Chicken-Derived Humanized Antibody Targeting a Novel Epitope

Jan 31, 2019 - National Research Institute of Chinese Medicine, Ministry of Health and ... ∥TMU Research Center of Cancer Translational Medicine, âŠ...
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Article Cite This: ACS Omega 2019, 4, 2387−2397

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Chicken-Derived Humanized Antibody Targeting a Novel Epitope F2pep of Fibroblast Growth Factor Receptor 2: Potential Cancer Therapeutic Agent Keng-Chang Tsai,†,‡,¶ Chao-Di Chang,§,¶ Ming-Hui Cheng,◆ Tsai-Yu Lin,∥ Yan-Ni Lo,∥ Tz-Wen Yang,∥ Fu-Ling Chang,⊥ Chen-Wei Chiang,§ Yu-Ching Lee,*,∥,⊥,# and Yun Yen*,∥,⊥,∇,○ †

National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 11221, Taiwan Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, §Ph.D. Program in Biotechnology Research and Development, College of Pharmacy, ∥TMU Research Center of Cancer Translational Medicine, ⊥Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, #TMU Biomedical Commercialization Center, ∇ Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, and ○Cancer Center, Wan Fang Hospital, Taipei Medical University, Taipei 11031, Taiwan ◆ Department of Laboratory Medicine, Lo-Hsu Medical Foundation, Lotung Poh-Ai Hospital, Yilan 26546, Taiwan

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‡

S Supporting Information *

ABSTRACT: Fibroblast growth factors (FGFs) and their receptors control various biological functions. Dysregulated FGF signaling has been implicated in the pathogenesis of human cancers. Aberrant activation of FGF receptor 2 (FGFR2) signaling caused by FGFR2 overexpression has been observed in various human cancers, such as gastric cancer. Antibodies are highly suitable for target therapy because of their specificity toward FGFR2. Patients with cancer and aberrantly activated FGFR2 signaling can benefit from therapeutic intervention with FGFR2targeting antibodies. In this study, we produced the anti-FGFR2 single-chain variable fragment (scFv) from immunized chickens through the phage display method. The isolated scFv S3 targeted designed epitope F2pep of FGFR2 and prevented the access of FGFs; thus, it exhibited the ability to inhibit cell growth in gastric cancer. Furthermore, scFv S3 recognized endogenous FGFR2 on the cancer cells and inhibited downstream cell signaling. To study the inhibition effect of the humanized immunoglobulin G (IgG) hS3 in vivo, nonobese diabetic/severe combined immunodeficiency mice were inoculated with SUN16 cancer cells. An intravenous injection of hS3 inhibited tumor growth in the mice. Moreover, the hS3 also inhibited angiogenesis in the matrix gel-assisted angiogenesis model. After using site-directed mutagenesis to identify the key residue on the hS3-targeting site of FGFR2, molecular modeling was used to determine the interaction between hS3 and FGFR2. Rational molecular docking analysis results revealed that two ionic interactions caused the interaction between scFv hS3 and the peptide F2pep of FGFR2. The results showed that antibody hS3 has high potential in cancer therapy in the future.

1. INTRODUCTION Fibroblast growth factors (FGFs) regulate the basic developmental signals in an organism, such as those regulating the early development of embryos and subsequent organ system maturation, through FGF receptors (FGFRs).1,2 FGF signaling is involved in many physiological functions in adults, including the proliferation of blood vessels and wound healing. The extensive effects of FGFs are executed mainly through four types of receptor tyrosine kinases (FGFR1−FGFR4) and subsequent signal transduction.3 FGFRs are expressed in many types of cells and affect cell behaviors, such as proliferation, differentiation, and survival; consequently, cancer cells take advantage of signal transduction mediated by FGFs.4 In particular, most cellular responses caused by targeting of FGFRs by FGFs are tissue specific. Disorders of auto and © 2019 American Chemical Society

peripheral secretion induced by activated FGFRs were reported to be correlated with the development of many human cancers.3 Evidence indicates that abnormal signaling of FGFR2 results mainly from the overexpression of FGFR2 and its ligands as well as mutations of FGFR2.3 A correlation between FGFR2 gene expression and tumorigenesis has been observed; approximately 10% of patients with gastric cancer exhibit high FGFR2 gene expression, which leads to poor prognosis and diffuse symptoms.5,6 Moreover, multivariate analysis results showed that high FGFR2 gene expression is an Received: November 4, 2018 Accepted: January 22, 2019 Published: January 31, 2019 2387

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Figure 1. Generation of anti-FGFR2-specific monoclonal antibodies. (A) Complex structure of PDB: 3OJM showing the position of designed peptide F2pep. Red indicates the F2pep site, light blue indicates the structure of extracellular domain of receptor FGFR2, and green indicates FGF1 targeting the active site of FGFR2. (B) Recombinant proteins F2pep, FGFR2, and FGFR3 were visualized through Coomassie blue staining or analyzed by Western blotting, which was probed using purified polyclonal IgY from chicken after the fourth immunization. Red arrows indicate the molecular weight of the recombinant protein F2pep (approximately 25 kDa). (C) The number of eluted phages was counted after each round of panning. The M13 wild-type phage was denoted as the negative control (NC) in the panning. (D) Binding reactivity and specificity of randomly selected scFvs expression cell lysate toward FGFR2 protein was examined through enzyme-linked immunosorbent assay (ELISA).

future, the antibody hS3 may become a crucial therapeutic agent in patients with cancer who exhibit abnormal activation or overexpression of FGFR2.

