T cell-dependent bispecific antibody (TDB)-induced T cell activation, which can eliminate tumor cells independent of MHC engagement, is expected to be a novel breakthrough immunotherapy against refractory cancer. However, the mechanism of action of TDBs has not been fully elucidated thus far. We focused on TDB-induced T cell-tumor cell contact as an important initial step in direct T cell-mediated tumor cell killing via transport of cytotoxic cell proteases (e.g., granzymes) with or without immunological synapse formation. Using an anti-EGFR/CD3 TDB, hEx3, we visualized and quantified T cell-tumor cell contact and demonstrated a correlation between the degree of cell contact and TDB efficacy. We also found that cytokines, including interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) secreted by activated T cells, damaged tumor cells in a cell contact-independent manner. Moreover, therapeutic experiences clearly indicated that hEx3, unlike conventional anti-EGFR antibodies, was effective against colorectal cancer (CRC) cells with mutant KRAS, BRAF, or PIK3CA. In a pharmacokinetic analysis, T cells spread gradually in accordance with the hEx3 distribution within tumor tissue. Accordingly, we propose that TDBs should have four action steps: 1st, passive targeting via size-dependent tumor accumulation; 2nd, active targeting via specific binding to tumor cells; 3rd, T cell redirection toward tumor cells; and 4th, TDB-induced cell contact-dependent (direct) or-independent (indirect) tumor cell killing. Finally, our TDB hEx3 may be a promising reagent against refractory CRC with an oncogenic mutation associated with a poor prognosis. Keywords Antibody therapeutics • Bispecific antibody (BsAb) • T cell-dependent bispecific antibody (TDB) • Immunological synapse • Immunotherapy • Colorectal cancer Abbreviations BsAb Bispecific antibody CRC Colorectal cancer EPR Enhanced permeability and retention IS Immunological synapse MTA Molecular targeted agent TDB T cell-dependent bispecific antibody Electronic supplementary material The online version of this article (

As a breakthrough immunotherapy, T cell bispecific antibodies (T-BsAbs) are a promising antibody therapy for various kinds of cancer. In general, T-BsAbs have dual-binding specificity to a tumor-associated antigen and a CD3 subunit forming a complex with the TCR. This enables T-BsAbs to crosslink tumor cells and T cells, inducing T cell activation and subsequent tumor cell death. Unlike immune checkpoint inhibitors, which release the brake of the immune system, T-BsAbs serve as an accelerator of T cells by stimulating their immune response via CD3 engagement. Therefore, they can actively redirect host immunity toward tumors, including T cell recruitment from the periphery to the tumor site and immunological synapse formation between tumor cells and T cells. Although the low immunogenicity of solid tumors increases the challenge of cancer immunotherapy, T-BsAbs capable of immune redirection can greatly benefit patients with such tumors. To investigate the detailed relationship between T-BsAbs delivery and their T cell redirection activity, it is necessary to determine how T-BsAbs deliver antitumor immunity to the tumor site and bring about tumor cell death. This review article discusses T-BsAb properties, specifically their pharmacokinetics, redirection of anticancer immunity, and local mechanism of action within tumor tissues, and discuss further challenges to expediting T-BsAb development.

(Background and purpose) A bispecific antibody (BsAb), as a next-generation therapeutic antibody, has two antigen binding sites in an antibody molecule. Many formats have been exploited for decades. Among them, we focused on T cell dependent BsAb (TDB), which can engage and redirect cytotoxic T cells against tumor cells and is expected to be effective in treating refractory cancer. However, although many studies revealed its potent antitumor effect in animal models, it has not been applied to solid tumors yet in clinics. In this context, we think that it is necessary to clarify pharmacokinetics (PK), pharmacodynamics (PD) and mechanism of action (MOA) of TDB, especially within tumor tissue. For this aim, we have decided to develop the molecular imaging-based systems for the evaluation of TDB in order to visualize antigen binding to two target antigens, BsAb delivery or immunological synapse formation. (Materials a& Methods) We used hEx3 which is a humanized IgG-like antibody with bivalent Fv regions against each EGFR and CD3. We applied the TDB to colorectal cancer (CRC) cells with or without KRAS or BRAF mutation. In the first part, we visualized the BsAb binding to two antigens, EGFR on CRC cells and CD3 on T cells. Next, immunological synapse formation as a cutting-edge mechanism involving T cells-killing tumor cells, which is an important MOA of TDB, was visualized in combination with Granzyme staining. In the second part, we evaluated in vitro cytotoxicity and the in vivo antitumor effect of hEx3 in NOD-SCID mice bearing CRC xenografts with KRAS or BRAF mutation following human PBMC administration. (Results) We found that CRC cells were damaged via Granzyme accumulation through immunological synapse. In the ELISA data, we also found that released gamma interferons can damage CRC cells. In vitro and In vivo studies showed that the hEx3 demonstrated a significant cytocidal effect and antitumor activity not only for KRAS wild but also mutant. (Conclusion) we established a molecular imaging-based TDB evaluation system and visualized antigen-binding and immunological synapse formation as a MOA of TDB. Present data indicated that TDB can damage CRC cells in two distinct manners, namely immunological synapse-dependent and -independent manners using cytokines including a gamma interferon. TDB may thus be a promising next generation antibody therapy against CRC regardless KRAS status. Citation Format: Daisuke Kamakura, Masahiro Yasunaga, Ryutaro Asano, Yasuhiro Matsumura. Development of bispecific antibodies using molecular imaging [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2274.