Genetic Engineering of Phages with a Specific Focus on Phage Display Technology in Cancer

Abstract

Genetic modification of phages has made significant contributions to cancer diagnostics and therapy in modern research. The development of these molecular tools has enabled a broad spectrum of applications, ranging from vaccine development to biomarker discovery, with significant implications in both medical and biotechnological fields. Among the various phage-based methods, phage display stands out as the most widely used. This technique presents novel concepts with vast potential. In this review, we provide an update on the engineering of M13 bacteriophage and its use in phage display technology in the context of cancer diagnosis and treatment. Additionally, we discuss phage-focused applications that involve the genetic modifications of phages themselves, which contribute to the improvement of cancer biotechnology. The effectiveness of phage display technology in identifying high-affinity ligands for substrates, such as cancer cells and tumor-associated molecules, further enhances its value in biomedical applications.

Introduction

Phage display has appeared as a powerful technique for identifying new ligands that specifically bind to desired targets. In this method, billions of peptides and antibodies are displayed on the genetically engineered bacteriophages, providing a vast sequence screening for a given target. In 1985, George Smith first described the phage display approach by inserting a 57-mer peptide into the bacteriophage genome.¹ Following his discovery, numerous attempts have been made to apply phage display in biotechnological and medical research.^2–4

Phage display technology has been widely adopted to design tumor-specific molecules for targeted delivery to cancerous tissues.⁵ Initially, antibody libraries were favored as targeting agents due to their high affinity and specificity for desired targets. On the other hand, recently, peptides have gained popularity because of their small size, low immunogenicity, and rapid tissue penetration, which has shifted the focus of research toward the discovery of peptide-based targeting agents. In 2018, George Smith and Gregory Winter were awarded the Nobel Prize for “phage display of peptides and antibodies,” respectively, owing to the widespread use of this valuable tool in numerous applications.⁶ Since then, several peptides and antibodies have been discovered and used in biotechnological and biomedical fields, including epitope mapping, targeted delivery, vaccine development, and biomarker discovery. This article reviews the applications of phage display technology in cancer research, with a specific focus on cancer biomarkers identified through this method.

M13 Structure and Vector Engineering

The first experimental study on phage display was conducted using the M13 bacteriophage by George Smith.¹ M13 bacteriophage, which has a circular 6.4 kb single-stranded DNA (ssDNA) genome, is approximately 800 nm in length and 6.6 nm in width. It is the most widely used vector due to several advantages. Its well-defined genome is packaged into five capsid proteins (pIII, pVI, pVII, pVIII, and pIX), which play a key role in determining the phage’s size and structure.⁷ Peptides and antibodies are displayed on the tip of the pIII or pVIII coat proteins, conferring the targeting ability. The high replication capacity and ability to accommodate large foreign DNA inserts make M13 a preferred choice for researchers.⁸ Furthermore, the bacteriophage infects bacteria non-lytically with a small number of proteins assembled around a single-stranded DNA, and it is non-infective to humans, making it particularly suitable for research purposes.⁹

Recombinantly constructed phage libraries are engineered by inserting random NNK codons, where N represents any of the four nucleotides (A, T, G, or C) and K stands for G or C, into the M13 phage vector.² The randomly synthesized oligonucleotides are amplified via PCR, purified, and digested with restriction enzymes before being ligated into the M13 vector system. After ligation, the vector is introduced into Escherichia coli via electroporation, aiming to achieve a diversity of at least 10⁹ to construct large libraries.¹⁰ The resulting phage pool expresses billions of peptides and antibodies with random sequences at the pIII or pVIII coat proteins. These biomolecules are then used in biopanning to select the best binders for the target of interest.