independent biomarker of a poor overall survival in patients with gastric cancer.7 Pathological mutations in the FGFR2 gene have been reported to be correlated with various cancers, including gastric, breast, lung, and colorectal cancers.3,8−10 The structure of the FGFR2 molecule shows that the immunoglobulin-like D3 functional domain near the carboxyl terminus is selectively spliced to produce the two subtypes of IIIb or IIIc, and different subtypes interact with different series of FGFs. Different cells have unique receptor subtypes that affect downstream cell growth signaling.3,11 Some recently developed FGFR tyrosine kinase inhibitors (TKIs) that are ATP-competitive vascular endothelial growth factor receptor (VEGFR) inhibitors are in the early stages of clinical trials.12−14 Because of the high similarity in the structures of the VEGFR and FGFR kinase domains, these TKIs typically exhibit dual-target inhibition. Simultaneous interference with two growth factors enables the inhibition of angiogenesis and tumor cell proliferation.12,14,15 However, the nonspecificity of these drugs reduces their efficacy against FGFR2; consequently, only low drug concentrations can be used because serious side effects are possible. Therefore, specific antibody drugs are advantageous. In this study, a humanized antibody hS3 was generated through the phage display method and was designed for directly targeting the active site of the FGFR2 molecule. Thus, the feasibility of developing chicken-derived therapeutic antibodies was confirmed and the antibody−antigen interaction was elucidated. In

2. RESULTS 2.1. Target Peptide Design and Chicken Immunization. To generate antibodies that bind directly to the functional active site of the FGFR2 molecule, the X-ray complex structure of the PDB id: 3OJM was used to design the peptide target, as shown in the Figure 1A. On the basis of the complex structure formed by the interactions of FGFR2 (cyan) and FGF1 (green), an amino acid sequence TNTEKMEKRLHAV (red), F2pep, at the activation site was designed as the target. Antibody targeting to this position was expected to affect ligand binding. Peptide F2pep was expressed as a recombinant protein with eight continuous repeats and fused with some protein tags showing the molecule weight around 25 kDa (Figure 1B). The recombinant peptide F2pep was subsequently used for chicken immunization. To confirm the peptide-induced humoral response in chickens, polyclonal IgY purified from the immunized chicken was used for Western blot analysis. IgY only showed antibody response to the recombinant peptide F2pep and FGFR2 but not FGFR3. The results confirmed that the structure of the peptide F2pep was consistently presented on the FGFR2 molecule. Moreover, the selectivity of IgY, indicated by its ability to distinguish between FGFR2 and FGFR3 molecules, was confirmed (Figure 1B). 2388

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Figure 2. Characterization of chicken scFv S3 binding to FGFR2. (A) Four cell lysates were extracted for Western blotting analysis. Samples were from three gastric cancer cells N87, MKN45, SNU16, and one was from the downregulation of FGFR2 by siRNA in the cell SNU16. The proteintransferred nitrocellulose membrane was probed using scFv S3 to test binding reactivity. (B) The endogenous FGFR2 of the purified membrane protein of MKN45 and SNU16 cells was immunoprecipitated by scFv S3 and then detected by commercial anti-FGFR2 antibody (left) and antiHis tag antibody (right). Red arrows indicated the molecular weight of FGFR2 and scFv. An irreverent scFv was used as the negative control in the experiment. (C) Endogenous cell surface FGFR2 molecules of the MKN45 and SNU16 cells were detected using scFv S3 through flow cytometry. An irrelevant chicken scFv was used as the negative control (NC), and a commercial rabbit anti-FGFR2 polyclonal antibody was used as the positive control. (D) Immunocytochemical staining of endogenous FGFR2 on the SNU16 cell membrane was detected using scFv S3. The nucleus (blue) was visualized through 4′,6-diamidino-2-phenylindole (DAPI) staining. Red arrows indicate the cell extension site of SNU16 cell membrane recognized by the scFv S3. (E) The competitive effect of scFv S3 was confirmed by interfering in the ligand−receptor interaction. The amount of ligand (FGF7 or bFGF) bound to the receptor (FGFR2 IIIb or FGFR2 IIIc) in the presence of free scFv S3 competitor was measured as a percentage of the binding of the ligand in the absence of a competitor.

2.2. Construction of scFv Antibody Library and Biopanning. The immunized chickens were sacrificed and the spleen cells were harvested to construct an scFv antibody library with a high complexity of 4 × 108 by using phage display. The library was used for four rounds of biopanning against the recombinant FGFR2 protein to isolate specific antiFGFR2 scFv binders. After each round of biopanning, the total number of phage particles bound to FGFR2 was eluted and counted. The number of phage particles bound to FGFR2 showed an increasing trend (Figure 1C). The number of the eluted phages in the final (fourth) round of panning was approximately 645 times higher than that in the first round of panning. The result demonstrated a successful selection process, and specific binders were enriched after panning. Furthermore, the enriched clone in the library was selected randomly for individual scFv expression and used for ELISA analysis. As shown in Figure 1D, almost all isolated scFvs (9/ 10) specifically recognized the recombinant FGFR2 protein but not FGFR3 or bovine serum albumin (BSA). After sequencing, nine positive clones exhibited identical gene sequences. The result was obtained from panning to isolate specific binders from the original scFv library using a complicated combination. Therefore, scFv S3 was used as the representative clone for further analysis. 2.3. Characterization of Anti-FGFR2 Chicken scFv S3. To confirm the ability of scFv S3 to recognize endogenous FGFR2 on cancer cells, the cell lysate of three gastric cancer cells SNU16, MKN45, and N87 were prepared individually for

