A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo

Authors

  • Hong Lin,

    1. Huadong Medical Institute of Biotechniques, Nanjing, China
    2. The Key Laboratory of Cancer Biomarkers, Prevention & Treatment Cancer Center and The Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing, China
    3. Department of Otolaryngology-Head and Neck Surgery, Jiangsu Provincial Hospital, Nanjing, China
    4. Jiangsu Provincial Blood Center, Nanjing, China
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  • Huiling Zhang,

    1. Department of Gynecology and Obstetrics, Nanjing Maternal and Children Care Hospital Affiliated to Nanjing Medical University, Nanjing, China
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  • Jun Wang,

    1. Department of Hematology and Oncology, Jiangsu Provincial Hospital, Nanjing, China
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  • Meiping Lu,

    1. Department of Otolaryngology-Head and Neck Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China
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  • Feng Zheng,

    1. Huadong Medical Institute of Biotechniques, Nanjing, China
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  • Changjun Wang,

    1. Huadong Medical Institute of Biotechniques, Nanjing, China
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  • Xiaojun Tang,

    1. The Key Laboratory of Cancer Biomarkers, Prevention & Treatment Cancer Center and The Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing, China
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  • Ning Xu,

    1. The Key Laboratory of Cancer Biomarkers, Prevention & Treatment Cancer Center and The Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing, China
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  • Renjie Chen,

    1. Department of Otolaryngology-Head and Neck Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China
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  • Dawei Zhang,

    1. Department of Otolaryngology-Head and Neck Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China
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  • Ping Zhao,

    1. Van Andel Research Institute, Grand Rapids, MI, USA
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  • Jin Zhu,

    1. Huadong Medical Institute of Biotechniques, Nanjing, China
    2. The Key Laboratory of Cancer Biomarkers, Prevention & Treatment Cancer Center and The Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing, China
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  • Yuan Mao,

    Corresponding author
    1. Department of Otolaryngology-Head and Neck Surgery, Jiangsu Provincial Hospital, Nanjing, China
    • Correspondence to: Dr. Jin Zhu, Huadong Medical Institute of Biotechniques, Nanjing 210002; The Key Laboratory of Cancer Biomarkers, Prevention & Treatment Cancer Center and The Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing 210029, People's Republic of China, Tel.: 86-25-8686-3100, Fax: 86-25-8686-3100, E-mail: zhujin1968@njmu.edu.cn or Dr. Yuan Mao, Department of Otolaryngology-Head and Neck Surgery, Jiangsu Provincial Hospital, Nanjing 210029, People's Republic of China, Tel.: 86-25-8371-2838, Fax: 86-25-8371-2838, E-mail: ymaoent@yahoo.com or Dr. Zhenqing Feng, The Key Laboratory of Cancer Biomarkers, Prevention & Treatment Cancer Center and The Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing 210029, People's Republic of China, Tel.: 86-25-8686-3100, Fax: 86-25-8686-3100, E-mail: fengzhenqing@njmu.edu.cn

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  • Zhenqing Feng

    1. The Key Laboratory of Cancer Biomarkers, Prevention & Treatment Cancer Center and The Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing, China
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  • Conflict of interest: The authors declare no conflict of interest.

Abstract

Human trophoblastic cell surface antigen 2 (Trop2) has been suggested as an oncogene, which is associated with the different types of tumors. In this study, a human Fab antibody against Trop2 extracellular domain was isolated from phage library by phage display technology, and characterized by ELISA, FACS, fluorescence staining and Western blotting analysis. MTT, apoptosis assay and wound healing assay were employed to evaluate the inhibitory effects of Trop2 Fab on breast cancer cell growth in vitro, while tumor-xenograft model was employed to evaluate the inhibitory effects on breast cancer growth in vivo. The results showed that Trop2 Fab inhibited the proliferation, induced the apoptosis and suspended the migration of MDA-MB-231 cells in a dose dependent manner. The expression caspase-3 was activated, and the expression of Bcl-2 was reduced while that of Bax was elevated in MDA-MB-231 cells by treating with Trop2 Fab. In addition, Trop2 Fab inhibited the growth of breast cancer xenografts and the expression of Bcl-2 was reduced while that of Bax was elevated in xenografts. Trop2 Fab, which was isolated successfully in this research, is a promising therapeutic agent for the treatment of Trop2 expressing breast cancer.

