Radiolabeling of polypeptides

Patent metadata

Field	Value
Publication number	US-12240790-B2
Application number	US-201816955094-A
Country	US
Kind code	B2
Filing date	Dec 17, 2018
Priority date	Dec 18, 2017
Publication date	Mar 4, 2025
Grant date	Mar 4, 2025

Abstract

Improved methods of radiolabeling antibodies using click chemistry are described. Also described are pharmaceutical compositions and uses related to the radiolabeled antibodies produced by the methods.

First claim

We claim:

1. A method of labeling a polypeptide with a radiometal ion, the method comprising:
a. providing an antibody or an antigen binding fragment thereof that is covalently linked to a first click reaction partner, wherein the first click reaction partner comprises an azide, and wherein the antibody or antigen binding fragment thereof that is covalently linked to a first click reaction partner is obtained by a site-specific incorporation of the first click reaction partner through a reaction with an azide-labeled sugar or an azido amine;
b. providing a radiocomplex comprising the radiometal ion and a chelating moiety coordinated with the radiometal ion, wherein the chelating moiety comprises a chelant covalently linked to a second click reaction partner, wherein the second click reaction partner comprises a strained alkyne group; and
c. contacting the antibody or antigen binding fragment thereof that is covalently linked to a first click reaction partner with the radiocomplex, wherein the contacting is performed without a copper catalyst, to allow the first click reaction partner to react with the second click reaction partner to thereby label the antibody or an antigen binding fragment thereof with the radiometal ion;
wherein the chelant comprises a macrocycle having the structure of formula (I):

wherein each of R 1 , R 2 , R 3 and R 4 is independently CHQCO 2 X, wherein
Q is independently hydrogen, C 1 -C 4 alkyl or (C 1 -C 2 alkyl) phenyl, and
X is independently hydrogen, benzyl, C 1 -C 4 alkyl; and
Z is (CH 2 ) n Y, wherein
n is 1-10, and
Y is a linker that covalently links the second click reaction partner and the chelant, and wherein Y is a bond, C(O)—(CH 2 ) m —C(O), (CH 2 ) p —C(O)—NH—(CH 2 ) q —C(O), (O—CH 2 —CH 2 ) r —O, (CH 2 ) p [C(O)—NH—(CH 2 ) q ] t , (CH 2 ) p —S—S—(CH 2 ) q , or valine-citrulline-PAB, wherein p=0-4, q=1-4, r=1-4, t=1-3, m=1-4;

alternatively, Z is hydrogen; and, when Z is hydrogen then
each of R 1 , R 2 , R 3 and R 4 is independently CHQCO 2 X, wherein
Q is a linker that covalently links the second click reaction partner and the chelant, and
wherein Q is a bond, C(O)—(CH 2 ) m —C(O), (CH 2 ) p —C(O)—NH—(CH 2 ) q —C(O), (O—CH 2 —CH 2 ) r —O, (CH 2 ) p [C(O)—NH—(CH 2 ) q ] t , (CH 2 ) p —S—S—(CH 2 ) q , or valine-citrulline-PAB,
wherein p=0-4, q=1-4, r=1-4, t=1-3, and
X is independently hydrogen, benzyl, or C 1 -C 4 alkyl;

or alternatively, the chelant comprises deferoxamine-YY, wherein YY is a linker that covalently links the second click reaction partner and the chelant, and wherein YY is a bond, C(O)—(CH 2 ) m —C(O), (CH 2 ) p —C(O)—NH—(CH 2 ) q —C(O), (O—CH 2 —CH 2 ) r —O, (CH 2 ) p [C(O)—NH—(CH 2 ) q ] t , (CH 2 ) p —S—S—(CH 2 ) q , or valine-citrulline-PAB, wherein p=0-4, q=1-4, r=1-4, t=1-3.

2. The method of claim 1 , wherein the antibody is an antibody that binds to human prostate-specific membrane antigen (PSMA) or an antigen binding fragment thereof, comprising a heavy chain (HC) complementarity-determining region(CDR)1 sequence of SEQ ID NO: 3, a HC CDR2 sequence of SEQ ID NO: 4, a HC CDR3 sequence of SEQ ID NO: 5, a light chain (LC) CDR1 sequence of SEQ ID NO: 6, a LC CDR2 sequence of SEQ ID NO: 7, and a LC CDR3 sequence of SEQ ID NO:8.

