The EGFR and TGFβ signaling pathways are important mediators of tumorigenesis, and cross-talk between them contributes to cancer progression and drug resistance. Therapies capable of simultaneously targeting EGFR and TGFβ could help improve patient outcomes across various cancer types. Here, we developed BCA101, an anti-EGFR IgG1 mAb linked to an extracellular domain of human TGFβRII. The TGFβ “trap” fused to the light chain in BCA101 did not sterically interfere with its ability to bind EGFR, inhibit cell proliferation, or mediate antibody-dependent cellular cytotoxicity. Functional neutralization of TGFβ by BCA101 was demonstrated by several in vitro assays. BCA101 increased production of proinflammatory cytokines and key markers associated with T-cell and natural killer–cell activation, while suppressing VEGF secretion. In addition, BCA101 inhibited differentiation of naïve CD4+ T cells to inducible regulatory T cells (iTreg) more strongly than the anti-EGFR antibody cetuximab. BCA101 localized to tumor tissues in xenograft mouse models with comparable kinetics to cetuximab, both having better tumor tissue retention over TGFβ “trap.” TGFβ in tumors was neutralized by approximately 90% in animals dosed with 10 mg/kg of BCA101 compared with 54% in animals dosed with equimolar TGFβRII-Fc. In patient-derived xenograft mouse models of head and neck squamous cell carcinoma, BCA101 showed durable response after dose cessation. The combination of BCA101 and anti-PD1 antibody improved tumor inhibition in both B16-hEGFR–expressing syngeneic mouse models and in humanized HuNOG-EXL mice bearing human PC-3 xenografts. Together, these results support the clinical development of BCA101 as a monotherapy and in combination with immune checkpoint therapy. Significance: The bifunctional mAb fusion design of BCA101 targets it to the tumor microenvironment where it inhibits EGFR and neutralizes TGFβ to induce immune activation and to suppress tumor growth.

The incidence of aggressive and resistant breast cancers is growing at alarming rates, indicating a necessity to develop better treatment strategies. Recent epidemiological and preclinical studies detected low serum levels of vitamin D in cancer patients, suggesting that vitamin D may be effective in mitigating the cancer burden. However, the molecular mechanisms of vitamin D3 (cholecalciferol, vit-D3)-induced cancer cell death are not fully elucidated. The vit-D3 efficacy of cell death activation was assessed using breast carcinoma cell lines in vitro and a widely used Ehrlich ascites carcinoma (EAC) breast cancer model in vivo in Swiss albino mice. Both estrogen receptor-positive (ER+, MCF-7) and -negative (ER-, MDA-MB-231, and MDA-MB-468) cell lines absorbed about 50% of vit-D3 in vitro over 48 h of incubation. The absorbed vit-D3 retarded the breast cancer cell proliferation in a dose-dependent manner with IC50 values ranging from 0.10 to 0.35 mM. Prolonged treatment (up to 72 h) did not enhance vit-D3 anti-proliferative efficacy. Vit-D3-induced cell growth arrest was mediated by the upregulation of p53 and the downregulation of cyclin-D1 and Bcl2 expression levels. Vit-D3 retarded cell migration and inhibited blood vessel growth in vitro as well as in a chorioallantoic membrane (CAM) assay. The intraperitoneal administration of vit-D3 inhibited solid tumor growth and reduced body weight gain, as assessed in mice using a liquid tumor model. In summary, vit-D3 cytotoxic effects in breast cancer cell lines in vitro and an EAC model in vivo were associated with growth inhibition, the induction of apoptosis, cell cycle arrest, and the impediment of angiogenic processes. The generated data warrant further studies on vit-D3 anti-cancer therapeutic applications.

Breast cancer (BC) continues to be one of the major causes of cancer deaths in women. Progress has been made in targeting hormone and growth factor receptor-positive BCs with clinical efficacy and success. However, little progress has been made to develop a clinically viable treatment for the triple-negative BC cases (TNBCs). The current study aims to identify potent agents that can target TNBCs. Extracts from microbial sources have been reported to contain pharmacological agents that can selectively inhibit cancer cell growth. We have screened and identified pigmented microbial extracts (PMBs) that can inhibit BC cell proliferation by targeting legumain (LGMN). LGMN is an oncogenic protein expressed not only in malignant cells but also in tumor microenvironment cells, including tumor-associated macrophages. An LGMN inhibition assay was performed, and microbial extracts were evaluated for in vitro anticancer activity in BC cell lines, angiogenesis assay with chick chorioallantoic membrane (CAM), and tumor xenograft models in Swiss albino mice. We have identified that PMB from the Exiguobacterium (PMB1), inhibits BC growth more potently than PMB2, from the Bacillus subtilis strain. The analysis of PMB1 by GC-MS showed the presence of a variety of fatty acids and fatty-acid derivatives, small molecule phenolics, and aldehydes. PMB1 inhibited the activity of oncogenic legumain in BC cells and induced cell cycle arrest and apoptosis. PMB1 reduced the angiogenesis and inhibited BC cell migration. In mice, intraperitoneal administration of PMB1 retarded the growth of xenografted Ehrlich ascites mammary tumors and mitigated the proliferation of tumor cells in the peritoneal cavity in vivo. In summary, our findings demonstrate the high antitumor potential of PMB1.