Peptide Phage Research Articles

Screening Intrinsically Disordered Regions for Short Linear Binding Motifs.

The intrinsically disordered regions of the proteome are enriched in short linear motifs (SLiMs) that serve as binding sites for peptide binding proteins. These interactions are often of low-to-mid micromolar affinities and are challenging to screen for experimentally. However, a range of dedicated methods have been developed recently, which open for screening of SLiM-based interactions on large scale. A variant of phage display, termed proteomic peptide phage display (ProP-PD), has proven particularly useful for the purpose. Here, we describe a complete high-throughput ProP-PD protocol for screening intrinsically disordered regions for SLiMs. The protocol requires some basic bioinformatics skills for the design of the library and for data analysis but can be performed in a standard biochemistry lab. The protocol starts from the construction of a library, followed by the high-throughput expression and purification of bait proteins, the phage selection, and the analysis of the binding-enriched phage pools using next-generation sequencing. As the protocol generates rather large data sets, we also emphasize the importance of data management and storage.

Identification of Plasmodium knowlesi Merozoite Surface Protein-119 (PkMSP-119 ) novel binding peptides from a phage display library.

Plasmodium knowlesi, the fifth human malaria parasite, has caused mortality in humans. We aimed to identify P.knowlesi novel binding peptides through a random linear dodecapeptide phage display targeting the 19-kDa fragment of Merozoite Surface Protein-1 protein. rPkMSP-119 protein was heterologously expressed using Expresso® Solubility and Expression Screening System and competent E.cloni® 10G cells according to protocol. Three rounds of biopanning were performed on purified rPkMSP-119 to identify binding peptides towards rPkMSP-119 using Ph.D.™-12 random phage display library. Binding sites of the identified peptides to PkMSP-119 were in silico predicted using the CABS-dock web server. Four phage peptide variants that bound to PkMSP-119 were identified after three rounds of biopanning, namely Pkd1, Pkd2, Pkd3 and Pkd4. The sequences of both Pkd1 and Pkd2 consist of a large number of histidine residues. Pkd1 showed positive binding signal with 6.1× vs. BSA control. Docking results showed that Pkd1 and Pkd2 were ideal binding peptides for PkMSP-119 . We identified two novel binding peptides of PkMSP-119 , Pkd1 (HFPFHHHKLRAH) and Pkd2 (HPMHMLHKRQHG), through phage display. They provide a valuable starting point for the development of novel therapeutics.

Diagnostic Profiling of the Human Public IgM Repertoire With Scalable Mimotope Libraries.

Specific antibody reactivities are routinely used as biomarkers, but the antibody repertoire reactivity (igome) profiles are still neglected. Here, we propose rationally designed peptide arrays as efficient probes for these system level biomarkers. Most IgM antibodies are characterized by few somatic mutations, polyspecificity, and physiological autoreactivity with housekeeping function. Previously, probing this repertoire with a set of immunodominant self-proteins provided a coarse analysis of the respective repertoire profiles. In contrast, here, we describe the generation of a peptide mimotope library that reflects the common IgM repertoire of 10,000 healthy donors. In addition, an appropriately sized subset of this quasi-complete mimotope library was further designed as a potential diagnostic tool. A 7-mer random peptide phage display library was panned on pooled human IgM. Next-generation sequencing of the selected phage yielded 224,087 sequences, which clustered in 790 sequence clusters. A set of 594 mimotopes, representative of the most significant sequence clusters, was shown to probe symmetrically the space of IgM reactivities in patients' sera. This set of mimotopes can be easily scaled including a greater proportion of the mimotope library. The trade-off between the array size and the resolution can be explored while preserving the symmetric sampling of the mimotope sequence and reactivity spaces. BLAST search of the non-redundant protein database with the mimotopes sequences yielded significantly more immunoglobulin J region hits than random peptides, indicating a considerable idiotypic connectivity of the targeted igome. The proof of principle predictors for random diagnoses was represented by profiles of mimotopes. The number of potential reactivity profiles that can be extracted from this library is estimated at more than 1070. Thus, a quasi-complete IgM mimotope library and a scalable representative subset thereof are found to address very efficiently the dynamic diversity of the human public IgM repertoire, providing informationally dense and structurally interpretable IgM reactivity profiles.

