 5 , terminal C 2 -C 24 alkenyl groups, C 3 -C 5 cycloalkenyl groups, C 4 -C 8 alkadienyl groups, terminal C 3 -C 24 allenyl groups and amino groups. The invention further relates to a glycoprotein obtainable by the process according to the invention, to a bioconjugate that can be obtained by conjugating the glycoprotein with a linker-conjugate, and to β-(1,4)-N-acetylgalactosaminyltransferases that can be used in preparing the the glycoprotein according to the invention.

First claim

The invention claimed is:

1. A process for the modification of a glycoprotein, the process comprising:
contacting a glycoprotein comprising a glycan comprising a terminal GlcNAc moiety, with a sugar-derivative nucleotide Su(A)-Nuc in the presence of a glycosyltransferase,
wherein:
(i) the glycosyltransferase is, or is derived from, Ascaris summ β-(1,4)-N-acetylgalactosaminytransferase or Trichoplusia ni β-(1,4)-N-acetylgalactosaminyltransferase;
(ii) the glycan, comprising a terminal GlcNAc-moiety, is according to formula (1) and optionally (2)

wherein:
b is 0 or 1;
d is 0 or 1;
e is 0 or 1; and
G is a monosaccharide, or a linear or branched oligosaccharide comprising 2 to 20 sugar moieties; and
(iii) the sugar-derivative nucleotide Su(A)-Nuc is according to formula (3):

wherein:
a is 0 or 1;
f is 0 or 1;
g is 0 or 1;
Nuc is a nucleotide;
U is [C(R 1 ) 2 ]n or [C(R 1 ) 2 ] p —O—[C(R 1 ) 2 C(R 1 ) 2 O] o —[C(R 1 ) 2 ] q , wherein n is an integer in the range of 1 to 24; o is an integer in the range of 0 to 12; p and q are independently 0, 1 or 2; and R 1 is independently selected from the group consisting of H, F, Cl, Br, I, OH and an unsubstituted or substituted C 1 -C 24 alkyl group;
T is a C 3 -C 12 (hetero)arylene group, wherein the (hetero)arylene group is optionally substituted;
A is selected from the group consisting of:
(a) —N 3
(b) —C(O)R 3
wherein R 3 is an unsubstituted or substituted C 1 -C 24 alkyl group;

(c) (hetero)cycloalkynyl group or a (CH 2 ) i C≡C—R 4 moiety
wherein i is 0-10 and R 4 is hydrogen or an unsubstituted or substituted C 1 -C 24 alkyl group;

(d) —SH
(e) —SC(O)R 8
wherein R 8 is an, unsubstituted or substituted, C 1 -C 24 alkyl group or phenyl group;

(f) —SC(V)OR 8
wherein V is O or S, and R 8 is an, unsubstituted or substituted, C 1 -C 24 alkyl group or phenyl group;

(g) —X
wherein X is selected from the group consisting of F, Cl, Br and I;

(h) —OS(O) 2 R 5
wherein R 5 is selected from the group consisting of C 1 -C 24 alkyl groups, C 6 -C 24 aryl groups, C 7 -C 24 alkylaryl groups and C 7 -C 24 arylalkyl groups, groups, aryl groups, alkylaryl groups and arylalkyl groups being optionally substituted;

(i) R 12
wherein R 12 is selected from the group consisting of, unsubstituted or substituted, terminal C 2 -C 24 alkenyl groups, C 3 -C 5 cycloalkenyl groups and C 4 -C 8 alkadienyl groups;

(j) R 13
wherein R 13 is unsubstituted or substituted terminal C 3 -C 24 allenyl group; and

(k) N(R′) 2

wherein R 17 is independently selected from the group consisting of H and C 1 -C 12 alkyl groups;
Z is CH 2 , CF 2 or C(O); or Z is CHOH with the proviso that g is 0, f is 1 and a is 0 or 1;
Y is selected from the group consisting of O, S, N(R 15 ), N(R 15 )C(O), N(R 15 )C(O)N(R 15 ), N(R 15 )C(O)O, OC(O)N(R 15 )S(O) 2 N(R 15 ) and N(R 15 )C(O)N(R 15 )S(O) 2 O, wherein R 15 is independently selected from the group consisting of H, C 1 -C 12 alkyl groups and (U) f -(T) a -A wherein f, a, U, T and A are as defined above; and
R 14 is selected from the group consisting of:

wherein:
a, f, T, A and U are as defined above;
h is 0 or 1; and
W is selected from the group consisting of O, S, NR 15 , NHS(O) 2 O and NHS(O) 2 NR 15 , wherein R 15 is as defined above.

2. The process according to claim 1 , wherein the β-(1,4)-N-acetylgalactosaminyltransferase is Trichoplusia ni β-(1,4)-N-acetylgalactosaminyltransferase.

3. The process according to claim 1 , wherein A is selected from the group consisting of —N 3 , —C(O)CH 3 , —SH, —CH═CH 2 and —CH 2 C≡CH.

4. The process according to claim 1 , wherein the sugar-derivative nucleotide Su(A)-Nuc has formula (15), (16), (17) or (18):

5. The process according to claim 1 , wherein sugar-derivative nucleotide Su(A)-Nuc has formula (19), (20), (21), (22), (23), (24), (25), (26), (65), (66), (67), (68) or (69):

wherein:
R 14 and R 15 are as defined in claim; and
R 16 is independently selected from the group consisting of H and F.

6. The process according to claim 1 , wherein the sugar-derivative nucleotide Su(A)-Nuc has formula (27):

7. The process according to claim 1 , wherein the nucleotide in sugar-derivative nucleotide Su(A)-Nuc is uridine diphosphate (UDP).

8. The process according to claim 1 , wherein the glycan, comprising a terminal GlcNAc moiety, has formula (1), (9), (10) or (11):

9. The process according to claim 1 , wherein the glycoprotein comprising a glycan, comprising a terminal GlcNAc moiety, is according to B formula (7) or (8b):

wherein:
b, d, e and G are as defined in claim 1 ;
y is independently an integer in the range of 1 to 24; and
Pr is a protein.

10. The process according to claim 1 , wherein the glycoprotein, comprising a glycan comprising a terminal GlcNAc moiety, is an antibody.

11. The process according to claim 1 , wherein R 14 is —NHC(O)Me.

Assignees

• Synaffix Bv

Inventors

• Van Berkel Sander Sebastiaan
• Van Geel Remon
• Wijdeven Maria Antonia
• Van Delft Floris Louis

Classifications

• C07K2317/41

  Glycosylation, sialylation, or fucosylation · CPC title

• C07K16/32

  against translation products of oncogenes · CPC title

• A61K47/48561

  Human Necessities · mapped topic

• C12Y204/01092

  (N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase (2.4.1.92) · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title