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

Inventors

Zhong, Xiaotian • Prashad, Amarnauth Shastrie • Kriz, Ronald William • He, Tao • Somers, Will • Wang, Wenge • Letendre, Leo Joseph

Assignees

Pfizer Inc

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.

Core Innovation

The invention relates to producing a labeled antibody in a cell culture by forming at least one covalent sulfur-containing bond. The covalent bond is formed by reaction of an exposed thiol group from at least one unpaired cysteine residue on the Fc region of the antibody with at least one predetermined capping moiety.

The predetermined capping moiety is selected from maleimido trioxa-4-formyl benzamide (MTFB), dibenzocyclooctyl-polyethylene maleimide (DBCO-PEG4-Maleimide), maleimide-PEG2-Biotin (MPB), and azido-PEG3-maleimide. This capping approach supports selective cysteine chemistry for generating a labeled antibody through covalent linkage at the engineered Fc unpaired cysteine thiols.

In dependent forms, the method further includes conjugating the predetermined capping moiety to a payload, where the payload is a cytotoxic payload. The cytotoxic payload is selected from listed cytotoxic agents, including auristatins, calicheamicins, maytansinoids, spliceostatins, and specific cyclopropabenz[e]indole (CBI) dimer, cyclopropa[e]pyrrolindolone (CPI) dimer, and CTI dimer members.

Claims Coverage

The partial claims provided include one independent claim and several dependent claims that add further limitations. The independent claim covers producing a labeled antibody in cell culture through covalent bonding at exposed thiols on unpaired Fc cysteines using a predetermined capping moiety selected from named capping reagents.

Overall, the claim set provided focuses on labeling an antibody in cell culture by covalently coupling a predetermined capping moiety to exposed thiol groups on unpaired Fc cysteines, with dependent claims narrowing the payload to cytotoxic payloads and further restricting cytotoxic payload selection, including auristatin-specific members.

Stated Advantages

Documented Applications