Site-specific labeling methods and molecules produced thereby

Patent metadata

Field	Value
Publication number	US-9585970-B2
Application number	US-201313906584-A
Country	US
Kind code	B2
Filing date	May 31, 2013
Priority date	Jun 4, 2012
Publication date	Mar 7, 2017
Grant date	Mar 7, 2017

Abstract

The present invention provides methods of site-specific labeling of antibodies, using proteins having 4′-phosphopantetheinyl transferase activity that catalyze post-translational modification of peptide sequences (“peptide tags”) incorporated into one or more specific sites of an antibody of interest. Enzymatic labeling enables quantitative and irreversible covalent modification of a specific serine residue within the peptide tags incorporated into the antibody, and thus creates desirable antibody conjugates.

First claim

What is claimed is:

1. A modified antibody or antigen binding fragment thereof, wherein said wherein said antibody comprises an amino acid sequence selected from SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:139, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:178, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:277, SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:359, SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:373, SEQ ID NO:374, SEQ ID NO:380, SEQ ID NO:384, SEQ ID NO:386, SEQ ID NO:387, or SEQ ID NO:388.

2. An immunoconjugate comprising the modified antibody or antigen binding fragment thereof of claim 1 , and a terminal group, wherein said terminal group is attached to the modified antibody or antigen binding fragment by a linker having the structure according to Formula (I-b):

wherein:
a)

L 1 is -A 1 X 2 —, wherein A 1 is —C(═O)NH(CH 2 ) n S— and X 2 is

L 2 is -A 2 -, wherein A 2 is —(CH 2 ) n C(═O;
L 3 is -A 3 -, wherein A 3 is

and
L 4 is

b) L is a -A 1 X 2 —, wherein A 1 is —C(═O)NH(CH 2 ) n S— and X 2 is —(CH 2 ) n C(═O)NH—;

L 2 is a bond-; L 3 is -A 3 -, wherein A 3 is —(CH 2 ) n C(═O)—, and L 4 is a bond;
c) L 1 is a -A 1 X 2 —, wherein A 1 is —C(═O)NH(CH 2 ) n S—, X 2 is —CHR 4 (CH 2 ) n C(═O)NH— and R 4 is —C(═O)OH;

L 2 is a bond; L 3 is -A 3 -, wherein A 3 is —(CH 2 ) n C(═O)— and, L 4 is a bond;
d) L 1 is -A 1 X 2 —, where A 1 is —C(═O)NH(CH 2 ) n S— and X 2 is —(CH 2 )C(═O)NH—;

L 2 is a bond; L 3 is a bond, and L 4 is -A 4 - wherein A 4 is —(CH 2 ) n NHC(═O)—;
e) L 1 is -A 1 X 2 —, wherein A 1 is —C(═O)NH(CH 2 ) n S— and X 2 is —(CH 2 )C(═O)NH—;

L 2 is a bond; L 3 is a bond; L 4 is -A 4 -, wherein A 4 is —(CH 2 ) n C(═O)—;
f) L 1 is -A 1 X 2 —, wherein A 1 is —C(═O)NH(CH 2 ) n S— and X 2 is —(CH 2 )C(═O)NH—;

L 2 is -A 2 -, wherein A 2 is —(CH 2 ) n C(═O;
L 3 is -A 3 -, wherein A 3 is

and
L 4 is

g) L 1 is a -A 1 X 2 —, wherein A 1 is —C(═O)NH(CH 2 ) n S— and X 2 is —(CH 2 )C(═O)NH—;

L 2 is a bond-; L 3 is -A 3 -, wherein A 3 is —(CH 2 ) n C(═O)—, and L 4 is a bond; or
h) L 1 is a -A 1 X 2 —, wherein A 1 is —C(═O)NH(CH 2 ) n S—, X 2 is —CHR 4 (CH 2 ) n C(═O)NH— and R 4 is —C(═O)OH;

L 2 is a bond; L 3 is -A 3 -, wherein A 3 is —(CH 2 ) n C(═O)—, and L 4 is a bond.

3. The immunoconjugate of claim 2 , wherein the terminal group is a moiety selected from an anti-inflammatory agent, an anticancer agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent and an anesthetic agent.

4. The immunoconjugate of claim 3 , wherein the terminal group is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizers, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, an inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, an EG5 inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder, a proteasome inhibitor, and a DHFR inhibitor.

5. The immunoconjugate of claim 2 , wherein the terminal group is selected from a fluorophore, a chromophore, a quantum dot, a magnetic probe, a radioactive probe, an imaging reagent, or a contrast reagent.

6. A method of treating a Her2 positive cancer comprising administering to a human in need thereof an effective amount of the immunoconjugate of claim 2 or a pharmaceutical composition thereof.

Assignees

• Geierstanger Bernhard Hubert
• Grunewald Jan
• Bursulaya Badry
• Novartis Ag

Inventors

• Geierstanger Bernhard Hubert
• Grunewald Jan
• Bursulaya Badry

Classifications

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K2319/90

  containing a motif for post-translational modification · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61K47/6817

  Toxins · CPC title

• A61K47/6815

  Enzymes · CPC title