Despite substantial advances in the treatment of acute myeloid leukemia (AML), only 30% of patients survive more than 5 years. Therefore, new therapeutics are much needed. Here, we present a novel therapeutic strategy targeting PR1, an HLA-A2 restricted myeloid leukemia antigen. Previously, we have developed and characterized a novel T-cell receptor-like monoclonal antibody (8F4) that targets PR1/HLA-A2 and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC). To improve the potency of 8F4, we adopted a strategy to link T-cell cytotoxicity with a bi-specific T-cell-engaging antibody that binds PR1/HLA-A2 on leukemia and CD3 on neighboring T-cells. The 8F4 bi-specific antibody maintained high affinity and specific binding to PR1/HLA-A2 comparable to parent 8F4 antibody, shown by flow cytometry and Bio-Layer Interferometry. In addition, 8F4 bi-specific antibody activated donor T-cells in the presence of HLA-A2+ primary AML blasts and cell lines in a dose dependent manner. Importantly, activated T-cells lysed HLA-A2+ primary AML blasts and cell lines after addition of 8F4 bi-specific antibody. In conclusion, our studies demonstrate the therapeutic potential of a novel bi-specific antibody targeting the PR1/HLA-A2 leukemia-associated antigen, justifying further clinical development of this strategy.

CD20 monoclonal antibodies (CD20 mAb) induce cellular cytotoxicity, which is traditionally measured by antibody-dependent cellular cytotoxicity (ADCC) assays. However, data suggest that antibody-dependent cellular phagocytosis (ADCP) is the primary cytotoxic mechanism. We directly compared in vitro ADCP versus ADCC using primary human cells. After establishing the primacy of ADCP, we examined next-generation CD20 mAbs, including clinically relevant drug combinations for their effects on ADCP. ADCP and ADCC induction by rituximab, ofatumumab, obinutuzumab, or ocaratuzumab was measured using treatment-naïve chronic lymphocytic leukemia (CLL) target cells and either human monocyte-derived macrophages (for ADCP) or natural killer (NK) cells (for ADCC). Specific effects on ADCP were evaluated for clinically relevant drug combinations using BTK inhibitors (ibrutinib and acalabrutinib), PI3Kδ inhibitors (idelalisib, ACP-319, and umbralisib), and the BCL2 inhibitor venetoclax. ADCP (∼0.5-3 targets/macrophage) was >10-fold more cytotoxic than ADCC (∼0.04-0.1 targets/NK cell). ADCC did not correlate with ADCP. Next-generation mAbs ocaratuzumab and ofatumumab induced ADCP at 10-fold lower concentrations than rituximab. Ofatumumab, selected for enhanced complement activation, significantly increased ADCP in the presence of complement. CD20 mAb-induced ADCP was not inhibited by venetoclax and was less inhibited by acalabrutinib versus ibrutinib and umbralisib versus idelalisib. Overall, ADCP was a better measure of clinically relevant mAb-induced cellular cytotoxicity, and next-generation mAbs could activate ADCP at significantly lower concentrations, suggesting the need to test a wide range of dose sizes and intervals to establish optimal therapeutic regimens. Complement activation by mAbs can contribute to ADCP, and venetoclax, acalabrutinib, and umbralisib are preferred candidates for multidrug therapeutic regimens. Cancer Immunol Res; 6(10); 1150-60. ©2018 AACR.
©2018 American Association for Cancer Research.

