Development of a MET-targeted single-chain antibody fragment as an anti-oncogene targeted therapy for breast cancer

The usage of monoclonal antibodies (mAbs) and antibody fragments, as a matter associated with the biopharmaceutical industry, is increasingly growing. Harmonious with this concept, we designed an exclusive modeled single-chain variable fragment (scFv) against mesenchymal-epithelial transition (MET) oncoprotein. This scFv was newly developed from Onartuzumab sequence by gene cloning, and expression using bacterial host. Herein, we examined its preclinical efficacy for the reduction of tumor growth, invasiveness and angiogenesis in vitro and in vivo. Expressed anti-MET scFv demonstrated high binding capacity (48.8%) toward MET-overexpressing cancer cells. The IC50 value of anti-MET scFv against MET-positive human breast cancer cell line (MDA-MB-435) was 8.4 µg/ml whereas this value was measured as 47.8 µg/ml in MET-negative cell line BT-483. Similar concentrations could also effectively induce apoptosis in MDA-MB-435 cancer cells. Moreover, this antibody fragment could reduce migration and invasion in MDA-MB-435 cells. Grafted breast tumors in Balb/c mice showed significant tumor growth suppression as well as reduction of blood-supply in response to recombinant anti-MET treatment. Histopathology and immunohistochemical assessments revealed higher rate of response to therapy. In our study, we designed and synthetized a novel anti-MET scFv which could effectively suppress MET-overexpressing breast cancer tumors.