Breast cancer is the most frequent neoplasm in women, accounting for nearly 25% of all cancers in females.[1] In 2012, nearly 226,870 new cases of invasive breast cancer and 39,510 breast cancer deaths occurred in USA.[2, 3] Although breast cancer is recognized as a common disease in Western countries, the incidence and mortality rates of breast cancer have been increasing rapidly in less developed regions. In China, breast cancer ranks the first among top 10 most common cancer.[4] Although there have been notable advances in early diagnosis and clinical treatment of breast cancer,[5, 6] the outcome of breast cancer treatment is still not satisfied. At present, the majority of available chemotherapeutic agents lack high efficacy and selectivity. Therefore, it is important to identify novel targets or biomarkers to evaluate the prognosis and predict the response to treatment of breast cancer.[7]

Human trophoblastic cell surface antigen 2 (Trop2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), belongs to the TACSTD family.[8-10] Trop2 has been suggested as an oncogene, which is remarkably associated with the development, progression and metastasis of various types of malignant tumors.[10-18] Our previous study demonstrated that the high expression of Trop2 was correlated with poor prognosis in breast cancer patients.[19] Thus, Trop2 is an ideal target for targeted therapy of breast cancer.

In this study, the extracellular domain (EMD) of Trop2 was chosen as the antigen to isolate human naive Fab from phage library. Next, we characterized its binding specificity and affinity to native Trop2 as well as breast cancer cells overexpressing Trop2. Furthermore, we evaluated the anticancer effects of Fab in vitro and in vivo.

Material and Methods

Phage library, helper phage and bacterial strains

A human naive Fab phage library was generated as described previously.[20] Before the first-round panning, the phage library was titrated and 1 × 1013 phage clones were collected for panning.

Cell lines, purified EMDs of Trop2 and biopanning methods

Mouse fibroblast cell line NIT3T3 and two breast cancer cell lines (SK-BR-3 and MDA-MB-231) were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) where they were characterized by DNA fingerprinting, isozyme detection and cell vitality detection. These cell lines were immediately expanded and frozen to restarting about every three months from a frozen vial of the same batch of cells. Two cell lines were used for biopanning and Fab characterization, as well as for in vitro bioassays NIH3T3 is a Trop2-negative cell line, and SK-BR-3 is a Trop2-positive cell line. The initial characterization of SK-BR-3 and NIH3T3 cells has been described previously.[21, 22] All cell lines were cultured in RPMI-1640 medium (Gibco, San Francisco, CA) supplemented with 10% fetal bovine serum (FBS; Gibco), penicillin (100 U/mL) and streptomycin (100 μg/mL). EMD antigen of Trop2 was purchased from R&D (R&D Systems, Minneapolis, MN). For screening the phages that specifically bind to Trop2 on the cell surface, subtractive panning was performed in both NIH3T3 and SK-BR-3 cell lines, as well as on immobilized Trop2 EMD as described previously.[23, 24]

Phage ELISA

Single phage clones from the E. coli XL1-Blue infected by the seventh round of eluted phage were picked up and grown in 1 mL super broth (SB) medium containing 50 mg/mL carbenicillin and 1% glucose. VCSM13 helper phage (1 × 109) was then added to each vial. Fifty microliters of supernatant from each vial was added to each well of 96-well plates coated with 100 ng EMDs of Trop2 that had been preblocked with 5% milk blocking buffer. After incubation and washing, 50 μL of horseradish peroxidase (HRP)-conjugated anti-M13 antibody (Amersham Pharmacia Biosciences, Piscataway, NJ; 1:4,000 dilution in blocking buffer) was added to each well, followed by incubation with 50 μL of HRP substrate solution (Pierce, Rockford, IL). The absorbance value at 450 nm was read by Multiskan Spectrum Microplate (Thermo Electron Corporation, MA). The ELISA assays were repeated at least three times.

Expression and purification of Fab fragment

A single clone was reinoculated in SB medium with carbenicillin, induced by 1 mmol/L isopropyl-β-d−1-thiogalactopyranoside (IPTG) in the presence of 4% sucrose at 25°C and harvested 24 hr later. Both bacteria lysate and sonicated supernatant were detected by Western blotting analysis for the expression of Trop2 Fab induced by IPTG. The soluble Trop2 Fab was purified from the periplasm of the bacteria using Protein L (GenScript Corporation, Piscataway, NJ) affinity purification.

Immunoprecipitation (IP) and mass spectrometry analysis

Twenty microliters of Protein L Plus-Agarose (Santa Cruz Biotechnology, CA) and 40 μg Trop2 Fab was mixed and incubated at 4°C for 3 hr, then incubated with 100 μg of cell lysate from NIH3T3 and SK-BR-3 cells at 4°C overnight. The precipitates were washed with phosphate Buffered Saline Tween-20 and resolved on a SDS-PAGE and transferred to PVDF membranes. The membranes were probed with mouse anti-Trop2 antibody (Abcam, Cambridge, England) followed by a secondary HRP-conjugated goat anti-mouse antibody (Santa Cruz) at 1:1,000 dilution. The reaction was detected by ECL Western Blotting Substrate (Pierce, Rockford, IL) following the manufacturer's instruction. Target protein band was excised for mass spectrometry analysis. Peptide mass fingerprinting (PMF) was performed on an ABI 4700 proteomics analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA). PMF data were compared with the Swiss-Prot database using the MASCOT search engine. The confidence of identification was indicated by the number of matching and total peptides and the protein sequence coverage by the matching peptides.

