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Despite the revolution of immunotherapy in cancer treatment, patients eventually progress due to the emergence of resistance. In this scenario, the selection of the tumor antigen can be decisive in the success of the clinical response. T cell bispecific antibodies (TCBs) are engineered molecules that include binding sites to the T cell receptor and to a tumor antigen. Using gastric CEA+/HER2+ MKN45 cells and TCBs directed against CEA or HER2, we show that the mechanism of resistance to a TCB is dependent on the tumor antigen. Acquired resistant models to a high-affinity-CEA-targeted TCB exhibit a reduction of CEA levels due to transcriptional silencing, which is reversible upon 5-AZA treatment. In contrast, a HER2-TCB resistant model maintains HER2 levels and exhibit a disruption of the interferon-gamma signaling. These results will help in the design of combinatorial strategies to increase the efficacy of cancer immunotherapies and to anticipate and overcome resistances.

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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.

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T-cell Bispecific Antibodies (TCBs) elicit anti-tumor responses by cross-linking T-cells to tumor cells and mediate polyclonal T-cell expansion that is independent of T-cell receptor specificity. TCBs thus offer great promise for patients who lack antigen-specific T-cells or have non-inflamed tumors, which are parameters known to limit the response of checkpoint inhibitors. The current study deepens the understanding of TCB mode of action and elaborates on one of the adaptive resistance mechanisms following its treatment in vivo in humanized mice and syngeneic pre-clinical tumor models. Single-agent TCB treatment reduced tumor growth compared with controls and led to a 2-10-fold increase in tumor-infiltrating T-cells, regardless of the baseline tumor immune cell infiltration. TCB treatment strongly induced the secretion of CXCL10 and increased the frequency of intra-tumor CXCR3+ T-cells pointing to the potential role of the CXCL10-CXCR3 pathway as one of the mechanisms for T-cell recruitment to tumors upon TCB treatment. Tumor-infiltrating T-cells displayed a highly activated and proliferating phenotype, resulting in the generation of a highly inflamed tumor microenvironment. A molecular signature of TCB treatment was determined (CD8, PD-1, MIP-a, CXCL10, CXCL13) to identify parameters that most robustly characterize TCB activity. Parallel to T-cell activation, TCB treatment also led to a clear upregulation of PD-1 on T-cells and PD-L1 on tumor cells and T-cells. Combining TCB treatment with anti-PD-L1 blocking antibody improved anti-tumor efficacy compared to either agent given as monotherapy, increasing the frequency of intra-tumoral T-cells. Together, the data of the current study expand our knowledge of the molecular and cellular features associated with TCB activity and provide evidence that the PD-1/PD-L1 axis is one of the adaptive resistance mechanisms associated with TCB activity. This mechanism can be managed by the combination of TCB with anti-PD-L1 blocking antibody translating into more efficacious anti-tumor activity and prolonged control of the tumor outgrowth. The elucidation of additional resistance mechanisms beyond the PD-1/PD-L1 axis will constitute an important milestone for our understanding of factors determining tumor escape and deepening of TCB anti-tumor responses in both solid tumors and hematological disorders.

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Monoclonal antibody-based therapeutics are an integral part of treatment of different human diseases, and the selection of suitable antibody candidates during the discovery phase is essential. Here, we describe a novel, cellular screening approach for the identification and characterization of therapeutic antibodies suitable for conversion into T cell bispecific antibodies using chimeric antigen receptor (CAR) transduced Jurkat-NFAT-luciferase reporter cells (CAR-J). For that purpose, we equipped a Jurkat-NFAT reporter cell line with a universal CAR, based on a monoclonal antibody recognizing the P329G mutation in the Fc-part of effector-silenced human IgG1-antibodies. In addition to scFv-based second generation CARs, Fab-based CARs employing the P329G-binder were generated. Using these anti-P329G-CAR-J cells together with the respective P329G-mutated IgG1-antibodies, we established a system, which facilitates the rapid testing of therapeutic antibody candidates in a flexible, high throughput setting during early stage discovery. We show that both, scFv- and Fab-based anti-P329G-CAR-J cells elicit a robust and dose-dependent luciferase signal if the respective antibody acts as an adaptor between tumor target and P329G-CAR-J cells. Importantly, we could demonstrate that functional characteristics of the antibody candidates, derived from the anti-P329G-CAR-J screening assay, are predictive for the functionality of these antibodies in the T cell bispecific antibody format.

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We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3+ T cell/myeloma cell crosslinking, followed by CD4+/CD8+ T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA+ cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration.

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