Dual conjugation process for preparing antibody-drug conjugates

Inventors

Coumans, Rudy Gerardus Elisabeth

Assignees

Byondis BV

Abstract

The present invention relates to a process for preparing antibody-drug conjugates wherein therapeutic moieties are conjugated to one or more engineered cysteines as well as to one or more reduced interchain cysteines via a cleavable or non-cleavable linker.

Core Innovation

The document describes a process for preparing an antibody-drug conjugate in which an antibody and therapeutic moieties are conjugated to engineered cysteines. The process begins by selectively reducing a cysteine-engineered antibody using a compound according to formula (I), (II), (III), (IV), (V), (VI) or (VII) or a salt thereof. The selective reducing step uses engineered cysteine positions located on heavy chain 40, 41, 89, heavy chain 152, 153, 155, 171, light chain 40, 41, and light chain 165, 168 according to Kabat and Eu numbering systems.

After selective reduction, the process further reduces the selectively reduced antibody with an interchain disulfide bond reducing agent to form one or more reduced interchain cysteines. The document then conjugates therapeutic moieties via cleavable or non-cleavable linkers to the engineered cysteines from the selectively reduced antibody and to at least one reduced interchain cysteine from the further reduced antibody.

The document links the dual-reduction/conjugation strategy to reported ADC characteristics including low DAR0 and reduced formation of higher molecular weight species and reduced hydrophobicity, and it describes tunability of average DAR by varying the interchain reducing-agent input. Experimental characterization is described using HIC/SEC along with in vitro and in vivo assessments that include evaluation of deconjugation behavior compared to ADCs prepared with interchain-cysteine-only approaches.

Claims Coverage

The independent claim covers a dual-reduction ADC preparation sequence: selective reduction of engineered cysteines using a specific phosphine/sulfonic-acid compound set, further reduction of interchain disulfides, followed by conjugation of therapeutic moieties through cleavable or non-cleavable linkers to both engineered and reduced interchain cysteines. The independent claim includes at least three main inventive features: selective reduction with formulas (I)-(VII), further reduction of interchain disulfides with an interchain disulfide reducing agent, and conjugation of therapeutic moieties to both engineered and interchain cysteines via cleavable or non-cleavable linkers.

Across the independent claim, the inventive concept is a two-step reduction strategy (selective reduction using a formula (I)-(VII) compound followed by interchain disulfide reduction) coupled to conjugation of therapeutic moieties to both engineered cysteines and reduced interchain cysteines using cleavable or non-cleavable linkers.

Stated Advantages

Documented Applications