Cell ELISA

SK-BR-3 and NIH3T3 cells were cultured in complete medium in 96-well plates to form a subconfluent monolayer and further incubated overnight in RPMI-1640 medium. Nonspecific binding was blocked with 3% bovine serum albumin (BSA). After washing, HRP-conjugated antihuman IgG(Fab)′ (Sigma, St Louis, MO) was added. The cells were washed and incubated with TMB substrate solution. The color reaction was suspended by adding 1 mol/L H2SO4 and the intensity was read at 450 nm in a Multiskan Spectrum Microplate.

Fluorescence-activated cell sorting (FACS)

SK-BR-3 and NIH3T3 cells were blocked using 1% BSA-PBS at 4°C for 30 min, then incubated with 100 μg/mL Trop2 Fab and stained using 1:50 diluted fluorescein isothiocyanate (FITC)-labeled human anti-Fab IgG (Sigma). Fluorescence intensity was analyzed with CellQuest software (BD Bioscience, San Diego, CA).

Fluorescence staining

SK-BR-3 and NIH3T3 cells were cultured in 6-well plates. At 80% confluence, cells were fixed using equal volumes of acetone and methanol and incubated with 50 μg/mL Trop2 Fab followed by incubation with 1:50 FITC-labeled human anti-Fab IgG in the dark. At the same time, the cells were stained by 4′-6-diamidino-2-phenylindole (DAPI, Biotium, Hayward, CA).

MTT assay

MTT assay was performed on MDA-MB-231 and NIH3T3 cells on a 96-well plate according to the method described previously.[23, 24] MDA-MB-231 is another Trop2-positive breast cancer cell line.[25]

Apoptosis assay and caspase-3 activity assay

MDA-MB-231 and NIH3T3 cells treated with 2.5–40 μg/mL Trop2 Fab for 12–48 hr were collected, washed with PBS and lysed in a lysis buffer. Apoptosis assay was assessed by measuring membrane redistribution of phosphatidylserine using an Annexin V-FITC apoptosis detection kit (Biouniquer Technology, Shanghai, China) described previously.[26] Furthermore, the activity of caspase-3 was determined by caspase colorimetric assay kit (R&D systems, Minneapolis, MN) according to the manufacturer's protocol. The cell lysates were inspected for protease activity using a caspase-specific peptide conjugated with the color reporter molecule p-nitroaniline. The chromophore p-nitroaniline, cleaved by caspases, was quantitated with a spectrophotometer at a wavelength of 405 nm. As the negative control, PBS was used.

Wound healing assay

MDA-MB-231 and NIH3T3 cells were treated with Trop2 Fab with different concentration and unrelated Fab (2.5–40 μg/mL), and grown to 90% confluency in RPMI-1640 complete medium, then incubated with medium containing 2% FBS overnight. PBS was used as negative control. The cells were visualized by light microscopy at 0, 24 and 48 hr after scratch. The migration rate was calculated using Image Pro Plus software by measuring the distance traveled by cells, using the following formula: n hr (n hr) migration rate = the distance from the edge (0 hr) − the distance from the edge (n hr)/the distance from the edge (0 hr). All experiment was repeated three times.

Detection of Bcl-2 and Bax expression by immunofluorescence assay and Western blotting analysis in vitro

MDA-MB-231 and NIH3T3 cells were treated with Trop2 Fab in different concentration (2.5–40 μg/mL) and unrelated Fab and grown on 12-well plate in RPMI-1640 complete medium overnight. PBS was used as negative control. For immunofluorescence assay, all cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100 and nonspecific binding was blocked by incubation with PBS containing 1% BSA and 0.05% Tween-20. Anti-Bcl-2 and anti-Bax antibodies (Abcam) labeled with FITC-conjugated goat anti-rabbit IgG and anti-mouse IgG, respectively, (Santa Cruz) were used to detect the expression of Bcl-2 and Bax. DAPI was employed for nuclei staining. Immunofluorescence assay was performed after the cells were cultured for 24 hr. Samples were mounted with Fluoromount-G (Southern Biotech, Birmingham, CA) and subjected to fluorescence microscopy (ZEISS Axioskop 40, Germany). Moreover, Western blotting analysis was further performed to detect the expression of Bcl-2 and Bax. Briefly, 1 × 106 cells after treating were washed and lysed with cell lysis buffer. Equal amounts of proteins were separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were first incubated with primary antibodies then for secondary antibody then was detected by ECL kit and autoradiography using X-ray film. β-tublin was used as an internal control.