Ovarian Cancer Targeting Phage for In Vivo Near-Infrared Optical Imaging.

Ovarian cancer is often diagnosed at late stages due to current inadequate detection. Therefore, the development of new detection methods of ovarian cancer is needed. This may be achieved by phage nanoparticles that display targeting peptides for optical imaging. Here, two such phage clones are reported. Ovarian cancer binding and specificity of phage clones (pJ18, pJ24) and peptides (J18, J24) were investigated using fluorescent microscopy and modified ELISA. Further, AF680-labeled phage particles were subjected to biodistribution and optical imaging studies in SKOV-3 xenografted mice. Fluorescent microscopy and ELISA of phage and peptides showed significantly increased binding to SKOV-3 cells compared to controls. Additionally, these studies revealed that J18 exhibits specificity for ovarian cancer SKOV-3 and OVCAR-3 cell lines. Further, peptides displayed increased SKOV-3 binding compared to N35 (non-relevant peptide) with EC50 values of 22.2 ± 10.6 μM and 29.0 ± 6.9 (mean ± SE), respectively. Biodistribution studies of AF680-labeled phage particles showed tumor uptake after 4 h and excretion through the reticuloendothelial system. Importantly, SKOV-3 tumors were easily localized by optical imaging after 2 h and 4 h and displayed good tumor-to-background contrast. The fluorescent tumor signal intensity was significantly higher for pJ18 compared to wild type (WT) after 2 h.

Tumor Specific Peptides Selected for Targeted Delivery of Therapeutic Agents to Glioma Human Cells

Brain tumors are among the most intractable types of malignant neoplasms. Despite advances in the treatment of cancer, in particular, the development of new approaches in surgery, radiotherapy and chemotherapy, the incidence remains high with a low 5-year survival rate. Targeted therapy (TT) may be a solution to the problem of low efficacy of the applied cancer treatment methods. TT is based on the use of drugs that specifically affect specific types of tumors, which allows one to increase the effectiveness of treatment and minimize toxic effects on healthy tissues of the body. The combination of the unique properties of cancer cells allows one to find specific ligands that interact directly with the tumor, and to conduct TT for malignant tumors. Phage display technology is one of the promising approaches to the search for tissue- and/or organ-specific molecules [1]. Combinatorial phage peptide libraries make it possible to obtain highly specific peptides, including for various types of tumors. Currently, such tumor-targeting peptides are considered as means of targeted delivery of therapeutic genes, cytokines, imaging agents, pro-apoptotic peptides, and cytotoxic drugs. The purpose of this work was to obtain from the phage peptide library of bacteriophages exposing peptides, which ensure the accumulation of phage particles in human glioblastoma cells U-87 MG, and to assess the specificity of the selected peptides for cells of the primary cultures of human gliomas. During the research, the following tasks were solved: (1) Tumor-targeting peptides were selected using human glioblastoma cells U-87 MG in vitro and on a U-87 MG tumor in a xenograft model in vivo; amino acid sequences of selected peptides (SWTFGVQFALQH (26), HPSSGSA (92), PVSNKMS (83)) were determined; (2) primary cultures of human gliomas AS2, MG1, MG2, MG3, MG4 cells were obtained and characterized; (3) specificity of the binding of bacteriophages exposing selected tumor-targeting peptides to cells of primary cultures of human gliomas was evaluated using immunocytochemical analysis. The specific binding of selected tumor-targeting peptides with cells of the primary cultures AS2, MG1 was shown.