Biopanning

Phage display is an affinity screening approach known as “biopanning,” which involves selecting biomolecules with high diversity against a target protein, followed by an enrichment process. In the first step of biopanning, a target protein (such as a cancer biomarker or a whole cell) is coated onto a 96-well plate for in vitro selection. This is followed by the binding of the constructed phages to the target through incubation. Unbound and low-affinity phages are removed during a washing step, which uses a detergent-containing buffer. The bound phages are then collected using a low-pH elution buffer or by competitive binding, followed by enrichment through infection of E. coli. In this step, the phages utilize the host’s enzymatic systems to replicate their DNA and express coat proteins, thereby forming new virion particles. The amplified phage library is then used for additional rounds of biopanning (typically 3–5 rounds), selecting phages with higher affinity while eliminating low-affinity phages in each round (Fig. 1).

FIG. 1.

Schematic illustration of biopanning. (A) The M13 bacteriophage, 800 nm in length and 6.6 nm in width is engineered to display peptides or antibodies fused to the pIII/pVIII coat proteins. (B) In the first step of biopanning, phages are incubated with antigens or patient sera from patients with cancer. Unbound phages are removed through washing steps, and bound phages are eluted using a low pH buffer or competitive binding. The phages are then amplified by infecting Escherichia coli. Biopanning is performed for 3–5 rounds.

In an alternative selection process, known as in vivo selection, biopanning is performed in living animals by injecting the phage library into the organism. The process continues by harvesting the tissue of interest, followed by washing, elution, and amplification of the phages for the next round.⁹ This approach is one step closer to clinical applications, considering the complexity and heterogeneity of the living organism. However, there are limitations, as animal models do not always precisely mimic human disease conditions.^7,10 Another concern with in vivo screening is the administration of phages into the biological circulation. While intravenous delivery is commonly used, this method may not be suitable for brain-targeting peptides due to the blood–brain barrier. However, Wan et al. demonstrated that intranasal administration of phage libraries could improve brain-targeting peptide binding by 50-fold.¹¹

Taken together, regardless of the screening method, several peptide and antibody ligands have been discovered through phage display technology, showing great potential for future applications.

Phage-Focused Applications in Cancer

Screening random peptide and antibody libraries displayed on phages offers an efficient method for identifying ligands that bind to specific target molecules and modulate their function.¹² Phage display-derived techniques have numerous applications in biomedical fields, particularly in cancer treatment and diagnosis.¹³ Several biomolecules such as peptides, antibodies, enzymes, and other biomolecules are identified and characterized through phage display technology by far. Furthermore, the phage particles themselves can be engineered and repurposed as nanocarriers, providing a promising platform for the targeted delivery of therapeutic agents. These modified phages have demonstrated significant potential in gene therapy, immunotherapy, vaccine development, epitope mapping, and cancer diagnosis, leveraging their unique properties for precise and efficient interventions¹⁴ (Fig. 2).

FIG. 2.

Phage-focused applications in cancer treatment and diagnosis.

Cancer vaccines

The host immune system is activated upon encountering antigens, triggering the production of antibodies. In this context, the inherent phage machinery could be modified to display antigens on their surface, showing affinity for the target receptor. Indeed, phages are highly attractive candidates for use as vaccination vehicles. Variable studies have demonstrated that the antigens displayed on the phage surface can be loaded into either MHC‐I or MHC‐II, inducing cytotoxic T lymphocyte and antibody-mediated immune responses upon phage engulfment by antigen-presenting cells.¹⁵ Moreover, because phages infect only prokaryotic cells, their use in humans is considered safe. In a study reported by Wang et al., cytokine GM-CSF was displayed on the pVIII coat protein of the M13 filamentous phage. Experiments using a murine colorectal cancer model showed that GM-CSF phage activated STAT5 signaling in murine macrophages and led to a reduction of over 50% in tumor size compared with the unmodified phage.¹⁶

An alternative approach involves using phages as DNA vaccines in which a eukaryotic promoter is inserted into the phage genome to express the cancer antigen and start a response in the adaptive immune system.¹³ After the phage genome is packaged into the E.coli system, the vaccine phage is introduced into the patient. Importantly, phage-based DNA vaccines may offer advantages over naked DNA vaccines as a large antigen DNA up to 20 kb could be inserted into the phage genome.^17,18 Moreover, phages are promising tools since phage particles themselves are also recognized as foreign antigens and trigger an immune response. Taken all, the ability of phages to stimulate both innate and adaptive immunity makes them attractive for vaccine development.