Western blot analysis. In addition, a downregulation of FGFR2 in the SNU16 cell by siRNA was generated (Supporting Information, Figure S1) and also included as the regulation control in the experiment. As shown in the Figure 2A, FGFR2 in three gastric cancer cell lysates was clearly identified by scFv S3 and the binding signal of scFv S3 to the FGFR2 downregulation SUN16 cell is decreased. Moreover, in the immunoprecipitation assay, FGFR2 in the two gastric cancer cell lysates (from cancer cell MKN45 and SNU16) can be pulled down by the scFv S3 and identified by the anti-FGFR2 antibody in the Western blotting (Figure 2B). Further, the reactivity of scFv S3 in binding to cancer cells was also confirmed through flow cytometry. As shown in the Figure 2C, scFv S3 recognized endogenous FGFR2 molecules on two of the gastric cancer cell lines, MKN45 and SNU16; the binding reactivity was similar to that of a commercially available antiFGFR2 antibody, which was used as the control. The immunocytochemical staining results showed that scFv S3 exhibited significant staining reactivity at the membrane of the SNU16 cells (Figure 2D). The binding signal at the cell extension site of cancer cells revealed the scFv S3 binding on the membrane. Because peptide F2pep was located at the active site of FGFR2, scFv S3 targeting FGFR2 probably affected ligand binding. Through sequence alignment of the FGFR2 isoforms IIIb and IIIc, peptide F2pep was located in the consensus region, and both isoform IIIb and IIIc were targeted, which resulted in a neutralizing effect (Supporting Information, Figure S2). Therefore, a competitive binding 2389

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Table 1. Rate Constants kon and koff of scFv S3 Targeting FGFR2a ligand

TA (s)

TD (s)

kon (104 M−1 s−1)

koff (10−3 s−1)

KD (10−8 M)

χ2 (signal2)

FGFR2

23.56

259.23

2.37 ± 0.0069

1.79 ± 0.0013

7.53 ± 0.0275

46.18

TA: time of association; TD: time of dissociation; kon: association rate; koff: dissociation rate; KD: affinity binding constants; χ2: Chi-squared test.

a

Figure 3. Growth inhibition in the gastric cancer cells by the anti-FGFR2 antibody. (A) The gastric cancer cell lines SNU16 and MKN45 were incubated with serially diluted scFv S3 for 5 days to analyze the inhibition effects on cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide (MTT) assay. The values represent mean ± standard deviation (SD) in all panels. (B) SNU16 cells were treated with the indicated concentrations of scFv S3 and incubation times to analyze the inhibition effects. (C) SNU16 cells were treated with the indicated concentrations of scFv S3 for 5 days to analyze molecule signaling through Western blotting analysis. (D) Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice bearing subcutaneously established SNU16 xenograft tumors were randomized into three groups (n = 5 per group) and received the indicated treatments. Humanized immunoglobulin G (IgG) hS3 was administered at 5 or 15 mg/kg through intravenous injection twice weekly. The curves of the tumor growth volume were expressed as the mean ± SD, and the percentage of tumor growth inhibition (TGI %) was determined. **P < 0.01 compared with the untreated control group. (E) Body weights were measured daily in week 1 and twice weekly thereafter. The body weights were expressed as mean ± SD.

2.4. Cancer Cell Growth Inhibition by scFv S3. The SNU16 and MKN45 cells were treated with scFv S3 in vitro at different concentrations for 5 days to confirm the inhibitory effect of scFv S3 on cell growth. The SUN16 cells were significantly inhibited in a dose-dependent manner (Figure 3A). In the SUN16 cells, the maximal inhibition by scFv S3, 80%, was observed at 125 μg/mL. However, the inhibition effect on MKN45 cells was statistically nonsignificant. The SNU16 cells treated with the indicated concentrations of scFv S3 exhibited inhibition effects on days 4 and 5; moreover, the cells exhibited a dose-dependent response (Figure 3B). Treatment with scFv S3 also exerted an inhibitory effect on the signaling cascades in the SUN16 cells. After scFv S3 treatment, tyrosine phosphorylation of FRS2, which is a key molecule associated with FGFR2 downstream signaling, was attenuated. Moreover, scFv S3 also effectively inhibited the activation of Akt and Erk1/2 (Figure 3C). 2.5. Antitumor Effect of Humanized Antibody hS3 in a Xenograft Model. Chicken scFv S3 was subjected to humanization. Furthermore, the complementarity-determining

assay was conducted to confirm the ability of scFv S3 to interfere with the interaction between FGF7 and FGFR2 IIIb as well as that between bFGF and FGFR2 IIIc. As shown in Figure 2E, the competition level in the two interactions increased in a dose-dependent manner. The competition level between scFv S3 and FGF7 (for FGFR2 IIIb) was higher than that between scFv S3 and bFGF (for FGFR2 IIIc). The competition level between scFv S3 and FGF7 (for FGFR2 IIIb) was as high as 70% at scFv S3 concentrations exceeding 1 μg/mL; however, the competition level between scFv S3 and bFGF (for FGFR2 IIIc) was 40% at a scFv S3 concentration of 100 μg/mL (Figure 2E). Furthermore, the binding affinity of chicken scFv S3 to recombinant FGFR2 molecules was measured using the surface plasmon resonance system (Supporting Information, Figure S3). On the basis of the association (kon) and dissociation (koff) rates, the affinity (KD) was determined to be 7.5 × 10−8 M. The average of the squared residuals (χ2) was 46.18, which indicated rational fitting confidence (Table 1). 2390