MDA-MB-231 xenografts

Female 3-week-old BALB/c nude mice with a body weight of ∼20 g were purchased from SLAC Laboratory Animal (Shanghai, China), and kept under specific pathogen-free conditions. For MDA-MB-231 xenograft model establishment, the mice were injected with 1 × 107/mL MDA-MB-231 cells subcutaneously in a volume of 0.1 mL into the flank. After inoculation, tumor-bearing mice were divided randomly into 5 treatment groups (8 mice per group) and treatment initiated when the xenografts reached a volume of about 100 mm3. Each mouse was injected intravenously on day 1, 3, 5 and 7 with different drugs: group I: high concentration of Trop2 Fab (30 mg/kg); group II: medium concentration of Trop2 Fab (5 mg/kg); group III: low concentration of Trop2 Fab (1 mg/kg); group IV: unrelated Fab (30 mg/kg); and group V: PBS as negative control. The animal studies were conducted in accordance with Public Health Service Policy and approved by the Animal Care and Use Committee of Nanjing Medical University. After xenograft transplantation, mice bearing tumors were observed and tumor size was measured once every 3 days with vernier caliper. The tumor volume was estimated according to the following formula: tumor volume (mm3) = L × W2/2 (where L was the length and W was the width) with the final measurement taken on day 50. At the end of the experiments (on day 50), the mice were anaesthetized by CO2 and killed. Tumors from each mouse were removed, measured and weighed individually. Inhibition rate of tumor growth was calculated by the following formula: inhibition rate (%) = (tumor weight of control group − tumor weight of experimental group)/tumor weight of control group × 100%. The tumor tissues were dissected and collected for further analyses.

Detection of Bcl-2 and Bax expression by immunohistochemistry (IHC) and Western blotting analysis in vivo

The tumors from xenografted nude mice were excised and then paraffin wax-embedded at Department of Pathology, Nanjing Medical University (Jiangsu, China). Sections (5 μm) were deparaffinized with xylene and then dehydrated in decreasing concentrations of alcohol. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxidase in Tris-buffered saline. Tissue sections were then incubated with primary antibodies of anti-Trop2 (Abcam), anti-Bcl-2 (Abcam), anti-Bax (Abcam), followed by incubation with EnVision HRP complex (DAKO, Carpinteria, CA). Negative controls were included by the replacement of the primary antibody with PBS. The results were analyzed according to the IHC score (IHS) as described previously.[26-28] Briefly, IHS was determined by the evaluation of both staining density and intensity. Multiplication of the intensity and the percentage scores gave rise to the ultimate IHS: samples with a sum score below 3 (IHS ≤ 3) were judged as low protein expression, and those with a sum score above 4 (IHS ≥ 4) as high protein expression. Furthermore, Western blotting analysis was employed to evaluate the expression of Bcl-2 and Bax in tumors from xenografts in nude mice.

Results

Isolation, purification and characterization of Trop2 Fab

After seven rounds of panning, 60 single phage clones were randomly picked up and amplified to test for specific binding to Trop2 EMDs by phage ELISA. A ratio of sample OD450 versus the blank of >0.5 was set as the standard for selecting positive clones and 26 clones became candidates. Number 23 clone showed the strongest binding to Trop2 EMDs (Fig. 1a), and its sequence was shown in Figure 1b (the sequences of No. 3, 13 and 23 clone were same). Soluble expression of Trop2 Fab was induced by IPTG at 25°C overnight. The expressed Trop2 Fab was found mainly in the periplasmic space of E. coli Top 10F′ (Fig. 1c). SDS-PAGE confirmed the expression of heavy and light chains of Trop2 Fab. The purity was above 95% after Protein L affinity purification (Fig. 1d). After performing IP with Trop2 Fab and SK-BR-3 cell lysate, mass spectrometry was used to identify the proteins that bound to Trop2 Fab. A representative example of mass spectrograph was shown in Figure 1e and the identified proteins were shown in Figure 1f, with the sequence corresponding to Trop2.

Figure 1.

