Purpose: CEA TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody, engaging CD3e upon binding to carcinoembryonic antigen (CEA) on tumor cells. Containing an engineered Fc region, conferring an extended blood half-life while preventing side effects due to activation of innate effector cells, CEA TCB potently induces tumor lysis in mouse tumors. Here we aimed to characterize the pharmacokinetic profile, the biodistribution, and the mode of action of CEA TCB by combining in vitro and in vivo fluorescence imaging readouts.Experimental Design: CEA-expressing tumor cells (LS174T) and human peripheral blood mononuclear cells (PBMC) were cocultured in vitro or cografted into immunocompromised mice. Fluorescence reflectance imaging and intravital 2-photon (2P) microscopy were employed to analyze in vivo tumor targeting while in vitro confocal and intravital timelapse imaging were used to assess the mode of action of CEA TCB.Results: Fluorescence reflectance imaging revealed increased ratios of extravascular to vascular fluorescence signals in tumors after treatment with CEA TCB compared with control antibody, suggesting specific targeting, which was confirmed by intravital microscopy. Confocal and intravital 2P microscopy showed CEA TCB to accelerate T-cell-dependent tumor cell lysis by inducing a local increase of effector to tumor cell ratios and stable crosslinking of multiple T cells to individual tumor cells.Conclusions: Using optical imaging, we demonstrate specific tumor targeting and characterize the mode of CEA TCB-mediated target cell lysis in a mouse tumor model, which supports further clinical evaluation of CEA TCB.

2Abbreviations: 2D, two-dimensional; 3D, three-dimensional; AFS, atomic force spectroscopy; ECM, extracellular matrix; H2B, histone-2B; MMP matrix metalloproteinase; NII, nuclear irregularity index; NT, non-targeting control; PDMS, polydimethylsiloxane SummaryNuclear deformability during cancer cell invasion and metastasis is critically regulated by lamin A. Here, researchers showed that lamin B2 also contributes to nuclear mechanics, implicating cooperating lamin networks regulating nuclear integrity, migration efficacy, and metastatic tumor progression. AbstractInterstitial tumor cell invasion depends upon complex mechanochemical adaptation of both cell body and the rigid nucleus in response to extracellular tissue topologies. Nuclear mechanics during cell migration through confined environments is controlled by A-type lamins, however, the contribution of B-type lamins to the deformability of the nucleus remains unclear. Using systematic expression regulation of different lamin isoforms, we applied multi-parameter wet-lab and in silico analysis to test their impact on nuclear mechanics, shape regulation, and cancer cell migration. Modulation of lamin A/C and B2 but not B1 isoforms controlled nuclear deformation and viscoelasticity, whereby lamin B2 generally followed lamin A/C-mediated effects. Cell migration rates were altered by 5 to 9fold in dense collagen environments and synthetic devices, with accelerated rates after lamin downregulation and reverse effects after lamin upregulation, with migration rates strongly depending on nuclear shape change. These findings implicate cooperation of lamin B2 with lamin A/C in regulating nuclear mechanics for shape adaptation and migration efficacy.

<p>2P time-lapse acquisition of LS174T / human PBMC cografts on day 4 after implantation (baseline), 24 h after control and 24 h after CEA TCB treatment. LS174T RFP tumor cells (red), CFSE labeled T-cells (green). Scale bar indicates 50 μm.</p>