Markus Muenz

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 55: Generation of half-life extended anti-CD33 BiTE® antibody constructs compatible with once-weekly dosing

T cell engaging bispecific antibody constructs (BiTE®), such as blinatumomab which targets CD19-positive cells, have shown great promise for treating certain CD19-positive hematological malignancies. Blinatumomab comprises a single chain Fv (scFv) that binds CD19 and a scFv that targets the T cell CD3 protein. The molecular weight of this “canonical” BiTE® is ~ 55 kDa, making it susceptible to kidney-mediated clearance and resulting in a short serum half-life (~ 4 hours). To maintain effective serum concentrations, canonical BiTE® antibody constructs must be administered by continuous IV (cIV) infusion. While there are many advantages associated with cIV administration (e.g., safety and uniform PK profile), patient convenience could be enhanced if the BiTE® antibody construct were compatible with once-weekly administration. To achieve this, the serum half-life of the BiTE® antibody construct would need to be extended. A canonical BiTE® targeting CD33 (AMG 330) is currently being evaluated in a phase I clinical trial. Like blinatumomab, AMG 330 is dosed cIV. To extend the serum half-life of AMG 330 and enable once-weekly dosing, several approaches were evaluated including fusion of AMG 330 to human albumin and Fc-containing moieties. Each of these half-life extended (HLE) constructs was evaluated in vitro, in mouse xenograft models and in non-human primates. In vitro assays evaluated 1) binding to both human and cynomolgus CD33 and CD3 proteins, and 2) cytotoxicity using human and cynomolgus target and effector cells. In each of these assays the canonical and HLE BiTE® antibody constructs demonstrated similar activity: single-digit nM binding and single digit pM cytotoxicity. Canonical and HLE BiTE® antibody constructs were subsequently evaluated in an orthotopic mouse model in which MOLM13 cells were administered IV and activated human T cells were administered IP two days later. The Fc-based HLE BiTE® antibody constructs provided a similar survival advantage when administered Q4D or Q5D as the canonical BiTE® when administered QD. However, the albumin fusion–based HLE BiTE® was less efficacious when administered Q4D than the QD- administered canonical BiTE®. Lastly, the PK/PD relationship was evaluated for each of the constructs in non-human primates. The serum half-lives varied from 6 hours for the canonical BiTE® to 44-167 hours for the HLE BiTE® antibody constructs. Each of the HLE BiTE® antibody constructs showed on-target depletion of CD33-positive monocytes and neutrophils in the blood and depletion of CD33-positive cells in the bone marrow. These data demonstrate that half-life extended BiTE® antibody constructs can be generated that retain comparable in vitro and in vivo activity as a canonical BiTE® and achieve a serum half-life compatible with once weekly dosing. Citation Format: Tara L. Arvedson, Mercedesz Balazs, Pamela Bogner, Kurt Black, Kevin Graham, Anja Henn, Matthias Friedrich, Patrick Hoffmann, Roman Kischel, Peter Kufer, Ralf Lutterbuese, Markus Muenz, Tobias Raum, Benno Rattel, Karen Rex, Dan Rock, Oliver Thomas, Joachim Wahl, Andreas Wolf, Angela Coxon. Generation of half-life extended anti-CD33 BiTE® antibody constructs compatible with once-weekly dosing [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 55. doi:10.1158/1538-7445.AM2017-55

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