(a) ELISA of 26 candidate phage clones picked up from the eluted phage pool after the seventh round of biopanning. The last NC column on the right was negative control phage that served as blanks. (b) DNA and amino acid sequences of the variable regions of Trop2 Fab.(c) Western blotting analysis of Trop2 Fab fragment expressed in E. coli. M: marker; lane 1: Top10F′ cell lysate control; lane 2: cell lysates after sonication; lane 3: cell sedimentation after sonication. (d): Purified Trop2 Fab fragment was separated on 10% SDS-PAGE gel and stained with Coomassie blue. The double bands were heavy chain Fd (top) and light chain L (bottom), and light chain L was thicker than heavy chain Fd. (e) Peptide mapping fingerprint of Trop2 binding proteins. Top Score: 100 for gi166795236, tumor-associated calcium signal transducer 2 precursor [Homo sapiens]. Protein scores greater than 65 were significant (p < 0.05). (f) Sequence blast of Trop2 binding protein. Matched peptides were underlined.

Binding of Trop2 Fab to the EMD of Trop2

Further analysis of Trop2 Fab was performed by comparing the binding ability of the Fab fragments to Trop2-positive (SK-BR-3) breast cancer cells and Trop2-negative (NIH3T3) cells. Cell surface ELISA showed that the purified Trop2 Fab recognized Trop2 present in SK-BR-3 cells but not in NIH3T3 cells (Fig. 2a). Flow cytometric assay showed that the population of Trop2 Fab-treated SK-BR-3 cells was separated from untreated cells by fluorescent intensity, whereas no difference was observed between Trop2 Fab-treated and untreated NIH3T3 cells (Fig. 2b), suggesting that Trop2 Fab binds to Trop2-positive cells only. To provide morphologic evidence that Trop2 Fab binds to the cell membrane, fluorescence staining experiment was conducted and Trop2 Fab stained only Trop2-positive SK-BR-3 cells but not Trop2-negative NIH3T3 cells (Fig. 2c). Taken together, these data demonstrate the high affinity and specificity of Trop2 Fab was isolated.

Figure 2.

Trop2 Fab binds the EMD of Trop2. (a) SK-BR-3 cells were Trop2 positive; NIH3T3 cells were Trop2 negative. The unrelated Fab was used as the negative control. * Indicated significant difference. (b) FACS analysis of SK-BR-3 and NIH3T3 cells. The fluorescent intensity was significantly different between Trop2 positive SK-BR-3 cells (b1) and Trop2 negative NIH3T3 cells (b2). Background staining (black line) was obtained by the unrelated Fab. (c) Fluorescence staining of SK-BR-3 and NIH3T3 cells. (c1) Staining by Trop2 Fab in SK-BR-3 cells; (c2) staining by commercial Trop2 antibody in SK-BR-3 cells; (c3) DAPI staining for the nuclei in SK-BR-3 cells; (c4) cell morphology under light microscopy; (c5) overlapping of c1–c4; (c6) no staining by Trop2 Fab in NIH3T3 cells. (×200, Olympus digital camera). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Trop2 Fab inhibits breast cancer cell growth in vitro

To determine the efficacy of Trop2 Fab to inhibit specifically the growth of Trop2 expressing breast cancer cells, we performed MTT assay. The results showed that at 40 μg/mL of Trop2 Fab, the growth of MDA-MB-231 cells was inhibited by 45.78%. Furthermore, Trop2 Fab inhibited MDA-MB-231 cell growth in a dose-dependent manner. In contrast, Trop2 Fab, unrelated Fab or PBS exhibited very modest or no effects on the proliferation of NIH3T3 cells, which did not express Trop2 (Fig. 3a). Annexin V-FITC apoptosis assay showed significant increases in apoptotic rate of MDA-MB-231 cells in a concentration- and time- dependent manner. As the Trop2 Fab concentration and duration of drug exposure increased, the apoptotic rate of MDA-MB-231 cell was substantially augmented. By contrast, apoptotic rates in NIH3T3 cells were low and showed no differences after treatment with Trop2 Fab, unrelated Fab or PBS (Figs. 3b1, 3b2, 3c1 and 3c2). To explore the activation of the caspase cascade during Trop2 Fab-induced apoptosis, we investigated caspase-3 activities after the cells were treated with 2.5–40 μg/mL Trop2 Fab for 12–48 hr. Colorimetric assay revealed that caspase-3 activity increases remarkably in a concentration- and time-dependent manner (Figs. 3d1 and 3d2). Next, we performed wound healing assay to examine the effects of Trop2 Fab on the migration ability of breast cancer cells. The results showed that the migration rate was 51.2, 79.1 and 85.4% after 24 hr of scratch in MDA-MB-231 cells treated with 40 μg/mL Trop2 Fab, unrelated Fab and PBS, respectively, and the difference was of statistical significance (Fig. 3e1). In addition, the migration rate was 46.1, 92.1 and 93.7% after 48 hr of scratch in MDA-MB-231 cells treated with 40 μg/mL Trop2 Fab, unrelated Fab and PBS, respectively, and the difference was also of statistical significance (Fig. 3e2). For NIH3T3 cells, the wound closure rates in Trop2 Fab group, unrelated Fab group and negative control group showed no significant difference. The representative images of the migration of MDA-MB-231 cells treated with Trop2 Fab, unrelated Fab and PBS were shown in Figure 3f.

