Abstract B070: Characterization of covariables modulating CD33/CD3 BITE® antibody construct mediated cytotoxicity against primary AML cells
Authors: Krupka Christina , Kufer Peter , Kischel Roman , Zugmaier Gerhard , Köhnke Thomas , Lichtenegger Felix , Altmann Torben , Spiekermann Karsten , Vick Binje , Jeremias Irmela , Hiddemann Wolfgang , Riethmüller Gert , Subklewe Marion 
Published: May 2016

Abstract
Antibody-based immunotherapy represents a promising strategy to target and eliminate chemoresistant leukemic cells in acute myeloid leukemia (AML). In our previous work, we were able to show effective elimination of primary AML cells by CD33/CD3 BiTE® antibody construct (AMG 330)-activated and -expanded residual autologous T cells. The goal of the present study was to characterize parameters that interfere with AMG 330-mediated lysis of AML cells. A long-term culture system supporting the growth of primary AML cells for up to 36 days ex vivo was used to determine covariables of AMG 330 cytolytic activity. One dominant variable of cytotoxicity was the expression level of the target antigen CD33 on primary AML cells. After 4 days of culture, a significantly higher lysis of target cells with CD33BRIGHT surface expression levels (n=38, p=0.01) was detected. However, after 12–15 days of culture, we could demonstrate similar lysis between samples with CD33DIM and CD33BRIGHT surface expression level, indicating that AMG 330 is also active at low antigen density (n=38, p=0.7). The CD33 expression level might not only be relevant in inter-patient comparison, but also in intra-patient comparison of AML subpopulations. In particular for CD34+/CD38- leukemia initiating cells (LICs), we have shown a significantly lower expression level of CD33 compared to AML bulk cells from the same donor {Krupka et al, Blood 2014}. To test if AMG 330 can also eliminate LICs, we performed in vivo engraftment experiments in NOD/SCID gamma null (NSG) mice. Patient-derived AML cells were co-cultured with healthy donor T-cells and either AMG 330 or control BiTE® antibody construct for 7 days. Residual CD3- cells were injected into NSG mice and monitored for AML outgrowth. Mice from the control group rapidly developed leukemia, and had to be sacrificed starting from 47 days post injection. In contrast, residual cells from AMG 330 treated cultures did not initiate leukemia in NSG mice in the entire observation period (6/6 mice, p=0.0003; n=2).
Another covariable in response to treatment with AMG 330 might be the induction of immune escape mechanisms due to continuous T-cell activation. The latter induces a proinflammatory environment, favoring the induction of immune checkpoint molecules on cancer cells. We therefore tested the relevance of immune checkpoint molecules in AMG 330 mediated lysis of primary AML cells. Although not constitutively expressed on primary AML cells (n=123), PD-L1 was strongly upregulated upon the addition of AMG 330 to ex vivo cytotoxicity experiments (p&lt;0.0001; n=27). This phenomenon was cytokine-mediated as the sole addition of IFN-γ and TNF-α also induced expression (n=6). Furthermore, we observed a significant upregulation of PD-1 on activated T cells (p=0.0002, n=18).
In ex vivo cytotoxicity experiments blocking of the PD-1/PD-L1 interaction significantly enhanced AMG 330 mediated lysis efficacy (n=9, p=0.03) which was accompanied by a significant increase in T cell proliferation (n=9, p=0.01) and a markedly increase in IFN-γ secretion (n=8, p=0.008).
In summary we demonstrate that CD33DIM cells are lysed with an equal efficacy compared to CD33BRIGHT cells through a prolongation in culture time. The data suggest that the relevance of the CD33 expression level can be compensated by an increased duration of AMG 330 exposure.
Our work further provides evidence that cytokine mediated PD-L1 upregulation is a relevant mechanism of primary AML cells to escape T cell driven immune responses. We demonstrate that AMG 330 mediated cytotoxicity is enhanced by blockade of inhibitory receptors on AML cells. Our results support the use of combinatorial approaches of BiTE® antibody constructs with immune checkpoint blockade.
Citation Format: Christina Krupka, Peter Kufer, Roman Kischel, Gerhard Zugmaier, Thomas Köhnke, Felix Lichtenegger, Torben Altmann, Karsten Spiekermann, Binje Vick, Irmela Jeremias, Wolfgang Hiddemann, Gert Riethmüller, Marion Subklewe. Characterization of covariables modulating CD33/CD3 BITE® antibody construct mediated cytotoxicity against primary AML cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B070.