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Figure 4. Angiogenesis inhibition by humanized IgG antibody hS3. (A) The HUVECs were pretreated with IgG hS3 and incubated with 10 ng/mL bFGF for 48 and 72 h to analyze the inhibition of cell growth through BrdU assay. Different concentrations of IgG hS3 were used to determine whether the HUVECs exhibited a dose-dependent response. The cell growth percentage was normalized to the untreated control group. (B) Whole-cell lysates were collected and tested for detecting molecular changes using Western blot analysis. (C) Subcutaneous injection of growth factor (including bFGF)-containing matrix gel into nude mice was used to establish an angiogenesis model in vivo. Humanized scFv hS3 was included in the gel to test the antiangiogenic effect. (D) Hb levels in the resected matrix gels were determined to assess the degree of angiogenesis.

regions (CDRs) of chicken scFv S3 were grafted within a stabilized human antibody scaffold derived from the PDB. Then, humanized heavy-chain (VH) and light-chain (VL) variable region genes were cloned into an IgG expression vector to express the IgG1 molecule using the Expi293 mammalian expression system. To confirm the inhibition of tumor growth by IgG hS3 in vivo, SNU16 tumor xenografts were first established in the NOD/SCID mice. The mice were then treated with 5 mg/kg (low dose) or 15 mg/kg (high dose) of intravenous IgG hS3 twice a week. The tumor growth inhibition (% TGI) of IgG hS3 was found to be 22% under the high-dose condition (P < 0.01) (Figure 3D). The mice tolerated the treatments without exhibiting overt signs of toxicity, significant changes in body weight, or other adverse side effects (Figure 3E). 2.6. Angiogenesis Inhibition Effect of IgG hS3. FGFR2 has been confirmed to play a crucial role in angiogenesis; therefore, antibodies that neutralize FGFR2 are expected to inhibit tumor angiogenesis. The cell growth response of human umbilical vein endothelial cells (HUVECs) induced by bFGF

can be inhibited by IgG hS3 (Figure 4A). Treatment with 500 μg/mL IgG hS3 reduced growth in HUVECs, which was driven by 10 ng/mL bFGF, to 40−50%. Furthermore, the HUVECs were treated with IgG hS3 in identical conditions to determine variations in their molecular profiles. IgG hS3 effectively inhibited downstream p-Akt and p-Erk signaling in a dose-dependent manner (Figure 4B). Moreover, the antiangiogenic effect of humanized scFv hS3 was confirmed through in vivo experiments. Subcutaneous injection of growth factor (including bFGF)-containing matrix gel into nude mice resulted in a significant subcutaneous angiogenesis reaction (Figure 4A). Angiogenesis was evidently inhibited after treatment with humanized scFv hS3. When the matrix gel was observed, neovascularization disappeared because of atrophy and these effects resulted from the neutralizing effect of the anti-FGFR2 antibody hS3. The Hb levels in the matrix gels from the humanized scFv hS3-treated mice were similar to the basal control levels (Figure 4D). These data demonstrate that the antibody hS3 evidently blocked FGFR2 downstream signaling and inhibited angiogenesis. 2391

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Figure 5. Key residue definition of antibody hS3 binding site. (A) Nine mutant peptides, each with 1 or 2 site-directed mutations, were displayed on the phage individually and used for the definition of key residues of the antibody binding site. (B) Microtiter wells were precoated with scFv hS3, and mutant peptide-displaying phage solutions were added individually into the wells for binding detection. F2pep-wt denotes the wild-type peptide F2pep-displaying phage. NC denotes an irrelevant human scFv used as a coating antibody control. Ctrl-pep denotes the scrambled peptidedisplaying phage.

Figure 6. Molecular modeling of the interaction between scFv hS3 and peptide F2pep. (A) Schematic representation of all interactions between the scFv hS3 and F2pep of FGFR2. The interface residues of scFv hS3 and F2pep are focused on the CDR-H1 (green), CDR-H2 (blue), CDR-H3 (magenta), CDR-L1 (red), CDR-L2 (orange), and CDR-L3 (yellow) loops in this panel. The F2pep epitope sequence is shown in the green, magenta, and black dotted lines, indicating hydrogen bonding, ion interactions, and hydrophobic interactions, respectively. (B) The colors of the six CDRs of scFv hS3 are the same as in panel (A). The frameworks of scFv hS3 and FGFR2 are gray and cyan, respectively. (C) The secondary structure of the F2pep (TNTEKMEKRLHAV) sequence of FGFR2 is shown in the illustration, and two major positively charged lysine residues have been labeled.

2.7. Key Residue Definition on Antibody hS3 Binding Site. Although the target site (peptide F2pep) of the antibody hS3 was clear, we additionally confirmed the key residues of the F2pep affecting the interaction between the antibody hS3 and the FGFR2 molecule. Therefore, nine peptide mutants including 6 residues (Glu160, Lys161, Glu163, Lys164, Arg165, and Lue166) were constructed through site-directed mutagenesis and displayed on the phage (Figure 5A). The importance of each amino acid position was confirmed by determining the binding reactivity of individual mutant peptide-displaying phage against hS3 scFv. The mutant peptides 1, 3, 5, and 6 did not exhibit effects on the binding reactivity (Figure 5B). Compared with the wild-type peptide F2pep, the single-mutation peptide 2 (Lys161Ala) and peptide 4 (Lys164Ala) exhibited attenuated binding reactions and the double-mutation peptide 7 (Lys161Ala and Lys164Ala) exhibited the maximum attenuation. However, the double-

mutation peptides 8 (Lys161Ala and Arg165Ala) and 9 (Lys164Ala and Arg165Ala) did not exhibit a visible additive effect; therefore, the two positively charged amino acids, Lys161 and Lys164, were thought to play roles in the interaction. These results can explain the interaction between antibody hS3 and peptide F2pep. 2.8. Molecular Docking of Interaction between Antibody hS3 and FGFR2. Antibody−antigen docking revealed that the CDRs of scFv hS3, namely L1, L3, H1, H2, and H3 (but not the L2 loop), were involved in the interaction between the specific epitope F2pep and scFv hS3 (Figure 6A). The peptide F2pep has 13 residues (amino acids 157−169 of the F2pep region); Thr157 through Val169 are key residues of the FGFR2 epitope. Eight residues of the CDR loops of the scFv hS3 were in contact with the F2pep epitope. The mechanism of epitope recognition involves eight hydrogen 2392

