Abstract 4622: A novel CD33/CD3-bispecific BiTE antibody can effectively recruit autologous T cells from AML-patients for in vitro cell lysis of CD33+ blasts
Authors: Feulner Julian , Aigner Michael , Kischel Roman , Kufer Peter , Baeuerle Patrick A. , Mackensen Andreas , Krause Stefan W. 
Published: August 2012

Abstract
Bispecific T cell-engaging (BiTE®) antibodies are single-chain antibody constructs which combine dual specifity against CD3 on T cells and against a tumor-associated antigen. Phase 1 and 2 clinical trials with the CD19/CD3-bispecific BiTE antibody blinatumomab have shown very high response rates in patients suffering from non-Hodgkin's lymphoma and acute lymphoblastic leukemia. Here, we report on the potential of a novel BiTE antibody targeting CD33 in redirecting autologous T cells for in vitro lysis of blasts from acute myeloid leukemia (AML) patients. Initial antibody titration experiments showed that CD33 BiTE concentrations at low picomolar ranges mediate effective lysis of leukemic cell lines. We next analysed primary samples from AML patients, which were co-cultured with mononuclear cells (MNC) from healthy donors in presence or absence of specific BiTE antibodies at an E:T ratio of 1:1. After 48h of incubation in the presence of 1ng/ml CD33 BiTE we observed a decrease of both CD33+ AML blasts as well as CD33+ monocytes when compared to samples with control BiTE or without BiTE. 1ng/ml CD33 BiTE specifically induced strong upregulation of activation markers CD25 and CD69 on the vast majority of all CD4+ and CD8+ T cells. We furthermore investigated whether T cells from AML patients were able to mediate lysis of CD33+ leukemia cells by CD33 BiTE. T cells purified from peripheral blood or bone marrow of newly diagnosed AML patients were tested for BiTE mediated lysis of U-937 target cells. Redirected T cells from AML patients were capable of eliminating leukemic cells in the presence of CD33 BiTE as effectively as T cells from healthy controls. In consequence of this we studied blast lysis and T cell behaviour in a completely autologous setting using cryo-conserved samples from AML patients. Despite strong activation of T cells only moderate lysis of myeloid cells was observed after 48h, possibly due to low E:T ratios in these samples. Therefore, to allow autologous T cell expansion, cells were cultured for up to 144h. After this prolonged incubation, CD33 BiTE-mediated redirected lysis of AML blasts and myeloid cells was observed in most but those patient samples that had a very low initial effector-to-target (E:T) cell ratio. To adress this problem, we stimulated autologous T cells in a first step by incubating AML samples in presence of 1ng/ml CD33 BiTE for 7 days. After purification of the expanded T cells we performed autologous cultures with high initial E:T ratio (1:1). This led to fast, effective and almost complete lysis of CD33+ target cells. Taken together, autologous T cells from AML patients are effectively engaged and activated by the novel CD33 BiTE and redirected for the elimination of myeloblasts at low picomolar concentrations in vitro and may thereby constitute a promising novel therapeutic option for the treatment of patients with CD33+ acute myeloid leukemia.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4622. doi:1538-7445.AM2012-4622