Switchable assembly and function of antibody complexes in vivo using a small molecule

The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody–drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody’s intended function in vivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities in vivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine’s half-life, and activation of bispecific, T cell–engaging antibodies.

Antibodies are multidomain proteins that have become an important therapeutic platform for a wide range of diseases (1). The key feature of antibodies that enables broad therapeutic application is their ability to couple selective molecular targeting to a functional output in a single molecule with long serum half-life. In the example of natural antibodies, targeting of pathogen-associated antigens is coupled to functions that facilitate an immune response, such as antibody-dependent cellular cytotoxicity via the recruitment of natural killer cells. In the case of antibody-based therapeutics, targeting of disease-specific antigens can be linked in a modular way to a myriad of desired natural or synthetic effector domains. These can include radionuclides, cytotoxic payloads, immune cell engagers, cytokines, and engineered cells (2–6).Historically, genetic and chemical techniques have been used to generate synthetic combinations of multiple domains that collectively impart both targeting and function into a single therapeutic molecule. This is exemplified by the wide array of antibody and protein fusion formats with unique specificities and therapeutic mechanisms that have entered the clinic to date (3–5, 7, 8). Despite the diversity of formats, the functional properties of these synthetic proteins are intrinsically defined by their chemical structures (Fig. 1A). Thus, once an antibody-based drug is infused into a patient, the clinician relinquishes any control over its activity, potentially for a period of weeks. The long half-lives can make it challenging to manage toxicities or to rapidly adjust drug activity in response to efficacy or pharmacodynamic biomarkers.Open in a separate windowFig. 1.LITE enables antibody complexes with switchable assembly and activity. (A) Most biologic drugs use a targeting domain to localize a functional, therapeutic moiety, but these molecular components are inextricably linked. (B) LITE is a technology to enable switchable assembly of individual antibody components into an active complex.Several classes of antibody-based drugs could benefit from a method of rapidly controlling drug activity and exposure. One such class is therapeutically active proteins that have been fused to the Fc-region of human IgG, which imparts a longer serum half-life (5, 8). The Fc fusion strategy has extended the half-lives of diverse proteins, including cytokine receptors (e.g., etanercept and aflibercept), hormones (e.g., dulaglutide), and cytokine mimetics (e.g., romiplostim) but at the risk of prolonging their potentially toxic activity for weeks after injection. Another class of antibody-based drugs, bispecific T cell–engaging antibodies (bsTCEs), can potently redirect the immune system to attack cancer cells but are also associated with unmanageable toxicities that have been observed in early clinical trials (4, 7, 9). First-generation bsTCEs had short half-lives (<5 h) that allowed for rapid termination of treatment in response to toxicities but necessitated the use of burdensome continuous infusion pumps. Extended half-life bsTCEs were explored as a way to achieve more convenient dosing, but physicians lack a mechanism to quickly terminate activity in response to adverse events. Thus, efforts to increase the half-life of this powerful modality must be paired with approaches for managing toxicity. An ideal solution to address these challenges would combine the long half-life advantages of biologic drugs with the precise temporal control of activity associated with small molecules. An antibody-based drug with these features would allow for convenient dosing but also enable a clinician to quickly respond to a patient''s needs by increasing drug activity or reversing toxicity.Here, we demonstrate a general approach for ligand-induced transient engagement (LITE) of multiple antibody domains, whereby chemically induced dimerization is applied to enable switchable antibody activity by modulating the dose of an Food and Drug Administration (FDA)-approved small molecule (Fig. 1B). We provide three examples demonstrating the broad utility of this approach. First, we show the induced association of a tumor-targeting domain to reversibly control the biodistribution and tumor localization of an antibody in vivo. Second, we demonstrate that the inducible transient engagement of a therapeutically relevant cytokine to an Fc domain dramatically increases its half-life in vivo. Finally, we show small-molecule–regulated formation of a functional, bispecific T cell engager complex capable of redirecting T cells to kill tumor cells in vitro and in vivo. In summary, the LITE platform enables a new class of biologic drugs with functions that can be precisely switched on and off after intravenous (i.v.) administration.