Site-specific covalent chemical ligation to monoclonal and polyclonal immunoglobulin

Patent metadata

Field	Value
Publication number	US-10588982-B2
Application number	US-201715727490-A
Country	US
Kind code	B2
Filing date	Oct 6, 2017
Priority date	Apr 8, 2015
Publication date	Mar 17, 2020
Grant date	Mar 17, 2020

Abstract

Methods and compositions are described herein for covalently linking an antibody to a molecular payload. Compositions are described herein containing an antibody covalently linked to a molecular payload.

First claim

What is claimed is:

1. A compound comprising:
i) a targeting moiety that specifically binds a nucleotide binding pocket of an antibody;
ii) a cross-linking agent;
iii) an active agent or a conjugating agent; and
iv) a linker, wherein the linter comprises an amino acid sequence, and the amino acid sequence comprises negatively charged amino acids, and wherein the linker covalently links:
a) the targeting moiety,
b) the cross-linking agent, and
c) the active agent or conjugating agent.

2. The compound of claim 1 , wherein the amino acid sequence has affinity for a nucleotide binding pocket of an antibody.

3. The compound of claim 1 , wherein the linker comprises: i) an amino acid sequence; and ii) an ethylene glycol dimer or a PEG polymer.

4. The compound of claim 3 , wherein the linker comprises a lysine-aspartate-serine amino acid sequence and the ethylene glycol dimer.

5. The compound of claim 4 , wherein the compound has a formula of:

wherein,
R 1 comprises the targeting moiety that specifically binds to the nucleotide binding pocket of an antibody;
R 2 comprises the cross-linking agent; and
R 3 comprises the active agent or conjugating agent.

6. The compound of claim 1 , wherein the targeting moiety that specifically binds a nucleotide binding pocket of an antibody comprises a purine or a purine analogue.

7. The compound of claim 6 , wherein the purine or purine analogue comprises an indole.

8. The compound of claim 6 , wherein the purine or purine analogue is indole-3-butyrate.

9. The compound of claim 1 , wherein the compound comprises a formula of:

wherein L 1 and L 2 are independently a C 1 -C 10 alkylene, and R 4 is represented by the following formula:

wherein R 5 is an amino acid side chain, m and n are independently from 1 to 10, and R 3 comprises the active agent or conjugating agent.

10. The compound of claim 1 , wherein the compound comprises a formula of:

wherein L 1 and L 2 are independently a C 1 -C 10 alkylene, and R 4 is represented by the following formula:

wherein R 5 is an amino acid side chain, m and n are independently from 1 to 10, and R 3 comprises the active agent or conjugating agent.

11. The compound of claim 9 , wherein m is at least 2,and the at least two amino acid side chains comprise a dipeptide affinity element that increases the affinity of the targeting moiety for the nucleotide binding pocket of the antibody.

12. The compound of claim 9 , wherein the compound has a formula of:

wherein R 3 is the active agent or conjugating agent.

13. The compound of claim 10 , wherein the compound has a formula of:

wherein R 3 is the active agent or conjugating agent.

14. The compound of claim 1 , wherein the compound comprises a formula of:

15. The compound of claim 1 , wherein the compound comprises a formula selected from the group consisting of:

16. A method for covalently conjugating an antibody to a molecular payload, wherein the molecular payload comprises a compound of claim 1 , the method comprising:
a) forming a reaction mixture comprising the antibody and the molecular payload under conditions suitable to form a non-covalent binding interaction between a nucleotide binding pocket of the antibody and a targeting moiety of the molecular payload, wherein the reaction mixture is an aqueous solution having a pH of less than about 7.5; and
b) raising the pH of the reaction mixture above about 8.0, under conditions suitable to form a covalent bond between the antibody and the cross-linking agent.

17. The method of claim 16 , wherein the method comprises
a) forming the reaction mixture comprising the antibody, a 5-fold molar excess of the compound of claim 12 relative to nucleotide binding pockets of the antibody, and PBS 7.0 or PBS 7.5;
b) incubating the reaction mixture of a) for at least about 0.25 h at a temperature of from about 4° C. to about 37° C.;
c) removing unbound compound of claim 12 by dialysis or size exclusion chromatography;
d) raising the pH of the reaction mixture to about 8.5, under conditions suitable to form a covalent bond between the antibody and the compound of claim 12 by adding a basic solution comprising 0.1 M sodium bicarbonate pH 8.5, 0.1 N NaOH or 0.1 N NH 4 OH; and
e) incubating the reaction mixture of d) for at least about 0.25 h at a temperature of from about 4° C. to about 37° C.

18. The method of claim 17 , wherein R 3 of the compound of claim 17 comprises a conjugating agent comprising an alkyne or azide and the method further comprises:
f) introducing into the reaction mixture a copper (I) catalyst and an active agent comprising an azide or alkyne that is reactive to the alkyne or azide of the conjugating agent, thereby conjugating the active agent to the conjugating agent.

19. The method of claim 17 , wherein R 3 of the compound of claim 17 comprises a conjugating agent comprising a 1,2-dihydroxybenzene moiety, and the method further comprises:
f) introducing into the reaction mixture an active agent comprising a boronic acid moiety that is reactive to the 1,2-dihydroxybenzene moiety of the conjugating agent, thereby conjugating the active agent to the conjugating agent.

20. A compound of claim 1 comprising the following formula:

wherein R 6 is an antibody and R 3 comprises an active agent or conjugating agent.

21. A compound of claim 1 comprising the following formula:

wherein R 6 is an antibody and R 3 comprises an active agent or conjugating agent.

Assignees

• Univ California

Inventors

• Lac Diana
• Feng Chun
• Bhardwaj Gaurav
• Chandrasekaran Siddarth
• Lam Kit S

Classifications

• A61K47/6867

  the tumour determinant being from a cell of a blood cancer · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61K47/6855

  the tumour determinant being from breast cancer cell · CPC title

• C07D403/12

  linked by a chain containing hetero atoms as chain links · CPC title

• A61K47/6849

  the antibody targeting a receptor, a cell surface antigen or a cell surface determinant · CPC title