Anti-CD96 mAb (TH-111) showed only limited reactivity with blood and bone marrow nucleated cells but stained a major (78.3%) subset of T-ALL (ALL), representing a potential tool for clinical translation but still awaiting for translational development.23 In this scenario, the advantage of here presented UMG1-epitope is the lack of expression by hematopoietic stem cells as well as by normal tissues, excluding cortical thymocytes and a minority of T lymphocytes. or monovalent (2+1) CD3 arms. Antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and redirected T-cell cytotoxicity assays were analysed PF-4191834 by circulation cytometry. In vivo antitumor activity of ahUMG1 and UMG1-BTCEs was investigated in NSG mice against subcutaneous and orthotopic xenografts of human T-ALL. == Results == Among 110 T-ALL patient-derived samples, 53 (48.1%) stained positive (24% of TI/TII, 82% of TIII and 42.8% of TIV). Importantly, no expression of UMG1-epitope was found in normal tissues/cells, excluding cortical thymocytes and a minority (<5%) of peripheral blood T lymphocytes. ahUMG1 induced strong ADCC and ADCP on T-ALL cells in vitro, which translated in antitumor activity in vivo and significantly extended survival of treated mice. Both UMG1-BTCEs exhibited highly effective killing activity against T-ALL cells in vitro. We exhibited that this effect was specifically exerted by engaged activated T cells. Moreover, UMG1-BTCEs effectively antagonized tumor growth at concentrations >2 log lower as compared with ahuUMG1, with significant mice survival advantage in different T-ALL models in vivo. == Conclusion == Altogether our findings, including the safe UMG1-epitope expression profile, provide a framework for the clinical development of these innovative immune-therapeutics for this still orphan disease. Keywords:hematologic neoplasms; immunotherapy; translational medical research; antibodies; antigens; neoplasm,; hematological malignancies; T-ALL; T-cell engagers; translational research == Background == T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived from the abnormal proliferation of aberrant intra-thymic T-cell progenitors.1 2Although CTLA1 T-ALL was historically associated with a substantially worse outcome as compared with B-cell ALL (B-ALL), intensive chemotherapy regimens have recently improved the prognosis of T-ALL patients.36However, approximately 20% of pediatric and 50% of adult patients experience disease relapse/progression after first-line chemotherapy with a dismal end result.7 8In fact, in these patients, the only approved agent is nelarabine, which can provide temporary benefit in a minority of cases only (30%),9while PF-4191834 few eligible patients can benefit from allogeneic hematopoietic cell transplantation and induction of graft-versus-leukemia.10 11 Unfortunately, while groundbreaking immunotherapeutic advancements have been achieved based on the targeting of B-cell antigens, such as CD19, CD20 and CD22, via chimeric antigen receptors (CAR-T) or bispecific T-cell engagers (BTCEs), and have dramatically empowered the treatment of relapsed/refractory B-ALL patients, the treatment scenery of relapsed/refractory T-ALL is still completely orphan and lacks immunotherapeutic options. Therefore, the development of innovative immunotherapeutics is usually urgently awaited. We present here a encouraging experimental therapeutic approach based on the targeting of a unique epitope of CD43 (UMG1), which is usually highly expressed in cortical-derived PF-4191834 T-ALL cells. We developed an afucosylated form of the humanized mAb UMG1 (ahuUMG1) and two PF-4191834 different BTCEs that, respectively, simultaneously bind UMG1-epitope on T-ALL cells and CD3 (by bivalent or monovalent arm) to induce cell-mediated killing of epitope-expressing leukemic cells. We performed an extensive analysis of the epitope expression on normal tissue/cells, and we investigated the in vitro and in vivo activity of these agents in different models of human T-ALL. The final aim of our study was the translational development of UMG1-based immune-therapeutics in the poor therapeutic scenery of T-ALL. == Material and methods == For a more detailed description of the methods used, seeonline supplemental data. jitc-2020-002026supp001.pdf(3.9MB, pdf) == Cell lines == Ke-37, PF-382, TALL-1, HPB-ALL, DND-41, MOLT-4, JURKAT, p12-ichikawa and ALL-SIL were purchased by DSMZ. CCRF-CEM cell lines was obtained by ATCC. Ke-37, PF-382, TALL-1, DND-41, ALL-SIL, CCRF-CEM, MOLT-4, JURKAT, p12-ichikawa were cultured in RPMI-1640 (Gibco, Life.