3. The method of claim 1 , wherein the radiometal ion is 225 Ac, 111 In or 89 Zr.

4. The method of claim 1 , wherein the antibody or antigen binding fragment thereof that is covalently linked to a first click reaction partner is obtained by a method comprising trimming an antibody or antigen binding fragment thereof with a bacterial endoglycosidase specific for the β-1,4 linkage between a core GlcNac residue in a Fc-glycosylation site of the antibody to obtain a trimmed antibody or antigen binding fragment thereof, and reacting the trimmed antibody or antigen binding fragment thereof with an azide-labeled sugar, in the presence of a sugar transferase to thereby obtain the antibody or antigen binding fragment thereof that is covalently linked to the first click reaction partner.

5. The method of claim 4 wherein the azide-labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido 6-deoxy GalNAc.

6. The method of claim 4 wherein the sugar transferase is GalT galactosyltransferase or GalNAc transferase.

7. The method of claim 1 , wherein the antibody or antigen binding fragment thereof that is covalently linked to a first click reaction partner is obtained by a method comprising deglycosylating an antibody or antigen binding fragment thereof with an amidase to obtain a deglycosylated antibody or antigen binding fragment thereof, and reacting the deglycosylated antibody or antigen binding fragment thereof with an azido amine in the presence of a microbial transglutaminase to thereby obtain the polypeptide that is covalently linked to a first click reaction partner.

8. The method of claim 7 wherein the azido amine is selected from 3-azido propylamine, 6-azido hexylamine, O-(2-Aminoethyl)-O′-(2-azidoethyl)tetraethylene glycol, O-(2-Aminoethyl)-O′-(2-azidoethyl)pentaethylene glycol, and O-(2-Aminoethyl)-O′-(2-azidoethyl)triethylene glycol.

9. The method of claim 1 , wherein the chelating moiety comprises the structure of formula (II):

or the structure of formula (III):

10. The method of claim 3 , wherein the antibody or antigen binding fragment thereof that is covalently linked to the first click reaction partner is obtained by a method comprising trimming an antibody or antigen binding fragment thereof with a bacterial endoglycosidase specific for the β-1,4 linkage between a core GlcNac residue in a Fc-glycosylation site of the antibody to obtain a trimmed antibody or antigen binding fragment thereof, and reacting the trimmed antibody or antigen binding fragment thereof with an azide-labeled sugar in the presence of a sugar transferase, to thereby obtain the modified antibody or antigen binding fragment thereof that is covalently linked to the first click reaction partner.

11. The method of claim 10 wherein the azide labeled sugar is UDP-N-azidoacetylgalactosamine (UDP-GalNaz) or UDP-6-azido 6-deoxy GalNAc.

12. The method of claim 10 wherein the sugar transferase is selected from GalT galactosyltransferase or GalNAc transferase.

13. The method of claim 3 , wherein the antibody or antigen binding fragment thereof that is covalently linked to the first click reaction partner is obtained by a method comprising deglycosylating an antibody or antigen binding fragment thereof with an amidase to obtain a deglycosylated antibody or antigen binding fragment thereof, and reacting the deglycosylated antibody or antigen binding fragment thereof with an azido amine, in the presence of a microbial transglutaminase to thereby obtain the modified antibody or antigen binding fragment thereof that is covalently linked to the first click reaction partner.

14. The method of claim 13 wherein the azido amine is selected from 3-azido propylamine, 6-azido hexylamine, O-(2-Aminoethyl)-O′-(2-azidoethyl)tetraethylene glycol, O-(2-Aminoethyl)-O′-(2-azidoethyl)pentaethylene glycol, and O-(2-Aminoethyl)-O′-(2-azidoethyl)triethylene glycol.

15. The method of claim 3 , wherein the chelating moiety comprises the structure of formula (II):

Assignees

• Janssen Biotech Inc

Inventors

• Dudkin Vadim
• Goldberg Shalom
• Erhardt Joseph
• Salter Rhys
• Mcdevitt Theresa

Classifications

• C07K2317/41

  Glycosylation, sialylation, or fucosylation · CPC title

• C07K16/3069

  Reproductive system, e.g. ovaria, uterus, testes, prostate · CPC title

• C07B2200/05

  Isotopically modified compounds, e.g. labelled · CPC title

• A61K2039/505

  comprising antibodies · CPC title

• A61K51/1096 Primary

  radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications · CPC title