Background
The use of monoclonal antibodies (mAbs), as an important branch of the biopharmaceutical industry, is increasingly developing. This includes a wide range of applications such as protein purification, protein-based in vitro diagnostic tests, and treatment of a large variety of human diseases in particular as a therapeutic approach against cancer cells [1,2]. More recently, smaller antibody binding fragments have become the topic of interest, since they offer significant advantages over the full-length mAbs, such as higher affinity and specificity, less immunogenicity, better solubility, and stability as well as less expensive production [1,3]. Three types of fragments, including the single V-type domain, antigen-binding fragment (Fab), and single-chain variable fragment (scFv) are representing consecutive waves of antibody fragment technologies [4,5]. The scFv consists of variable regions of heavy and light chains of antibodies, which are assembled by a flexible linker [6]. Although this unit of antibody is the smallest format of immunoglobulin, it still retains antigen-binding activity and specificity [6,7]. Small size of scFv (∼30 kDa), is a desirable trait for tissue infiltration, especially in cancer therapy [8,9]. The monovalency of this antibody fragment made it desirable for targeting receptor tyrosine kinases to overcome antibodymediated receptor dimerization [10].
Receptor tyrosine kinases (RTKs) as transmembrane proteins are expressed on different normal cell types as well as tumor cells [11]. Their substantial role in tumorigenesis made them targets for the development of promising anticancer drugs [12]. Multifarious antibodies inhibiting RTKs are being used in clinical practice [13]. A subfamily of RTKs known as hepatocyte growth factor receptor (HGFR) or mesenchymal-epithelial transition (MET or c-Met) is expressed on the epithelial cell surface. Upon binding of HGF to this receptor, MET homodimerization and phosphorylation occur, triggering a signaling cascade that is responsible for early embryonic development, which is generally quiescent in adult tissues, except for tissue repair [14]. Aberrant activation of the MET/HGF signaling pathway in cancer cells, as a result of overexpression, mutation, or amplification of the MET gene, plays a pivotal role in growth factor-mediated proliferation, epithelial to mesenchymal transition, metastasis, and survival, leading to neoplastic transformation in a variety of cancers, including breast cancer [10,[15][16][17]. This signaling axis, as one of the hallmarks of malignancy in initiating invasion and metastasis, has become a favorite target for developing monoclonal antibodies for immunotherapeutic purposes [14]. MET augments proliferation, migration, invasion, protection from apoptosis, and angiogenesis in malignant cells [18]. Of note, MET overexpression acts as a 'stress-response gene' which leads to transcriptional adaptation of malignant cells to the undesirable microenvironment, including hypoxia [19]. MET amplification and overexpression have been correlated with lymph node metastasis, depth of tumor invasion, advanced stage, and decreased survival rate [20]. Exposure to antiangiogenic therapy bevacizumab results in upregulation of MET [21]. Thus, activation of the MET axis has known as one of the mechanisms of resistance to the bevacizumab [22]. Besides the ability of the oncogenic driver, MET also contributes to targeted therapy resistance during using approved EGFR and Her2 inhibitors [23,24].
Although various clinical trials are ongoing in this field, none of the HGF/MET-targeted antibodies or antibody fragments could complete phase 3 of the clinical trial with satisfactory results [10,[25][26][27][28]. The challenging complication of ordinary monoclonal antibodies is the undesirable MET activation rather than anatomization, which happens as a result of RTK dimerization, leading to kinase auto-activation [27]. For example, DN30 which is a monoclonal antibody against an epitope on the extracellular domain behaves as a partial agonist [13]. But this antibody induced MET downregulation by the mechanism of shedding the ectodomain of the receptor [13]. Another conventional monoclonal antibody against MET increases MET activity by imitating HGF-mediated dimerization, even so, it could mediate endo-lysosome-associated degradation [28]. To overcome this problem, monovalent antibodies were replaced by traditional bivalent antibodies to target MET without dimerization-induced activation [10]. In this regard, Onartuzumab (Genentech) has been engineered as a "one-armed" monovalent antibody with native-like architecture which lacks antibody-induced dimerization function with resultant antagonism of the HGF\MET axis [27]. This specific Met-MAb is made up of a single humanized antigen-binding fragment (Fab) assembled to the constant domain fragment (Fc) expressed in E. coli [27]. The Onartuzumab (Genetech), was successful in avoiding MET dimerization but, the phase III study did not confirm the efficacy results observed in the phase II study for non-small cell lung cancer [29]. Furthermore, this drug was not successful in phase 2 of the clinical trial in triple-negative breast cancer [30]. Another inhibitor of MET signaling is AMG 337, a selective smallmolecule kinase repressor [31]. The preclinical studies of AMG 337 showed, inhibition of MET phosphorylation and MET-dependent cell growth in several MET-overexpressing cell lines as well as induction of apoptosis and decreasing tumor growth in MET-dependent xenograft models [32]. This study also showed anti-tumor activity in MET-amplified adenocarcinoma in phase 1 and 2 clinical trials [20,33]. Other small molecule kinase inhibitors targeting MET, such as Tivantinib (ArQule), have been found unsuccessful [34]. Different studies were designed anti-MET scFv to target MET RTK [35][36][37][38]. The anti-MET scFv could also be used for drug delivery approaches [39]. A more recent anti-MET scFv showed apoptotic and anti-proliferative potency in the human colorectal carcinoma cell line [38].
Further efforts should be made to overcome the current difficulties of MET targeted therapy, which is considered an important immunotherapeutic program in epithelial neoplasms.
Congruent with this notion, here we developed a novel modeled anti-MET scFv, which was synthesized by gene cloning and expression in a bacterial host. Designing, construction, and purification of this antibody fraction were then followed by functional assessment as well as in vivo assays.

Cell culture & bacteria strains
Cloning and gene expression were accomplished using E. coli strains NovaBlue GigaSingles™ (Novagen-USA) as the primary cloning host and BL21-DE3 (Novagen-USA) as the expressing host. For gene cloning, plasmid pET-32 LIC vector (Novagen-USA) was utilized. The cell surface expression of MET is abundant in the MDA-MB-435 cell line whereas this protein has no expression in the BT-483 cell line [40]. Thus, authenticated human ductal carcinoma cell lines (MDA-MB-435 and BT-483), as well as mouse breast cancer line (4T1), were purchased from Pasteur Institute of Iran (IPI, Iran). All cell lines were cultured as adherent cells in high glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 1% non-essential amino acid (Gibco-USA), 2 mM L-glutamine (Gibco, USA), 1 mM sodium pyruvate (Gibco, USA), and 1% penicillin-streptomycin (Gibco, USA) in gamma radiated sterile polystyrene plates and flasks then stayed at 37 °C in a 5% CO2 humidified atmosphere incubator.

