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The recent development of immune checkpoint inhibitors for many types of cancers has prompted us to identify markers that predict patients with clinical benefits. Several trials on nivolumab for the treatment of esophageal squamous cell carcinoma (ESCC) have been performed worldwide, and the identification of markers specific to ESCC is urgently needed. We conducted a clinical trial on nivolumab for advanced ESCC (JapicCTI-No.142422) and investigated markers using peripheral blood collected from 20 patients enrolled in our institute, including 1 with a complete response (CR), 5 with a partial response (PR), 6 with a stable disease (SD), and 8 with a progressive disease (PD) as clinical responses. The expression of surface molecules and cytokine production by T cells were analyzed using flow cytometry, and clinicopathological factors and general blood parameters were examined. Albumin, neutrophils, %Tim3, %OX40, %CD103, %CD45RA - CD27 - , and IL-1b after the first cycle of nivolumab treatment, but not at baseline, distinguished CR/PR from SD/PD patients. When markers to distinguish longer survivors with nivolumab therapy were analyzed, changes in these levels between baseline and after the first cycle of nivolumab treatment, but not levels at each period, were indicative, similar to the tumor burden. Among them, elevations in %Tim-3 + CD4 had a marked impact on survival rates. In conclusion, dynamic elevations in %Tim-3 in T cells in the early period of nivolumab therapy have potential as a marker for the clinical responses and prognosis of advanced ESCC patients.

Background: The PD-1-blocking antibody nivolumab persists in patients several weeks after the last infusion. However, no study has systematically evaluated the maximum duration that the antibody persists on T cells or the association between this duration and residual therapeutic efficacy or potential adverse events., Methods: To define the duration of binding and residual efficacy of nivolumab after discontinuation, we developed a simplified strategy for T cell monitoring and used it to analyze T cells from peripheral blood from 11 non-small cell lung cancer patients previously treated with nivolumab. To determine the suitability of our method for other applications, we compared transcriptome profiles between nivolumab-bound and nivolumab-unbound CD8 T cells. We also applied T cell monitoring in 2 nivolumab-treated patients who developed progressive lung tumors during long-term follow-up., Results: Prolonged nivolumab binding was detected more than 20 weeks after the last infusion, regardless of the total number of nivolumab infusions (2-15 doses) or type of subsequent treatment, in 9 of the 11 cases in which long-term monitoring was possible. Ki-67 positivity, a proliferation marker, in T cells decreased in patients with progressive disease. Transcriptome profiling identified the signals regulating activation of nivolumab-bound T cells, which may contribute to nivolumab resistance. In 2 patients who restarted nivolumab, T cell proliferation markers exhibited the opposite trend and correlated with clinical response., Conclusions: Although only a few samples were analyzed, our strategy of monitoring both nivolumab binding and Ki-67 in T cells might help determine residual efficacy under various types of concurrent or subsequent treatment., Trial Registration: University Hospital Medical Information Network Clinical Trials Registry, UMIN000024623., Funding: This work was supported by Japan Society for the Promotion of Science KAKENHI (JP17K16045, JP18H05282, and JP15K09220), Japan Agency for Medical Research and Development (JP17cm0106310, JP18cm0106335 and JP18cm059042), and Core Research for Evolutional Science and Technology (JPMJCR16G2).

Immunotherapy with CD123-specific T-cell engager proteins or with T cells expressing CD123-specific chimeric antigen receptors is actively being pursued for acute myeloid leukemia. T cells secreting bispecific engager molecules (ENG-T cells) may present a promising alternative to these approaches. To evaluate therapeutic potential, we generated T cells to secrete CD123/CD3-bispecific engager molecules. CD123-ENG T cells recognized primary acute myeloid leukemia (AML) cells and cell lines in an antigen-dependent manner as judged by cytokine production and/or tumor killing, and redirected bystander T cells to AML cells. Infusion of CD123-ENG T cells resulted in regression of AML in xenograft models conferring a significant survival advantage of treated mice in comparison to mice that received control T cells. At high effector to target ratios, CD123-ENG T cells recognized normal hematopoietic stem and progenitor cells (HSPCs) with preferential recognition of HSPCs from cord blood compared to bone marrow. We therefore introduced the CD20 suicide gene that can be targeted in vivo with rituximab into CD123-ENG T cells. The expression of CD20 did not diminish the anti-AML activity of CD123-ENG T cells, but allowed for rituximab-mediated ENG-T cell elimination. Thus, ENG-T cells coexpressing CD20 suicide and CD123 engager molecules may present a promising immunotherapeutic approach for AML.

T cells expressing chimeric antigen receptors (CARs) or the infusion of bispecific T-cell engagers (BITEs) have shown antitumor activity in humans for CD19-positive malignancies. While BITEs redirect the large reservoir of resident T cells to tumors, CAR T cells rely on significant in vivo expansion to exert antitumor activity. We have shown that it is feasible to modify T cells to secrete solid tumor antigen-specific BITEs, enabling T cells to redirect resident T cells to tumor cells. To adapt this approach to CD19-positive malignancies we now generated T cells expressing secretable, CD19-specific BITEs (CD19-ENG T cells). CD19-ENG T cells recognized tumor cells in an antigen-dependent manner as judged by cytokine production and tumor killing, and redirected bystander T cells to tumor cells. Infusion of CD19-ENG T cells resulted in regression of leukemia or lymphoma in xenograft models and a survival advantage in comparison to control mice. Genetically modified T cells expressing engager molecules may present a promising addition to current CD19-targeted immunotherapies.

Adoptive immunotherapy with antigen-specific T cells has shown promise for the treatment of malignancies. However, infused T cells are unable to redirect resident T cells, limiting potential benefit. While the infusion of bispecific T-cell engagers can redirect resident T cells to tumors, these molecules have a short half-life, and do not self amplify. To overcome these limitations, we generated T cells expressing a secretable T-cell engager specific for CD3 and EphA2, an antigen expressed on a broad range of human tumors (EphA2-ENG T cells). EphA2-ENG T cells were activated and recognized tumor cells in an antigen-dependent manner, redirected bystander T cells to tumor cells, and had potent antitumor activity in glioma and lung cancer severe combined immunodeficiency (SCID) xenograft models associated with a significant survival benefit. This new class of tumor-specific T cells, with the unique ability to redirect bystander T cells, may be a promising alternative to current immunotherapies for cancer.