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hS3. The result also appeared to imply that treatment inhibiting only FGFR2 is inadequate and must be combined with other therapies. Notably, the FGFR2 molecule has been considered the target because it plays a crucial role in the mechanism of angiogenesis23,24 through FGFR2-specific ligand binding, overexpression of FGFR2 IIIb, enhancement of angiogenesis, and adhesion to type-IV collagen in cancer development.25,26 In the in vivo matrix gel-assisted angiogenesis experiment, the antibody hS3 exhibited high antiangiogenic ability, which resulted in the significant inhibition of microvascular production. The suppressed response was also confirmed on HUVEC growth and downstream signaling. We have tried to use IgG hS3 for treatment in the other xenograft-containing HCT116 cells, which are colorectal cancer cells. HCT116 cells have been confirmed to first exhibit low FGFR2 expression, but after IgG hS3 treatment, they still exhibited a tumor growth inhibition effect (24% TGI, data not shown). The inhibition effect may be caused by the antiangiogenic response resulting from antibody treatment. Moreover, staining with the angiogenesisrelated marker CD31 significantly decreased in the immunohistochemistry experiments, which indicated that angiogenesis was inhibited in the tumor after antibody treatment. Furthermore, the staining of apoptosis-associated markercleaved caspase 3 also significantly increased (Supporting Information, Figure S4). These results indicated the relatively wide range of therapeutic applications of the antibody hS3. In the peptide F2pep docking analysis, the antibody− peptide interaction indicated that the structure of 13 specific amino acids from the F2pep epitope formed a bowlike structure (Figure 6B,C). The epitope-binding sites (CDR-L1, -L3, -H1, -H2, and -H3) of the scFv hS3 formed a fingerlike shape. The scFv hS3 interacted with F2pep through three binding interactions: hydrogen bonds, hydrophobic interactions, and ionic interactions. We observed that two positively charged lysine residues in the F2pep (TNTEKMEKRLHAV) sequence dominated the ionic interactions. Two lysine molecules in the middle of the sequence of F2pep corresponded to two negatively charged aspartate residues on the CDR-H1 and CDR-H3 loops of the scFv hS3 (Asp38 and Asp111Y). The docking model showed that the two ionic interactions were the driving force for the interactions between scFv hS3 and F2pep. However, no studies have reported that the specific sequence of F2pep dominates the antibody− antigen interaction. Moreover, we observed hydrogen bonds at the binding site around the antibody−F2pep interface. Hydrophobic interactions in the unexposed binding site between the scFv hS3 and F2pep might have further stabilized the antibody−F2pep complex. In conclusion, our study revealed an excellent binding ability of the anti-F2pep scFv hS3 antibody. The results of our molecular modeling structural investigation were consistent with the experiments.

bonds, three hydrophobic interactions, and two ionic interactions. The CDR-L loop interacted with the F2pep epitope at three residues, namely, Thr157, Thr159, and Glu160. The side chain of Tyr37 on the CDR-L1 loop simultaneously interacted with the main chain of Thr157, and the side chain of Thr159, through two hydrogen bonds. The side chain of Glu160 also interacted with the main chain of Tyr37 and Gly38 through hydrogen bonds. The interaction on CDR-L3 was a hydrogen bond between the main chain of Ser110 and the side chain of Thr159. In addition, the side chains of Asp38 and Asp111Y on the CDR-H loop interacted with the side chains of Lys164 and Lys161, respectively, through two ionic interactions. The side chain of Asn58 on the CDR-H2 loop interacted with the side chain of Lys164. The main chain and side chain of Tyr111 on the CDR-R3 loop interacted with the side chain of Lys164 and the main chain of Lue166, respectively, through two hydrogen bonds. Moreover, Tyr111, Trp111B, and Tyr113 on the CDRH3 loop interacted with the side chains of Val169, His167, and Glu163, respectively, through hydrophobic interactions.

3. DISCUSSION Many human malignancies have been reported to be associated with dysregulated FGFR signaling and development. Therefore, the development of antibody drugs against individual FGFRs can have various direct clinical applications. However, the high degree of sequence similarity of FGFRs between mammals, particularly FGFR2 and FGFR4, may result in a lack of immunogenicity for immunization, which is required for the production of effective antibodies. Although Bai et al. used FGFR2 D2−D3 domain fusion proteins in several attempts for mouse immunization and successfully generated and isolated specific antibodies against the FGFR2 IIIb isoform through the hybridoma method,16 the development of a high-efficiency method for targeting antibodies is necessary. In this study, we used chicken immunization and phage display to isolate specific antibodies for targeting and defining the epitope of the FGFR2 active site directly. The use of chickens to overcome the protein similarity among mammals or improve immunogenicity and produce highly specific antibodies has been previously reported.17−20 The CDRs of chicken antibodies are usually longer and have higher variability than those of mousederived antibodies, which is advantageous in inducing somatic mutations with neutralizing effects.21,22 Humanization is a key technology for generating antibodies for clinical use. The humanization of chicken-derived antibodies is a feasible strategy for the generation of highly effective and neutralizing antibodies and has great potential to have clinical benefits. In this study, chicken antibody S3 was humanized successfully and retained its neutralizing effect against FGFR2 molecules. The generation of antibodies through phage display is associated with concerns regarding structural instability and interfaces of isolated antibodies caused by the recombination of the VH and VL regions. However, the binding affinity of the isolated scFv was 75 nM, and the recombination did not exhibit significant effects on stability and ability loss. Furthermore, because the antibody gene was cloned, the binding affinity could be further enhanced conveniently through affinity maturation engineering. In this study, the tumor suppression effect of antibody hS3 on the SNU16 xenograft animal model was 22%. Although the results presented a meaningful inhibitory response, there’s still room for improvement of therapeutic performance of antibody