Construction of recombinant anti-MET scFv expression cassette
The DNA sequences of the variable heavy chain (VH), as well as variable light chain (VL) domains of anti-MET Onartuzumab Fab, were obtained from GenBank (Accession number: 4K3J-H and 4K3J-L respectively). The detailed process of construction of Onartuzumab antibody has been described elsewhere [27]. A glycine-rich 15 amino acid peptide was inserted between variable chains as a flexible linker. After the synthesis of this codon (Cinnagen, Inc. Tehran, Iran), it was amplified by PCR, followed by 3 sequential PCR runs to add the following sequences to the reading frame by use of specific primers listed in Table 1: a stII signal peptide coding sequence at the 3' end to localize anti-MET protein in the periplasmic space of expressing bacteria which can then be easily harvested in its native structure after disrupting the bacterial wall, a 6 constitutive histidine tag moiety at 5' to isolate the protein by Ni-NTA affinity chromatography and also for protein characterization by immunoblotting and the compatible overhang sequence compatible to pET-32 Ek-LIC vector at both ends of the codon. (Fig. 1A) The primers were designed by Gene Runner Software v3.05 (Hastings Software Inc. Las Vegas, USA) and then synthesized by Pishgam Biotech Co. (Tehran, Iran). The PCR reaction mix was comprised of 5 µl dNTP mix (0.2 mM), 5 µl of 10× reaction buffer with 1.5 mM MgCl2, 2 µl of each primer (12.5 mM/µl), 0.4 µl Taq DNA polymerase (1 Unit) and 2 µl template (50 ng/ml) and 8.6 µl ddH2O in a total volume of 50 µl in 30 cycles. Each cycle was started with an initial denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 1 min and a final extension at 72 °C for 5 min. Then the final PCR product was harvested from the 2% agarose gel after the electrophoresis and concentrated with the Qiagen gel extraction kit (Qiagen, Germany). Theoretical isoelectric point was estimated 8.57 ± 1 using Expasy (https://web. expasy.org/compute_pi/).

Cloning and expression of recombinant anti-MET scFv
The achieved DNA amplicons were cloned within the ligation-independent cloning (LIC) site of the pET-32 EK/LIC vector based on the protocol of manufacturer (Novagen, USA). Transforming the constructed plasmid, pET-32 Ek-LIC/Anti-MET, into E-coli Nova Blue GigaSingles™ competent cells (Novagen, USA), was performed by the calcium chloride method [41]. Transformants were then cultured on LB agar containing ampicillin (50 µg/ml). The recombinant clones were submitted randomly to colony-PCR and clones with desirable insertion sizes were characterized by sequencing (Gene Fanavaran Co, Iran) to confirm successful cloning. The accuracy of the sequence was confirmed using Sequence Alignment Editor Software Bio Edit. Harvested plasmids were then used to transform into proteinexpressing E-coli strain, BL21 (DE3) competent cells (Novagen, USA), and transformants were cultures on LB agar plates containing 50 µg/ml ampicillin. Single colonies of the E. coli BL21 DE3 containing pET32Ek/LIC-anti-MET plasmids were then grown in 5 ml LB broth containing 50 µg/ml ampicillin and incubated at 37 °C and 180 rpm in a shaker incubator for 16 h. Subsequently, the culturing tube was poured into 200 mL ampicillin containing terrific broth to reach an optical density (OD) 600 of at least 0.4. At this point, isopropyl thiogalactoside (IPTG) was added (1mM) to induce protein expression at 27 °C for 16 h in a shaker incubator (150 rpm).  were finally visualized using 0.05% 3, 3′-Diaminobenzidine (DAB; Sigma, Germany) in 100 mM Tris-HCl, pH 7, containing 0.03% hydrogen peroxide.

Cell surface antigen-binding assay via flow cytometry
A human metastatic breast carcinoma cell line BT-483 and a putative breast/melanoma cell line MDA-MB-435 were used as rarely expressing (as a control cell line) and constitutively expressing MET, respectively [40]. The cells were incubated with 50 µg/ml of purified concentrated anti-MET scFv for 1 h at 4 °C. FBS-treated cells were used as a negative control. The cells were then washed three times with ice-cold PBS and 4% FBS and were incubated with 10 µg/ ml FITC conjugated anti-His specific antibody (Abcam, UK) for 1 h at 4 °C (1:500). Subsequently, antigen-binding to the cell surface was assessed using the FACScalibur Flow Cytometer (FC) device (Becton Dickinson, USA) at the FL1 channel.

