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Microbiology and Biotechnology Letters

Research Article(보문)

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Molecular and Cellular Microbiology (MCM)  |  Microbial Genetics, Physiology and Metabolism

Microbiol. Biotechnol. Lett. 2024; 52(4): 441-447

https://doi.org/10.48022/mbl.2408.08008

Received: August 9, 2024; Revised: September 20, 2024; Accepted: September 27, 2024

Selected D7 Antibody Inhibit Cancer Cell Growth

Chi Hun Song and Kyung Ho Han*

Department of Biological Sciences and Biotechnology, Hannam University, Daejeon 34054, Republic of Korea

Correspondence to :
Kyung Ho Han,    kyungho1@hnu.kr

Abstract

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We developed to screen for antibodies that promote cell growth in serum-free conditions using a human antibody library. The antibody library was engineered to be anchored near the cell surface and delivered to cells using a lentivirus to introduce the antibody genes. When treated cells cultured under serum-free conditions, viable cells were obtained and antibodies were selected by PCR. Although selected D7 antibody did not induce cell growth and proliferation under serum-free conditions as initially expected, we unexpectedly discovered that D7 antibody significantly inhibits MCF cancer cells in 10% FBS condition. Overall, our findings suggest that D7 antibody, discovered through this novel method, is a promising candidate for future cancer therapies.

Keywords: Antibody, serum, antibody library, cancer

Graphical Abstract


Introduction

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Antibodies are protein that specifically recognize and bind to antigens such as bacteria, viruses, toxins and other foreign substances. Antibodies perform to remove antigens by neutralization, opsonization and complement activation. Antibodies are produced by B cells through adaptive immune system. They are composed of two heavy and light chains and recognize foreign antigens through antigen binding sites (Fabs). Monoclonal antibodies (mAbs) are produced by B cells, hybridoma technology, and recombinant technology [1, 2].

B cell antibody production is a vital aspect of the adaptive immune response, involving recognize antigens on B cell receptors, activation through helper T cells, clonal expansion and differentiate into plasma cells, which secrete large amounts of antibodies. Hybridoma technology involves immunizing an animal, typically a mouse, with the target antigen, fusing the animal's B cells with myeloma cells to create hybridomas, and selecting those that produce the desired antibody. Recombinant antibody production using technologies like pFUSE vectors involves cloning the genes encoding the antibody's variable regions into the pFUSE plasmid vector, which is then transfected into mammalian host cells such as CHO or HEK cells. These cells are cultured and selected for high antibody production, and the chosen clones are expanded in large-scale bioreactors. The secreted antibodies are harvested from the culture medium and purified using chromatography techniques [3−6].

To select mAbs, phage display is a powerful method. In phage display, a library of antibody fragments is expressed on the surface of bacteriophages, and those that bind to a target antigen are selected through multiple rounds of biopanning. Other methods include ribosome display, where antibody-mRNA-ribosome complexes are selected, yeast display, where yeast cells display antibody fragments and are sorted using FACS, and using transgenic mice engineered to produce human antibodies. Each method offers unique advantages for generating specific and high-affinity monoclonal antibodies.

There are 17 phage display and 19 transgenic mouse-derived human antibodies currently approved by the US FDA for the treatment of human disease, demonstrating the reliability of this technique as a platform for antibody discovery [3, 7, 8]. mAb market was valued at approximately $138.6 billion in 2024 and is expected to continue to grow in the future [4].

Combinatorial antibody libraries are developed by amplifying the variable regions of heavy (VH) and light (VL) chains from B cells by PCR method, followed by ligating these regions into a phage display vector (phagemid). This results in the expression of antibody fragments on the phage surface, linking genotype to phenotype. These libraries are then screened against immobilized antigens to select for high-affinity antibodies. Recent advancements have enabled the isolation of functional antibodies in cellular environments, influencing cell fate and providing insights into cellular biology, particularly in stem cells and cancer research [9− 14].

