6). leukemia (AML) is regarded as a stem cell disorder affecting blood and bone marrow. Although the cancer stem cell model is still under debate, there is strong evidence for the existence of cancer stem Midodrine cells, especially in AML. AML tumor cells display a clonal hierarchy consisting of a small population of leukemic stem cells (LSCs) with self renewing capacity and their progeny[1],[2],[3]. In contrast to AML blasts, AML-LSCs are able to produce a heterogeneous leukemic xenograft in immunodeficient mice[4]. Due to their slow proliferation and characteristic gene expression profiles AML-LSCs are more resistant to chemotherapeutic agents compared to the more differentiated blasts[5],[6],[7]. Consequently most currently used chemotherapeutic agents kill the majority of AML blasts but are not able to efficiently eliminate AML-LSCs[8]. Therefore, outgrowth of remaining AML-LSCs may eventually lead to relapse of the disease[9]. Targeted therapies directed against AML-LSCs may represent effective therapeutic approaches Midodrine for AML therapy and, in combination with conventional therapies, may ultimately cure AML patients[8],[10]. For efficient targeting via monoclonal antibodies, identification of cell surface markers preferentially expressed on AML-LSCs but being absent on most non-malignant tissue, especially on normal HSCs, is essential[11]. Several potential target structures on AML-LCSs have been suggested, including CD33[12],[13], CD44[14], CD123[15],[16], CLL-1[17],[18], CD47[19]and TIM3[20]. Recently, expression of CD96 (TACTILE) has been reported on AML-LSCs while only very low expression levels have been found on a small subset of normal HSCs[21]. CD96 is a member of the Ig gene superfamily and its expression has been described on NK-cells and activated T cells[22],[23]. In AML, CD96 expression was detected on the majority of blasts in about 30% of patients[22]. Besides its expression in AML, CD96 was found on a major subset of T-cell acute lymphoblastic leukemias Midodrine (T-ALL)[22],[24]. The function of CD96 on AML-LSCs or AML blasts is widely unknown. CD96 expressed on NK-cells has been identified as a receptor for CD155 (polio virus receptor) and mediates adhesion of NK-cells to tumor cells, thereby modulating effector function of NK-cells[25]. As demonstrated for other adhesion molecules, CD96 expressed on AML-LSCs may be involved in cell-cell interaction in the bone marrow[26],[27], but further studies are necessary to clarify its role in the pathophysiology of AML. Together, these findings suggest that CD96 may represent a promising target structure for the development of antibody-based therapeutic strategies directed against AML-LSCs. Especially for the treatment of AML, therapeutic antibodies may be used in different clinical settings, including ex vivo purging of AML-LSCs from autologous stem cell grafts or for the targeting of AML-LSCsin vivo[21]. Monoclonal antibodies are able to mediate anti-tumor activity by various mechanisms of action[28]. Several findings from mouse models and clinical trials point to an important role for Fc receptors suggesting that indirect effector mechanisms such as antibody dependent cell-mediated cytotoxicity (ADCC) may represent important effector mechanismsin vivo[29],[30],[31]. Although therapeutic antibodies were successfully added in many clinical settings there is still room for improvement. Therefore, strategies are developed to increase the antitumor efficacy of monoclonal antibodies by enhancing ADCC activity[32]. Binding affinity to the Midodrine target antigen as well as Fc binding to activating Fc-receptors has been identified as critical parameters for the ADCC activity of monoclonal antibodies[33],[34]. In addition, the use of recombinant antibody technology may allow design of Rabbit Polyclonal to Cyclin E1 (phospho-Thr395) novel antibody derivatives. Single chain fragment of the variable regions (ScFv) -Fc fusion proteins (mini-antibodies) may represent one promising new molecule format[35],[36]. These molecules display functional characteristics similar to that of complete antibodies but have a molecular weight Midodrine that is reduced by roughly one third, probably improving tissue or tumor penetration. Recently it has been demonstrated that Fc engineering strategies such as introducing amino acid substitutions[37],[38]or altering the glycosylation profile of antibodies[39],[40],[41], that have been frequently applied to enhance the lytic activity of complete IgG1 antibodies, can also be used to enhance mini-antibody-mediated ADCC[42]. In the present study the V-regions of TH-111, a CD96 mouse monoclonal antibody previously generated in our lab[22], were cloned and used to generate scFv-based mini-antibodies. The effect of affinity.