Cell viability assay; MTT assay
To appraise the potentiality of anti-MET scFv cytotoxicity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) assay was performed. Briefly, 5 × 10 3 MBA-MD-435 and BT-483 cells were seeded in every well of a 96-well plate and the cells were cultured with 0.2 ml medium at 37 °C for 24 h. Different concentrations (0-300 µg/ml) of anti-MET scFv were added to wells followed by 48 h incubation. Cells were then incubated with the MTT dye at 37 °C for 3 h. After washing out the MTT with PBS, formazan which was formed from MTT in viable cells by dehydrogenase enzyme was dissolved using DMSO. Finally, absorbance rate was read at 570 nm by a microplate absorbance reader (BioRad, USA), and 50% inhibitory concentration values (IC 50 ) were calculated from triplicate data as the concentration of the protein inducing a 50% reduction in viability when compared to untreated cells.

Apoptosis assay
The percentage of the total, late and early apoptotic cells was determined using flow cytometric Annexin V-FITC)/ propidium iodide (PI) assay (Biolegend, San Diego, USA) according to the manufacturer' s guidelines. For this purpose, 4 × 10 5 cells were seeded into each well of a 6-well plate, treated with anti-MET scFv at different concentrations (18, 32, and 50 µg/ml), then incubated for 48 h at 37 °C. These concentrations were selected based on the average of 25% and 75% growth inhibitory activity of our protein in the MTT assay amongst all cell lines. Then Cells

Protein purification and concentration
For the purification of anti-MET scFv, immobilized metal affinity chromatography (IMAC) was performed through denaturing method and by using a Ni-NTA-Agarose column (Qiagen, Germany). For this purpose, expressing bacterial cells were first collected by centrifugation (9000 rpm, 4 °C, and 30 min). After resuspension of pellets in the ice-cold denaturing buffer (500mM NaCl, 8 mM Urea, Tris-HCl pH 7.0, and imidazole 10 mM), all soluble periplasmic proteins were extracted utilizing sonication (1mA for 10 min) with subsequent centrifugation (4 °C, 15,000 rpm). The supernatant, containing His-tagged anti-MET scFv and the host soluble proteins were collected and then submitted for IMAC. First, the column was prepared by passing the binding buffer (300 mM NaCl, 6 mM Urea, Tris-HCl pH:8.0, and imidazole 2 mM) through 5 mg Ni-NTA HisBind Resin (Qiagen, Germany). Afterward, the extracted protein was added to the column, incubated at 26 °C for 1 h, then washed by three sequential wash buffers to reduce the urea concentration gradually (wash 1: 500 mM NaCl, 4 mM Urea, Tris-HCl pH 7.0; wash 2: 500 mM NaCl, 2 mM Urea, Tris-HCl pH 7.0; wash 3: 500 mM NaCl, Tris-HCl pH 7.0). In the last stage, the bound anti-MET was eluted by 5 column volumes of elution buffer (300 mM NaCl, 300 mM imidazole, and Tris-HCl pH 7.0). The imidazole of the elute was eliminated by diafiltration. Then purified proteins were concentrated to 500 micrograms per milliliter on an Amicon (Merck, Germany) concentrator using sterile PBS, pH 7.0, and kept at 4 °C for experiments. The concentration was measured by a spectrophotometer (WPA Biowave II, Bichrom, England).

Characterization of the purified protein; SDS-PAGE, Western blotting
Eluted fractions of IPTG positive, IPTG negative, and purified anti-MET fragments were electrophoresed on a 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel), in the presence of 2-mercaptoethanol as a reducing agent. The gels were then stained with Coomassie brilliant blue to evaluate the protein bands. Isolated proteins by SDS-PAGE were then submitted to the polyvinylidene difluoride (PVDF; Millipore, Merck, Germany) membrane using a semi-dry blot transfer device (Bio-Rad, Hercules, USA) based on the standard protocol [42]. To improve the signal-to-noise ratio, the membrane was blocked with a blocking buffer comprising 5% w/v non-fat dried milk (Merck, Germany) in 0. Tumor volume was measured non-invasively through the US imaging every 3 days according to the following formula: V (mm 3 ) = 1/6 π × x × y × z. In which x, y, and z represent tumor height, width, and length (mm), respectively. The results were then compared to the PBS receiving control group. At the end of the treatment period, mice were sacrificed, then tumors were surgically excised, weighed, and submitted to 10% formaldehyde (NPF; Sigma-Aldrich, HT501128) for histopathology assessment.

