Valentina Fiori

Acute Myeloid Leukemia with FLT3 internal tandem duplication mutations ( FLT3 -ITD mut AML) is an aggressive leukemia characterized by heterogeneous genetic landscape [1]. Despite signi fi cant advances in AML therapy, the relapse rate remains high, due to the emergence of resistant clones, possibly involving leukemic stem cells (LSCs) [2]. This highlights the unmet need for deeper understanding of the molecular and immunophenotypic landscape of LSCs in FLT3 -ITD mut AML. We previously identi fi ed a population of leukemic precursor cells (LPCs), present at the time of initial AML diagnosis, characterized by the CD34/CD123/CD25/ CD99 + immunophenotype, and predictive for FLT3 -ITD mut positivity [3]. In particular, these cells overexpress CD99 antigen, which may represent a target for monoclonal antibody (mAb) treatment [4]. CD99 is a transmembrane

Acute myeloid leukemia (AML) with FLT3-ITD mutations (FLT3-ITDmut) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts for FLT3-ITD-positivity. The aim of this study was to characterize the distribution of FLT3-ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture of FLT3-ITDmut AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients with FLT3-ITDmut AML (n = 12). A higher FLT3-ITDmut load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/−,CD25−,CD99low/−) (p = 0.0005) and mononuclear cells (MNCs) (p 

Ewing's sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and "up-scalable" process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60 % soluble fraction, we obtained a yield of 25 mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic incl...

Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enha...

The paucity of new drugs for the treatment of Ewing sarcoma (EWS) limits the cure of these patients. CD99 has a strong membranous expression in EWS cells and, being also necessary for tumor survival, is a suitable target to aim at. In this article, we described a novel human monospecific bivalent single-chain fragment variable diabody (dAbd C7) directed against CD99 of potential clinical application. In vitro and in vivo evaluation of cell death and of the molecular mechanisms triggered by anti-CD99 agents were performed alone or in combination with doxorubicin to demonstrate efficacy and selectivity of the new dAbd C7. The dAbd C7 induced rapid and massive EWS cell death through Mdm2 degradation and p53 reactivation. Mdm2 overexpression as well as silencing of p53 in p53wt EWS cells decreased CD99-induced EWS cell death, whereas treatment with nutlin-3 enhanced it. Furthermore, cell death was associated with induction of p21, bax, and mitochondrial depolarization together with subs...

Carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) is a cell surface glycoprotein involved in intercellular binding, belonging to the immunoglobulin superfamily. It is involved in cell-cell recognition and modulates cellular processes that range from vascular angiogenesis to the regulation of insulin homeostasis and T-cell proliferation. Aberrant expression of CEACAM1 is often associated with progression and metastatic potential in melanoma, lung carcinoma and other types of tumor. Tumor-specific antigens such as CEACAM1 are ideal targets for cancer immunotherapy because they are over-expressed by the cancer cell and not on non-malignant tissues, minimizing the risk of autoimmune destruction. Many of the limitations of therapeutic use of rodent monoclonal antibodies (mAbs) can now be overcome by exploiting the use of recombinant antibody fragments and the advances in antibody engineering methods to improve tumor retention, reduce immunogenicity and modulate pharmaco...

ABSTRACT Ewing sarcoma is the second most frequent primary tumor of bone, preferentially occurring in children and adolescents. Despite significant improvements have been achieved in localized tumors thanks to dose intensification of chemotherapy, outcome of patients with metastasis at diagnosis remains grim. In addition, even in the most favorable situations, patients must face with important side effects which significantly impact their quality of life. Either scientific and patient communities are now very sensitive on the need of new drugs, which may reduce chemotherapy toxicity while maintaining effectiveness of current regimens. We present here a new engeneered human bivalent single chain fragment variable diabody (C7 scFv diabody) directed against CD99, a transmembrane protein whose high expression characterizes Ewing sarcoma. The triggering of CD99 with C7scFv diabody induces rapid and massive Ewing sarcoma cell death through MDM2 ubiquitination and p53 reactivation. Accordingly, the most CD99-responsive Ewing sarcoma cells have transcriptional active p53 and greatly benefit from MDM2 degradation. CD99 triggering also potentiates the cytotoxic effect of doxorubicin in vitro and in vivo and reactivates p53 to a much greater degree, which in turn markedly increased expression of pro-apoptosis genes. Evaluation of Ki-67 labelling and apoptosis rate by TUNEL confirms the efficacy of the treatment in xenografts. In contrast, mesenchymal stem cells, though expressing high levels of CD99, show no p53 activation and escape death induced by CD99 C7scFv diabody. No sign of toxicity was observed in mice treated with anti-CD99 scFV C7 diabody. Overall, our data provide the rationale for the use of this newly developed anti-CD99 diabody in the treatment of patients with Ewing sarcoma.