Generating a Novel Bispecific Nanobody to Enhance Antitumor Activity

Abstract Tumor cells express high levels of human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor receptor 2 (VEGFR2), which are closely related to their proliferation and survival. Cancer treatments that target a single signaling pathway may result in immune pathway escape or drug resistance. Based on the correlation between the HER2 and VEGFR2 signaling pathways, we speculated that targeting the two pathways simultaneously may produce a synergistic effect and avoid occurrence of drug resistance, resulting in improved efficacy. Anti-VEGFR2 nanobody 3VGR19–3 and anti-HER2 nanobody 2D3 were combined to construct a bispecific nanobody (Bi-Nb). They can recognize both HER2 and VEGFR2 (both highly expressed in HT-29 cells) to simultaneously block the two signaling pathways. We verified the affinity of the Bi-Nb to its targets using the surface plasmon resonance technology, and test its effects to inhibit tumor cell growth and promote cell apoptosis in vitro by the Cell Counting Kit-8 assay and apoptosis assay. In summary, we have successfully constructed a Bi-Nb, and verified its tumor-suppressing effects in vitro. Compared with a single monospecific nanobody, our Bi-Nb showed superior antitumor effect, which provides a new perspective for treatment of tumors with high HER2 and VEGFR2 expression.


Introduction
Colorectal cancer (CRC) is a common malignancy of the digestive tract, which accounts for a significant portion of cancerrelated deaths around the world. 1 While human epidermal growth factor receptor 2 (HER2), a member of the ErbB family, is expressed on the surface of normal endothelial cells, it is often overexpressed on tumor cells, 2 causing tumors with elevated HER2 expression highly invasive. Vascular endothelial growth factor receptor 2 (VEGFR2) is involved in the regulation of angiogenesis, therefore, inhibiting VEGFR2 activity, and the downstream signaling pathway has become an important approach in cancer treatment. [3][4][5] Overexpression of HER2 can upregulate VEGF through the PI3K signaling pathway, so damage to normal tissue. [9][10][11][12] Antibodies and bispecific antibodies have become the most important part of targeted tumor therapy. [13][14][15][16] Bispecific Antibody by Protein Trans-Splicing (BAPTS) has been reported to synthesize BsAbs with natural human immunoglobulin G (IgG) structure and no chain mispairing. 17,18 Nanobodies, such as camel-derived antibodies, that naturally lack the light chains and contain only the variable region of the heavy chain are the smallest antigenbinding fragments. 19 Compared with traditional monoclonal antibodies, nanobodies have the advantages of small size, high solubility, high stability, high affinity to their targets, low immunogenicity, and excellent tissue penetration. 20,21 Based on the characteristics of nanobodies and the special structure of VHH of the natural heavy-chain antibody, 21,22 previously our laboratory has shown that nanobodies combined with immunotoxin improve antitumor activity. 23 In this study, we developed a bispecific nanobody (Bi-Nb) using anti-HER2 and anti-VEGFR2 nanobodies. This Bi-Nb targets simultaneously HER2 and VEGFR2 in tumor cells overexpressing these two receptors, 24,25 which holds greater potential in medical research and clinical treatment of cancer.

Cell Lines and Reagents
The HT-29 cells used in this study were preserved in our laboratory and cultured on the McCoy's 5A medium (McCoy's 5A; Gibco, United States), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, USA) and 1% (v/v) penicillin/streptomycin (P/S; Hyclone, United States). All the cultures were kept in a plastic flask and incubated at 37°C in 5% CO 2 . The Cell Counting Kit-8 (CCK-8) kit was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., BCA kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., and Ni-NTA gel from Nanjing Kingsley Co., Ltd. Yeast extract, tryptone, and peptone were purchased from OXOID Co., Ltd. Flow cytometry used in this study was CytoFlex from Beckman. VEGFR2 antibody (2C6) was purchased from Novus Biologicals (United States). VEGF165 and PE-conjugated VEGFR2 antibody were purchased from Sino Biological (Beijing, China). FITCconjugated rat anti-mouse IgG1 secondary antibody and FITCconjugated 6 Â His tag mAb (AD1.1.10) were purchased from Thermo Fisher (United States).

Construction and Expression of Bispecific Nanobody
The 2D3 and Bi-Nbs were synthesized by Synbio Technologies (Soochow, China). The sequence information of the anti-HER2 2D3 nanobody was obtained from Ablynx. 25 The sequence information of the anti-VEGFR2 3VGR19-3 nanobody was provided by our laboratory. Then they were cloned into plasmid pET-22b(þ) between restriction sites NcoI and XhoI. Primers used were T7 and T7 terminals. The above three nanobodies were confirmed by sequencing. The constructed plasmid was transformed into Escherichia coli strain BL21 (DE3). Inoculum 1% (v/v) was inoculated to 1 L culture, and 1 mmol/L IPTG inducer was added when the OD 600 value of the bacterial liquid reached 0.5 to 0.6. After centrifugation (12,000 r/min, 10 minutes), periplasmic protein was extracted by osmotic shock and purified by an affinity metal chromatography column (nickel column), then analyzed by 15% SDS-PAGE and Western blotting with anti-His tag mouse antibody.

