Antibody chemically induced dimerizer (AbCID) as molecular switches for regulating cellular therapies
Inventors
Wells, James A • HILL, Zachary B. • MARTINKO, Alexander J.
Assignees
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Publication Number
US-11939379-B2
Publication Date
2024-03-26
Expiration Date
Abstract
Chemically induced dimerizers (AbCIDs) have emerged as one of the most powerful tools to artificially regulate signaling pathways in cells; however, no facile method to identify or design these systems currently exists. The present invention provides a methodology to rapidly generate antibody-based chemically induced dimerizers (AbCIDs) from known small-molecule-protein complexes by selecting for synthetic antibodies that recognize the chemical epitope created by the bound small molecule. Success of this strategy is demonstrated by generating ten chemically-inducible antibodies against the BCL-xL/ABT-737 complex. Three of the antibodies are highly selective for the BCL-xL/ABT-737 complex over BCL-xL alone. Two exemplary important cellular applications of AbCIDs are demonstrated by applying them intracellularly to induce CRISPRa-mediated gene expression and extracellularly to regulate CAR T-cell activation with the small molecule, ABT-737. ABT-737 is not toxic at the concentrations used to activate AbCIDs in cells. AbCIDs provided by this invention are new and orthogonal AbCIDs, expanding the limited toolbox of available CIDs.
Core Innovation
The invention provides a system comprising a first chemically-induced dimer (CID) component and a second CID component, where the two CID components dimerize in the presence of a small molecule. The first CID component includes a first binding moiety that is a protein capable of interacting with a small molecule to form a complex, and a first adapter moiety linked to the first binding moiety.
The second CID component includes a second binding moiety that is a protein capable of binding to a chemical-epitope formed between the small molecule and the first binding moiety, thereby creating a dimer between the second CID component and the first CID component. The second CID component binds to the complex at a site comprising at least a portion of the small molecule and a portion of the first binding moiety.
The system links immune cell engagement to target cell binding by using adapter moieties, where one adapter moiety comprises a T cell antigen-binding moiety and the other adapter moiety comprises a target cell antigen-binding moiety, in either arrangement. The target cell antigen-binding moiety is an extracellular antigen-binding moiety, and the first CID component includes an ABT-199 binding domain comprising the amino acid sequence of SEQ ID NO:315.
Claims Coverage
The independent claims cover a two-component CID system in which dimerization is created by binding of a second binding moiety to a complex formed between a small molecule and a protein first binding moiety. The claims further require adapter moieties that provide T cell antigen-binding and extracellular target cell antigen-binding in either arrangement, while fixing the first CID component to an ABT-199 binding domain (SEQ ID NO:315) and the second CID component to an antibody moiety defined by specific heavy-chain and light-chain CDR sets from selected FAB-AZ clones.
Two chemically-induced dimer components driven by complex-epitope binding
A system comprising a first chemically-induced dimer (CID) component and a second CID component, where the first binding moiety forms a complex with a small molecule and the second binding moiety binds to the complex between the small molecule and the first binding moiety at a site of the complex comprising at least a portion of the small molecule and a portion of the first binding moiety, such that binding creates a dimer between the second CID component and the first CID component.
Adapter assignment for T cell and target cell antigen binding in either arrangement
The system wherein the first adapter moiety comprises a T cell antigen-binding moiety and the second adapter moiety comprises a target cell antigen-binding moiety, or wherein the second adapter moiety comprises a T cell antigen-binding moiety and the first adapter moiety comprises a target cell antigen-binding moiety.
Extracellular antigen-binding target moiety
The system wherein the target cell antigen-binding moiety is an extracellular antigen-binding moiety.
ABT-199 binding domain and antibody moiety defined by selected CDRs
The system wherein the first CID component comprises an ABT-199 binding domain comprising the amino acid sequence of SEQ ID NO:315, and wherein the second CID component comprises an antibody moiety comprising three heavy chain complementarity determining regions (CDRs) and three light chain CDRs of an antibody clone selected from antibody clone FAB-AZ11 through FAB-AZ43.
The independent claims cover a two-component CID system in which dimerization is created by binding of an antibody moiety to a complex formed between a small molecule and a protein first binding moiety. The claims further require adapter moieties that provide T cell antigen-binding and extracellular target cell antigen-binding in either arrangement, while fixing the first CID component to an ABT-199 binding domain (SEQ ID NO:315) and the second CID component to an antibody moiety defined by specific heavy-chain and light-chain CDR sets from selected FAB-AZ clones.
Stated Advantages
Selective, orthogonal AbCIDs that reduce binding to the apo protein.
Fewer adverse effects than conventional CAR T-cell approaches or other conventional T-cell engagers using corresponding (non-CID) signaling/engager domains.
Documented Applications
Intracellular CRISPRa gene activation via dCas9 recruitment.
Extracellular regulation of CAR T-cell activation using ABT-737.
Therapeutic methods using modified T cells and CID components together with administration of a small-molecule dimerizer to modulate and/or treat disease.
Routes in which modified T cells express CID cytoplasmic signaling domains with separately administered extracellular antigen-binding CID components and a small molecule, or in which CID components are administered.
Embodiments including nucleic acids encoding CID components and T cells comprising the CID components, including CAR T cells.
Transcriptional regulation via dCas9/VPR adapters.
Apoptosis kill switch.
Heterodimeric CAR.
Bispecific T-cell engagement with CD3 and target antigens.
Immune modulation.
ABT-737/BCL-xL AbCID development referenced in an examples section.
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