Tobias Raum

Preclinical Characterization of AMG 330, a CD3/CD33-Bispecific T-Cell–Engaging Antibody with Potential for Treatment of Acute Myelogenous Leukemia

There is high demand for novel therapeutic options for patients with acute myelogenous leukemia (AML). One possible approach is the bispecific T-cell–engaging (BiTE, a registered trademark of Amgen) antibody AMG 330 with dual specificity for CD3 and the sialic acid–binding lectin CD33 (SIGLEC-3), which is frequently expressed on the surface of AML blasts and leukemic stem cells. AMG 330 binds with low nanomolar affinity to CD33 and CD3ϵ of both human and cynomolgus monkey origin. Eleven human AML cell lines expressing between 14,400 and 56,700 CD33 molecules per cell were all potently lysed with EC50 values ranging between 0.4 pmol/L and 3 pmol/L (18–149 pg/mL) by previously resting, AMG 330–redirected T cells. Complete lysis was achieved after 40 hours of incubation. In the presence of AML cells, AMG 330 specifically induced expression of CD69 and CD25 as well as release of IFN-γ, TNF, interleukin (IL)-2, IL-10, and IL-6. Ex vivo, AMG 330 mediated autologous depletion of CD33-positive cells from cynomolgous monkey bone marrow aspirates. Soluble CD33 at concentrations found in bone marrow of patients with AML did not significantly affect activities of AMG 330. Neoexpression of CD33 on newly activated T cells was negligible as it was limited to 6% of T cells in only three out of ten human donors tested. Daily intravenous administration with as low as 0.002 mg/kg AMG 330 significantly prolonged survival of immunodeficient mice adoptively transferred with human MOLM-13 AML cells and human T cells. AMG 330 warrants further development as a potential therapy for AML. Mol Cancer Ther; 13(6); 1549–57. ©2014 AACR.

Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110.

Background Bispecific T cell engager (BiTE®) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells. Methods The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells. Findings An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-βand PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not. Conclusions Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct.

Bispecific T cell engaging antibody constructs targeting a universally conserved part of the viral M2 ectodomain cure and prevent influenza A virus infection

ABSTRACT The ectodomain of the influenza A matrix protein 2 (M2e) is highly conserved amongst all influenza virus A subtypes. M2e is present on the surface of influenza A virus‐infected cells, and therefore a suitable target for broadly protective therapies. We designed bispecific T cell engaging (BiTE®) antibody constructs specific for M2e by genetically fusing a single chain variable fragment (scFv) derived from an M2e‐specific murine monoclonal antibody with a CD3&egr;‐specific scFv. These so‐called FLU BiTE® antibody constructs selectively mediate T cell dependent lysis of M2‐expressing and influenza A virus infected cells and protect BALB/c mice against challenge with different influenza A virus subtypes. By humanizing the M2e‐binding scFv, we generated human‐like FLU BiTE® antibody constructs, with increased in vitro cytotoxic activity and in vivo protective capacity against influenza A virus infection. FLU BiTE® antibody constructs represent a promising new curative and prophylactic treatment option for influenza disease. HighlightsBispecific T cell engaging (FLU BiTE®) antibody constructs targeting the M2 ectodomain of influenza A were constructed.FLU BiTE® antibody constructs show strong cytotoxic activity to M2‐expressing cells in vitro.In vivo, FLU BiTE® antibody constructs protect mice against influenza A virus, providing memory T cells are present.Humanized FLU BiTE® antibody constructs display increased affinity for M2e, resulting in stronger protective effects.

Abstract 585: Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy

BiTE® therapeutics are single chain antibody constructs harboring two binding moieties: one directed at a tumor associated antigen and one directed at the CD3e protein, which trigger T cell dependent cellular cytotoxicity (TDCC) against targeted cancer cells. Here, we evaluated the suitability of ENPP3 as a potential BiTE® target. The ENPP3 mRNA is highly differentially expressed in clear cell Renal Cell Carcinoma (ccRCC) and the ENPP3 protein is detected with high uniformity and intensity in ccRCC tumor samples by immunohistochemistry. We demonstrated the surface expression of this protein in Renal Cancer cell lines and confirmed that the ENPP3 protein was highly restricted to the luminal side of normal epithelial layers in which it was detected (proximal renal tubules, bronchial epithelium, salivary glands). We developed highly potent anti-ENPP3 BiTE® molecules and demonstrated the in vitro TDCC activity of these molecules. Two anti-ENPP3 BiTE® molecules further demonstrated tumor formation inhibition activity in a human T cell / human target cell admixture mouse xenograft model. Citation Format: Olivier Nolan-Stevaux, Flordeliza Fajardo, Lily Liu, Suzanne Coberly, Patricia McElroy, Aaron Nazarian, Franziska Bott, Elisabeth Nahrwold, Tobias Raum, Roman Kischel, Ralf Lutterbuese, Patrick Hoffmann, Peter Kufer. Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 585.