To date, CD5 expression and its role in acute T cell lymphoblastic leukaemia (T-ALL) have not been studied closely. We observed a significant reduction in surface expression of CD5 (sCD5) on leukaemic T cells compared to autologous non-leukaemic T cells. In this study, we have shown the molecular mechanism regulating the expression and function of CD5 on leukaemic T cells. A total of 250 patients suffering from leukaemia and lymphoma were immunophenotyped. Final diagnosis was based on their clinical presentation, morphological data and flow cytometry-based immunophenotyping. Thirty-nine patients were found to be of ALL-T origin. Amplification of early region of E1A and E1B transcripts of CD5 was correlated with the levels of surface and intracellular expression of CD5 protein. Functional studies were performed to show the effect of CD5 blocking on interleukin IL-2 production and survival of leukaemic and non-leukaemic cells. Lack of expression of sCD5 on T-ALL blasts was correlated closely with predominant transcription of exon E1B and significant loss of exon E1A of the CD5 gene, which is associated with surface expression of CD5 on lymphocytes. High expression of E1B also correlates with increased expression of cytoplasmic CD5 (cCD5) among leukaemic T cells. Interestingly, we observed a significant increase in the production of IL-2 by non-leukaemic T cells upon CD5 blocking, leading possibly to their increased survival at 48 h. Our study provides understanding of the regulation of CD5 expression on leukaemic T cells, and may help in understanding the molecular mechanism of CD5 down-regulation.
© 2017 British Society for Immunology.

The role of Notch signaling in human innate lymphoid cell (ILC) differentiation is unclear, although IL-7 and IL-15 promote differentiation of natural cytotoxicity receptor (NCR) NKp44+ group 3 ILCs (NCR+ILC3s) and conventional NK (cNK) cells from CD34+ hematopoietic progenitor cells (HPCs) ex vivo. In this study, we analyzed the functions of Notch in the differentiation of NCR+ILC3s and cNK cells from human HPC subpopulations circulating in peripheral blood by limiting dilution and clonal assays using high-throughput flow cytometry. We demonstrated that Notch signaling in combination with IL-7 induced NCR+ILC3 differentiation, but conversely suppressed IL-15-dependent cNK cell generation in CD45RA+Flt-3-c-Kitlow, a novel innate lymphocyte-committed HPC subpopulation. In contrast, Notch signaling induced CD45RA-Flt-3+c-Kithigh multipotent HPCs to generate CD34+CD7+CD62Lhigh, the earliest thymic progenitor-like cells, which preserved high cNK/T cell potential, but lost NCR+ILC3 potential. These findings implicate the countervailing functions of Notch signaling in the fate decision between NCR+ILC3 and cNK cell lineages at different maturational stages of human HPCs. Inhibition of Notch functions by Abs specific for either the Notch1 or Notch2 negative regulatory region suggested that both Notch1 and Notch2 signals were involved in the fate decision of innate lymphocyte-committed HPCs and in the generation of earliest thymic progenitor-like cells from multipotent HPCs. Furthermore, the synergistic interaction between Notch and IL-7 in NCR+ILC3 commitment was primarily explicable by the induction of IL-7 receptor expression in the innate lymphocyte-committed HPCs by Notch stimulation, suggesting the pivotal role of Notch in the transcriptional control required for human NCR+ILC3 commitment.
Copyright © 2017 by The American Association of Immunologists, Inc.

Mantle cell lymphoma (MCL) is one of the most aggressive lymphoid neoplasms whose pathogenesis is not fully understood. The neural transcription factor SOX11 is overexpressed in most MCL but is not detected in other mature B-cell lymphomas or normal lymphoid cells. The specific expression of SOX11 in MCL suggests that it may be an important element in the development of this tumor, but its potential function is not known. Here, we show that SOX11 promotes tumor growth in a MCL-xenotransplant mouse model. Using chromatin immunoprecipitation microarray analysis combined with gene expression profiling upon SOX11 knockdown, we identify target genes and transcriptional programs regulated by SOX11 including the block of mature B-cell differentiation, modulation of cell cycle, apoptosis, and stem cell development. PAX5 emerges as one of the major SOX11 direct targets. SOX11 silencing downregulates PAX5, induces BLIMP1 expression, and promotes the shift from a mature B cell into the initial plasmacytic differentiation phenotype in both primary tumor cells and an in vitro model. Our results suggest that SOX11 contributes to tumor development by altering the terminal B-cell differentiation program of MCL and provide perspectives that may have clinical implications in the diagnosis and design of new therapeutic strategies.