Serum plays a vital role in cell culture by providing essential nutrients, growth factors, and hormones that support cell metabolism, growth, and differentiation in animal cell culture system. It contains attachment factors that help cell adhesion, binding proteins like albumin that stabilize various components, and detoxifying agents that neutralize toxins and contaminants. Serum also contributes to the buffering capacity of the culture medium, maintaining optimal pH levels, and contains protease inhibitors to protect cells from enzymatic damage [15, 16].

Despite the major roles of serum-containing media, serum-free media continues to be in high demand for animal cell culture. Serum-free media has several advantages in cell culture: they provide consistency and reproducibility by eliminating the variability inherent in serum, which contains a complex mix of growth factors and proteins that can differ between batches. Additionally, serum-free media reduce the risk of contamination from viruses, mycoplasma, and prions, aligning with ethical and regulatory standards that favor animal welfare. These media can be optimized for specific cell types, improving viability and productivity, and simplify downstream processing in biopharmaceutical production by reducing the complexity and cost of purification [17, 18].

In this study, we developed a new antibody selection method in serum free media condition. We found that D7 antibody demonstrated effective inhibition of tumor growth in MCF7 cells. This finding suggests that D7 antibody has the potential to be developed as a new therapeutic candidate for cancer treatment.

Materials and Methods

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Cell lines and culture conditions

DU145 cells were cultured in RPMI 1640 medium (Corning, USA) with 0% or 10% FBS and 1% penicillin/streptomycin. MCF7 cells were cultured in DMEM (Corning) with 0% or 10% FBS and 1% penicillin/streptomycin. In serum-free conditions, cells were cultured in media with antibiotics but without FBS. All cells were maintained at 37℃ in a 5% CO2 incubator.

Lentivirus production

HEK293T cells were seeded at a density of 8 × 105 cells per well in a 6-well plate. After overnight incubation lentiviral plasmid, pCMVD plasmid, and pVSVG plasmid (1.3 μg each) were incubated with 10 μl of Lipidofect-P (Lipidomia, Republic of Korea) for 20 min and then added to HEK293T cells. After 4 h the medium was replaced with 2 ml of fresh media, and the cells were incubated for 3 days. After 3 days the media was filtered and stored at -80℃.

Cell viability assay

DU145 and MCF7 cells were seeded at a density of 3 × 103 cells per well in a 96-well plate. Cells were incubated with D7 lentivirus and 10 μg/ml polybrene reagent (Millipore, USA). After 24 h the media was replaced with 100 μl of fresh media and incubated for 3 days. For MTS assay 20 μl of CellTiter 96 AQueous One Solution (Promega, USA) was added and incubated for 3 h at 37℃. MTS titer was measured at 490 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, USA).

Wound healing assay

MCF7 cells were seeded at a density of 1.3 × 105 cells per well in a 12-well plate. After 24 h a wound was created using a 100 μl pipette tip. Cells were then incubated with fresh media containing lentivirus and 10 μg/ ml polybrene reagent (Millipore). After 24 h the media was replaced with fresh media, and cells were observed for an additional 48 h. Images of wound healing were taken every 24 h. Cell migration was quantified using ImageJ software from 0 to 72 h in 24 h intervals.

Colony formation assay

MCF7 cells were seeded at a density of 2 × 103 cells per well in a 12-well plate. After 24 h, the media was replaced with fresh media containing lentivirus and 10 μg/ml polybrene reagent (Millipore). The media was changed every 3 days. After 10 days cells were washed with PBS fixed with 4% paraformaldehyde for 20 min and stained with 5% crystal violet for 20 min. The experiment was performed in triplicate and colonies were quantified using ImageJ software.

Immunoblotting

MCF7 cells were washed with cold PBS and lysed with RIPA lysis buffer. The lysates were centrifuged at 13,000 rpm for 30 min at 4℃. Protein concentrations were quantified using the BCA protein assay kit (Thermo Fisher Scientific, USA). Lysates were analyzed by SDS-PAGE and denatured with SDS-page loading buffer at 70℃ for 5 min. Proteins were transferred to nitrocellulose (NC) membranes and blocked with 5% skim milk solution (5% skim milk powder in 0.2% Tween20 TBST). Membranes were incubated overnight with primary antibodies against PARP (Cell Signaling Technology, USA), p53 (Millipore), and Bax (Cell Signaling Technology). After washing with TBST membranes were incubated with HRP-conjugated antirabbit and anti-mouse IgG secondary antibodies (Cell Signaling Technology) for 1 h. Bands were visualized using SuperSignalTM West Dura substrate (Thermo Fisher Scientific).