Gene therapy

Recent research has explored various approaches that leverage the natural interactions between phages and mammalian cells.¹⁹ One such approach is phage-assisted cancer gene therapy, which employs phages to deliver therapeutic genes specifically to cancer cells. In fact, traditional gene therapy typically relies on viral vectors derived from mammalian viruses, which raises safety concerns due to the potential risk of gene integration into the host genome. On the other hand, with the discovery of phages’ capacity to interact with mammalian cells in the 1940s, phages were shown to aggregate in malignant cells and inhibit tumor growth.²⁰ Recent research further supports the regular interactions between phages and mammalian immune cells. Building on this, phage-based gene therapy involves engineering phages to deliver therapeutic genes directly to cancer cells by modifying their surface proteins to recognize cancer-specific markers or receptors. For instance, proteins displayed on phage surfaces can induce apoptosis in cancer cells, inhibit tumor progression, or enhance immune responses targeting tumor cells. In a study, a cationic nanoparticle (NP) was linked to an integrin αvβ3-targeting ligand and used to deliver genes to angiogenic blood vessels in tumor-bearing mice. NPs were conjugated with a mutant Raf gene, ATP^μ-Raf, which inhibits endothelial signaling and angiogenesis triggered by various growth factors. Significantly, administration of the NPs in mice led to apoptosis in tumor-associated endothelial cells followed by tumor cell death and regression of primary and metastatic tumors.²¹ These findings highlight the potential of phage-based platforms as efficient vehicles for targeted gene delivery in cancer therapy. Phages are relatively simple to manipulate in the laboratory, and their ability to infect a wide variety of bacteria allows for the creation of diverse phage variants with unique tumor-targeting properties, making them a promising tool for advancing cancer gene therapy.¹⁴

Epitope mapping

Epitope mapping refers to the identification of the specific sites where antibodies bind to antigens, providing essential insights into the pathogenesis of infections and cancer.¹² Understanding epitopes is critical for improving diagnostic assays and developing therapeutic antibodies. Epitopes derived from cancer antigens are particularly valuable as they are recognized by anti-cancer immune cells.

During immune responses, the innate immune system detects neoantigens, commonly known as tumor-associated antigens (TAAs). When these TAAs interact with the immune system, specific B lymphocyte clones are activated, leading to the production of high levels of antibodies against TAAs.²² Therefore, TAAs have become highly attractive targets, with their activation being facilitated through the administration of either full-length TAAs, their antigenic fragments, or TAA mimotopes (peptides recognized by anti-TAA antibodies). In addition to several popular approaches such as site-directed mutagenesis of the antigen,²³ high-throughput mutagenesis,²⁴ array-based oligopeptide scanning²⁵ and X-ray co-crystallography,²⁶ phage display using random peptide libraries or single-gene libraries is currently one of the most widely used methods for epitope mapping.²⁷ In this approach, phage libraries are created by cloning randomly fragmented peptide libraries derived from tumor antigens. These libraries are then subjected to biopanning against antibodies obtained from patients with cancer, allowing for the identification of specific B-cell epitopes recognized by polyclonal antibodies. Pérez-Martínez et al. exploited phage display of antigenic versions of HER-1 and HER-2 domains to accomplish domain-level epitope mapping. The antibodies isolated from immunized mice were utilized for the recognition of domains I, III, and IV of both antigens. Moreover, the combination of phage display and site-directed mutagenesis enabled the identification of polyclonal antibodies that recognize mutant receptor escape variants, demonstrating the potential use of phage-displayed HER domains as anti-cancer vaccines in the future.²⁸ Consequently, peptide-based antigens are identified as promising candidates for serological diagnosis, while others are considered suitable for vaccine development.

Biomarker detection

Proteomic strategies that enable the unbiased identification of proteins, along with their post-transcriptional and post-translational modifications, play a crucial role in complementing genomic approaches in biomarker detection. While genomics provides valuable insights into gene activity, it often lacks the comprehensive understanding that proteomics offers. However, the complexity of the proteome and current limitations in proteomic techniques make it challenging to isolate disease-relevant targets. To address these challenges, phage display has become an increasingly utilized method for discovering specific ligands that bind to cancer cells or tissues. This technology has proven instrumental in biomarker detection by identifying peptides and antibodies that selectively bind to cancer-specific proteins.

