T Lymphocytes Can Be Effectively Recruited for Ex Vivo and in Vivo Lysis of AML Blasts by a Novel CD33/CD3-Bispecific Bite Antibody Construct
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Leukemia (2013) 27, 1107–1115 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu ORIGINAL ARTICLE T lymphocytes can be effectively recruited for ex vivo and in vivo lysis of AML blasts by a novel CD33/CD3-bispecific BiTE antibody construct M Aigner1,3, J Feulner1,3, S Schaffer1, R Kischel2, P Kufer2, K Schneider2, A Henn2, B Rattel2, M Friedrich2, PA Baeuerle2, A Mackensen1 and SW Krause1 Patients with acute myelogenous leukemia (AML) are in high need of novel targeted therapies. Here we explored the ex vivo activity of AMG330, a novel T-cell-engaging BiTE (bi-specific T-cell engagers) antibody (Ab) construct, that is bispecific for the myeloid differentiation antigen, CD33 and CD3, in primary samples from AML patients (N ¼ 23) and AML cell lines. KG-1 and U937 cells were lysed in co-culture with healthy donor T-cells at AMG330 concentrations as low as 0.1 ng/ml (1.8 pM). T-cells derived from AML patient samples were found to be as active in redirected lysis by AMG330 as T-cells from healthy donors. In an autologous setting, AMG330 could activate and expand T-cells in primary AML patient samples, and effectively mediated the redirected lysis of AML blasts and normal myeloid cells. A deficiency in target-cell lysis was only observed in samples with very low initial effector-to-target (E:T) ratio. However, this could be overcome if previously stimulated autologous T-cells were tested in patient samples at a higher E:T ratio. In vivo experiments in immunodeficient mice demonstrated significant inhibition of tumor growth by AMG330 and an inducible infiltration of human T-cells into subcutaneous HL60 tumors. The activities of the CD33/CD3-bispecific BiTE Ab construct AMG330 warrant further development for the treatment of AML. Leukemia (2013) 27, 1107–1115; doi:10.1038/leu.2012.341 Keywords: bispecific antibodies; tumor immunology; cytotoxic T-cells; T-cell mediated immunity; CD33; acute myeloid leukemia INTRODUCTION A clinically validated format of bispecific Abs are the so called The search for new agents and new treatment modalities in acute BiTE (bi-specific T-cell engagers) molecules, which are based on 4 myeloid leukemia (AML) has been stimulated by the lack of two fused single-chain mAbs. A CD19/CD3-bispecific BiTE significant improvements in outcome over the last 10 years.1 construct called blinatumomab has demonstrated high anti- Among the several new agents that offer potential therapeutic use lymphoma and -leukemia activity at very low dose levels in 13–15 in AML, considerable attention has been focused on monoclonal clinical studies. BiTE Ab constructs specific for solid tumor antibodies (mAbs), which target antigens expressed primarily on targets EpCAM and CEA are currently under early clinical myeloid and derived-malignant cells including CD33, CD123 and investigation, and many others have been shown to have CD135. The frequent expression of CD33 on both AML blasts and potent activity in co-culture experiments and xenograft 16–18 AML stem cells has made it a target antigen of choice for models. By transiently tethering effector T-cells with target developing mAb-based approaches in the past.2,3 However, all cells, BiTE Abs elicit T-cell-mediated cellular cytotoxicity three major developments of CD33-targeting Abs in AML faced independent of the specificity of the T-cell receptor or the problems. The CD33-specific mAb drug conjugate (ADC), presence of MHC/peptide complexes on target cells. gemtuzumab ozogamicin (Mylotarg, Wyeth, NJ, USA), recently The mechanism of BiTE action has been shown to rely on the 17 lost market approval in the United States owing to an inferior risk/ activity of perforin, granzymes and caspases in target cells. Other benefit profile.4 Recent studies, however, showed potential for immunotherapeutic strategies for AML patients such as adoptive benefical use in distinct patient subpopulations.5,6 The humanized T-cell transfer and vaccination may prove difficult as many anti-CD33 IgG1 mAb, lintuzumab, showed insufficient efficacy in a immunological defects are known to occur in AML. For instance, phase-3 trial in combination with cytarabine,7 as did monotherapy loss of MHC class-I expression is frequent in AML cells, and some with the CD33-specific ADC, AVE9633, in a phase-1 study.8 studies indicate that virtually all AML blasts have defects in their 19,20 Therefore, for the treatment of AML, the target antigen CD33 antigen processing and presenting machinery. As BiTE Ab requires to be used with drug formats that have a more potent or constructs are known to induce strong proliferation and activation safer mode of action than IgG1 or ADCs, respectively. This may be of T-cells in a polyclonal fashion and lyse target cells achieved by mAb-based constructs that selectively engage