Ultrasonography techniques
Percutaneous real-time ultrasonography (B-mode and color Doppler) was performed using a multi-frequency (6-13 MHz) linear transducer with a small footprint (SLAx/6-13 Ultrasound Transducer), connected to a portable ultrasound unit (Micromaxx, Sonosite, USA, 2009). The transducer was aligned to the center of tumor and the maximum depth of 19 mm was set to gain the sagittal and transverse planes. The tumor's height, width, and length were measured using the unit's caliper. In addition, tumor's shape, margin (smooth or irregular), parenchymal echogenicity (hypoechoic or hyperechoic), and echotexture (homogeneous or heterogeneous) were evaluated. Also, probable invasion to regional structures, severity (mild, moderate and severe), and the pattern of the vascularization (centrally, peripherally or both) were assessed. The ultrasonography was performed every 3 days with all the above parameters checked to analyze the amount of tumor outgrowth, spreading and invasion.
For the best attachment of the probe to the skin, the tumor site was shaved and an ultrasonography jelly was applied. Image orientation was performed using landmarks to procure standardized planes and correct measurements. In Color Doppler technique, high-quality images and accurate measurements of optimum velocity were obtained by setting the Color Doppler map to 13 mm/s and by changing were harvested utilizing 1X trypsin-EDTA (Gibco, USA) and washed with PBS. Cells were then centrifuged and the pellet was suspended in 100 µl binding buffer of the assay kit and then stained by FITC-labeled Annexin V and PI. Cells were then submitted to flow cytometry analysis (BD FACS, Calibur) on the FL1/FL3 channels and the double staining quadrant was gated on targeted cells. The single positive FL1 population (Annexin V labeled cells) and double-positive cells (FLs1/FL3) as respective early and late apoptosis, were summed up to obtain the total apoptotic cell percentage.

Invasion assay
Given the role of MET in metastasis, we examined whether anti-MET scFv affects cancer cell invasion and migration. The Cultrex BME Cell Invasion Assay (R&D Systems) (Trevigen Inc., Gaithersburg, MD, USA) was used based on manufacturer protocol using MBA-MD-435 and BT-483 lines. 50 µL basement membrane extract (BME) solution was added to each well of the top chamber and incubated at 37 °C. 50,000 cells/ 50 µL serum-free medium containing 50 µg/ml anti-MET were plated on the coated BME. Control wells contained PBS instead of anti-MET. 150 µL of the medium was then loaded into the lower chambers. The Cells were then incubated for 24 h, at 37 °C. 100 µL of Calcein AM solution/cell dissociation solution was added to the bottom chamber and incubated for 1 h, at 37 °C. Relative fluorescence units (RFU) of the samples were determined at 485 nm excitation and 520 nm emission with an ELISA reader (BioTek-VT, USA). The data was compared to the standard curve to measure the number of cells that have invaded through BME.

Migration assay
Migration assay was performed utilizing Cultrex BME Cell Migration Assay (R&D Systems) (Trevigen Inc., Gaithersburg, MD, USA) similar to the above-mentioned invasion assay. On the second day, the upper chamber was carefully aspirated and washed with 1X cell wash buffer. Afterward, 100 µL of cell dissociation solution/Calcein-AM was added to the bottom chamber of a black assayed 96-well microplate. The cell migration chamber was assembled and incubated for 1 h, 37 °C. The top chamber was eliminated and absorbance was read at 485 nm excitation and 520 emissions compared to the standard curve. [6][7] week-old female inbred BALB/c mice, weighing 16-17 gram (Faculty of Veterinary Medicine, University was performed using primers specific to the recombinant anti-MET fraction (Fig. 1B). Based on our results, DNA sequences were successfully incorporated into the plasmids and subsequent sequencing results showed no point mutation or rearrangement.

Protein expression and characterization
To recognize the expression of our desired recombinant anti-MET scFv among all of the harvested periplasmic proteins, a comparison between the protein panel of IPTGpositive and IPTG-negative bacteria was done using 12% SDS-PAGE. Figure 1 C depicts the occurrence of a ~ 35 kDa protein band among other bacterial proteins which is absent in the negative control. This molecular weight is consistent with the anti-MET scFv and the histidine tag. The signal peptide moiety detaches during the expression. Purified anti-MET scFv demonstrated only a single band with the same molecular weight (Fig. 1D) while having a lower concentration. The final concentration of purified protein after passing through the Amicon (Merck, Germany) concentrator was 500 micrograms per milliliter. The concentration was also shown by SDS-page (Fig. 1D), with the purified band being then confirmed through western blotting (Fig. 1E).

