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Abstract

The migration of effector lymphocytes from the blood stream into the tumor microenvironment is believed to be a crucial step for the anti-tumor defense of the immune system. A high density of infiltrating CD8+ T cells in the tumor bed has been correlated with a better prognosis in a variety of malignancies. Poorly infiltrated tumors often show an abnormal and dysfunctional vasculature with reduced expression of adhesion molecules such as E-selectin, ICAM1/2 and VCAM1 that are involved in the extravasation of immune cells. Therefore, it seems desirable to develop new strategies leading to tumor vessel activation that consequently can increase the transendothelial migration of effector T cells into the tumor. We hypothesized that tumor endothelial cell activation could be facilitated either by direct activation through soluble factors or indirectly by T cell activation in contact with endothelial cells. In the direct activation approach, recombinant bifunctional VEGFR2-binding fusion proteins were used to deliver activating cytokines to vascular endothelial cells. This targeted delivery of cytokines, that could be useful for future in vivo approaches, successfully achieved up-regulation of adhesion molecules, E-selectin, ICAM1 and VCAM1, and consequently increased CD3+ T cell binding to activated human umbilical vein endothelial cells (HUVEC). In the T cell activation approach, as an indirect means to activate vascular endothelium, tetravalent bispecific monoclonal antibodies (BiMAb) in the (scFv-Fc-scFv)2 format were used. These BiMAb bind VEGFR2 or TIE2 endothelial cell (EC) growth factor receptors with the N-terminal single-chain variable fragment (scFv) antibodies, and the stimulatory/co-stimulatory T cell molecules CD3ε or CD28 with the C-terminal scFv antibodies. Local CD3ε-mediated T cell activation is expected to result in the release of cytokines (e.g., TNF-α and IFN-γ) that in turn induce the expression of adhesion molecules in endothelial cells. Antibody-mediated blocking of VEGF binding to VEGFR2 could concomitantly exert anti-angiogenic effects. Also this approach could allow the in situ activation of endothelial cells within tumor tissues. To assess the activation capacity of self-produced bispecific antibodies, CD3+ T cells were co-cultured with HUVEC in the presence of BiMAbs for 24 h. The treatment with αVEGFR2–αCD3 BiMAb resulted in a strong T cell activation as well as up-regulation of adhesion molecules on endothelial cells, as detected by cell surface staining and cytokine ELISA. This effect was slightly enhanced by the addition of an αTIE2–αCD28 BiMAb. A transwell assay was established to assess the migration capacity of T cells through an EC monolayer in the presence or absence of BiMAbs. Compared to a monospecific αVEGFR2 scFv-Fc control antibody that elicited no EC or T cell activation, the migration of T cells was significantly increased in the presence of αVEGFR2–αCD3 BiMAb. Although additional treatment with the co-stimulatory αTIE2–αCD28 BiMAb augmented T cell activation, the migration rate of T cells was not increased. In order to prioritize CD8+ T cell transmigration αEC antigen–αCD8 BiMAbs were tested in combination with stimulatory and/or co-stimulatory BiMAbs mentioned above, however, without significant changes in the migration rate of cytotoxic T cells. To study the killing capacity of transmigrated T cells against tumor cells, transmigrated T cells were transferred onto a monolayer of MCF-7 breast cancer cells in the presence or absence of BiMAbs mediating tumor cell targeting. Tumor cell killing was quantified using an LDH release assay. We observed that T cells that had been pre-activated with αVEGFR2–αCD3 and subsequently traversed an EC monolayer, still required the subsequent addition of tumor-reactive αHER2–αCD3 BiMAb in order to become cytotoxic. Killing was more prominent if migrated cells were pre-treated with a combination of stimulatory and co-stimulatory BiMAbs, αVEGFR2–αCD3 and αPD-L1–αCD28. In conclusion, T cells activated by αVEGFR2–αCD3 BiMAb can indirectly activate EC in situ resulting in a better T cell migration. This treatment can be combined with other BiMAbs that target T cells towards tumor cells and induce tumor cell killing.