Universal immune receptors represent a rapidly emerging form of adoptive T-cell therapy with the potential to overcome safety and antigen escape challenges faced by conventional chimeric antigen receptor (CAR) T-cell therapy. By decoupling antigen recognition and T-cell signaling domains via bifunctional antigen-specific targeting ligands, universal immune receptors can regulate T-cell effector function and target multiple antigens with a single receptor. Here, we describe the development of the SpyCatcher immune receptor, the first universal immune receptor that allows for the post-translational covalent attachment of targeting ligands at the T-cell surface through the application of SpyCatcher-SpyTag chemistry. The SpyCatcher immune receptor redirected primary human T cells against a variety of tumor antigens via the addition of SpyTag-labeled targeting ligands, both in vitro and in vivo. SpyCatcher T-cell activity relied upon the presence of both target antigen and SpyTag-labeled targeting ligand, allowing for dose-dependent control of function. The mutational disruption of covalent bond formation between the receptor and the targeting ligand still permitted redirected T-cell function but significantly compromised antitumor function. Thus, the SpyCatcher immune receptor allows for rapid antigen-specific receptor assembly, multiantigen targeting, and controllable T-cell activity.
Bispecific
antibodies (BsAb) refer to a class of biomacromolecules
that are capable of binding two antigens or epitopes simultaneously.
This can elicit unique biological effects that cannot be achieved
with either individual antibody or two unlinked antibodies. Bispecific
antibodies have been used for targeting effector cells to tumor cells,
preferential targeting of cells expressing two target biomarkers over
cells expressing either target biomarker individually, or to couple
two molecular targets on the same cell surface to trigger unique intracellular
signaling pathways. Here, we present two related methods that enable
direct, rapid assembly of bispecific antibodies from any two “off-the-shelf”
Immunoglobulin G (IgG) antibodies, in as little as 1 day. Both workflows
can be summarized into two steps: (1) attach a small photoreactive
antibody binding domain (pAbBD) fused to SpyCatcher or SpyTag (peptide–protein
partners derived from the S. pyogenes fibronectin-binding
protein FbaB) to each component IgG, respectively; (2) assemble the
BsAb through the spontaneous isopeptide bond formation that occurs
between SpyTag and SpyCatcher. These approaches enable production
of BsAbs from any two IgG molecules without the need to elucidate
their amino acid sequences or genetically alter their structure. Binding
assays and T cell-mediated cytolysis assays were performed to validate
the binding and functional properties of Trastuzumab × Cetuximab
BsAb and Cetuximab × OKT3 BsAb, respectively. This approach enables
rapid, low-cost production of highly homogeneous tetravalent BsAbs
in a modular fashion, presenting an opportunity to quickly evaluate
antibody pairs in a BsAb format for unique or synergistic functionalities.
Extensive antibody engineering and cloning is typically required to generate new bispecific antibodies. Made-to-order genes, advanced expression systems, and high-efficiency cloning can simplify and accelerate this process, but it still can take months before a functional product is realized. We developed a simple method to site-specifically and covalently attach a T cell–redirecting domain to any off-the-shelf, human immunoglobulin G (IgG) or native IgG isolated from serum. No antibody engineering, cloning, or knowledge of the antibody sequence is required. Bispecific antibodies are generated in just hours. By labeling antibodies isolated from tumor-bearing mice, including two syngeneic models, we generated T cell–redirecting autoantibodies (TRAAbs) that act as an effective therapeutic. TRAAbs preferentially bind tumor tissue over healthy tissue, indicating a previously unexplored therapeutic window. The use of autoantibodies to direct the tumor targeting of bispecific antibodies represents a new paradigm in personalized medicine that eliminates the need to identify tumor biomarkers.
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