Kamiya (Kyushu University) for flow cytometer. to suppress tumor growth in a mouse model. Thus, Fc-ARMs have the potential to be a novel class of cancer immunotherapeutic agents. Fc-binding antibody-recruiting molecules provide strong and sufficient opportunities to employ endogenous antibodies for anti-tumor immune responses. == Introduction == Immune cells, such as natural killer (NK) cells, recognize antibodies bound to target proteins and lyse target cells, and this process is usually termed antibody-dependent cell-mediated cytotoxicity (ADCC). Based on immune-mediated mechanisms of action including ADCC, therapeutic IgG antibodies have revolutionized the clinical outcomes for multiple types of cancers.1However, the potential immunogenicity of monoclonal antibodies (mAbs) is one of their limitations, causing side effects such as IgE-mediated anaphylaxis and the production of anti-drug antibodies in the clinic.2,3 As a potential solution to this issue, antibody-recruiting small molecules (ARMs) have been developed to induce antibody-mediated immune responses while circumventing antibody administration.4ARMs are composed of an antibody-binding terminus (ABT) and a target-binding terminus (TBT) (Fig. 1A), and redirect endogenous antibodies CeMMEC13 in the bloodstream to target cells such as tumor cells. Subsequently, recruited antibodies trigger immune responses to eliminate the target (Fig. 1B, left panel). Notably, ARMs are thought to be less immunogenic due to their small molecular size.5,6To date, various types of TBTs have been developed.722By contrast, only a few types of ABTs have shown potential utility: galactose–1,3-galactose (-Gal),12,13rhamnose,21nitroarenes,1416,20,22and phosphorylcholine.23This is because ABTs have so far been limited to antigens against endogenous antibodies present in a wide range of population. == Fig. 1. Fc-binding antibody-recruiting molecule (Fc-ARM) concept in comparison with conventional ARMs. (A) Antibody-recruiting small molecule (ARM). ABT = antibody-binding terminus; TBT = target-binding terminus. (B) Conventional ARM-mediated (left) and Fc-ARM-mediated (right) induction of ADCC. (C) Molecular design of Fc-ARM1 and Fc-ARM2. == However, these types of ABTs suffer from some shortcomings that limit their therapeutic application. For example, endogenous antibodies against ABTs are generally in short supply (e.g.anti–Gal accounts for 1% of endogenous IgG in human serum24), limiting the probability of encountering ARMs and antibodies in the human body. In addition, the efficacy of ARM-based therapy may be influenced by the polyclonal affinity of endogenous antibodies to the ABT. EMR2 4Although immunization of patients with ABTs may help to improve the characteristics of anti-ABT endogenous antibodies,4,9,17,20,22it increases the complexity of treatment procedures, and may also induce additional side effects. Thus, new approaches that enable the exploitation of a larger proportion of pre-existing endogenous antibodies by constant affinity would be a breakthrough for the clinical translation of ARMs. The Fc region of an antibody is usually characterized by its conserved molecular structure, as well as a variety of biological functions. It CeMMEC13 is the Fc module that is responsible for ADCC in the development of biopharmaceuticals.25,26A recent study revealed that this Fc region of an antibody binds to virus-infected cells and NK cells simultaneously, resulting in ADCC against the infected cells.27This report indicated that ADCC could be induced independently of antigenFab interactions. Previously, we reported a new class of ARMs, namely Fc-ARMs, in which the Fc-binding cyclic peptide (Fc-III)28is used as an ABT (Fig. 1B, right panel).29Use of the Fc-binding peptide enables Fc-ARMs to at least access IgG1 and IgG2 with constant affinity.30These two IgG subclasses account for more than 80% of the total IgG in humans.31,32Thus, Fc-ARMs can theoretically exploit the majority of endogenous antibodies through their constant affinity to IgG-Fc. We previously exhibited that Fc-ARMs can recruit human IgG on the surface of cancer cells in a selective manner.29However, recruited antibodies did not mediate ADCC under the previous experimental conditions. Given that ADCC is usually regulated by the overall affinity of the antibody to its antigen33and CD16a,34we hypothesized that this alternative of the Fc-III peptide with a recently reported peptide, Fc-III4C, which has higher affinity for IgG-Fc,35would strengthen the recruited antibody ability to activate effector cells such as NK cells for target cell destruction. We conducted quantitative evaluations of antibody recruitment and decided the anti-tumor effects of Fc-ARM bothin vitroandin vivoto show the concept of the Fc-ARM strategy. == Results and discussion == == Fc affinity and antibody recruitment == We used an Fc-III peptide or Fc-III4C peptide35as an ABT and folic acid (FA) as a TBT (Fig. 1C). FA has sub-nanomolar affinity to folate receptor-a (FR-)36and is usually therefore used for targeted drug delivery and imaging CeMMEC13 of cancers overexpressing FR-.37We connected the ABT and TBT using a hexaethylene glycol linker and synthesized two types of Fc-ARMs, namely.