Christiane Jaeger

Development of tetravalent IgG1 dual targeting IGF-1R–EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.

Abstract 2481: CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors

T cell bispecific antibodies (TCBs) are potent molecules that upon simultaneous binding to tumor cells and T cells trigger strong T cell activation resulting in the killing of tumor cells. CEA TCB (RG7813) is a novel bispecific antibody targeting carcinoembryonic antigen (CEA), often overexpressed on solid tumors (e.g. colorectal, gastric, pancreatic, lung carcinoma etc.), and the CD3 epsilon chain present on T cells. CEA TCB bears several innovative technological features that distinguish it from other bispecific antibodies currently in (pre-)clinical development: (a) bivalency for tumor antigen translating into higher avidity, superior potency and better differentiation between high and low antigen-expressing cells; (b) head-to-tail fusion geometry for anti-tumor and CD3-binding domains, resulting in higher potency compared to conventional IgG-based TCBs; (c) extended half-life compared to non-Fc-based TCBs; (d) fully silent Fc ensuring lower risk of FcgR-mediated infusion reactions; and (e) robust production using standard manufacturing processes (enabled by “CrossMAb” and knob-into-hole bispecific antibody technologies). In vitro, CEA TCB mediates potent target-dependent T cell cytotoxicity, T cell activation, proliferation, and cytokine release in killing assays, exclusively in the presence of CEA-expressing target-cells. CEA TCB activity correlates with CEA expression level, showing higher potency against tumor cells with high expression of CEA. In vivo, CEA TCB induces dose- and time-dependent regression of CEA-expressing tumors with variable amounts of immune cell infiltrate. In fully humanized NOG mice, CEA TCB is efficacious in poorly-infiltrated tumors and converts non-inflamed into highly-inflamed tumors. Histological and FACS analyses revealed that CEA TCB recruits new T cells into tumors and/or expands pre-existing ones and is able to induce T cell re-localization from the tumor periphery into the tumor bed. Surprisingly, CEA TCB treatment also qualitatively alters the composition of intratumoral T cells resulting in an increased frequency of activated (CD69, CD25), proliferating (Ki67) and differentiated T cells (having effector memory phenotype) that are ready to kill (express high levels of Granzyme B). Taken together, these preclinical data show that CEA TCB is a novel tumor-targeted T cell bispecific antibody with promising anti-tumor activity and the novel ability to modify the tumor microenvironment. Phase 1 clinical trials with CEA TCB are currently ongoing. Future studies will focus on identification of combination partners that inhibit T cell suppression and unleash the full potential of T cell activity. Citation Format: Marina Bacac, Tanja Fauti, Sara Colombetti, Johannes Sam, Valeria Nicolini, Nathalie Steinhoff, Oliver Ast, Peter Bruenker, Ralf Hosse, Thomas Hofer, Ekkehard Moessner, Christiane Jaeger, Jose Saro, Vaios Karanikas, Christian Klein, Pablo Umana. CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2481. doi:10.1158/1538-7445.AM2015-2481