Crossing the blood-brain-barrier with nanoligand drug carriers self-assembled from a phage display peptide

The filamentous bacteriophage fd bind a cell target with exquisite specificity through its few copies of display peptides, whereas nanoparticles functionalized with hundreds to thousands of synthetically generated phage display peptides exhibit variable and often-weak target binding. We hypothesise that some phage peptides in a hierarchical structure rather than in monomeric form recognise and bind their target. Here we show hierarchial forms of a brain-specific phage-derived peptide (herein as NanoLigand Carriers, NLCs) target cerebral endothelial cells through transferrin receptor and the receptor for advanced glycation-end products, cross the blood-brain-barrier and reach neurons and microglial cells. Through intravenous delivery of NLC-β-secretase 1 (BACE1) siRNA complexes we show effective BACE1 down-regulation in the brain without toxicity and inflammation. Therefore, NLCs act as safe multifunctional nanocarriers, overcome efficacy and specificity limitations in active targeting with nanoparticles bearing phage display peptides or cell-penetrating peptides and expand the receptor repertoire of the display peptide.

Identification of a Specific Inhibitor of Human Scp1 Phosphatase Using the Phosphorylation Mimic Phage Display Method

Protein phosphatases are divided into tyrosine (Tyr) phosphatases and serine/threonine (Ser/Thr) phosphatases. While substrate trapping mutants are frequently used to identify substrates of Tyr phosphatases, a rapid and simple method to identify Ser/Thr phosphatase substrates is yet to be developed. The TFIIF-associating component of RNA polymerase II C-terminal domain (CTD) phosphatase/small CTD phosphatase (FCP/SCP) phosphatase family is one of the three types of Ser/Thr protein phosphatases. Defects in these phosphatases are correlated with the occurrence of various diseases such as cancer and neuropathy. Recently, we developed phosphorylation mimic phage display (PMPD) method with AlF4−, a methodology to identify substrates for FCP/SCP type Ser/Thr phosphatase Scp1. Here, we report a PMPD method using BeF3− to identify novel substrate peptides bound to Scp1. After screening peptide phages, we identified peptides that bound to Scp1 in a BeF3−-dependent manner. Synthetic phosphopeptide BeM12-1, the sequence of which was isolated at the highest frequency, directly bound to Scp1. The binding was inhibited by adding BeF3−, indicating that the peptide binds to the active center of catalytic site in Scp1. The phosphorylated BeM12-1 worked as a competitive inhibitor of Scp1. Thus, PMPD method may be applicable for the identification of novel substrates and inhibitors of the FCP/SCP phosphatase family.

Screening and Identification of PLK1-Polo Box Binding Peptides by High-Throughput Sequencing of Phage-Selected Libraries.

Human proteome contains a plethora of short linear peptide motifs that is crucial for signaling and other cellular processes. These motifs are difficult to identify due to lack of systematic approach for their detection. Here we demonstrate the use of peptide phage display in combination with high throughput next generation sequencing to identify enriched peptide sequences through biopanning process against polo box domain (PBD) of mitotic polo like kinase 1 (Plk1). Purified recombinant Plk1 and two unrelated controls namely B-lymphocyte antigen (CD20) and fluorescent protein (mCherry) were subjected to peptide phage display analysis. Bacterially-propagated phage DNA was amplified by PCR using triplet bar coded primers to tag the pool from each amplicon. Proteomic peptide phage display along with next generation sequencing and Bioinformatics analysis demonstrated several known and putative novel interactions which were potentially related to Plk1-PBD. With our strategy, we were able to identify and characterize several Plk1-PBD binding peptides, as well as define more precisely, consensus sequences. We believe that this information could provide valuable tools for exploring novel interaction involved in Plk1 signaling as well as to choose peptides for Plk1 specific drug development.

Characterization of Monoclonal Antibodies against σA Protein and Cross-Reactive Epitope Identification and Application for Detection of Duck and Chicken Reovirus Infections.

