Orthogonal mutations for heterodimerization
Inventors
DAVIS, Jonathan Harry • Marshall, Nicholas M.
Assignees
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Publication Number
US-12312418-B2
Publication Date
2025-05-27
Expiration Date
Abstract
Heterodimerizing domains with orthogonal mutations that drive heterodimerization, in particular heterodimerizing antibody CH3 domains, heterodimeric polypeptides comprising such heterodimerizing domains, and antibody constructs comprising such heterodimeric polypeptides, are provided.
Core Innovation
The disclosure describes engineered orthogonal mutations for driving specific heterodimerization of antibody CH3 domains, including heterodimeric polypeptides and multivalent or multispecific antibody constructs. The approach uses a CH3-interface mutation framework to promote pairing between selected CH3 domains while maintaining orthogonality between different CH3 interfaces.
A CH3-interface mutation framework is defined using engineered disulfide bridges and additional substitutions, including Y349C and S354C with E357 substitutions, together with further cascade mutations such as E357W, K370R, and S364R. The disclosure also describes orthogonalization strategies including knob-in-hole (KIH) and charge-pair mutation concepts to support multiple distinct heterodimerization pairings.
The disclosure further provides antibody architectures for bivalent bispecific B-Body constructs, including BC1, BC6, BC28, BC44, and an exemplary BC96 construct, each defined by particular combinations of CH3 mutations across antibody components. SEC and SDS-PAGE results are reported to indicate improved purity and assembly characteristics when E357W and K370R co-occur in opposing CH3 domains, and reduced homodimer contaminants when E357W and S364R are included together with Y349C.
Claims Coverage
One inventive feature is identified across the provided claims content.
Four-chain antibody construct with specified domain ordering and two antigen binding sites
An antibody construct comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the domains are ordered from N terminus to C terminus as V_L-CH3-CH2-CH3 for the first chain and V_H-CH3 for the second chain, such that the V_L domain of the first polypeptide chain and the V_H domain of the second polypeptide chain associate to form a first antigen binding site; the third polypeptide chain is ordered from N terminus to C terminus as V_L-C_L-CH2-CH3 and the fourth polypeptide chain is ordered from N terminus to C terminus as V_H-CH1, such that the V_L domain of the third polypeptide chain and the V_H domain of the fourth polypeptide chain associate to form a second antigen binding site, with CH1/CH2/CH3/CL domains specified by SEQ ID NO: 5 through SEQ ID NO: 12.
The claim content centers on a four-chain antibody construct with specified domain ordering that forms two antigen binding sites.
Stated Advantages
Improved purity and assembly, as indicated by SEC/SDS-PAGE findings, when E357W and K370R co-occur in opposing CH3 domains.
Reduced homodimer contaminants when E357W and S364R are included together with Y349C.
Documented Applications
Bivalent bispecific antibody constructs (B-Body), including BC1, BC6, BC28, BC44, and BC96, with engineered CH3 domain heterodimerization architectures.
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