Dual conjugation process for preparing antibody-drug conjugates
Inventors
Coumans, Rudy Gerardus Elisabeth
Assignees
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Publication Number
US-11696958-B2
Publication Date
2023-07-11
Expiration Date
Abstract
The present invention relates to a process for preparing antibody-drug conjugates and to 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 invention relates to an antibody-drug conjugate comprising an antibody and therapeutic moieties conjugated to cysteine residues in the antibody via a cleavable or non-cleavable linker. The therapeutic moieties are conjugated to one or more engineered cysteines and to one or more reduced interchain cysteines of the antibody, and the antibody is defined by engineered cysteine placement at a heavy chain position 41 and/or a light chain position 40 or 41 according to the Kabat numbering system.
The antibody-drug conjugate is characterized by an average DAR of from 2.0 to 6.0, including an embodiment having an average DAR of 2.0 to about 3. The conjugation scheme combines engineered cysteines with reduced interchain cysteines, while the therapeutic moieties are conjugated through cleavable or non-cleavable linkers to provide controlled DAR and defined cysteine conjugation sites.
A process for making the antibody-drug conjugate includes selectively reducing the cysteine-engineered antibody using a compound according to formula (I), (II), (III), (IV), (V), (VI) or (VII) or a salt thereof, to form one or more selectively reduced species. The selectively reduced antibody is further reduced with an interchain disulfide bond reducing agent to form one or more reduced interchain cysteines, and therapeutic moieties are then conjugated via cleavable or non-cleavable linkers to the engineered cysteines and to at least one reduced interchain cysteine formed after the further reduction.
Claims Coverage
The independent claims are clm-00001 and clm-00002. Across these claims, there are two main inventive feature groupings: a dual cysteine conjugation architecture combining engineered cysteines at specified Kabat positions with reduced interchain cysteines, and a defined manufacturing process using a selective reduction compound of formulas (I)–(VII), followed by interchain disulfide reduction and subsequent linker-mediated conjugation, with DAR constraints.
Engineered cysteines at Kabat positions and dual conjugation to reduced interchain cysteines
An antibody-drug conjugate comprising an antibody and therapeutic moieties conjugated to cysteine residues in the antibody via a cleavable or non-cleavable linker, wherein the therapeutic moieties are conjugated to one or more engineered cysteines and to one or more reduced interchain cysteines of the antibody, and the antibody has an engineered cysteine at heavy chain position 41, or light chain position 40 or 41 according to the Kabat numbering system, or a combination thereof.
Average DAR limited to 2.0 to 6.0
The antibody-drug conjugate has an average DAR of from 2.0 to 6.0, including an embodiment having an average DAR of 2.0 to about 3.
Selective reduction using a compound of formulas (I)–(VII) followed by interchain disulfide reduction and conjugation
The antibody-drug conjugate is made by selectively reducing the cysteine-engineered antibody with a compound according to formula (I), (II), (III), (IV), (V), (VI) or (VII) or a salt thereof, further reducing the selectively reduced antibody with an interchain disulfide bond reducing agent to form one or more reduced interchain cysteines, and conjugating the therapeutic moieties via cleavable or non-cleavable linkers to the one or more engineered cysteines and to at least one reduced interchain cysteine of the further reduced antibody.
Single engineered cysteine at Kabat heavy chain 41 or light chain 40/41 and conjugation to engineered plus reduced interchain cysteines
An antibody-drug conjugate comprising an antibody and therapeutic moieties conjugated to cysteine residues in the antibody via a cleavable or non-cleavable linker, wherein the antibody comprises one engineered cysteine at heavy chain position 41, light chain position 40 or 41 according to the Kabat numbering system, and the therapeutic moieties are conjugated via the cleavable or non-cleavable linker to the engineered cysteine as well as to one or more reduced interchain cysteines of the antibody.
The independent claims define an antibody-drug conjugate in which therapeutic moieties are conjugated via cleavable or non-cleavable linkers to engineered cysteines positioned at Kabat heavy chain 41 and/or Kabat light chain 40/41 and also to reduced interchain cysteines, with average DAR constraints. One independent claim further requires a manufacturing process that includes selective reduction using compounds of formulas (I)–(VII), followed by interchain disulfide bond reduction and then linker-mediated conjugation to both engineered and reduced interchain cysteines.
Stated Advantages
negligible DAR0
reduced HMW species
tunable average DAR (2.0–6.0)
improved homogeneity
reduced hydrophobicity
less deconjugation in PK
Documented Applications
in vivo efficacy and PK comparisons using xenograft in ces1c KO nude mice with tumor volume measurements
apoptosis/caspase 3/7 assessment (ApoTox-Glo Triplex)
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