Surface Plasmon Resonance Kinetics Measurements
Affinity constants for the binding between Bi-Nb and VEGFR2 or HER2 were determined by surface plasmon resonance (SPR) analysis using the Biacore T200 system. The VEGFR2 or HER2 was immobilized on the CM-5 sensor chip. There are four channels on the CM-5 sensor chip, two coupling VEGFR2 or HER2 interaction detection channels and two nonfixed VEGFR2 or HER2 as blank reference channels. At a flow rate of 30 μL/min, HBS solution was used as the working solution, and the gradient concentrations of nanobodies were injected respectively, each concentration was detected twice. All Biacore kinetics experimental data were obtained using the Biacore T200 evaluation software to estimate the association rate constant (k a ) and dissociation rate constant (k d ).

Flow Cytometry Analysis
After washing three times with phosphate buffered saline (PBS)-2% FBS, 2Â 105 HT-29 cells were incubated at 4°C for 30 minutes with 1 mg/mL nanobodies (2D3, 3VGR19-3, and Bi-Nb) in PBS-2% FBS. After three times washing with PBS-1% BSA (w/v), cells were incubated with anti-His-tag FITC antibody for 30 minutes on ice. Excess fluorescein-labeled antibody was removed by washing with PBS-2% FBS. Then, the cells were fully suspended with 400 μL PBS and analyzed by flow cytometry.

CCK-8 Cell Proliferation Assay
The CCK-8 assay kit (Sigma-Aldrich) was used to detect the effect of nanobodies on the cell viability of HT-29 cells. Briefly, the starved cells were seeded into separate 96-well plates at 3,000 cells/well and cultured overnight at 37°C. At the end of incubation, 10 μL CCK-8 was added to each well, and the cells were counted by absorbance measurements at a wavelength of 450 nm. The cell survival rate (%) of the target cells was calculated using the following formula:

Apoptosis Assay
Apoptosis assay was performed with an Annexin V-FITC/ propidium iodide (PI) kit (Lianke, Hangzhou, China). A total of 5 Â 10 5 HT29 cells were seeded in a 6-well plate. After adding the apoptosis-inducing agent, the cells were incubated at 37°C for 48 hours, then stained with PI and Annexin V-FITC for apoptosis assay on a flow cytometer. Anenxin V-FITC/PI double staining can detect cell apoptosis and differentiate into early apoptotic cells (annexin V þ /PI À ), late apoptotic cells (annexin V þ /PI þ ) and necrotic cells (annexin V À /PI þ ) Apoptosis. The percentage of apoptotic cells was calculated as the total percentage of early apoptotic cells and late apoptotic cells.

Construction, Expression, and Purification of the Bi-Nb
The construction of the mono-Nb and Bi-Nb is illustrated in ►Fig. 1 and ►Table 1. We designed forward and reverse primers for plasmid construction and subcloned them into the vector pET-22b (þ). The target gene sequence was confirmed by DNA sequencing. The sequence information of 2D3, 3VGR19-3, and Bi-Nb was confirmed by sequencing. The genes of interest were expressed in E. coli; six anti-His tags were added and purified with a nickel column. 2D3 and 3VGR19-3 were present as a single band of 14kDa (►Fig. 1A). Bi-Nbs were constructed by linking two nanobodies, anti-VEGFR2 and anti-HER2, with a (G4S)3 linker. The recombinant protein was present as a single band of $28 kDa (►Fig. 1B). The final yield was 5 mg/L for 2D3, 1 mg/L for 3VGR19-3, and 4 mg/L for the Bi-Nb.

Bi-Nb Binding to VEGFR2/HER2 Analyzed by SPR
Evaluation of the binding of antigens VEGFR2 or HER2 with Bi-Nb was performed by SPR analysis, as shown in ►Fig. 2. The SPR technology was used to detect the binding affinity of 2D3 to HER2 (►Fig. 2A) and 3VGR19-3 to VEGFR2 (►Fig. 2B). The detailed kinetic parameters are listed in ►Table 2. A hyperbolic curve was formed by the combination of the two antigens with the Bi-Nb (►Fig. 2E). Based on the results of the affinity constant, we can find that the ability of Bi-Nb to recognize antigens is weaker than that of single-target nanobody. We consider that the high molecular weight of Bi-Nbs limits its ability to bind to antigens.

