Enzymatic conjugation of polypeptides

Patent metadata

Field	Value
Publication number	US-9717803-B2
Application number	US-201213725382-A
Country	US
Kind code	B2
Filing date	Dec 21, 2012
Priority date	Dec 23, 2011
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

Abstract

The present application relates to methods for the functionalization of immunoglobulins, in particular with drugs. Also disclosed herein are linking reagents, functionalized antibodies, pharmaceutical compositions, and method of treating disease and/or conditions.

First claim

The invention claimed is:

1. A composition comprising a population of antibodies or antibody fragments, wherein each member of the population has the same primary amino acid sequence, and wherein at least 90% of the antibodies or antibody fragments in the composition have (m) functionalized acceptor glutamine residues (Q) per antibody or fragment, wherein m is an integer selected from 2 or 4, and wherein each of the functionalized acceptor glutamine residues has the structure of Formula IVb,
(Q)-NH-(C) n -X-L-(V—(Y-(M) z ) q ) r   Formula IVb

or a pharmaceutically acceptable salt or solvate thereof,
wherein:
Q is a glutamine residue present within or substituted into the primary amino acid sequence of a constant region of the antibodies or antibody fragments;
(C)n is a substituted alkyl chain, an unsubstituted alkyl chain, a substituted heteroalkyl chain, or an unsubstituted heteroalkyl chain;
n is an integer selected from among the range of 2 to 20;
X is NH, O, S, absent, or a bond;
L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 1 to 200 atoms substituted at one or more atoms;
r is an integer selected from among 1, 2, 3 or 4;
q is an integer selected from among 1, 2, 3 or 4;
z is an integer selected from among 1, 2, 3 or 4;
V is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety;
Y is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers;
M is (RR′)-L′-(V′—(Y′—(Z)z′)q′)r′, wherein
R is a reactive moiety;
(RR′) is an addition product between R and a complementary reactive moiety R′;
L′ is independently a carbon comprising framework of 1 to 200 atoms substituted at one or more atoms;
V′ is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety;
Y′ is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers;
Z is selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mytomycins, combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamycins, duocarmycins, tubulysines, amatoxins, dolastatins, auristatins, enediynes, pyrrolobenzodiazepines, and ethylenimines; and
z′, q′ and r′ are each independently an integer selected from among 1, 2, 3 or 4,
wherein the antibodies or antibody fragments specifically bind to a tumor antigen.

2. The composition of claim 1 , wherein RR′ is a thio-maleimide (or halo-acetamide) addition product, a Staudinger ligation product, a Huisgen 1,3-cycloaddition product, a Diels-Alder cycloaddition adduct, or any high yield selective amidation or imidization reaction product.

3. The composition of claim 1 , wherein said acceptor glutamine residue is flanked at the +2 position by a non-aspartic acid residue.

4. The composition of claim 1 , wherein L comprises a (CH2-CH2-O-)x group, wherein x is an integer from among the range of 1 to 24.

5. The composition of claim 1 , wherein the groups —(C)n-X-L- collectively comprise a structure (CH2-CH2-O-)x, wherein x is an integer from among the range of 3 to 24.

6. The composition of claim 1 , wherein m is 4.

7. A composition comprising a population of antibodies or antibody fragments each comprising at least one acceptor glutamine on each heavy chain, wherein at least 90% of the antibodies or antibody fragments in the composition comprise on each heavy chain two functionalized acceptor glutamine residues (Q) having the structure of Formula IVb of claim 1 and wherein each member of the population has the same primary amino acid sequence.

8. A composition comprising a population of antibodies or antibody fragments, wherein at least 90% of the antibodies or antibody fragments in the composition comprise on each heavy chain at least one functionalized acceptor glutamine residues having the structure of Formula IVb,
(Q)-NH—(C) n -X-L-(V—(Y-(M) z ) q ) r   Formula IVb

or a pharmaceutically acceptable salt or solvate thereof,
wherein:
Q is a glutamine residue present within or substituted into the primary amino acid sequence of a constant region of the antibodies or antibody fragments;
(C)n is a substituted alkyl chain, an unsubstituted alkyl chain, a substituted heteroalkyl chain, or an unsubstituted heteroalkyl chain;
n is an integer selected from among the range of 2 to 20;
X is NH, O, S, absent, or a bond;
L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 1 to 200 atoms substituted at one or more atoms;
r is an integer selected from among 1, 2, 3 or 4;
q is an integer selected from among 1, 2, 3 or 4;
z is an integer selected from among 1, 2, 3 or 4;
V is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety;
Y is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers;
M is (RR′)-L′-(V′—(Y′—(Z)z′)q′)r′, wherein
R is a reactive moiety;
(RR′) is an addition product between R and a complementary reactive moiety R′;
L′ is independently a carbon comprising framework of 1 to 200 atoms substituted at one or more atoms;
V′ is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety;
Y′ is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers;
Z is selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mytomycins, combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamycins, duocarmycins, tubulysines, amatoxins, dolastatins and auristatins, enediynes, pyrrolobenzodiazepines, and ethylenimines; and
z′, q′ and r′ are each independently an integer selected from among 1, 2, 3 or 4 wherein at least 90% of the antibodies or antibody fragments in the composition have (m) functionalized acceptor glutamine residues (Q) per antibody or fragment, wherein m is an integer selected from 2 or 4,
wherein the antibodies or antibody fragments specifically bind to a tumor antigen and wherein each member of the population has the same primary amino acid sequence.

9. A pharmaceutical formulation comprising the composition of claim 1 , and a pharmaceutically acceptable carrier.

10. A method of treating a disease comprising administering to a mammal a composition of claim 9 .

11. The composition of claim 1 , wherein Z is a pyrrolobenzodiazepine.

12. The composition of claim 1 , wherein R is a reagent capable of undergoing a Huisgen 1,3-cycloaddition.

13. The composition of claim 12 , wherein RR′ is 1,4-disubstituted-1,2,3-triazole.

14. The composition of claim 2 , wherein the thio-maleimide addition product is N,S-disubstituted-3-thio-pyrrolidine-2,5-dione.

15. The composition of claim 2 , wherein the Staudinger ligation product is N,3-substitued-5-dipenylphosphinoxide-benzoic amide or N,4-substitued-5-dipenylphosphinoxide-benzoic amide.

16. The composition of claim 2 , wherein the Huisgen 1,3-cycloaddition product is selected from the group consisting of N,S-disubstituted-3-thio-pyrrolidine-2,5-dione, 1,4-disubstituted-1,2,3-triazole, 3,5-disubstituted-isooxazole, and 3,5-disubstituted-tetrazol.

17. The composition of claim 2 , wherein the Diels-Alder cycloaddition adduct is the 2,4-cycloaddition product between a 9-substituted anthracene or 3-substituted 1,2,4,5-tetrazine with a compo

Assignees

• Innate Pharma
• Scherrer Inst Paul

Inventors

• Bregeon Delphine
• Dennler Patrick
• Belmant Christian
• Fischer Eliane
• Gauthier Laurent
• Romagné Francois
• Schibli Roger

Classifications

• C07C247/04

  being saturated · CPC title

• C12N9/1044

  Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII · CPC title

• A61K47/6817

  Toxins · CPC title

• C07K19/00

  Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} · CPC title

• C07C323/60

  with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms · CPC title