Results

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Selection of antibodies for inducing cell growth in serum free media condition

We considered the possibility that antibodies could replace one the essential roles of serum, specifically promoting cell growth in cell culture conditions. To investigate this, we developed a novel antibody screening method in serum-free medium conditions. (Fig. 1A). The method uses a human single-chain fragment variable (ScFv) phage library to construct a human ScFv lentiviral combinatorial antibody library has 108 unique antibody genes. The antibodies were displayed on the cell membrane using a previously published method [9−14]. Compared to normal cells, cancer cells typically grow faster and have stronger attachment properties. Therefore, we used a specific cancer cell line, DU145. The expressed antibody could bind to the membrane protein of prostate cancer cell line DU145 and trigger a cascade of cell signaling. After antibody library transfection, DU145 cells were cultured for three days. Interestingly, the DU145 cells treated with the antibody library showed better growth in the 0% FBS condition compared to the 0% FBS control group (Fig. 1B). Subsequently, the treated cells on the surface of the plate were harvested, genomic DNA was extracted, and PCR was performed to obtain the ScFv DNA candidates (Fig. 1A). ScFv DNA bands were selected by PCR (Fig. 1B). The results showed a novel antibody in 4 out of 20 samples, with D7 antibody identified in one of them.

Figure 1.Antibody selection for promoting cell growth under 0% FBS condition. (A-B) To select antibodies that increase cell growth, genes from a human ScFv phage library containing 108 members were cloned into a lentiviral vector, creating a lentiviral antibody library. In this library, antibody molecules are anchored to the plasma membrane and displayed on the cell surface. DU145 cells were infected with the antibody library in vitro and cultured for three days. This system operates on an autocrine basis, as each cell expresses a unique antibody. After three days of culturing, the cells were harvested, collected, and analyzed using PCR to identify the antibody genes.

Selected D7 antibody regulate cancer cell growth

Our study aimed to determine whether the selected D7 antibody could help in cell growth which is normally supported by serum. To investigate the D7 potential of cell growth, we incubated DU145 cells with D7 antibody for three days in 0% FBS cell culture conditions and analyzed cell growth using the MTS assay. The result indicated that D7 treated cells grew slightly but not as much as the 10% FBS control. We also examined breast cancer cell line MCF7 under 0% FBS conditions. MCF7 showed the same results as DU145 (Fig. 2A). These results suggest that D7 antibody was not promoting the proliferation of DU145 and MCF7.

Figure 2.D7 antibody suppress MCF7 cell growth. (A-B) Proliferation of DU145 and MCF7 cells treated with D7 under 0% FBS or 10% FBS, measured using the MTS assay. Data are presented as mean ± s.e.m. from a typical experiment performed independently at least three times. The control is used as a non-treatment condition and GFP is used as a control in pseudotype lentivirus experiments to express green fluorescent protein (GFP). Statistical analysis was conducted using multiple t-tests and one-way ANOVA: NS, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

To determine if D7 antibody regulate cell growth in 10% FBS cell culture conditions, DU145 and MCF7 were incubated with D7 antibody under normal 10% FBS for three days and assessed differences in cell growth between the treated and control groups by MTS assay. The results demonstrated that under 10% serum culture conditions, DU145 cells showed no significant difference between the D7 treatment and the control. Remarkably, MCF7 cells showed reduced growth compared to the control (Fig. 2B). These finding indicates that while D7 did not significantly increase cell proliferation in 0% FBS condition, it inhibits the growth of MCF7 cells in 10% FBS condition.

