Capped and uncapped antibody cysteines, and their use in antibody-drug conjugation

• US 10,653,794 B2
• Filed: 08/09/2016
• Issued: 05/19/2020
• Est. Priority Date: 08/12/2015
• Status: Active Grant

• Current Assignee: Pfizer Inc.
• Original Assignee: Pfizer Inc.
• Inventors: Zhong, Xiaotian, Prashad, Amarnauth Shastrie, Kriz, Ronald William, He, Tao, Somers, Will, Wang, Wenge, Letendre, Leo Joseph
• Primary Examiner(s): Huynh, Phuong
• Application Number:
  US15/748,268
• Publication Number:
  US 20190030183A1
• Time in Patent Office: 1,379 Days
• CPC Class Codes: A61K 38/05 Dipeptides
• CPC Class Codes: A61K 47/68 the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
• CPC Class Codes: A61K 47/6817 Toxins
• CPC Class Codes: A61K 47/6851 the antibody targeting a determinant of a tumour cell
• CPC Class Codes: A61K 47/6855 the tumour determinant being from breast cancer cell
• CPC Class Codes: A61K 47/6889 Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• 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 16/30 from tumour cells
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

An antibody production process in mammalian cells in which engineered unpaired cysteine residues are post-translationally modified and capped with particular chemical entities, which capped antibodies are well suited to further site-specific conjugation steps to form antibody-drug conjugates (ADCs) or protein drug conjugates; ADCs produced using these capped antibodies including in particular ADCs formed by the selective reduction of the capped antibodies'"'"' cysteine residues, and ADCs formed using chemical handles such as aldehyde/azide/alkyne biorthogonal groups, which permit additional drug conjugation chemistry; and uncapped antibodies produced by cells in low cysteine, cysteine and glutathione media, and ADCs produced via direct conjugation to these uncapped antibodies.

Claims

1. A method of producing an antibody drug conjugate (ADC) comprising the steps:

(a) producing a capped antibody in a cell culture, wherein one or more unpaired cysteine residues on said antibody are covalently bonded through sulfur bonds to one or more predetermined capping moieties, wherein said predetermined capping moieties are selected from the group consisting of 5-thio-2-nitrobenzoic acid (TNB), 2-mercaptopyridine and dithiodipyridine (DTDP);

(b) exposing said capped antibody to a reducing agent capable of removing said capping moieties from said antibody without reducing antibody inter-chain sulfur bonds, wherein said reducing agent is tris (3-sulfophenyl) phosphine (TSPP);

2. The method of claim 1 wherein said predetermined capping moieties are 5-thio-2-nitrobenzoic acid (TNB).

3. The method of claim 1 wherein said reduction occurs primarily at unpaired cysteine residues.

4. The method of claim 1, wherein said auristatin is selected from (2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1 S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide);

(2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide);

(2-methyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3 oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, trifluoroacetic acid salt);

(2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide);

(2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide);

(2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, trifluoroacetic acid salt);

monomethyl dolastatin 10;

(N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine); and

(N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine).

5. The method of claim 1, wherein said cell culture contains a precursor of a capping moiety and wherein said precursor of said capping moiety is Ellman'"'"'s reagent.

6. A method of producing an antibody drug conjugate (ADC) comprising the steps:

(a) producing a capped antibody in a cell culture, wherein one or more unpaired cysteine residues on said antibody are covalently bonded through sulfur bonds to one or more predetermined capping moieties, wherein said predetermined capping moieties are selected from the group consisting of 5-thio-2-nitrobenzoic acid (TNB), 2-mercaptopyridine and dithiodipyridine (DTDP);

(b) exposing said capped antibody to a reducing agent capable of removing said capping moieties from said antibody without reducing antibody inter-chain sulfur bonds, wherein said reducing agent is tris (3-sulfophenyl) phosphine (TSPP);