In 2023, Heine et al. reported the ORFeome phage display approach, which describes the discovery of immunogenic proteins that are utilized as cancer biomarkers.²⁹ In this approach, a genome or metagenome is fragmented and integrated into the phagemid vector system where the fragments are fused into the N-terminal of the pIII protein. This results in the creation of a phage library enriched with open reading frames (ORFs). The next step involves biopanning against serum to identify relevant peptides or protein fragments that could serve as potential cancer biomarkers. This approach exemplifies the practicality of phage display in biomarker discovery.

In this section, we will discuss a number of cancer biomarkers identified through phage display, highlighting the importance of biomarker discovery in enhancing the precision of cancer diagnosis and treatment.

Antigen GRP78

The production of monoclonal antibodies (mAbs) is both costly and time-consuming. However, phage display technology offers a more cost-effective and rapid alternative for generating these antibodies.³⁰ In 2007, Jacobsen et al. isolated single-chain human mAbs from a large naive antibody phage display library by panning against a single-cell suspension of freshly isolated live cancer cells from a human breast cancer specimen.³¹ A human antibody fragment, Ab39, recognized a cell surface antigen expressed on a subpopulation of cancer cell lines from different origins. Through further analysis, the antigen was identified as GRP78, a heat shock protein 70 (HSP70), confirmed by a yeast two-hybrid screen of a large human testis cDNA library. The study was validated through colocalization studies and antibody competition experiments, which further identified the epitope recognized by Ab39 as being located at the COOH terminus of GRP78.

Membrane-localized plectin-1

In addition to phage display screening, modified immunoprecipitation and the development of novel molecularly targeted imaging agents have enabled the identification of membrane-localized plectin-1 as a potential biomarker for pancreatic ductal adenocarcinoma (PDAC).³² Kelly et al. utilized a randomized 7-amino acid peptide library to perform biopanning against PDAC cells, identifying clone 27 as a binder to tumor cells but not normal cells. Proteomic analysis revealed that plectin-1, which is localized both intracellularly and on the membrane of human and mouse PDAC cells, was the binding partner for peptide 27. Furthermore, they conjugated plectin-1-targeted peptides to magnetofluorescent NPs (PTP-NPs) and successfully visualized them in vivo using specialized microscopy and MRI in a mouse model of human PDAC.

Biomarker annexin-2

Proteomic approaches that enable the unbiased identification of proteins and their post-transcriptional and post-translational modifications are crucial complements to genomic strategies. Screening techniques, including phage display, systematic evolution of ligands by exponential enrichment (SELEX), and small-molecule combinatorial chemistry, have been extensively utilized to identify specific ligands that interact with these proteins. Reynolds et al. described a functional proteomics method based on phage display screening and biochemical techniques and identified binding partners of 15 peptides against pancreatic cancer.³³ Three new biomarkers, annexin-2, Vimentin, and plectin-2 were discovered as a result of pull-down assays followed by mass spectroscopy analyses. This study reveals the most abundant proteins on the membrane and their pot ency as cancer biomarkers offering new insights for further research into this disease.

Annexin XI-A

Autoimmune diseases are often characterized by the presence of autoantibodies that aberrantly target self-antigens (autoantigens).³⁴ In cancer cells, abnormal proteins can arise due to genetic mutations or cellular damage, leading the immune system to recognize these proteins as “foreign”. This recognition concludes with the generation of antibodies. In light of this information, Fernandez-Madrid et al. conducted a study using a T7 cDNA library containing 938 clones encoding potential breast cancer autoantigens. They performed biopanning against sera from patients with breast cancer and identified annexin XI-A, the p80 subunit of the Ku antigen, ribosomal protein S6, and other autoantigens as potential discriminators between cancerous and non-cancerous control sera.³⁵ Specifically, while 19% of all women with breast cancer exhibited IgG antibodies that interacted with the amino acid 41–74 sequence of annexin XI-A, 60% of patients with ductal carcinoma in situ (DCIS), an early stage of breast cancer, exhibited these autoantibodies. These findings suggest that annexin XI-A and related autoantibodies may serve as valuable biomarkers for early breast cancer detection.