Selected D7 antibody inhibits proliferation and migration in MCF7 cancer cell lines

We next performed wound healing assay and colony formation assay on MCF7 cells. D7 treated cells showed a slower cell proliferation rate compared to the untreated control group in the wound healing assay (Fig. 3A). We also observed fewer single colonies in the D7 treatment cells than in the control cells in the colony formation assay (Fig. 3B). These results suggest that D7 inhibits the growth, proliferation, and migration of MCF7 cells.

Figure 3.D7 antibody inhibits cell proliferation. (A) Wound healing assays showing the effect of D7 on the migration of MCF7 cells. Representative images depict scratched and healing wounded areas (marked by white dotted lines) on confluent layers of solid cancer cells transduced with D7. (B) Colony formation analysis of MCF7 cells, with the number of colonies quantified using ImageJ. Data are presented as mean ± s.e.m. from an experiment independently repeated at least three times. The control is used as a non-treatment condition and GFP is used as a control in pseudotype lentivirus experiments to express green fluorescent protein (GFP). Statistical analysis was conducted using multiple t-tests and one-way ANOVA: NS, not significant; *p< 0.05, **p < 0.01, ***p < 0.001.

Selected D7 antibody induces apoptosis

To determine if the inhibition of MCF cell growth by D7 antibody is regulated through apoptosis, we investigated the protein levels related to apoptosis using Western blot analysis. The results showed that MCF7 cells treated with the D7 antibody exhibited decreased levels of Poly(ADP-ribose) polymerase (PARP) and increased levels of cleaved PARP. High levels of cleaved PARP indicate that the cells are undergoing programmed cell death. Moreover, both p53 and Bax were significantly increased compared to control (Fig. 4). These findings indicate that the D7 antibody induces apoptosis in MCF7 cells.

Figure 4.D7 induces apoptosis in tumor cells.

Western blot analysis of apoptotic protein expression levels in MCF7 cells transduced with D7, GFP or control. Cell lysates were immunoblotted with PARP, cleaved PARP, p53, and Bax. The con is used as a non-treatment condition and GFP is used as a control in pseudotype lentivirus experiments to express green fluorescent protein (GFP).

Discussion

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Antibody library has been used in the screening of antibodies for various applications, including inflammatory diseases, cancer, brain disorders, and metabolic diseases. In the present research, we applied antibody library to select antibodies that promote cell growth and survival under serum-free cell culture conditions. While we did not identify any antibodies that could replace the serum, we found D7 antibody that inhibit the growth of MCF7 breast cancer cells.

Monoclonal antibodies in cancer therapy offer benefits like selective cancer cell apoptosis, precise targeting, extended action, and minimal side effects. Recent advancements of antibody-drug conjugates (ADCs) that combine antibodies with cytotoxic drugs to enhance treatment potency [19, 20]. In addition, cancer immunotherapy involves various stages aimed at eliciting the appropriate immune responses to eliminate cancer cells and inhibit oncogenesis. One effective strategy in this context is targeting immune checkpoints with monoclonal antibody (mAb) therapy. CTLA-4 is a key player in this process as it negatively regulates costimulatory signaling during T cell activation, which is crucial for maintaining peripheral tolerance. Tumor cells can exploit this by inducing peripheral tolerance and modifying their MHC I expression to avoid immune detection [4].

As demonstrated here, D7 antibody did not increase or decrease cell growth in the DU145 cells which we used for screening antibodies, but it inhibited cell growth and induced cell death in MCF7 cells under normal cell culture conditions. This effect might be due to the receptor being present only on specific MCF7 cells, which requires further investigation. Additional research is necessary to identify what antigen binds to D7 antibody, understand its cellular mechanism, and observe its effectiveness in animal models.

In this study, we developed a new approach to screen for antibodies and discovered that the D7 antibody effectively inhibited the growth of MCF7 breast cancer cells. This finding highlights D7 as a promising candidate for cancer therapy, with potential for further development as a new treatment for breast cancer.

Acknowledgments

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This work was supported by the Korea government (MFDS) (24202MFDS732).

Author Contributions

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KHH conceptualized and designed the study, CHS, KHH wrote draft paper and performed the experiments. CHS, KHH analyzed data. KHH reviewed and edited the paper. KHH acquired funding.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

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