Abstract
Although CD3 T cell redirecting antibodies have been successfully utilized for the treatment of hematological malignancies (blinatumomab), the T cell signaling pathways induced by these molecules are incompletely understood. To gain insight into the mechanism of action for T cell redirection antibodies, we created a novel murine CD3xEpCAM bispecific antibody that incorporates a silent Fc to dissect function and signaling of murine CD8 OT1 T cells upon stimulation. T cell-mediated cytotoxicity, cytokine secretion, expression of activation markers, and proliferation were directly induced in T cells treated with the novel CD3xEpCAM bispecific molecule in vitro in the presence of epithelial cell adhesion molecule (EpCAM) expressing tumor cells. Nanostring analysis showed that CD3xEpCAM induced a gene expression profile that resembled antigen-mediated activation, although the magnitude was lower than that of the antigen-induced response. In addition, this CD3xEpCAM bispecific antibody exhibited in vivo efficacy. This is the first study that investigates both in vitro and in vivo murine CD8 T cell function and signaling induced by a CD3xEpCAM antibody having a silent Fc to delineate differences between antigen-independent and antigen-specific T cell activation. These findings expand the understanding of T cell function and signaling induced by CD3 redirection bispecific antibodies and may help to develop more efficacious CD3 redirection therapeutics for cancer treatment, particularly for solid tumors.
Highlights
C D3 redirection bispecific antibodies represent a cancer immunotherapy strategy that redirects T cells to kill tumor cells regardless of antigen specificity through direct engagement of CD3e.(1–4) An example of successful clinical application of a bispecific T cell redirection antibody is the CD3xCD19 bispecific T cell engager (BiTE), blinatumomab,(5,6) which was approved by the Food and Drug Administration for treatment of acute lymphoblastic leukemia in 2014
The immune synapse triggered by the CD3 redirection bispecific antibodies resembles that induced by cognate antigen in the MHC/TCR interaction[1] in that clustering of CD3, Lck, and perforin takes place as well as ZAP70 translocation and CD45 exclusion.[14]. Subsequently, T cell proliferation is induced and is accompanied by acquisition of cytotoxic effector function via the perforin/granzyme B pathway supporting the hypothesis that the therapeutic effect of CD3 redirection bispecific antibodies is due to T cell activation, as has been demonstrated in vitro using T cell lines and primary T cells.[1,5,14,15,16] suggestive, the majority of these
Studies focused on synapse comparison for MHC/TCRactivated versus CD3-redirection-bispecific-antibody-activated T cell lines[1,14] rather than on bispecific antibody activation in primary T cells.[12,16,17] Only a few studies address T cell function mediated by CD3 redirection antibody compared with MHC/TCR-activated primary CD8 T cells.[12,16,17] To address this, a novel CD3xEpCAM T cell redirection bispecific antibody composed of an anti-mouse CD3e paired with an anti-human epithelial cell adhesion molecule (EpCAM) binding arm and a functionally silent Fc has been constructed to characterize murine CD8 T cell activation and function in the absence of Fc effector function
Summary
C D3 redirection bispecific antibodies represent a cancer immunotherapy strategy that redirects T cells to kill tumor cells regardless of antigen specificity through direct engagement of CD3e.(1–4) An example of successful clinical application of a bispecific T cell redirection antibody is the CD3xCD19 bispecific T cell engager (BiTE), blinatumomab,(5,6) which was approved by the Food and Drug Administration for treatment of acute lymphoblastic leukemia in 2014. Another T cell redirection antibody CD3xEpCAM, catumaxomab, was previously approved for clinical use in Europe in 2009, it has since been withdrawn from the market.[7,8] There are currently 45(9) CD3-based T cell redirection bispecific antibodies including BCMAxCD3, Her2xCD3, CEAxCD3, and PSMAxCD3(10–12) being tested in clinical trials for treatment of solid and hematological tumors. There is only one other CD3 redirection molecule with a silent Fc[17] amenable for use in syngeneic tumor models with wild-type mice
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