Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo

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.

Harnessing the hERG1/β1 Integrin Complex via a Novel Bispecific Single-chain Antibody: An Effective Strategy against Solid Cancers

“…at 160 g/ mouse, i.e. 8 mg/Kg,a dose compatible with that used in (33), into immunodeficient athymic Nude-Foxn1nu (nu/nu) mice and its plasma concentrations were determined by ELISA at different time points. A characteristic twophase PK behavior emerged, with a rapid distribution phase and a longer elimination phase (Figure 5B).…”

Recombinant expressing angiopep-2 fused anti-VEGF single chain Fab (scFab) could cross blood–brain barrier and target glioma

“…Currently, it is widely used to treat various types of cancers, including metastatic non-small-cell lung cancer (NSCLC), metastatic renal cell carcinoma (RCC), breast cancer, epithelial ovarian cancer, and glioblastoma (Ferrara and Adamis 2016). Anti-VEGF-antibody is widely applied in novel antibody-based drug development, such as anti-VEGF and anti-PD1 bi-specific antibody (Xiong et al 2018). However, almost all the large-molecule drugs and more than 98% of small-molecule drugs are unable to reach the brain parenchyma due to the presence of the blood–brain barrier (BBB).…”

Therapeutic targeting of VEGF and/or TGF-β to enhance anti-PD-(L)1 therapy: The evidence from clinical trials

“…Preclinical data showed that HB0025 could inhibit two pathways concurrently to enhance its anti-cancer activities, supporting further clinical studies (84). Xiong et al also generated a nonimmunogenic, bispecific antibody targeting both VEGF165 and PD-1, serving as a potential anti-tumor agent (85). And the safety and efficacy of another anti-PD-1/VEGF bispecific antibody AK112 are assessed in the several phase I/II trials (NCT04047290, NCT05116007, and NCT04900363).…”

“…Xiong et al. also generated a non-immunogenic, bispecific antibody targeting both VEGF165 and PD-1, serving as a potential anti-tumor agent ( 85 ). And the safety and efficacy of another anti-PD-1/VEGF bispecific antibody AK112 are assessed in the several phase I/II trials (NCT04047290, NCT05116007, and NCT04900363).…”