Anticancer activity of a humanized anti-VEGFR-1 monoclonal antibody and its derived antibody-drug conjugates targeting tumor cells and tumor-microenvironment components

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor for VEGF-A and placental growth factor (PlGF), involved in pathological angiogenesis, tumor invasiveness and infiltration by protumoral M2 macrophages. Aim of the study was to investigate the antitumor activity of the humanized anti-VEGFR-1 monoclonal antibody (mAb) hD16F7 and its antibody-drug conjugates (ADCs) derivatives. A unique property of this anti VEGFR-1 mAb is its ability of preserving the decoy/anti-angiogenic function of the soluble VEGFR-1, which sequesters VEGF-A/PlGF in the tumor microenvironment (TME). The humanized anti-VEGFR-1 mAb was initially tested for its ability to hamper migration of human melanoma, glioblastoma (GBM) and endothelial cells in response to PlGF. Thereafter, it was tested in patient-derived tumor xenografts (PDTXs) of melanoma and GBM organoids. Two anti-VEGFR-1 ADCs, derived from hD16F7 mAb by conjugation with monomethyl auristatin E (MMAE) or pyrrolobenzodiazepine (PBD), were also evaluated for their cytotoxic activity on VEGFR-1 expressing melanoma and GBM cells, as well as on cells of the TME (i.e., endothelial cells and M2 polarized macrophages). The hD16F7 mAb inhibited PlGF-induced migration of VEGFR-1 positive human melanoma (CR-Mel) and GBM (U87 and SJ-GBM) cell lines. Moreover, hD16F7 mAb strongly hampered migration of human endothelial cells (HUV-ST) in response to PlGF. Melanoma PDTXs were characterized for VEGFR-1 expression by RT-PCR and for the mutational status of BRAF gene by DNA sequencing. Treatment with hD16F7 mAb (10 mg/kg) significantly inhibited the growth of PDTXs transplanted in immunodeficient mice, both in the case of BRAF wild-type and mutated tumors and regardless of VEGFR-1 expression in melanoma cells. Indeed, hD16F7 mAb can also act on cells of the TME as it recognizes both the human and murine receptors. Moreover, in the case of BRAF mutated PDTXs, hD16F7 mAb also enhanced the efficacy of the BRAF inhibitor vemurafenib. The hD16F7-derived ADCs, hD16F7-MMAE and hD16F7-PBD, exerted a greater cytotoxicity on VEGFR-1 overexpressing melanoma cells (M14-MF5) than on their VEGFR-1 negative counterparts (M14-C2) with the following IC50 values: hD16F7-MMAE 5.84 μg/ml ± 0.59 vs 12.17 μg/ml ± 1.56; hD16F7-PBD 12.4 μg/ml ± 1.9 vs 19.2 μg/ml ± 0.8. The hD16F7-PBD also effectively hampered the viability of M2 macrophages and human endothelial cells (IC50 values: 5.8 µg/ml ± 0.3 and 12.2 µg/ml± 3.9, respectively). The cytotoxic effects of hD16F7-PBD were also demonstrated on GBM organoids by measuring the fluorescence intensity of calcein-AM (viable cells) and propidium iodide (dead cells). In conclusion, the humanized anti-VEGFR-1 mAb hD16F7 and its derived ADCs showed promising antitumor effects on preclinical tumor models that more closely reflect the clinical response.Funding: AIRC IG 2024-ID 30361 and Italian Ministry of Health NRR Plan, grant PNRR-MCNT2-2023-12377670 (CUP F93C24000250007)