Cell surface antigen-binding assay
To check the particular binding of anti-MET scFv to the HGFR, flow cytometry analysis was accomplished using MET-positive MDA-MB-435 and MET-negative BT-483 cancer cell lines. As exhibited in Fig. 2A, the MET-positive MDA-MB-435 line showed a high binding capacity with recombinant anti-MET scFv; however, a very low capacity of binding was observed with MET negative BT-483 cells ( Fig. 2A). The results showed 48.8% and 0.87% of binding to the MDA-MB-435 and BT-483 cancer cell lines, respectively. These findings propose that anti-MET scFv is specifically bound to HGFR-positive cancer cells.

Cell viability assay
The IC 50 value of anti-MET scFv against MET-positive human breast cancer cell line (MDA-MB-435) was 8.4 µg/ ml whereas this value was measured as 47.8 µg/ml in METnegative cell line BT-483 (Fig. 2B). The results relying on the dose of anti-MET in an increasing concentration show a gradual reduction in the proliferation of MDA-MB-435 in vitro (Fig. 2B). A negligible difference was seen in the cellular viability of the BT-483 cells upon treatment with the anti-MET antibody fragment.
its angle to 60 degrees. This was repeated in all animals to decrease the possible miscues.

Histopathology
At the end of the treatment period, tumors were excised and prepared for histopathological evaluation. The H&E staining was performed to study the percentage of the tumor necrosis, mitotic count (per 10 HPF), nuclear pleomorphism (scores 1, 2, and 3), and tumor-infiltrating lymphocytes (TILs) (number of lymphocytes in 10 HPF). Following antibodies were used for immunohistochemical staining: anti-CD8 monoclonal antibody (Catalog number: ab209775, Abcam, USA), marker of cytotoxic T cells; anti-Bax monoclonal antibody (Catalog number: ab263897, Abcam, USA), apoptosis regulator; anti-Ki-67 monoclonal antibody (ab15580, Abcam, USA), marker of proliferation; anti-VEGF receptor 2 (VEGF-R 2 ) monoclonal antibody (Catalog number: ab2349, Abcam, USA), indicator of angiogenesis, and antimatrix metalloproteinase-9 (MMP-9) monoclonal antibody (Catalog number: ab38898, Abcam, USA), a proteinase up-regulating in metastasis and angiogenesis. The specific HRP-conjugated secondary antibody was used. Gill's hematoxylin was used as counterstaining for all sections. Microvessel's density (MVD) was assessed as the average number of microvessels in 4 random fields after VEGF-R 2 staining. The percentage of positive nuclei for Ki-67 per 1000 malignant cells was also used to assess proliferation and a modified ALL-Red scoring system (proportion score and intensity score) was used to assess MMP-9-and BAXstained sections.

Statistics
The experiments were carried out in triplicate, and the data represent mean ± SD. Gaussian distribution was assessed by the Shapiro-Wilk test. Ordinary one-way ANOVA was used to analyze among 3 or more group differences and a Tukey test as post-hop. Two-tailed unpaired t-test was carried out to compare between two groups. Statistically significant, P-value ≤ 0.05 was considered (* p < 0.05, **p < 0.01, ***p < 0.001). Graphpad Prism version 9.0.0 and Excel 2016 were used for statistical analysis.

Gene cloning
Full-length anti-MET-scFv coding sequence was cloned into the pET32 /LIC vector. To screen the anti-MET-bearing colonies containing our desired sequence, colony PCR rough boundaries on the final day of treatment (Fig. 3D). Contrarily in anti-MET scFv receiving mice, the smooth form of tumors was generally notified.