Figure 3.

Trop2 Fab inhibits the proliferation of breast cancer cells. (a) MTT assay showing the dose-dependent inhibitory effects of Trop2 Fab (2.5–40 μg/mL) on the proliferation of MDA-MB-231 cells, while the inhibitory effect on NIH3T3 cells was bare and insignificant. * Significant difference in MDA-MB-231 cell lines with Trop2 Fab (40 μg/mL) compared with PBS treatment. (b1) Apoptotic rates in MDA-MB-231 cells treated with Trop2 Fab (2.5–40 μg/mL). * Significant difference of apoptotic rate in MDA-MB-231 cells compared with NIH3T3 cells. p < 0.05. (b2) Representative images of cell apoptosis with flow cytometry by Annexin V/PI double staining after treating with Trop2 Fab (2.5–40 μg/mL). (c1) Apoptotic rates in MDA-MB-231 cells treated with Trop2 Fab (12–48 hr). * Significant difference of apoptotic rate in MDA-MB-231 cells compared with NIH3T3 cells. p < 0.05. (c2) Representative images of cell apoptosis with flow cytometry by Annexin V/PI double staining after treating with Trop2 Fab (12–48 hr). (d1) Caspase-3 activity in MDA-MB-231 cells treated with Trop2 Fab (2.5–40 μg/mL). * Significant difference of caspase-3 activity in MDA-MB-231 cells compared with NIH3T3 cells. p < 0.05. (d2) Caspase-3 activity in MDA-MB-231 cells treated with Trop2 Fab (12–48 hr). * Significant difference of caspase-3 activity in MDA-MB-231 cells compared with NIH3T3 cells. p < 0.05. The data revealed that caspase-3 activity was increased remarkably in a concentration- and time-dependent manner. (e1) Migration rate of MDA-MB-231 cells treated with Trop2 Fab after 24 hr. * Significant difference of migration rate in MDA-MB-231 cells compared with NIH3T3 cells. (e2) Migration rate of MDA-MB-231 cells treated with Trop2 Fab after 48 hr. * Significant difference of migration rate in MDA-MB-231 cells compared with NIH3T3 cells. (f) Representative images of the migration of MDA-MB-231 cells treated with Trop2 Fab and unrelated Fab. PBS was negative control.

Trop2 Fab induces the downregulation of Bcl-2 and upregulation of Bax in breast cancer cells

Since Trop2 Fab promotes the apoptosis of MDA-MB-231 breast cancer cells, we evaluated the expression of Bcl-2 and Bax by immunofluorescence assay and Western blotting analysis, respectively. The results of immunofluorescence assay showed that Trop2 Fab treatment led to decreased staining of Bcl-2 and increased staining of Bax in MDA-MB-231 breast cancer cells. However, treatment with unrelated Fab or PBS, the immunofluorescence staining of both Bcl-2 and Bax remained stable. In contrast, immunofluorescence staining of Bcl-2 and Bax remained unchanged in NIH3T3 cells treated with Trop2 Fab, unrelated Fab or PBS (Figs. 4a and 4b). The results of Western blotting analysis also showed that Bcl-2 expression was dramatically downregulated while Bax expression was remarkably upregulated in MDA-MB-231 breast cancer cells upon treatment with Trop2 Fab. Using unrelated Fab or PBS resulted in no changes in Bcl-2 or Bax expression. By comparison, upon treatment with Trop2 Fab, unrelated Fab or PBS, no significant change in Bcl-2 or Bax expression was observed in NIH3T3 cells (Fig. 4c).

Figure 4.

Detection of Bcl-2 and Bax expression in MDA-MB-231 and NIH3T3 cells by immunofluorescence staining and Western blotting analysis. (a) The immunofluorescence staining of Bcl-2 was decreased in MDA-MB-231 cells treated with Trop2 Fab. (b) The immunofluorescence staining of Bax was enhanced in MDA-MB-231 cells treated with Trop2 Fab. (c) Bcl-2 expression was downregulated while Bax expression was upregulated in MDA-MB-231 breast cancer cells treated with Trop2 Fab by Western blotting analysis. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Trop2 Fab inhibits tumor growth in MDA-MB-231 xenograft model

