Method for generating multispecific antibodies from monospecific antibodies
Inventors
Brinkmann, Ulrich • Dengl, Stefan • Georges, Guy • Hoffmann, Eike • Mayer, Klaus • BORMANN, FELIX
Assignees
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Publication Number
US-12297290-B2
Publication Date
2025-05-13
Expiration Date
Abstract
Herein is reported a method for the generation of multispecific antibodies by a half-antibody exchange reaction between two 2/3-IgGs destabilized in one half by asymmetric perturbing mutations fostering the generation of correctly assemble full length bispecific antibodies. The method can be performed in the absence of reducing agents and does not require hinge region disulfide bonds in the starting 2/3-IgGs.
Core Innovation
The invention relates to producing heterodimeric exchanged polypeptides by incubating heterodimeric starting polypeptides with a second polypeptide and exchanging polypeptides to form heterodimeric exchanged polypeptides. The polypeptides comprise an immunoglobulin G (IgG) CH3 domain and at least one functional binding site or a part thereof, and the starting polypeptide is formed by the first and second polypeptides. The CH3 domains carry mutation patterns to control CH3 heterodimer formation, including knob-cys/hole and hole-cys/knob arrangements, with further mutations selected from a defined group. The exchanged polypeptides are used to generate additional heterodimeric exchanged polypeptides, and the fourth exchanged polypeptide is recovered.
The disclosure further relates to heterodimeric exchanged polypeptides and multimeric IgG-format architectures produced via CH3 knob/hole exchange. The assemblies include IgG1 CH1 domain and IgG1 CH2 domain arrangements with hinge regions and optional peptidic linkers, and may comprise scFv, scFab, or split binding-site parts. The CH3 domains comprise defined mutation patterns, including knob-cys and hole-cys, with wild-type residues preserved at interacting positions, and a set of perturbing CH3 mutations comprising D356K, E357K, K370E, and K439E.
The disclosure includes recovering exchanged heterodimeric polypeptides and optionally using affinity tags for chromatographic recovery. It further recites embodiments involving reducing-agent presence or absence, covalent disulfide linkage and dimer formation between CH3 variants, and an overall method for identifying multispecific polypeptides using combinatorial exchange and simultaneous target binding assays.
Claims Coverage
The claim coverage centers on production of heterodimeric exchanged polypeptides via CH3 knob/hole exchange and recovery of a selected exchanged heterodimer, with dependent claims further narrowing CH3 mutation sets, hinge-region composition, tag presence, and recovery-related conditions.
Knob-cys and hole ch3 domain patterning for heterodimeric starting polypeptides
Producing a heterodimeric exchanged polypeptide by incubating first and second polypeptides to form a first heterodimeric starting polypeptide, wherein the CH3 domain of the first polypeptide comprises mutations knob-cys and the CH3 domain of the second polypeptide comprises mutations hole, or the CH3 domain of the first polypeptide comprises mutations hole-cys and the CH3 domain of the second polypeptide comprises the mutation knob.
Selectable further ch3-domain mutations distinct from starting mutations
Including in the CH3 domain a further mutation selected from a defined group of mutations, and ensuring that in the third polypeptide the further mutation is different from the mutations present in the CH3 domain of the first polypeptide and the second polypeptide.
Exchange of second and third polypeptides to form third and fourth heterodimeric exchanged polypeptides
Forming a second heterodimeric starting polypeptide comprising a third polypeptide and a fourth polypeptide, and wherein the third and fourth polypeptide form a heterodimer with corresponding knob/cys and hole assignment rules; exchanging the second and the third polypeptide to form a third heterodimeric exchanged polypeptide comprising the second and the third polypeptide and a fourth heterodimeric exchanged polypeptide comprising the first and the fourth polypeptide, and recovering the fourth heterodimeric exchanged polypeptide.
Ch3-domain perturbing mutations and partner-different further mutations
The CH3 domains include further mutations selected from a specified group of mutations, including D356K, E357K, K370E, and K439E, with the further mutation of one polypeptide differing from the further mutation of the corresponding other polypeptide.
Exchange and recovery of the desired heterodimeric product
The method comprises exchanging the second and third polypeptides to form exchanged heterodimeric polypeptides and recovering the fourth heterodimeric exchanged polypeptide.
The claim set covers CH3-domain knob/hole mutation pairing, including knob-cys and hole-cys variants, combined with exchange between defined heterodimeric starting polypeptides and recovery of the desired exchanged heterodimeric product. Dependent claims further narrow the designs with additional CH3 mutations, hinge-region variants, optional tags, and reducing-agent-related conditions.
Stated Advantages
Preferential Fc-region matching heterodimers.
Enables exchange under physiological-condition exchange by omitting reduction/re-oxidation when Fc-region interchain hinge disulfide bonds are eliminated.
Enables reduction-free chain exchange for producing stable, isolatable exchanged products.
Enables exchange and correct assembly without hinge-region interchain disulfides as described in the summary content.
Allows avoidance of unreacted starting materials and aggregates via purification logic as described in the summary content.
Documented Applications
Generation of multiple bispecific antibody formats from exchanging 2/3-IgG-derived molecules while maintaining MHCFcRP and varying heavy-chain formats, producing different binding-site positions, valencies, and geometries.
Format-variant extension including non-antibody binders (affibody), Fab-extended 2/3-IgGs providing a third binding site, constrained binders, and hinge- and Fc-region variants affecting effector functions and FcRn binding.
Production of multispecific antibodies, notably bispecific antibodies, via half-antibody exchange using engineered 2/3-IgGs.
Generation and purification of stable, isolatable exchanged bispecific antibody products, including purification using affinity tags.
Identification of multispecific polypeptides using combinatorial exchange and simultaneous target binding assays.
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