Modified glycoprotein, protein-conjugate and process for the preparation thereof

Patent metadata

Field	Value
Publication number	US-10072096-B2
Application number	US-201415029123-A
Country	US
Kind code	B2
Filing date	Oct 14, 2014
Priority date	Oct 14, 2013
Publication date	Sep 11, 2018
Grant date	Sep 11, 2018

Abstract

The invention relates to a glycoprotein comprising an optionally fucosylated glycan according to formula (105) or (106), wherein Su(A)x is a modified sugar moiety comprising one or more functional groups A. Functional group A is independently selected from the group consisting of a thiol group, a halogen, a sulfonyloxy group, a halogenated acetamido group, a mercaptoacetamido group and a sulfonated hydroxyacetamido group. The invention also relates to a glycoprotein-conjugate wherein a glycoprotein according to the invention is conjugated to a molecule of interest. Said molecule of interest may for example be an active substance. The invention further relates to a process for the preparation of a modified glycoprotein, and to a method for the preparation of a glycoprotein-conjugate. The invention particularly relates to modified antibodies, antibody-conjugates, antibody-drug conjugates and methods for the preparation thereof.

First claim

The invention claimed is:

1. A glycoprotein-conjugate comprising the formula:
Pr[glycan-Su[(Q) p -S—Z-L(D) r ] x ] y

wherein:
Pr represents a protein;
glycan comprises the formula (101A) or (102A):

wherein G is a monosaccharide, or a linear or branched oligosaccharide comprising 2 to 20 sugar moieties;
b is 0 or 1;
d is 0 or 1; and
e is 0 or 1;

Su is a sugar or a sugar derivative;
Q is —N(H)C(O)CH 2 — or —CH 2 —;
Z is a connecting group selected from -succinimide, —CH 2 —C(O)—N(R 9 )— and —C(═CH 2 )—CH 2 —C(O)—N(R 9 )—, wherein the carbon atom is connected to and S and the nitrogen atom to L;
wherein R 9 is selected from the group consisting of L(D) r , hydrogen, 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, wherein said 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 are optionally substituted;

L is a linker;
D is a molecule of interest;
p is 0 or 1;
r is 1 to 20;
x is 1, 2, 3, or 4; and
y is 1 to 20.

2. The glycoprotein-conjugate of claim 1 , wherein said glycoprotein conjugate comprises the formula (121) or (122):

wherein: Pr, L, D, r, x, y, b, d, e, p, Q, Su, and G are as defined in claim 1 .

3. The glycoprotein-conjugate of claim 1 , wherein said glycoprotein conjugate comprises the formula (123) or (124):

wherein: Pr, L, D, r, x, y, b, d, e, p, Q, Su, G, and R 9 are as defined in claim 1 .

4. The glycoprotein-conjugate of claim 1 , wherein said glycoprotein conjugate comprises the formula (125) or (126):

wherein: Pr, L, D, r, x, y, b, d, e, p, Q, Su, G, and R 9 are as defined in claim 1 .

5. The glycoprotein-conjugate of claim 1 , wherein said glycoprotein conjugate is an antibody-conjugate.

6. The antibody-conjugate of claim 5 , wherein said antibody-conjugate comprises a monoclonal antibody.

7. The antibody-conjugate of claim 5 , wherein D is a pharmaceutically active substance.

8. A pharmaceutical composition comprising the antibody conjugate of claim 5 and a pharmaceutically acceptable excipient.

9. A process for the preparation of the glycoprotein-conjugate of claim 1 , said process comprising:
contacting a glycoprotein comprising the formula Pr-[glycan] y with a nucleotide-sugar derivative in the presence of a catalyst to provide a modified glycoprotein comprising the formula Pr-[glycan-Su(A) x ] y ;
wherein Pr is as defined in claim 1 ,
wherein said glycan comprises the formula (101) or (102):

wherein G, b, d, and e are as defined in claim 1 ,
wherein said catalyst is selected from the group consisting of β(1,4)-galactosyltransferases, β(1,3)-N-galactosyltransferases, β(1,4)-galactosyltransferases comprising a mutant catalytic domain and β(1,3)-N-galactosyltransferases comprising a mutant catalytic domain,

wherein said nucleotide-sugar derivative comprises the formula Su(A) x )-P, wherein x is as defined in claim 1 ,
A is a functional group independently selected at each occurrence from the group consisting of a thiol group or a precursor thereof, a halogen, a sulfonyloxy group, a halogenated acetamido group, a mercaptoacetamido group and a sulfonated hydroxyl acetamido group, and
P is a nucleotide;

reacting said modified glycoprotein with a linker-conjugate comprising the formula B-L-(D) r ;
wherein B is a functional group capable of reacting with said functional group A; and L, D, and r are as defined in claim 1 ;

thereby producing said glycoprotein-conjugate of claim 1 .

10. The process of claim 9 , wherein said catalyst is selected from the group consisting of a β(1,4)-galactosyltransferases comprising a mutant catalytic domain and a β(1,3)-galactosyltransferases comprising a mutant catalytic domain.

11. The process of claim 9 , wherein said catalyst comprises a mutant catalytic domain from a β(1,4)-galactosyltransferase selected from the group consisting of bovine β(1,4)-Gal-T1 GaIT Y289L, GaIT Y289N, GaIT Y289I, GaIT Y289F, GaIT Y289M, GaIT Y289V, GaIT, Y289G and GaIT Y289A.

12. The process of claim 9 , wherein said glycoprotein-conjugate is an antibody-conjugate.

13. The process of claim 9 , further comprising providing a glycoprotein comprising a terminal GlcNAc-moiety at the non-reducing end.

14. The process of claim 9 , wherein:
(a) when said modified glycoprotein is a halogen-modified glycoprotein or a halogenated acetamido-modified glycoprotein, functional group B comprises a thiol group, an alcohol group or an amine group;
(b) when said modified glycoprotein is a thiol-modified glycoprotein or a mercaptoacetamido modified glycoprotein, functional group B comprises an N-maleimide group or a halogenate acetamido group or an alkene group; or
(c) when said modified glycoprotein is a sulfonyloxy-modified glycoprotein or a sulfonated hydroxyacetamido-modified glycoprotein, functional group B comprises a thiol group, an alcohol group or an amine group.

15. The process of claim 9 , wherein said glycoprotein is a thiol-modified glycoprotein or a mercaptoacetamido-modified glycoprotein, and functional group B comprises an alleneamide group.

Assignees

• Synaffix Bv

Inventors

• Van Delft Floris Louis
• Van Geel Remon
• Wijdeven Maria Antonia
• Verkade Jorge Merijn Mathieu
• Heesbeen Ryan

Classifications

• C07K16/40 Primary

  against enzymes · CPC title

• C07K2317/41

  Glycosylation, sialylation, or fucosylation · CPC title

• C07K16/32

  against translation products of oncogenes · CPC title

• C12P21/00 Primary

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12P21/005

  Glycopeptides, glycoproteins · CPC title