Site-specific antibody-drug glycoconjugates and methods

• US 10,905,678 B2
• Filed: 04/08/2015
• Issued: 02/02/2021
• Est. Priority Date: 04/08/2014
• Status: Active Grant

• Current Assignee: The University of Georgia Research Foundation, Inc.
• Original Assignee: The University of Georgia Research Foundation, Inc.
• Inventors: Boons, Geert-Jan
• Primary Examiner(s): Blumel, Benjamin P
• Application Number:
  US15/300,479
• Publication Number:
  US 20170173175A1
• Time in Patent Office: 2,127 Days
• Field of Search: None
• CPC Class Codes: A61K 31/4188 condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/6809 Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 49/0043 Fluorescein, used in vivo
• CPC Class Codes: A61K 49/0058 Antibodies
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

Compounds, compositions, and methods are provided for covalently linking an antibody or an antibody fragment to a cargo molecule, such as a therapeutic or a diagnostic agent, using a combination of enzymatic glycan remodeling and click chemistry. The method allows a cargo molecule to be selectively and efficiently attached post-translationally to an antibody or an antibody fragment. Also provided are antibody drug conjugates, methods of making, and uses thereof.

Claims

1. An antibody-drug conjugate thereof, comprising at least one N-linked oligosaccharide comprising at least one terminal sialoside, said terminal sialoside covalently linked to a cytotoxic drug, wherein said N-linked oligosaccharide is glycosylated to an IgG-class antibody through an asparagine residue in the heavy chain,

wherein the linker comprises a cycloaddition product between a 1,3 dipole and strained alkyne or alkene.

2. The antibody-drug conjugate according to claim 1, comprising 1, 2, 3 or 4 terminal sialosides, each covalently linked to a cytotoxic drug through a linker.

3. The antibody-drug conjugate according to claim 2, wherein the linker comprises a triazole.

4. The antibody-drug conjugate according to claim 1 wherein the N-linked oligosaccharide is a biantennary glycan, wherein both arms of the biantennary glycan comprise a terminal sialoside covalently linked to a cytotoxic drug.

5. The antibody-drug conjugate according to claim 1, wherein the cytotoxic drug is selected from methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, calicheamicin, and docetaxel.

6. The antibody-drug conjugate according to claim 2, wherein the linker comprises an acid sensitive group, redox active group, or protease cleavable group.

7. The antibody-drug conjugate according to claim 1, prepared by a process comprising:

conducting a cycloaddition reaction between;

a) an antibody comprising an N-linked oligosaccharide comprising a functionalized terminal sialoside, wherein the antibody is of the IgG class and the N-linked oligosaccharide is glycosylated to the antibody through an asparagine residue; and

b) a functionalized cytotoxic drug molecule,

wherein one of the functionalized terminal sialoside or functionalized cytotoxic drug molecule comprises a 1,3 dipole or 1,2,4,5 tetrazine, and the other comprises a strained cyclooctyne or trans cyclooctene.

8. The antibody-drug conjugate according to claim 7, wherein the 1,3 dipole is selected from azide, nitrone, nitrile oxide, diazo, acyl diazo, and azoxy.

9. The antibody-drug conjugate according to claim 7, wherein the 1,3 dipole is an azide.

10. The antibody-drug conjugate according to claim 7, wherein the cyclooctyne comprises a dibenzylcyclooctyne moiety.

11. The antibody-drug conjugate according to claim 7, wherein the N-linked oligosaccharide comprising a terminal sialoside is prepared by glycosylating a antibody comprising an N-linked oligosaccharide comprising a terminal galactose with a functionalized CMP-sialic acid donor in the presence of a sialyl transferase.

12. The antibody-drug conjugate according to claim 11, wherein functionalized CMP-sialic acid compound is selected from:

13. The antibody-drug conjugate according to claim 7, wherein the cytotoxic drug is selected from methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustards, Cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, calicheamicin, and docetaxel.

14. The antibody-drug conjugate according to claim 1, wherein the antibody is of the IgG1 subclass.

15. The antibody-drug conjugate according to claim 1, wherein the antibody is of the IgG2 subclass.

16. The antibody-drug conjugate according to claim 1, wherein the asparagine is a conserved asparagine at the 297 position in the heavy chain, numbered according to the Kabat system.