ScFvs as Allosteric Inhibitors of VEGFR-2: Novel Tools to Harness VEGF Signaling

Ballmer-Hofer, Kurt; Hyde, Caroline A. C.; Schleier, Thomas; Avramovic, Dragana

doi:10.3390/ijms19051334

Cited by 5 publications

(3 citation statements)

References 48 publications

(58 reference statements)

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“…An important feature of antibodies, affinity, represents the binding capacity between an antibody and its target antigen. The results of the sandwich ELISA demonstrated BsDb can bind to PD-1 and VEGF165 simultaneously, and the K aff values of binding reactions detected by indirect ELISA between the BsDb and VEGF165 and the BsDb and PD-1 were 124.78 nM and 25.07 nM, respectively, illustrating that the BsDb could bind strongly to VEGF165 and PD-1 simultaneously, which was favorably in agreement with other reported antibody fragments [ 35 ].…”

Section: Discussionsupporting

confidence: 86%

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

Xiong

Mao

et al. 2018

IJMS

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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.

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Section: Discussionsupporting

confidence: 86%

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

Xiong

Mao

et al. 2018

IJMS

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“…The isolation of scFvs with apparent and predicted affinity in the subnanomolar range from a moderate diversity and synthetic phage scFv library (Tomlinson I, 3.8 × 10 8 ) in the current study is acceptable when compared with the affinity of anti-VEGFR-2 scFv candidates in the 137-6800 nM range, which were isolated from the highly diverse synthetic ETH-2 Gold library (3 × 10 9 antibody clones) through solid-phase biopanning. 36 In another study conducted by Böldicke et al, 37 an affinity of 3.8 nM was reported for one of the anti-VEGFR-2 scFvs (A7), isolated from a mouse immune library (undergoing in vivo affinity maturation through somatic mutation) and using a solid-phase selection approach. Taken together, these results underscore the capacity of SPB used in this study for the isolation of scFv clones, possibly with high affinity binding in the nanomolar range.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology

Kazemzadeh,

Bagheri,

Sepehri

et al. 2024

ACS Omega

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Angiogenesis, as a tumor hallmark, plays an important role in the growth and development of the tumor vasculature system. There is a huge amount of evidence suggesting that the vascular endothelial growth factor receptor (VEGFR-2)/ VEGF-A axis is one of the main contributors to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2 signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In this study, we employ phage display technology and solution-phase biopanning (SPB) to isolate specific single-chain variable fragments (scFvs) against VEGFR-2 and report on the receptor binding characteristics of the candidate scFvs A semisynthetic phage antibody library to isolate anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations of the VEGFR-2-His tag and VEGFR-2biotin. After successful expression and purification, the specificity of the selected scFv clones was further analyzed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. The competition assay was undertaken to identify the VEGFR-2 receptor-blocking properties of the scFvs. Furthermore, the molecular binding characteristics of candidate scFvs were extensively studied by peptide−protein docking. Polyclonal ELISA analysis subsequent to four rounds of biopanning showed a significant enrichment of VEGFR-2-specific phage clones by increasing positive signals from the first round toward the fourth round of selection. The individual VEGFR-2-reactive scFv phage clones were identified by monoclonal phage ELISA. The sequence analysis and complementarity-determining region alignment identified the four unique anti-VEGFR-2-scFv clones. The soluble and purified scFvs displayed binding activity against soluble and cell-associated forms of VEGFR-2 protein in the ELISA and flow cytometry assays. Based on the inference from the molecular docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and 3 on the VEGFR-2 protein and displayed competition with VEGF-A for binding to VEGFR-2. The competition assay confirmed that scFvs H1 and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells.

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“…Consistent with previous reports, we found that concentrations of VEGF were significantly lower in PDR patients pretreated with IVI. As VEGF inhibitors, anti-VEGF drugs prevent VEGF from binding to VEGF receptors such as vascular endothelial growth factor receptor-2 (VEGFR-2), thereby inhibiting endothelial cell proliferation and reducing vascular leakage, thus eliminating fragile neovascularization or inhibiting its formation [21][22] . A large number of studies have confirmed that preoperative IVI combined with PPV in the treatment of PDR can not only reduce intraoperative and postoperative complications, but also improve the visual prognosis of patients [11][12][13] .…”

Section: Discussionmentioning

confidence: 99%

Optimal timing of preoperative intravitreal anti-VEGF injection for proliferative diabetic retinopathy patients

Xu¹,

Yan²,

Wang³

et al. 2022

Int J Ophthalmol

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AIM: To analyze concentrations of vascular endothelial growth factor (VEGF) and fibrosis-related factors in vitreous fluid of proliferative diabetic retinopathy (PDR) patients pre-treated with intravitreal anti-VEGF injections (IVI) at different time periods prior to pars plana vitrectomy (PPV), and their correlation with the degree of vitreoretinal fibrosis and explore the optimal timing of preoperative IVI. METHODS: The prospective case-control study from January 2019 to July 2020 included 31 eyes with PDR-related complications (PDR group) and 21 eyes with non-diabetic ocular disease (control group) requiring PPV. PDR eyes were divided into four groups based on timing of PPV: 3d after IVI (3-day group); 5d after IVI (5-day group); 7 or more days after IVI (≥7-day group); and no IVI. Vitreous fluid samples (0.5-1.0 mL) were collected prior to switching on the infusion before routine 23-G PPV. Concentrations of VEGF, basic fibroblast growth factor (bFGF), periostin (PN), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α were measured by immunoassay, and concentration differences for each cytokine were compared among the groups. The degree of vitreoretinal fibrosis was graded intraoperatively, and the correlation between the changes in cytokine levels and the severity of vitreoretinal fibrosis was analyzed by univariate ordinal logistic regression analysis. RESULTS: PDR eyes without IVI had significantly higher VEGF, bFGF, PN, and IL-6 concentrations than non-diabetic eyes (all P<0.05), and had a significantly higher concentration of VEGF (P<0.05) and a significantly lower concentration of IL-8 (P<0.05) than PDR eyes with IVI. Statistically significant differences were also observed for concentrations of VEGF, bFGF, PN, IL-6, and IL-8 among 3-day, 5-day, and ≥7-day groups (all P<0.05); meanwhile there was no significant difference in TNF-α among groups (P=0.226). The 5-day group had the lowest concentration of VEGF and the ≥7-day group had the highest concentration of bFGF and PN. The degree of vitreoretinal fibrosis was significantly higher in the ≥7-day group compared to the 3-day (P=0.015) and 5-day group (P=0.039), and vitreoretinal fibrosis correlated significantly with concentrations of bFGF, PN, IL-6, and IL-8 (all P<0.05). Univariate ordinal logistic regression analysis showed that bFGF was an independent risk factor for the severity of vitreoretinal fibrosis in PDR patients pre-treated with IVI. CONCLUSION: The vitreous concentrations of VEGF, bFGF, PN, IL-6, and IL-8 change after pretreatment with IVI before PPV in PDR patients. The degree of vitreoretinal fibrosis is higher in patients with a longer time between IVI treatment and PPV, which may be related to the angio-fibrosis switch. The results suggest that PPV should be performed 5d after IVI administration in PDR patients.

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ScFvs as Allosteric Inhibitors of VEGFR-2: Novel Tools to Harness VEGF Signaling

Cited by 5 publications

References 48 publications

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

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

Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology

Optimal timing of preoperative intravitreal anti-VEGF injection for proliferative diabetic retinopathy patients

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