Abstract
Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.
Highlights
The development of antibody engineering has provided opportunities to generate second generation antibody drugs, including bispecific antibodies (BsAbs)
It is noteworthy that the Programmed death-1 (PD-1)-targeting monoclonal antibody (mAb), nivolumab, has received the Food and Drug Administration (FDA) approval for treating non-small-cell lung cancer (NSCLC), melanoma, and renal cell carcinoma (RCC) [9] and, recently, the FDA expanded the nivolumab for metastatic NSCLC [10]
This study focused on the preparation of a novel, fully human bispecific antibody (BsAb), which inhibited tumor angiogenesis, and activated immune responses
Summary
The development of antibody engineering has provided opportunities to generate second generation antibody drugs, including bispecific antibodies (BsAbs). Blinatumomab, a bispecific antibody fragment targeting CD3 and CD19, was approved in December 2014 by the U.S Food and Drug Administration (FDA) for the treatment of patients with Philadelphia chromosome-negative precursor B cell acute lymphoblastic leukemia (ALL) [2]. Neovascularization and the immune responses are hallmarks of cancer and play crucial roles in the progression and metastasis of tumor cells. Bevacizumab, a recombinant humanized anti-VEGF-A monoclonal antibody (mAb), was approved by the FDA to treat metastatic colorectal cancer in combination with standard chemotherapy in 2004. The beneficial effects of antiangiogenic monotherapy to control tumor progression lack satisfactory sustainability [6], and the resistance mediated by the adaptive and intrinsic mechanisms in anti-angiogenic therapy is a major obstacle for cancer treatment [7]. It must be reported that the administration of immune checkpoint inhibitors induced immune-related adverse events such as organic inflammation and persistent high fever [11,12]
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