4. MATERIALS AND METHODS 4.1. Cells and Animals. Three human gastric cancer cell lines, namely MKN45, SNU16, and N87, were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS). Human umbilical vein endothelial cells (HUVECs) were cultured in M199 medium supplemented with 20% FBS and 10 μg/mL endothelial cell growth supplement (BD Biosciences). These cell lines were purchased from American Type Culture Collection (Manassas, VA). The 2393

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cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Female white leghorn (Gallus domesticus) chickens, nude mice, and nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were purchased from the National Laboratory Animal Center, Taiwan, and maintained in the animal facility of the Taipei Medical University. The 8 week old male nude mice and NOD/SCID mice were provided water (reverse osmosis, 1 ppm chlorine) and Pico-Lab Rodent Diet (20.0% crude protein, 9.9% crude fat, and 4.7% crude fiber) ad libitum. The mice were housed in groups under conditions of constant photoperiod (12 h each of light and darkness) at 21−23 °C and 60−85% humidity. The chickens and mice were maintained carefully, and all animal experiments followed ethical standards. The protocols were reviewed and approved by the Animal Use and Management Committee of Taipei Medical University (IACUC number: LAC-20130139). 4.2. Antibody Library Construction, Panning, and scFv Expression. The peptide sequence of F2pep (TNTEKMEKRLHAV) of the FGFR2 molecule was designed, consisted of eight continual repeats, and was expressed as a recombinant protein. The full-length gene was synthesized (GENEWIZ, Inc.), cloned into the pET32a vector (Novagen, Inc.), and transformed into the Escherichia coli BL-21 (DE3) strain for expression. After purification by Ni2+-charged sepharose (GE Healthcare Life Sciences), recombinant F2pep protein was used for animal immunization. Female white leghorn chickens were immunized through intramuscular injection of 50 μg per time of recombinant F2pep protein mixed with an adjuvant. The immunization schedule had four times of immunization and was performed at an interval of 7 days. The spleens of the chickens were harvested 7 days after the final immunization to construct an antibody library. The library construction method followed published protocols with minor modifications.27 The full-length gene of recombinant FGFR2 extracellular domain (from GenBank accession no. AAA52449.1) was synthesized and cloned into the pET-21a expression vector. The resultant plasmids were transformed into the E. coli BL-21 (DE3) strain for protein expression. For biopanning, purified recombinant FGFR2 protein (1 μg/well) was precoated on the wells of a microtiter plate and blocked using 3% bovine serum albumin (BSA). Next, the recombinant scFv library phage solution (1011 phage particles) was added to the wells at room temperature for 2 h. Subsequently, the supernatants (unbound phage) were removed and the wells were washed 10 times by pipetting with phosphate-buffered saline with 0.05% Tween 20 (PBST). The elution buffer (0.1 M HCl/glycine [pH 2.2] and 0.1% BSA) was added to elute the bound phage particle, and then the eluted phage particle was neutralized by using a 2 M Tris-base buffer. The eluted phage was immediately used to infect the E. coli ER2738 strain for recombinant phage amplification. After precipitating and recovering, the amplified phage was used for the next round of panning. After the fourth panning, the amplified phage was recovered and the total library DNA in the phage-infected E. coli was purified by the EasyPrep Plasmid Extraction kit (BioTools, Inc., Taiwan). For scFv protein expression, the purified library DNA was transformed into a nonsuppressor E. coli strain TOP 10F′ (Invitrogen). Next, single colonies (scFv clone) were selected randomly for further analysis and expressed scFvs that fused with His tag were purified using Ni2+-charged sepharose according to the standard protocol in the instruction.