independently of the antigen processing and presenting cytotoxic T- or natural killer (NK)-cells. A number of bi- and tri- machinery, a BiTE Ab construct specific for a myeloid-specific specific mAb constructs with this property are currently in early target antigen could provide for an alternative mode of action as development.9–12 needed for a successful targeted therapy of AML. Based on the 1Department of Internal Medicine 5—Hematology/Oncology, University of Erlangen-Nuernberg, Erlangen, Germany and 2Amgen Research (Munich) GmbH, Munich, Germany. Correspondence: Dr SW Krause, Department of Internal Medicine 5-Hematology/Oncology, University of Erlangen-Nu¨rnberg, Ulmenweg 18, Erlangen D-91054, Germany. E-mail: [email protected] 3These authors contributed equally to this study. Received 27 February 2012; revised 17 October 2012; accepted 15 November 2012; accepted article preview online 26 November 2012; advance online publication, 14 December 2012 Recruitment of T-cells for ex vivo and in vivo lysis of AML blasts by BiTEs M Aigner et al 1108 positive experience with blinatumomab in the treatment of NHL CD20 þ and CD56 þ cells. The resulting population consisted of 490% and ALL patients, we were prompted to apply this novel CD8 þ T-cells, and was used as effector cell population. therapeutic principle to the treatment of AML by developing a CD33/CD3-bispecific BiTE Ab construct called AMG330. Here we characterized the biological activities of AMG330 Chromium release assay in vitro with human AML cell lines, ex vivo with peripheral-blood The cytotoxic activity of T-cells was measured by a 19-h 51Cr release assay mononuclear cell samples from AML patients, and in vivo in an as described.11 Effector-to-target (E:T) ratios were calculated based on the immunodeficient mouse xenograft model. number of T-cells in the effector population in order to accurately compare different T-cell populations. 51Cr-labeled target cells were CD33 þ cell lines. MATERIALS AND METHODS Cell lines BiTE Ab constructs CD33-expressing cell lines, U937 and KG-1, were obtained from ATCC. Cells AMG330 is a fusion protein consisting of an N-terminal single-chain Ab were cultured in RPMI 1640 (PAN Biotech, Aidenbach, Germany) specific for CD33, and a C-terminally fused single-chain Ab specific for the 1 supplemented with 10% FCS (PAA, Pasching, Austria) at 37 C and 5% CO2. epsilon chain of CD3. AMG330 was constructed by recombinant DNA technologies as described in detail for several other BiTE Ab constructs.16,21 Primary cells Binding affinities for CD33 and CD3 are in the low nM-range, and the Mononuclear cells (MNCs) collected from healthy volunteers’ leukapheresis apparent molecular weight is 55 kDa. AMG330 was selected from a larger products or from AML patients’ peripheral blood or bone marrow were panel of CD33-specific BiTE Ab constructs based on its superior separated by density gradient centrifugation (PAN-Biotech, Aidenbach, pharmacological and pharmaceutical properties (to be reported Germany). Informed consent was obtained from patients according to the elsewhere). BiTE Ab constructs were produced by a stably transfected Declaration of Helsinki. The study was approved by the local ethics Chinese hamster ovary cell clone, and the monomeric form in cell culture committee. Cells were cryopreserved in medium containing 90% FCS (PAA, supernatants purified to homogeneity by a three-step purification. The Pasching, Austria) and 10% DMSO (Sigma, Munich, Germany). Character- Control BiTE consisted of the same human anti-CD3 single-chain Ab as istics of AML patients are shown in Supplementary Table 1. AMG330, but recognized with the second binding site a herbizide instead of CD33. It was also produced by a stably transfected CHO cell clone and purified to homogeneity by a three-step procedure. Cell cultures All in vitro cultures were performed in 96-well plates (Falcon, BD, Heidelberg, Germany), containing RPMI 1640 (PAN-Biotech, Aidenbach, Animal tumor studies Germany) supplemented with 10% human serum (PAN-Biotech, Aiden- Seven to eight-week-old NOD/SCID mice were obtained from Charles River bach, Germany) at 37 1C and 5% CO2. BiTEs were added only once at the beginning of the culture at the concentrations indicated. (L’Arbresle Cedex, France) and housed in sterilized cages. All mice were fed ssniff R/M-H food pellets (ssniff, Soest, Germany) sterilized by 25 kGy irradiation, and autoclaved drinking water ad libitum. Animals were Beads and normalization maintained on a 12-h/12-h light/dark cycle at 22±2 1C, and a relative To normalize the cell numbers between wells and over time during in vitro humidity between 34 and 73%. All procedures were performed in culture, 2 ml Cytometer Setup and Tracking Beads (BD, Heidelberg, accordance with the German Animal Protection Law and with permission Germany) were added to each well of the culture directly before cell from the responsible local authorities. Animals were irradiated with a harvest.