Although σA is an important major core protein of duck reovirus (DRV), the B-cell epitopes of this protein remain unknown to reseacrhers. Six monoclonal antibodies (MAbs) (1A7, 3F4, 5D2, 4E2, 3C7, and 2B7) were developed by using prokaryotic-expressed recombinant His-σA protein. Five of six MAbs (1A7, 3F4, 4E2, 3C7, and 2B7) reacted with His-σA protein in a conformation-independent manner, while 5D2 reacted with σA in a conformation-dependent manner. Immunofluorescence assays showed that the MAbs could specifically bind to DRV infected BHK-21 cells. The MAbs were delineated as three groups by a competitive binding assay. By using 12-mer peptide phage display and mutagenesis, MAb 4E2 was identified to recognize minimal epitope 56EAPYPG61 and MAb 1A7 recognize 341WVV/MAGLI/V347, residues 341V/M and 347I/V are replaceable. Dot blotting and sequence analysis confirmed that EAPYPG and WVV/MAGLI/V are cross-reactive epitopes in both DRV and avian reovirus (ARV). An enzyme-linked immunosorbent assay (ELISA) based on two expressed EAPYPG and WVVAGLI as antigen demonstrated its diagnostic potential by specific reacting with serum samples from DRV- or ARV-infected birds. Based on these observations, an epitope-based ELISA could be potentially used for DRV or ARV surveillance. These findings provide insights into the organization of epitopes on σA protein that might be valuable for the development of epitope-based serological diagnostic tests for DRV and ARV infection.

485 Vaspin (serpin A12) increases melanoma cell invasion through extracellular matrix models

The development of targeted therapies and immune checkpoint inhibitors has revolutionised melanoma treatment over the past decade. However, there remains an urgent need to identify new pro-metastatic pathways in melanoma. Vaspin was identified in a phage display screen against two in vitro melanoma models. The effect of recombinant human vaspin (rhVaspin) was tested on a range of melanoma lines including. Phenotypic changes were assessed using 2D cell migration assays and monitoring cell invasion through Matrigel-coated Boyden chambers or fluorescently labelled gelatin-coated glass coverslips. 3D melanoma spheroids were transferred to gels composed of Matrigel or type I collagen before time-lapse imaging. Protein expression was examined by Western blotting and immunofluorescence microscopy. From the peptide phage display hits we identified vaspin as a candidate protein for further investigation. rhVaspin treatment of A375, M202, SKMEL5, SKMEL28 cell models caused a temporal increase in phospho-Akt levels and increased the invasion of A375 and M202 cells (1.4- and 2.0-fold, respectively) through Matrigel coated Boyden chambers, but not M229 cells. Overexpression of vaspin in melanoma cells did not significantly affect cell proliferation, cell cycle progression or adhesion. In contrast, time-lapse microscopy revealed that the invasion of A375 and SKMEL28 cells from 3D melanoma spheroids into Matrigel and collagen gels was significantly increased. The accelerated invasion of A375 cells through Matrigel coated Boyden chambers was reversible by treatment with the pan-MMP inhibitor CT1746 (10 μM). Moreover, the expression levels of MMP-2 and -3 were increased in A375 cells following treatment with rhVaspin. Our studies show that vaspin expression in melanoma models is associated with increased invasive capacity through an MMP-dependent mechanism. Ongoing work aims to examine the pro-invasive phenotype in primary melanoma cultures using in vivo models.

Promotion of angiogenesis by M13 phage and RGD peptide in vitro and in vivo

One of the most important goals of regenerative medicines is to generate alternative tissues with a developed vascular network. Endothelial cells are the most important cell type required in angiogenesis process, contributing to the blood vessels formation. The stimulation of endothelial cells to initiate angiogenesis requires appropriate extrinsic signals. The aim of this study was to evaluate the effects of M13 phage along with RGD peptide motif on in vitro and in vivo vascularization. The obtained results demonstrated the increased cellular proliferation, HUVECs migration, cells altered morphology, and cells attachment to M13 phage-RGD coated surface. In addition, the expression of Vascular Endothelial Growth Factor A (VEGF-A), VEGF Receptors 2 and 3, Matrix Metalloproteinase 9 (MMP9), and epithelial nitric oxide synthase (eNOS) transcripts were significantly upregulated due to the HUVECs culturing on M13 phage-RGD coated surface. Furthermore, VEGF protein secretion, nitric oxide, and reactive oxygen species (ROS) production were significantly increased in cells cultured on M13 phage-RGD coated surface.

