Composition and method for modifying polypeptides
Inventors
Krantz, Alexander • Wilczynski, Andrzej • Rymarczyk, Grzegorz
Assignees
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Publication Number
US-11400165-B2
Publication Date
2022-08-02
Expiration Date
Abstract
The present disclosure provides methods for site-selectively crosslinking payloads to antibodies and other proteins. This can be accomplished using traceless affinity labels designed to label target proteins with bio-orthogonally reactive entities (ORE) using the compositions and methods described herein.
Core Innovation
The invention is directed to site-selective antibody conjugation and traceless affinity labeling of proteins and antibodies by using site-selective acylation and bio-orthogonal reactive handles. The approach targets a conserved antibody light-chain site, including lysine 188 of a kappa light chain constant domain, while maintaining a linker attached without leaving a traceless affinity label. In one aspect, at least 90% of the linker is site selectively attached to lysine 188 based on Kabat numbering, providing a defined attachment point for subsequent conjugation.
Bio-orthogonal cycloaddition is used to create site-selective linkages between the installed handle and an appropriate corresponding reaction entity from that conserved attachment site. Site-selective acyl transfer agents install an orthogonally reactive entity, often an azide, and the document describes compound and formula frameworks that define variable reactive-handle types, linker structures including PEG/polyalkyleneoxy and ether/thioether variants, and electrophilic leaving-group motifs.
The invention further provides payload-linker-antibody conjugates that incorporate toxins, drugs, chelators, and multi-payload conjugate concepts. It also describes chelator-antibody conjugates using two-step bio-orthogonal labeling, including DIBAC and macrocyclic chelators such as H2macropa (MacroPa), and radiometal chelation with chelators such as macropa and DOTA, with radioisotopes such as actinium-225 and lutetium-177.
The document also describes characterization and biological validation demonstrating preserved HER2 binding after the site-selective modification, together with potent cytotoxicity when payloads such as alpha-amanitin are conjugated. The disclosed examples include in vivo tumor regression in xenograft models as reported in the document.
Claims Coverage
The patent includes two independent claims directed to a linker-antibody conjugate of Formula IA and a payload-linker-antibody conjugate of Formula IB, each requiring site-selective attachment at lysine 188. Across the independent claims, the coverage centers on bio-orthogonal reactive handle chemistry for Formula IA and payload, chelator, or tether structure selection for Formula IB, each constrained by the at least 90% site-selective attachment to lysine 188 (Kabat).
Site-selective linker attachment to lysine 188 of a kappa light chain constant domain
A linker-antibody conjugate where at least 90% of the linker is site selectively attached to lysine 188 of a kappa light chain constant domain of the antibody based on a Kabat numbering.
Bio-orthogonal reactive handle for cycloaddition chemistry
A linker-antibody conjugate where R is selected from alkyne, cycloalkyne, azide, benzyl azide, 1,3-diene, nitrile oxide, nitrone, tetrazine, and a trans-cyclooctene, or structurally related derivatives capable of undergoing bio-orthogonal cycloaddition to an appropriate corresponding reaction entity.
Payload-linker-antibody conjugate with defined linker moiety and payload selection
A payload-linker-antibody conjugate of Formula IB where m is 1-40, n is 1-20, o is 1-2, L is a linking moiety selected from specified —C(O)C1-C30-heteroaromatic- and —C1-C30-heteroaromatic-C(O)— forms, Z is selected from a toxin, a drug, and a chelator payload, and at least 90% of the linker is site selectively attached to lysine 188 of a kappa light chain constant domain based on a Kabat numbering.
Overall, the claim set focuses on antibody conjugates that achieve high site-selective attachment at lysine 188 (Kabat). Formula IA uses bio-orthogonal reactive handles, and Formula IB defines a payload-linker-antibody conjugate with a specified linker moiety and payload class while maintaining the lysine 188 attachment requirement.
Stated Advantages
Minimal antibody perturbation is stated relative to non-site-selective ADCs.
Improved capacity without destabilizing branching is stated.
Synergistic multi-payload delivery to target cells is stated.
Potential reduction of resistance by simultaneous delivery of heterogeneous toxins/radionuclides is stated.
Preserved HER2 binding is stated after site-selective modification.
Potent cytotoxicity is stated for alpha-amanitin ADCs.
In vivo tumor regression in xenograft models is stated.
Documented Applications
HER2-targeted ADC payload delivery and efficacy testing in HER2-expressing contexts, with preserved binding to HER2 and cytotoxicity reported for alpha-amanitin ADCs.
In vivo tumor regression studies in xenograft models, including JIMT-1 and NCI-N87 as described.
Chelator-antibody conjugate applications using chelators such as DIBAC and macrocyclic chelators such as H2macropa (MacroPa), as described for chelator-antibody conjugates.
Potential therapeutic and diagnostic applications of the resulting antibody conjugates.
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