Affinity Analysis of the Nanobodies to HT-29 Cell Surface Antigen
The ability of individual nanobody and Bi-Nb for the recognition of receptor on the HT29 cell surface was analyzed by flow cytometry. The results showed that 2D3, 3VGR19-3, and Bi-Nb bound to HER2 or VEGFR2 in HT-29 cells (►Fig. 3). 2D3, 3VGR19-3, and Bi-Nb had certain affinity to antigen in HT-29 cells; therefore, we chose to use HT29 cells to detect the efficacy of antibodies for against tumors.

Bi-Nb Inhibits the Proliferation of HT29 Cells
The biological activities of the nanobodies were tested by the CCK-8 kit assay, such as inhibition of cell proliferation, migration, and induction of cell apoptosis were verified. Inhibition of cell proliferation may indicate a possibility for cancer treatment. We measured cell viability in HT29 cells treated with different concentrations of nanobody or Bi-Nb using CCK-8 assays. The results are shown in ►Fig. 4. The IC 50 value of 2D3 was 168.2 nmol/L, that of 3VGR19-3 was 584.4 nmol/L, while that of the Bi-Nb was 101.2 nmol/L.

Bi-Nb Induces Apoptosis
As shown in ►Fig. 5, HT29 cells were treated with 200 nm nanobody, followed by apoptosis assay with the FITC-Annexin V/PI kit to determine cytotoxicity of the nanobodies to the cells, and then with annexin V-FITC and PI to distinguish populations of early apoptotic (annexin V þ /PI À ), late apoptotic (annexin V þ /PI þ ) cells. The results showed that the Bi-Nb was more effective in inducing apoptosis of the tumor cells.

Discussion and Conclusions
Studies on CRC risk have identified potential factors associated with the disease. [26][27][28][29][30] A close correlation has been detected between HER2 overexpression and elevated VEGF in several human tumors, especially breast cancer. 31,32 Simultaneously blocking HER2 and VEGFR2 signaling pathways with the Bi-Nb may overcome the drug resistance commonly seen after treatments targeting only one of them and produce synergistic antitumor effects, which would be of great clinical significance. [33][34][35] We chose to use the (G4S)3 linker to bind the two nanobodies together for constructing the novel Bi-Nb (►Fig. 1A). The results showed that (►Fig. 1B) the expression Fig.1 (A) Basic structure of anti-HER2 nanobody, anti-VEGFR2 nanobody, and bispecific nanobody. (B) Coomassie-stained SDS-PAGE and Western blotting analysis of the purified Bi-Nb and the Nbs. Expression and purification of 2D3 (1,2). Expression and purification of 3VGR19-3 (3,4). Expression and purification of bispecific nanobody (5,6). L is (G4S)3 (GGCGGCGGTGGTAGCGGCGGCGGCGGTTCCGGCGGTGGTGGTTCT) linker, M is size standards. PSP, periplasmic space protein; FT, flow through by IMAC; E20-E500, elution samples with 20-500 mmol/L imidazole.    level of Bi-Nb was the same as that of single nanobody and could be effectively secreted in periplasmic protein. The transient gene expression technology platform has been widely used in a variety of therapeutic proteins and monoclonal antibodies. [36][37][38] Compared with the traditional Bi-Nbs, this Bi-Nb not only has a high yield, but also is easy to be purified. We consider that the high yield of the Bi-Nb may be due to its small molecular weight, high solubility, and the high expression of target protein in the prokaryotic system. 39 In addition, in the process of purification, the proteins in the periplasmic space were used as the mother liquor for gradient elution, but there were fewer miscellaneous proteins in the cell periplasmic space, so it was easier to purify the target protein. The results of SPR and fluorescence-activated cell sorting showed that the Bi-Nb could target HER2 and  VEGFR2. Next, biological activities or anticancer activities in vitro were evaluated by cell proliferation assay and apoptosis assay. The Bi-Nb inhibited HT29 cell proliferation in a dosedependent manner, with an IC 50 of $100 nmol/L (►Fig. 4).
As the Bi-Nb has been successfully constructed and its efficacy against tumor cells has been preliminarily validated, the next stage of the study seeks to evaluate its efficacy in animal models. Generally, the Bi-Nb still has some limitations in clinical application. It is believed that blocking neovascularization would effectively inhibit tumor growth, but once the drug is withdrawn, relevant signaling pathways would be activated again, and the tumor would resume vascularization and continue growing. 40 As seen in renal cell carcinoma treated with bevacizumab alone, tumors grow rapidly during the intervals of treatment. To address this problem, we shall further combine toxins with nanobodies into immunotoxins, or with other antitumor drugs. This study provides a new perspective for clinical treatment of tumor.

Conflicts of Interest
The authors declare no conflict of interest.