Immunohistochemistry (IHC), quantification of microvessel's density and histopathology
Results of the hematoxylin and eosin staining (H&E) method showed a higher rate of response to therapy in the anti-MET scFv receiving group compared to the non-treated control one (Fig. 4). In parallel, the percentage of infiltrated lymphocytes was defined by an expert pathologist which was blinded from the experimental groups, based on their nuclear morphology. Our results showed no significant difference in the percentage of TILs in the anti-MET receiving tumors compared to the control tumors (less than 5% TILs in both groups) (Fig. 4A). Moreover, results of IHC analyses revealed a significantly reduced microvascular density in anti-MET receiving animals compared to the PBS receiving animals (Fig. 4B). Analyzing apoptosis by evaluation of Bax in the control and treated mice, a significantly declined apoptosis rate was spotted in the anti-MET receiving tumors compared to the PBS control tumors (Fig. 4B). Moreover, our results demonstrated that the expression level of Ki-67 was significantly decreased in the tumors harvested from the anti-MET receiving animals (Fig. 4B). However, no significant change was observed in MMP-9 and CD-8 markers in the anti-MET receiving group when compared with the control tumors (Fig. 4B).

Discussion
In the present study, we designed and synthesized a novel anti-MET scFv, and then investigated the functional properties and tumor-suppressive effects in vitro and in vivo. Since MET plays a dual role both as a necessary oncogene and also as a proper target in the acquired drug resistance, it could apply to target MET alone or concurrent with other therapies to prevent the advent of resistance [21,44]. However, targeting MET is still challenging so far, mainly due to the poor state of knowledge regarding MET kinase-independent function and the agonistic action of MET targeting for homo-dimerization [45,46]. The use of the bacterial expression of scFv resulted in lack of Fc effector function such as ADCC (Antibody-dependent cellular cytotoxicity) and high production yield as well as facilitating purification for further clinical use [47]. Results presented herein confirmed the usefulness of an E-coli expressed recombinant scFv with the preserved capacity of binding and blocking the MET. In our study, we used the scFv fragment to target MET which was proven to have rapid blood circulation,

Apoptosis assay
The potential of apoptosis induction by our anti-MET scFv was evaluated on MDA-MB-435 and BT-483 cell lines using Annexin V/PI assay. The results showed a considerably higher percentage of total apoptosis in the MDA-MB-435 cell line in comparison to the rare expressing cell line BT-483 (Fig. 2C). The apoptosis potency of the anti-MET scFv to MDA-MB-435 and BT-483 was measured as 52.5%, and 10.1%, respectively (Fig. 2C).

Migration and invasion assay
To evaluate the anti-invasive properties of anti-MET scFv, the Cultrex® BME cell invasion assay kit was used. This assay offers a standardized and flexible method for evaluation of the severity penetration of invasive cells through a barrier of basement membrane and extracellular matrix elements in vitro. The results showed significantly higher inhibition of migration and invasion of the anti-MET scFv treated MDA-MB-435 cell line. This effect was not seen in the BT-483 cells which express low levels of MET (Fig. 2D).