4.3. Western Blotting. After sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), the recombinant FGFR2 protein or the extracted cell lysates were transferred onto nitrocellulose membranes (GE Healthcare Life Sciences) to determine binding reactivity, which was measured using purified poly-immunoglobulin Y (IgY) or scFv antibodies. The membranes were blocked with 5% skim milk and then poly-IgY or scFv antibodies were added for incubation at room temperature for 1 h. Next, the membranes were washed and detected using the goat antichicken lightchain antibodies (Bethyl Laboratories, Inc.) followed by developing using the horseradish peroxidase (HRP)-conjugated donkey antigoat IgG antibodies (Jackson ImmunoResearch Laboratories, Inc.). Finally, 3,3′-diaminobenzidine was added for color development until the desired color intensity was reached. 4.4. Enzyme-Linked Immunosorbent Assay. In the enzyme-linked immunosorbent assay (ELISA), the recombinant FGFR2 protein (0.5 μg/well) was precoated in microtiter wells and incubated at 4 °C overnight. The wells were blocked with 5% skim milk, and then the scFv-expressing lysates or purified scFvs were added to the wells at room temperature for 1 h. After washing with PBST, the scFv binding reactivity was detected and developed by using secondary antibodies, as previously described. Finally, the substrate, 3,5,5-tetramethylbenzidine dihydrochloride (TMB) was added for signal development. After adding 1 N HCl to stop the reaction, the absorbance was measured by the optical density at 450 nm 4.5. Sequence Analysis. The orientation of scFv in the study is VL-linker-VH. A forward primer (5′-AAGACAGCTATCGCGATTGCAGTG-3′), namely, ompseq was used for scFv gene sequencing; the primer sequence is complementary to the signal sequence (outer membrane protein A) in front of the light-chain variable region (VL). Subsequently, the network program was used to compile and analyze sequence data in accordance with germline genes (International ImMunoGeneTics information system/V-QUEry and STandardization [http://imgt.cines.fr/]). 4.6. Flow Cytometry Analysis and Immunocytochemical Staining. Two gastric cancer cells MKN45 and SNU16 with endogenous FGFR2 molecule expression were analyzed through flow cytometry to confirm the binding reactivity of scFv S3. Freshly prepared cancer cells were harvested and washed twice with PBS before adding scFv for reaction. Bound scFv was visualized using goat antichicken light-chain antibodies and donkey antigoat antibodies conjugated with fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories, Inc.). An irrelevant scFv was used as the negative control, and commercially available rabbit anti-FGFR2 antibodies were used as the positive control (GeneTex, Inc.). The results were analyzed using a FACScan flow cytometer (BD Biosciences). The SNU16 cells (2 × 105 cells/mL) were individually seeded on a cover glass and fixed by incubating with freshly prepared 4% paraformaldehyde on ice for 10 min. Then, the cells were dehydrated using sequential treatments of 70, 95, and 99% methanol and rehydrated using 95 and 70% methanol. The slides were then blocked with 3% BSA at room temperature for 1 h. Following washing with PBS, the scFvs were incubated with the cells at room temperature for an additional 1 h. Finally, their binding to FGFR2 molecules was detected using the corresponding secondary antibodies, as previously described. Nuclei were counterstained with a 4′,6diamidino-2-phenylindole (DAPI) solution (Sigma). The 2394

DOI: 10.1021/acsomega.8b03072 ACS Omega 2019, 4, 2387−2397

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and N-hydroxysuccinimide and subsequently incubated at room temperature for 1 h. Next, the mixture solution was introduced into the sensor chip for ligand coating. After using blocking buffer to deactivate the remaining activated amino groups on the chip, the anti-FGFR2 scFv S3 (analyte) was prepared at different concentrations for detection. In the analysis, the chip was treated by 10 mM glycine−HCl (pH 2.2) buffer for each time of regeneration. The data were analyzed according to a 1:1 binding model by the Trace Drawer software. 4.11. Tumor Xenograft Model. For tumor implantation, the freshly prepared SNU16 cancer cells were harvested and resuspended in PBS at a concentration of 5 × 107 cells/mL. Each mouse was inoculated subcutaneously with 1 × 107 cells for tumor forming. When the tumors size was approximately 200 mm3, the animals were divided into groups (n = 6) for treatment. The tumor size was measured twice weekly, and the volume was calculated as follows: V = 0.5lw2, where w = width and l = length. At the end of the experiment, the antitumor effects were quantified by dividing the tumor volumes in the treatment groups by those in the control groups and multiplying by 100 for the calculation of tumor growth inhibition (TGI %). The mice were also examined frequently for overt signs of any adverse drug-related side effects. 4.12. Matrix Gel-Assisted Angiogenesis Model. Angiogenesis assay in vivo was determined as the blood vessel growth in the exogenous matrix gel plug injected into the male nude mice. Matrix gel (BD Bioscience) was mixed with heparin (10 units/mL), VEGF (40 ng/mL), insulin-like growth factor1 (40 ng/mL), epithelial growth factor (EGF, 40 ng/mL), bFGF (40 ng/mL), and hS3 antibody (not in the controls), and the resulting mixture was injected subcutaneously into the abdomens of the mice. After 7 days, the animals were sacrificed and the matrix gels were carefully dissected and photographed. To quantify blood vessel formation, the hemoglobin (Hb) levels were analyzed using Drabkin’s reagent kit (Sigma). 4.13. Binding Site Key Residue Definition. To define the key residues on peptide F2pep for antibody hS3 interaction, peptide phage display was used for the experiment, as described previously.20 Five mutagenesis peptides were designed and displayed on the surface of the phage through site-directed mutagenesis. In brief, the wells of microtiter plates were coated with humanized scFv hS3 in duplicate at room temperature for 1 h. After blocking with 3% BSA, a mutagenesis peptide-displaying phage solution was added into each well individually and incubated for 1 h at room temperature. Then, the wells were washed with PBST and the bound phages were detected using HRP-conjugated mouse anti-M13 phage antibodies (GE Healthcare Life Sciences). Finally, the TMB substrate was added for development and the reaction was stopped by adding 1 N HCl. The absorbance was measured by the optical density at 450 nm 4.14. Molecular Modeling. To further investigate the interaction between scFv hS3 and FGFR2, the scFv S3 was modeled as a scFv scaffold and the three-dimensional crystallographic structure of FGFR2 was derived from the Protein Data Bank (PDB, id: 3OJM). The homology model of the humanized scFv S3, scFv hS3 was generated using the MODELER program in Discovery Studio v. 2018 (BIOVIA Inc., San Diego, CA) using the crystallographic structure of the anti-VEGF antibody (PDB id: 2FJG) as a humanized structural template by complementarity-determining region (CDR)