Delineating Surface Epitopes of Lyme Disease Pathogen Targeted by Highly Protective Antibodies of New Zealand White Rabbits.

Lyme disease (LD), the most prevalent vector-borne illness in the United States and Europe, is caused by Borreliella burgdorferi No vaccine is available for humans. Dogmatically, B. burgdorferi can establish a persistent infection in the mammalian host (e.g., mice) due to a surface antigen, VlsE. This antigenically variable protein allows the spirochete to continually evade borreliacidal antibodies. However, our recent study has shown that the B. burgdorferi spirochete is effectively cleared by anti-B. burgdorferi antibodies of New Zealand White rabbits, despite the surface expression of VlsE. Besides homologous protection, the rabbit antibodies also cross-protect against heterologous B. burgdorferi spirochetes and significantly reduce the pathology of LD arthritis in persistently infected mice. Thus, this finding that NZW rabbits develop a unique repertoire of very potent antibodies targeting the protective surface epitopes, despite abundant VlsE, prompted us to identify the specificities of the protective rabbit antibodies and their respective targets. By applying subtractive reverse vaccinology, which involved the use of random peptide phage display libraries coupled with next-generation sequencing and our computational algorithms, repertoires of nonprotective (early) and protective (late) rabbit antibodies were identified and directly compared. Consequently, putative surface epitopes that are unique to the protective rabbit sera were mapped. Importantly, the relevance of newly identified protection-associated epitopes for their surface exposure has been strongly supported by prior empirical studies. This study is significant because it now allows us to systematically test the putative epitopes for their protective efficacy with an ultimate goal of selecting the most efficacious targets for development of a long-awaited LD vaccine.

Bi-specific tenascin-C and fibronectin targeted peptide for solid tumor delivery

Oncofetal fibronectin (FN-EDB) and tenascin-C C domain (TNC-C) are nearly absent in extracellular matrix of normal adult tissues but upregulated in malignant tissues. Both FN-EDB and TNC-C are developed as targets of antibody-based therapies. Here we used peptide phage biopanning to identify a novel targeting peptide (PL1, sequence: PPRRGLIKLKTS) that interacts with both FN-EDB and TNC-C. Systemic PL1-functionalized model nanoscale payloads [iron oxide nanoworms (NWs) and metallic silver nanoparticles] homed to glioblastoma (GBM) and prostate carcinoma xenografts, and to non-malignant angiogenic neovessels induced by VEGF-overexpression. Antibody blockage experiments demonstrated that PL1 tumor homing involved interactions with both receptor proteins. Treatment of GBM mice with PL1-targeted model therapeutic nanocarrier (NWs loaded with a proapoptotic peptide) resulted in reduced tumor growth and increased survival, whereas treatment with untargeted particles had no effect. PL1 peptide may have applications as an affinity ligand for delivery of diagnostic and therapeutic compounds to microenvironment of solid tumors.

Enhanced Biosorption of Nickel Ions on Immobilized Surface-Engineered Yeast Using Nickel-Binding Peptides.

Three nickel-binding peptides were screened from a phage peptide library and displayed separately or in combination with surface-engineered Saccharomyces cerevisiae EBY100. The sorption of nickel ions on the surface of yeast cells increased with the increasing number of nickel Ni(II)-binding peptides displayed. The combined expression of the three peptides by EBY100/pYD1-N123 demonstrated the highest sorption of Ni(II) (2.603 ± 0.004 g g−1, dry weight) and an enhanced sorption capacity of 60.15%, compared to S. cerevisiae EBY100. An orthogonal test for yeast immobilization was designed. A maximum sorption capability of 68.62% was observed for a treatment at 25°C with 2.0% calcium chloride and 3.0% sodium alginate.