In vivo tumor growth suppression and ultrasound imaging
After 14 days of anti-MET scFv administration in 4T1 tumor-bearing BALB/c mice, a decrease in tumor growth was recorded in comparison with the PBS receiving group (Fig. 3B, D). In this context, Fig. 3A represents the ultrasound imaging technique to gain transverse and longitudinal scans of tumor mass by changing the angle between the ultrasound waves and the direction of the animal. By this technique, three different dimensions were obtained from the ovoid mass (x, y, z) to calculate the tumor volume. As depicted in Fig. 3, enhancement of tumor growth suppression and lac of logarithmic phase of tumor growth was significant in the anti-MET receiving animals (Fig. 3B, D). No significant changes were observed in the bodyweight of mice during the treatment, confirming that the administered dosage of anti-MET scFv was safely tolerated (Fig. 3D). In addition, the tumor weight after the treatment period was significantly less in the anti-MET group than in the control group (Fig. 3D). Differences in tumor size became meaningful from the second injection and progressively increased until the last injection (Fig. 3D). In addition, inhomogeneity in the texture of tumor images, as well as the development of waved-shaped boundaries, is related to the invasiveness and metastatic behavior of tumors [43]. Results demonstrated a higher risk of invasion and metastasis in the control animals than in the anti-MET scFv receiving group. Overall, tumors of the PBS receiving mice were inhomogeneous and had acted as an antagonist for MET receptor in cell-based assays, including dose-dependent inhibition of cell viability and inhibition of migration in MET expressing cell lines providing good localization and tissue penetration of the drug within one hour after the administration [48,49]. Like the Onartuzumab, our newly designed anti-MET fragment protein kinase (MAPK) and Jun amino-terminal kinases (JNKs) [52,53]. Also the death receptor FAS (also known as CD95 and TNFRSF6) interacts the ectodomain of MET, which inhibits FAS self-aggregation and FAS receptor-FAS ligand (FASL) recognition, so preventing apoptosis through the extrinsic pathway [52]. As we showed here, blocking of MET results in decreased cell growth and enhances cell apoptosis. Our results demonstrated increased BAX expression which was in agreement with the in-vitro Annexin V/PI assay exhibiting significantly higher apoptosis in response to the anti-MET treatment. The pro-invasive and pro-migratory potency of MET has been shown in several human breast cancer cell lines [54]. Also, there is a positive [27]. High binding affinity has been demonstrated between the MET receptor and three different anti-MET scFv fragments [39]. Our study supports the aforementioned report in that it showed a significantly higher binding capacity to cancer cell lines over-expressing MET. As HGF/MET is known to play an important role in 4T1 tumor progression [50], the 4T1 breast cancer mice model was used for in-vivo experiments in the current study. MET RTK acts as an antiapoptotic mediator by increasing the BAX/BCL2 ratio [51]. MET-dependent cell growth is mediated by up-regulation of many signaling cascades including the extracellular signal-regulated kinase 1 (ErK1) and ErK, the phosphoinositide 3-kinase-Akt (Pi3K-Akt) axis, the mitogen-activated A Effect of Anti-MET scFv administration on TILs in tumor site compared to the control. TILs were counted in 10 high power fields (×40 magnification) and reported as percent ± SD, no significant change was noted. Scale bars are equal to 100 μm. B IHC staining of Bax (marker for apoptosis), Ki-67 (marker of proliferation), MMP-9 (marker for invasion), VEGF-R 2 (marker for angiogenesis) and CD-8 (marker for cytotoxic T-cell). Data are representative of mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars are equal to 100 μm Data Availability The authors declare that all data supporting the findings of current study are provided within the manuscript.

Declarations
Ethics approval and consent to participate This project was in accordance with the national norms, the ethical principles and standards for conducting medical research in Iran and evaluated by Motamed Cancer Institute-Academic Centre for Education, Culture and Research. This institution performed its reviews based on United States Public Health Service (USPHS) regulations and applicable federal and local laws. Protocols were also approved by the Animal Care Committee of the University of Tehran (7508017623).

Consent for publication
All authors have agreed to publish this manuscript.

Competing Interest
The authors declare no competing interests. association between the level of MET expression and the invasiveness-related genes (MMP-9 and MMP-2) [55]. In our study, we showed that the invasion and migration could be significantly inhibited by anti-MET treatment in the MET over-expressing cell line, MDA-MB-435. Nevertheless, MMP-9 expression was not affected by anti-MET treatment in 4T1 tumor graft mice. This could be attributed to the different pro-invasive effects of MET in different cell lines. The HGF/ MET signaling is also deemed as a potent trigger of endothelial cell growth [54]. The MET axis can increase VEGFR expression; with MET and VEGF-R 2 synergistically collaborating in inducing tumor angiogenesis [56]. It has been showed that consecutive administration of the VEGF-R tyrosine kinase inhibitors in human lung cancer leads to activation and upregulation HGF/MET signaling cascade in both the tumor cells and stromal cells [57]. In another study, inhibition of MET tyrosine phosphorylation in lung carcinoma was associated with effectively decreased MVD and reduced VEGF expression in cell extracts [58]. Our results showed the usefulness of the anti-MET scFv synthesized in this study to curb the MVD and down-regulation of VEGF-R 2 in experimental breast cancer.

Conclusion
Altogether, scFv antibody fragments targeting tumor oncogenes, in particular MET TKR, could be regarded as appropriate therapeutic tools in targeted therapy for MET expressing tumors. In our study, we developed and synthesized a novel anti-MET scFv that could effectively suppress MET-overexpressing breast cancer cells. Our results showed that the anti-MET scFv fragment could be a potent therapeutic agent to inhibit tumor proliferation and invasiveness. However, evaluation of this antibody fragment in mice breast cancer model shed more light on the details of tumor suppression activity of the anti-MET fragment. Therefore, we conclude that the anti-MET scFv could be a promising candidate for MET-targeted cancer therapy.