Selective reduction of cysteine-engineered antibodies
Inventors
Coumans, Rudy Gerardus Elisabeth • Spijker, Henri Johannes
Assignees
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Publication Number
US-10814009-B2
Publication Date
2020-10-27
Expiration Date
Abstract
The present invention relates to a process for the selective reduction of cysteine-engineered antibodies comprising reacting an antibody comprising one or more engineered cysteines at positions selected from HC40, HC41,HC42, HC89, HC152, HC153, HC155, HC171, LC40, LC41, LC165, and LC168 with a compound according to formula (I), (II), (III), (IV), (V), (VI) or (VII): (I) (II) (III) (IV) (V) (VI) (VII), and to a process for the preparation of antibody conjugates, including antibody-drug conjugates (ADCs).
Core Innovation
The document describes a selective reduction method for an antibody comprising one or more engineered cysteines located at specified heavy chain and light chain positions. The engineered cysteines are selected from heavy chain 40, 41, 42 and 89 according to the Kabat numbering system; heavy chain 152, 153, 155 and 171 according to the Eu numbering system; light chain 40 and 41 according to the Kabat numbering system; and light chain 165 and 168 according to the Eu numbering system. The antibody is reacted with a phosphine sulfonic-acid reducing agent of formula (I), (II), (III), (IV), (V), (VI) or (VII), or a salt thereof, to reduce the engineered cysteines.
A stated key outcome is that the reducing agents uncapped engineered cysteines in a Fab cavity while leaving native interchain disulfides intact. This selective reduction is described as eliminating mAb half-body and scrambled disulfides. The result is described as enabling more homogeneous antibody conjugates and antibody-drug conjugates through a two-step process that includes selective reduction followed by conjugation.
The document further characterizes the resulting antibody conjugates and ADCs with structural constraints via formula (VIII) and formula (IX), including parameters such as n, m (average DAR), y, and substituent selections for R1 and R2. It also specifies conjugation scope and example therapeutic moieties, including duocarmycin seco-DUBA, together with comparative experimental evidence using non-reducing SDS-PAGE and SEC/DAR/HMW/half-body comparisons to support improved monomer/HMW reduction and elimination of mAb half-body for a protocol described as protocol A versus protocol B.
Claims Coverage
The independent claim set includes one independent process claim, which is directed to reacting a cysteine-engineered antibody at specified Kabat/Eu positions with a phosphine sulfonic-acid reducing agent of formulas (I)–(VII) (or salts) to reduce the engineered cysteines. The coverage is refined in dependent claims by defining the reducing-agent stoichiometry, removal of unreacted reducing agent, and structural/DAR constraints for the resulting antibody conjugate via formulas (VIII) and (IX).
Reduction of engineered cysteines at specified Kabat/Eu positions using phosphine sulfonic-acid compounds
A process comprising reacting an antibody comprising one or more engineered cysteines at positions selected from heavy chain 40, 41, 42 and 89 according to the Kabat numbering system; heavy chain 152, 153, 155, and 171 according to the Eu numbering system; light chain 40 and 41 according to the Kabat numbering system; and light chain 165 and 168 according to the Eu numbering system, with a compound according to formula (I), (II), (III), (IV), (V), (VI) or (VII) or a salt thereof, to reduce said one or more engineered cysteines.
Stoichiometry of reducing agent relative to engineered cysteines
The process characterized in that the compound defined by formula (I), (II), (III), (IV), (V), (VI), or (VII) is present at 2 to 16 molar equivalents per engineered cysteine of the antibody.
Removal of unreacted reducing agent before conjugation
The process further includes removing any unreacted reducing agent before performing the conjugation reaction.
Removal without UF/DF or TFF
The process includes a removing step carried out without using ultrafiltration/diafiltration (UF/DF) or tangential flow filtration (TFF).
Antibody conjugate constrained by formula (VIII) and DAR-related parameters
A process limited such that the antibody conjugate conforms to formula (VIII) with specified values for n (0–3), m (average DAR 1–6), R1, y (1–16), and R2 selected as shown in the disclosed structures.
Selected anti-target antibody set
The process uses an antibody selected from the group of specific anti-target antibodies.
Overall, the claims cover a process that reduces engineered cysteines at defined Kabat/Eu positions using phosphine sulfonic-acid reducing agents of formulas (I)–(VII), with claim refinements that constrain reducing-agent stoichiometry, require removal of unreacted reducing agent, and limit the resulting antibody conjugate structure and DAR to formula (VIII) parameters. The antibody selection is further restricted to a specified set of anti-target antibodies.
Stated Advantages
Eliminating mAb half-body.
Eliminating scrambled disulfides.
Providing antibody conjugates and antibody-drug conjugates described as more homogeneous.
Improved monomer/HMW reduction described in comparative evidence.
Zero mAb half-body described for a protocol compared to another protocol.
Documented Applications
Antibody conjugates and antibody-drug conjugates (ADCs) produced using the selective reduction followed by conjugation approach, including conjugates directed to a range of anti-target antibodies and ADCs described with linkers, therapeutic moieties, and duocarmycin seco-DUBA.
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