Angiogenesis, the sprouting of capillaries from pre-existing blood vessels, is a fundamental process in the formation of the vascular system during embryonic development. In adulthood, angiogenesis takes place during corpus luteum formation and in pathological conditions such as wound healing, diabetic retinopathy, and tumor-igenesis. Vascularization is essential for solid tumour growth and is thought to be regulated by tumour cell-produced factors, which have a chemotactic and mitogenic effect on endothelial cells. Vascular endothelial growth factor (VEGF), a homodimeric glycoprotein of relative molecular mass 45,000, is the only mitogen, however, that specifically acts on endothelial cells, and it may be a major regulator of tumour angiogenesis in vivo. Its expression has been shown to be upregulated by hypoxia, and its cell-surface receptor, Flk-1, is exclusively expressed in endothelial cells. Here we investigate the biological relevance of the VEGF/Flk-1 receptor/ligand system for angiogenesis using a retrovirus encoding a dominant-negative mutant of the Flk-1/VEGF receptor to infect endothelial target cells in vivo, and find that tumour growth is prevented in nude mice. Our results emphasize the central role of the Flk-1/VEGF system in angiogenesis in general and in the development of solid tumours in particular.

BackgroundCheckpoint inhibitors as well as adoptive cell therapy hold great promise for cancer therapy and encouraging treatment responses have already been demonstrated in different cancer indications. Glioblastoma (GB) is the most common and aggressive primary brain tumor. Standard therapy has very limited efficacy in the majority of patients. Analysis of the GB tumor microenvironment (TME) has shown prominent immunosuppressive features including expression of PD-L1 on tumor cells and increased frequency of FOX-P3 positive regulatory T cells. While the surrounding brain is HER2-negative, GB tumors are frequently HER2-positive, suggesting HER2 as a promising target for adoptive immunotherapy. Previous results from mouse glioma models showed efficacy of CAR-NK cells (NK-92/5.28.z) targeted against HER2 as monotherapy with relatively small tumors, but not with advanced late-stage tumors.Materials and MethodsThe murine glioma cell line GL261 was transfected with HER2. Tumor cells were implanted either subcutaneously or orthotopically into C57BL/6 mice and treated either with HER2-specific NK-92/5.28.z cells alone or in combination with an anti-PD-1 antibody. Effects on tumor growth and survival were determined. Lymphocyte infiltration and immunosuppressive TME were characterized in high-dimensional high-throughput analysis via RNAseq and multiplex IHC.ResultsCombined treatment with NK-92/5.28.z cells and anti-PD-1 checkpoint blockade resulted in synergistic effects with tumor regression and long-term survival even of advanced-stage tumor bearing mice. Analysis of TME showed enhanced cytotoxic lymphocyte infiltration and altered profiles of exhaustion markers in tumor and immune cells, leading to an altered TME after combined treatment with NK-92/5.28.z cells and anti-PD-1 antibody.ConclusionsThese data demonstrate that efficacy of NK-92/5.28.z cells can be enhanced in combination with checkpoint blockade, resulting in successful treatment of advanced tumors refractory to NK-92/5.28.z monotherapy. Furthermore, the combination therapy induces a cytotoxic rather than immunosuppressive TME, leading to a primed immune system. To address this question in a clinical setting, we are preparing a combination therapy cohort as part of our ongoing phase I clinical study (CAR2BRAIN; NCT03383978).Disclosure InformationF. Strassheimer: None. M.I. Strecker: None. T. Alekseeva: None. J. Macas: None. M.C. Demes: None. I.C. Mildenberger: None. T. Tonn: None. P.J. Wild: None. L. Sevenich: None. Y. Reiss: None. P.N. Harter: None. K.H. Plate: None. W.S. Wels: None. J.P. Steinbach: None. M.C. Burger: None.

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The tumor microenvironment in brain metastases is characterized by high myeloid cell content associated with immune suppressive and cancer-permissive functions. Moreover, brain metastases induce the recruitment of lymphocytes. Despite their presence, T-cell-directed therapies fail to elicit effective anti-tumor immune responses. Here, we seek to evaluate the applicability of radioimmunotherapy to modulate tumor immunity and overcome inhibitory effects that diminish anti-cancer activity. Radiotherapyinduced immune modulation resulted in an increase in cytotoxic T-cell numbers and prevented the induction of lymphocyte-mediated immune suppression. Radio-immunotherapy led to significantly improved tumor control with prolonged median survival in experimental breast-to-brain metastasis. However, long-term efficacy was not observed. Recurrent brain metastases showed accumulation of blood-borne PD-L1 + myeloid cells after radio-immunotherapy indicating the establishment of an immune suppressive environment to counteract re-activated T-cell responses. This finding was further supported by transcriptional analyses indicating a crucial role for monocyte-derived macrophages in mediating immune suppression and regulating T-cell function. Therefore, selective targeting of immune suppressive functions of myeloid cells is expected to be critical for improved therapeutic efficacy of radio-immunotherapy in brain metastases.