Abstract 3632: BiTE® antibody constructs for the treatment of SCLC

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor with a poor prognosis and limited therapeutic options. Most patients present with extensive stage disease, where recent advances in immunotherapies, including bi-specific T cell engager (BiTE®) antibody constructs, represent a promising new therapeutic approach. BiTE® molecules have demonstrated durable complete responses in the clinic in hematological malignancies. Similar to hematological malignancies, SCLC is also a widely-disseminated malignancy that shows very high response rate to first line therapies with high rates of disease recurrence, features which may support efficacy of the BiTE® modality. Next generation sequencing (NGS) identified Delta-like Ligand 3 (DLL3) as a highly specific tumor associated antigen for SCLC, with consistent expression in tumors and very low expression in normal tissues. Tumor expression of DLL3 protein was confirmed by IHC, with 30 of 35 SCLC tumors staining positive for DLL3. DLL3 BiTE® antibody constructs showed low pM potency in vitro and also demonstrated significant inhibition of tumor growth in vivo in an orthotopic model of SCLC. A half-life extended (HLE) BiTE® targeting DLL3 demonstrated antibody-like pharmacokinetic properties in single-dose studies in non-human primates (NHP), with a half-life of 11 days. This is predicted to support every other week dosing in humans. The combination of high potency and excellent PK properties suggests that HLE BiTE® molecules may provide a useful tool for targeting residual disease in SCLC patients whose tumors express DLL3. Citation Format: Michael J. Giffin, Ed K. Lobenhofer, Keegan Cooke, Tobias Raum, Jennitte Stevens, Pedro J. Beltran, Angela Coxon, Paul E. Hughes. BiTE® antibody constructs for the treatment of SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3632. doi:10.1158/1538-7445.AM2017-3632

Abstract 4734: Impact of various immune escape mechanisms on redirected lysis by T cells engaged via EpCAM/CD3 bispecific BiTE® antibody AMG 110 .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC BiTE antibodies are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They are capable of temporarily mounting a polyclonal T cell response that is not restricted by T cell receptor specificity, presence of MHC class I, generation and presentation of peptide antigen, or the addition of T cell co-stimuli. Examples are the CD19/CD3-bispecific BiTE antibody blinatumomab, which has been reported to show high response rates in leukemia and lymphoma patients, and EpCAM/CD3-bispecific BiTE antibody solitomab (AMG 110), which is in a dose-escalating phase 1 study in patients with solid tumors. Because specific T cell responses against cancer cells are frequently hampered by a variety of immune escape mechanisms, we have here assessed to what extent surface expression of PD-L1 (B7-H1) and CD73, cytoplasmic expression of IDO and serpin PI-9, and secretion of TGF-s and IL-10 can protect target cells from redirected lysis by AMG 110-engaged T cells. The six human proteins, which are all known to be associated with immune escape of cancer cells by inhibition of T cell function, were stably expressed in human EpCAM-expressing Chinese hamster ovary (CHO) cells. In all cases, expression levels of the six proteins in stable CHO cell lines exceeded those found in human cancer cell lines. Characterized CHO cell lines and the parental EpCAM+ CHO line were used in co-culture assays as target cells for analysis of EC50 values for redirected lysis, and induction of proliferation of resting human peripheral T cells in the presence of various AMG 110 concentrations. High level cytosolic expression of the tryptophan metabolizing enzyme IDO had no effect on redirected lysis but reduced AMG 110-induced T cell proliferation, while expression of the granzyme inhibitor PI-9 increased the EC50 value for redirected lysis but had no effect on T cell proliferation. High level expression of the membrane-bound ligand for PD-1, PD-L1, and of secreted IL-10 and TGF-β all had no impact on AMG 110-induced T cell proliferation. The EC50 value for redirected lysis was however impacted by expression of TGF-s and PD-L1 as determined by a FACS-based cytotoxicity assays. In no case, transfected CHO cell lines became completely resistant to BiTE-induced lysis despite expressing the immune escape proteins at higher levels than observed in tumor cell lines. Our data suggest that BiTE-engaged T cells are relatively insensitive to proteins expressed by cancer cells to fend off T cells, and that inhibitory effects can be compensated by higher concentrations of the BiTE antibody. Citation Format: Wibke Deisting, Tobias Raum, Peter Kufer, Patrick A. Baeuerle, Markus Muenz. Impact of various immune escape mechanisms on redirected lysis by T cells engaged via EpCAM/CD3 bispecific BiTE® antibody AMG 110 . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4734. doi:10.1158/1538-7445.AM2013-4734