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Despite the promising clinical benefits of therapies targeting epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF) with antibodies in various cancers, resistance to these therapies will inevitably develop following treatment. Recent studies suggest that crosstalk between the EGFR and VEGF signaling pathways might be involved in the development of resistance. Therefore, simultaneous blockade of EGFR and VEGF signaling may be able to counteract this resistance and improve clinical outcomes. Here, we devised a fusion protein with two copies of VEGFR1 domain 2 connected to the C-terminus of cetuximab that can simultaneously bind to EGFR and VEGF and effectively inhibit target cell growth mediated by these two pathways. Furthermore, the fusion protein could bring soluble VEGF into target cells for degradation through internalization upon binding to EGFR. Tissue distribution in mice confirmed that the fusion protein effectively accumulated in tumors compared to its mAb counterpart cetuximab. These features resulted in stronger antitumor efficacies in vivo than the combination of bevacizumab and cetuximab. Thus, we provide a promising new strategy for the treatment of EGFR-overexpressing cancers.

Targeting PD1/PDL1 with blocking antibodies for cancer therapy has shown promising benefits in the clinic, but only approximately 20–30% of patients develop durable clinical responses to the treatment. Bispecific antibodies (BsAbs) that combine PD1/PDL1 blockade with the modulation of another immune checkpoint target may have greater potential to enhance immune checkpoint blockade therapy. In this study, we identified an anti-PD1 monoclonal antibody, 609A, whose heavy chain can pair with a variety of light chains from different antibodies while maintaining its PD1 binding/blocking activity. Taking advantage of this property and using a linear F(ab’)2 format, we successfully produced a series of tetravalent IgG-like BsAbs that simultaneously target PD1 and other immune checkpoint targets, including PDL1 and CTLA4. The BsAbs exhibited superior bioactivities in vitro and in vivo compared to their respective parental mAbs. Importantly, the BsAbs demonstrated the desired IgG-like physicochemical properties in terms of high-level expression, ease of purification to homogeneity, good stability and in vivo pharmacokinetics. In summary, we describe a novel and flexible plug-and-play platform to engineer IgG-like BsAbs with excellent development potential for clinical applications.

We developed a strategy to combine conventional targeted therapy with immune checkpoint blockade using a tumor-targeting bispecific antibody (BsAb) to treat solid tumors. The BsAb was designed to simultaneously engage a tumor-associated antigen, epidermal growth factor receptor (EGFR), and programed cell death protein 1 (PD1). In addition to its direct anti-tumor activity via EGFR inhibition, the BsAb mediated efficient antibody-dependent cellular cytotoxicity (ADCC) and activated T cell antitumor im munity through blockade of PD1 from interacting with its counterpart, programed cell death ligand 1 (PDL1). Further, the BsAb exhibited a potent direct tumor cell killing activity in the presence of PBMC, most likely, via activating and, at the same time, physically engaging T cells with tumor cells. Taken together, we here illustrate a new strategy in the design and production of novel BsAbs with enhanced therapeutic efficacy through both direct tumor growth inhibition and T cell activation via tumor-targeted immune checkpoint blockade.

Platelet-derived growth factor receptor β (PDGFRβ) is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRβ from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRβ and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRβ and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRβ antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.

Accumulating evidence suggests that multiple complex mechanisms may be involved, simultaneously or complementarily, in the emergence and development of multidrug resistance (MDR) in various cancers. Cell adhesion-mediated MDR is one such mechanism. In the present study, we initially observed increased cell adhesion to extracellular matrix proteins by the MDR human breast tumor cell line MCF-7/MX compared to its parental cells. We then used a strategy that combined antibody-based screening technique and mass spectrometry-based proteomics to identify membrane proteins that contribute to the enhanced adhesion of MCF-7/MX cells. Using MCF-7/MX cells as immunogen, we isolated a mouse monoclonal antibody, 9C6, that preferentially reacts with MCF-7/MX cells over the parental MCF-7 cells. The molecular target of 9C6 was identified as cytokeratin 8 (CK8), which was found to be overexpressed on the cell surface of MCF-7/MX cells. We further observed that down-regulation of cell surface levels of CK8 through siRNA transfection significantly inhibited MCF-7/MX cell adhesion to fibronectin and vitronectin. In addition, anti-CK8 siRNA partially reversed the MDR phenotype of MCF-7/MX cells. Taken together, our results suggest that alterations in the expression level and cellular localization of CK8 may play a significant role in enhancing the cellular adhesion of MDR MCF-7/MX cells.