Next, we successfully established MDA-MB-231 xenograft nude mice model to evaluate the in vivo tumor inhibitory effects of Trop2 Fab. As shown in Figure 5a, on day 50 the mean body weight of nude mice in all treated group showed no significant difference. The tumor size and weight of PBS control group was 1010.03 ± 77.12 mm3 and 808.23 ± 86.85 mg, respectively. However, the tumor size and weight decreased to 940.12 ± 92.78 mm3 and 752.69 ± 88.85 mg in the low concentration Trop2 Fab (1 mg/kg) treated group, to 886.44 ± 72.05 mm3 and 708.47 ± 55.13 mg in the medium concentration Trop2 Fab (5 mg/kg) treated group, and to 725.41 ± 102.28 mm3 and 580.76 ± 95.12 mg in the high concentration Trop2 Fab (30 mg/kg) treated group, respectively. In unrelated Fab treated group, the tumor size and weight was 962.41 ± 115.34 mm3 and 770.14 ± 53.85 mg, which showed no significant differences compared with PBS control group. In the high concentration Trop2 Fab (30 mg/kg) treated group, both the tumor size and weight exhibited statistical difference with PBS control group (Figs. 5b and 5c). The inhibition rate of tumor growth was 28.2% in high concentration Trop2 Fab group, 12.3% in moderate concentration Trop2 Fab group, 6.9% in low concentration Trop2 Fab group and 4.8% in unrelated Fab group (Fig. 5d).

Figure 5.

Trop2 Fab inhibits the growth of tumor xenografts. After the inoculation, 40 nude mice were randomly divided into five groups and treated as indicated. (a) Body weight of xenograft nude mice throughout the in vivo experiments. (b) Measurement of tumor volume of xenograft tumors. * p < 0.05 compared with control group. (c) Measurement of tumor weight of xenograft tumors. * p < 0.05 compared with control group. (d) Comparison of inhibition rates in treatment groups. The inhibition rates in unrelated Fab group, low concentration of Trop2 Fab (1 mg/kg) group, medium concentration of Trop2 Fab (5 mg/kg) group and high concentration of Trop2 Fab (30 mg/kg) group were 4.8, 6.9, 12.3 and 28.2%, respectively.

Morphologic observation and detection of Bcl-2 and Bax by IHC and Western blotting analysis

Positive expression of Trop2 on tumor xenografts was first confirmed by IHC (Fig. 6a). The morphology of the tumor tissue was observed by HE staining. The level of tumor necrosis varied in different treatment groups, tumor necrosis was seldom observed in PBS and unrelated Fab treated groups, but large areas of necrosis were observed in Trop2 Fab treated group (Figs. 6b1–6b3). We further detected the expression of Bcl-2 and Bax in xenografts in nude mice by IHC. Compared with control group, the expression of Bcl-2 was significantly reduced following the increasing dose of Trop2 Fab in nude mice, while the expression of Bax was elevated after treatment with Trop2 Fab (p < 0.05). The representative staining of Bcl-2 and Bax in xenografts in nude mice was shown in Figures 6b4–6b9. The IHC score of Bcl-2 and Bax expression in xenografts in nude mice was shown in Figure 6c. In addition, the results of Western blotting analysis also displayed that the expression of Bcl-2 was decreased while the expression of Bax was increased in nude mice following the treatment of Trop2 Fab (Fig. 6d).

Figure 6.

Morphologic observation and Bcl-2 and Bax expression in xenografts. (a) Positive expression of Trop2 on xenografts in nude mice was confirmed by IHC. (b1–b3) HE staining of xenografts in nude mice. Black arrow in b1 indicated the necrosis of tumor cells. (b4–b6) IHC analysis of Bcl-2 expression in xenografts in nude mice of different treatment groups. (b7–b9) IHC analysis of Bax expression in xenografts in nude mice of different treatment groups. (c) IHC score of Bcl-2 and Bax expression in xenografts in nude mice. Compared with PBS control group, Bcl-2 expression was significantly reduced while Bax expression was significantly increased after treatment with high dose of Trop2 Fab (30 mg/kg). * p < 0.05 compared with PBS control group. (d) Western blotting analysis showed that the expression of Bcl-2 was inhibited while the expression of Bax was elevated following the treatment of Trop2 Fab in xenografts in nude mice. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Discussion

Human antibodies are mostly desired for clinical diagnosis and treatment in various kinds of cancers. Several DNA recombination technologies have been developed to construct human antibodies. Among them, phage display technology has the advantage of being inexpensive and highly efficient. With phage display, much smaller antibody fragments (Fabs or scFvs) can be constructed, which exhibit great efficiency in penetrating into the targeted sites with high concentration and validity.[29] We have previously constructed a fully human Fab phage library.[20]