slides were examined using a confocal spectral microscope imaging system (TCS SP5, Leica). 4.7. Competitive Inhibition Assay. The competitive inhibition assay was used to determine the ability of scFv S3 to block FGF7−FGFR2 IIIb or basic FGF (bFGF)−FGFR2 IIIc interactions. In brief, the wells of microtiter plates were coated with human FGFR2 IIIb or FGFR2 IIIc recombinant proteins (0.5 μg/well). After blocking with 5% skim milk for 1 h at room temperature, plates were washed twice with PBST. Trxtag-fused recombinant FGF7 or bFGF was incubated first with various concentrations of scFv S3 (from 0.1 to 100 μg/mL) and then introduced into the wells to react with coated FGFR2 recombinant protein. After incubating the plates at room temperature for 1 h, the plates were washed six times with PBST. Rabbit anti-Trx-tag antibodies (GeneTex Inc.) were added to detect bound FGF7 or bFGF molecules. Then, HRPconjugated donkey antirabbit antibodies (Jackson ImmunoResearch Laboratories, Inc.) were used for the reaction. After washing, the TMB substrate was added to each well for development. The reaction was stopped with 1 N HCl, and signal intensity was measured at 450 nm. 4.8. Proliferation Analysis. In the cancer cell lines SNU16 and MKN45, cell proliferation was determined on the basis of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide (MTT) assay. The cells were seeded in a 96-well plate at a density of 1 × 104 cells/well and incubated overnight for attachment. The cells were then treated with scFv antibodies at the indicated concentrations in 2% FBSsupplemented medium. After treatment, the medium was removed and dimethyl sulfoxide was added to lyse the cells. Signal intensity was measured at 570 nm. In the HUVECs, cell proliferation was measured using the BrdU Cell Proliferation Assay Kit (Merck Millipore). Briefly, 5000 cells/well were seeded in a 96-well plate for attachment and subsequently treated with 50, 100, and 200 μg/mL hS3 IgG for 72 h. Then, the cells were treated using 10 ng/mL bFGF for additional 48 and 72 h incubations. Finally, the BrdU reagent was added to the wells and incubated for 24 h for development; the absorbance of each well was measured at 450 nm. 4.9. Molecule Signaling Determination. For the determination of molecule signaling in the SNU16 cells, the cells were cultured in 100 mm diameter culture dishes and treated using scFv antibodies at the indicated concentrations for 5 days. The cells were collected and lysed using a lysis buffer [50 mM Tris−HCl (pH 7.5), 50 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100] mixed with a mixture of proteinase inhibitors (Roche Applied Science). The protein concentration of the cell lysate was measured through the Coomassie Plus (Bradford) Protein Assay (Thermo Scientific). Samples were run on reducing SDS/ PAGE for Western blot analysis and detected using antibodies against Erk, Akt, p-Erk, and p-Akt (Cell Signaling Technology, Inc.). For HUVEC molecule signaling determination, the cells were cultured in culture dishes and pretreated with 50, 100, and 200 μg/mL hS3 IgG for 72 h. After incubation with 10 ng/ mL bFGF for 48 and 72 h, cell protein was collected and analyzed through Western blotting. 4.10. Surface Plasmon Resonance. Antibody binding affinity was analyzed and determined using the OpenSPR instrument (Nicoya Lifesciences Inc.). The FGFR2 protein was immobilized on an amine sensor chip and detected with fluid phase scFv. The FGFR2 protein (ligand) was mixed with one aliquot of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 2395

DOI: 10.1021/acsomega.8b03072 ACS Omega 2019, 4, 2387−2397

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PDB, protein data bank scFv, single-chain variable fragment SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis TGI, tumor growth inhibition TKIs, tyrosine kinase inhibitors TMB, 3,5,5-tetramethylbenzidine dihydrochloride VEGFR, vascular endothelial growth factor receptor VH, heavy-chain variable region VL, light-chain variable region

grafting. The details of the strategy of antibody−antigen docking are provided in the Supporting Information.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03072. Materials and methods, siRNA treatment, sequence alignment, the analysis of surface plasmon resonance, immunohistochemical staining and the strategy of antibody−antigen docking (PDF) (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +886-2-27361661 ext. 7279 (Y.-C.L.). *E-mail: [email protected]. Tel: +886-2-27361661 ext. 7682 (Y.Y.). ORCID

Chao-Di Chang: 0000-0002-1926-8344 Yun Yen: 0000-0003-0815-412X Author Contributions ¶

K.-C.T. and C.-D.C. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the “TMU Research Center of Cancer Translational Medicine” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan, the Health and welfare surcharge of tobacco products grant MOHW107-TDU-B-212-114020, MOHW107-TDU-B-212-114014, and MOHW107-TDU-B212-114026B, and the Ministry of Science and Technology in Taiwan under Grant MOST 105-2628-B-038-007-MY3.

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ABBREVIATIONS bFGF, basic FGF BSA, bovine serum albumin CDR, complementarity-determining region DAPI, 4′,6-diamidino-2-phenylindole ELISA, enzyme-linked immunosorbent assay E. coli, Escherichia coli FBS, fetal bovine serum FGFs, fibroblast growth factors FGFR2, FGF receptor 2 FGFRs, FGF receptors Hb, hemoglobin HRP, horseradish peroxidase HUVECs, human umbilical vein endothelial cells IgG, immunoglobulin G IgY, immunoglobulin Y MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide NOD/SCID, nonobese diabetic/severe combined immunodeficiency PBS, phosphate-buffered saline PBST, PBS with 0.05% Tween 20 2396

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