T7-1

Humoral and cellular components of the immune system have the capability to recognize tumors.³⁶ Among them, natural antibodies against cancer cells in the peripheral blood of tumor patients probably play a protective role against tumor development. The identification of immunogenic tumor proteins (TAAs) by these antibodies through the construction of recombinant cDNA libraries has been a recent popular approach.³⁷ In a study conducted by researchers screening phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines, 18 different antigens were identified after screening against sera from patients with breast cancer. Of these, three antigens (T7-1, T11-3, and T11-9) were found to be overexpressed in tumors compared with normal breast tissue.³⁵ In particular, T7-1 was found to be overexpressed in 50% of primary breast carcinomas and metastasis specimens, demonstrating its potential as a valuable biomarker for breast cancer.

Aminopeptidase N

Angiogenesis, the formation of new blood vessels, is a critical process for tumor growth and metastasis, providing the necessary nutrients and oxygen to sustain rapidly proliferating cancer cells.³⁶ Due to the significant role of the NGR motif in binding with integrins, a phage library displaying NGR peptide on the phage surface was selected against tumor vasculature in vivo. The findings indicated that NGR phage is specifically bound to immunocaptured aminopeptidase N (APN) and to cells engineered to express APN on their surface.³⁷ Moreover, antibodies against APN inhibited in vivo tumor homing. Immunohistochemical staining confirmed this result with up-regulated APN expression in endothelial cells within both mouse and human tumors.

Conclusion

Phage-focused research has given rise to significant advancements in biomedicine and biotechnology, establishing phage display as a promising tool for discovering specific ligands that target molecules of interest. The genetic engineering of phages has positioned them as potential carriers for targeted cancer therapies, providing an innovative method for delivering therapeutic agents directly to cancerous sites. Gene therapy by utilizing phages facilitates the delivery of therapeutic genes that induce cancer cell apoptosis, inhibit tumor growth, and enhance immune responses against cancer. Alongside these advancements, displaying cancer antigens on phage surfaces has sparked the development of cancer vaccines, stimulating immune responses to target and eliminate tumor cells.

In this context, phage display technology remains at the forefront of cancer research and therapeutic development. Its ability to identify novel biomarkers, engineer phages for targeted drug delivery, and develop cancer vaccines highlights its high potential in precision medicine.

Despite the significant progress, challenges persist in translating phage-based approaches to clinical settings, including issues such as scalability, stability, and immune response. Phage-induced immune responses, for instance, could limit its therapeutic potential. Phage engineering strategies could minimize immunogenicity to overcome this obstacle. Large-scale production of phages is also an issue that needs to be considered. The methods for the development of cost-effective and scalable manufacturing could enable the widespread use of phages. Another significant challenge is to screen the cancer antigens on the phage surface efficiently to elicit a robust and specific immune response against cancer cells. Moreover, organs such as the brain and pancreas are difficult for phage entry, and the dense fibrotic stroma surrounding tumors can impede phage penetration into tumor tissue. Despite all these challenges, recent advancements in phage engineering and delivery methods, including innovations such as intranasal administration for brain-targeting peptides, offer promising solutions to overcome these hurdles.

In conclusion, phage display and phage-based therapies hold immense promise in reshaping the landscape of cancer treatment. The continued exploration of these technologies offers the potential for more targeted, personalized, affordable, and effective interventions, ultimately improving the precision of cancer therapies and advancing the future of cancer care.

Footnotes

Acknowledgments

The author declares that no funds, grants, or other support were received during the preparation of this article.

Author’s Contributions

A.N.C.: Conceptualization, writing—original draft, writing—review and editing, visualization.

Author Disclosure Statement

The author declares no conflict of interest.

Funding Information

No funding was received for this article.

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