Although antibody-based targeting therapy is a well established treatment strategy for cancer,[23, 29, 30] a satisfactory therapeutic effect in breast cancer has not been accomplished. However, Trop2-related cancer treatments have been quickly developed in recent years and Trop2-targeted cancer therapy may offer novel and promising therapeutic strategies for cancer treatment.[31-34]

In this study, we employed the human Fab phage library to isolate a human Fab fragment that recognizes EMD of Trop2. The biopanning strategy with the repeated panning with living cells and coated Trop2 EMD protein in microtiter plates guaranteed the enrichment of specific Trop2 EMD-binding phage. After seven rounds of panning, one of selected 26 candidate phage clones showed significantly positive signal and its DNA sequencing was consistent with the phagemids that exhibited the strongest binding by ELISA. Western blotting and SDS-PAGE confirmed the appropriate expression of Fab, and mass spectrometry showed that Trop2 Fab binding proteins were identified as Trop2. In addition, the specifical binding of Trop2 Fab to Trop2 on the surface of breast cancer cells were confirmed by cell ELISA, FACS and fluorescence staining.

To the best of our knowledge, this is the first study to report the antitumor activity of Trop2 antibody. The results of MTT assay showed that Trop2 Fab was effective in inhibiting MDA-MB-231 cell proliferation in a dose-dependent manner. Wound healing assay showed that after 24 and 48 hr of scratch, the cell migration rate was significantly decreased after treating with Trop2 Fab. These data indicate that Trop2 Fab is able to inhibit the growth and migration of Trop2 expressing cancer cells.

Furthermore, Trop2 has been shown to mediate tumor-associated calcium-mediated signal cascades and activation of the ERK1/2-MAPK pathways, both of which modulate cell cycle progression and protect cancer cells from apoptosis.[35, 36] Caspase-3, in particular, is widely recognized as a key member of the caspase family, and its activation is a typical characteristic of apoptosis.[37] The Bcl-2 family, another crucial checkpoint in apoptosis, comprises antiapoptotic proteins, including Bcl-2, Bcl-xl and Mcl-1 and proapoptotic proteins, including Bax, Bak and Bim.[38] The expression of Bcl-2 and Bax may also be regulated by the MEK/ERK signaling pathway under certain circumstances.[39, 40] The coordinated regulation and function of all of the apoptosis-related factors specified above is essential for optimal responses to chemotherapeutics in cancer. Therefore, we proposed that Fab against Trop2 could inhibit the protection in cancer cells from apoptotic effects and induce apoptosis by affecting related signaling pathways, including those of caspase-3, Bcl-2 and Bax. Annexin V-FITC apoptosis assay showed that Trop2 Fab induced the apoptosis of MDA-MB-231 cells significantly and substantially elevated caspase-3 activity in a concentration- and time-dependent manner. The data indicate that the apoptosis induced by Trop2 Fab in MDA-MB-231 cells may correlate with the activation of caspase-3. Subsequently, we performed immunofluorescence staining and Western blotting analysis to investigate the expression of Bcl-2 and Bax in cancer cells treated with Trop2 Fab. We found that Trop2 Fab upregulated Bax expression while downregulated Bcl-2 expression. These results suggest that Trop2 Fab may induce apoptosis in MDA-MB-231 cells through regulating Bcl-2 and Bax expression.

The antitumor effect of Trop2 Fab was further evaluated in vivo. In xenograft model of breast cancer, we found that the highest tumor inhibition rate was 28% in the high concentration Trop2 Fab (30 mg/kg) treated group. We also performed IHC and Western blotting analysis in tumors excised from executed nude mice. Compared with control group, Bcl-2 expression was significantly downregulated while Bax expression was significantly upregulated after treatment with Trop2 Fab in nude mice. These results are consistent with previous immunofluorescence staining and Western blotting analysis results in vitro.

It is important to note that Trop2 is expressed at different levels in different type of breast cancer cells and tissues. Our previous study showed that high expression of Trop2 was only detected in 50% breast cancer patients.[19] In addition, we found that only high concentration of Trop2 Fab exerted significant tumor inhibitory effects. Therefore, further studies are needed to improve the anti-tumor efficacy of Trop2 Fab. Our next strategy is to conjugate some immunotoxin or liposomes with Trop2 Fab to enhance its efficacy and application in cancer therapy. Moreover, a series of investigations on the potential mechanism of Trop2 Fab in breast cancer is ongoing, and related signaling pathways involved in the Trop2 Fab reaction warrant thorough and further research.

In conclusion, we isolated Trop2 Fab by phage display technology, and characterized its affinity and selectivity to Trop2. The fully human Trop2 Fab fragment inhibits breast cancer growth both in vitro and in vivo, and may represent a promising chemotherapeutics for Trop2 expressing breast cancer.

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