Immunization with a peptide mimicking Lipoteichoic acid protects mice against Staphylococcus aureus infection

Lipoteichoic acid (LTA), a major component of the cell wall of Staphylococcus aureus (S. aureus), is not generally considered as an ideal vaccine candidate since it is a thymus-independent antigen. In this study, we screened a 12-mer phage peptide library and identified a series of peptide sequences that can mimic the epitope of LTA. A tetra-branched multiple antigenic peptide, named MAP2-3, comprising one of the positive peptide sequences (GHKEDRQWCQHS), was synthesized. Immunization with MAP2-3 induced LTA-specific IgG antibodies, prolonged the survival time, and decreased the bacterial burden in organs of mice infected with S. aureus. Moreover, passive immunization with polyclonal anti-MAP2-3 sera reduced bacterial load in organs of mice with bacteremia, alleviated acute lung injury in mice with pneumonia, and decreased the size of lesions in mice with skin infection. The number of LTA-specific antibody-secreting cells in the spleen of MAP2-3 immunized mice were significantly higher than that in the control mice. In summary, as a surrogate of LTA, vaccination with MAP2-3 elicited humoral immune response and protected mice from S. aureus infection. This study provides a new option to design vaccines against S. aureus.

A novel interleukin-13 receptor alpha 2-targeted hybrid peptide for effective glioblastoma therapy.

We previously designed and reported a novel class of drugs, namely hybrid peptides, which are chemically synthesized and composed of a targeted binding peptide and a lytic-type peptide containing cationic amino acid residues that cause cancer cell death. In the present study, we screened for peptides that bind to interleukin-13 receptor alpha 2 (IL-13Rα2) by using a T7 random peptide phage display library system and isolated several positive phage clones. The A2b11 peptide, which was one of the positive clones, was shown to bind to IL-13Rα2 protein by Biacore analysis and a binding assay using glioblastoma (GB) cell lines. This peptide was linked with a lytic peptide containing a linker sequence to form the IL-13Rα2-lytic hybrid peptide. The IL-13Rα2-lytic hybrid peptide showed cytotoxic activity against GB cell lines in vitro. The IL-13Rα2-lytic hybrid peptide also affected Akt and Erk1/2 activation following treatment with interleukin-13 and induced rapid ATP dynamics in GB cells. Anti-tumor activity of the IL-13Rα2-lytic hybrid peptide was observed in vivo after intratumoral injection in a mouse xenograft model of human GB cells. These results suggest that the IL-13Rα2-lytic hybrid peptide might be a potent therapeutic option for patients with GB.

Post-exposure administration of chimeric antibody protects mice against European, Siberian, and Far-Eastern subtypes of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted pathogen. It belongs to the Flaviviridae family and causes severe human neuroinfections. In this study, protective efficacy of the chimeric antibody chFVN145 was examined in mice infected with strains belonging to the Far-Eastern, European, and Siberian subtypes of TBEV, and the antibody showed clear therapeutic efficacy when it was administered once one, two, or three days after infection. The efficacy was independent of the TBEV strain used to infect the mice; however, the survival rate of the mice was dependent on the dose of TBEV and of the antibody. No enhancement of TBEV infection was observed when the mice were treated with non-protective doses of chFVN145. Using a panel of recombinant fragments of the TBEV glycoprotein E, the neutralizing epitope for chFVN145 was localized in domain III of the TBEV glycoprotein E, in a region between amino acid residues 301 and 359. In addition, three potential sites responsible for binding with chFVN145 were determined using peptide phage display libraries, and 3D modeling demonstrated that the sites do not contact the fusion loop and, hence, their binding with chFVN145 does not result in increased attachment of TBEV to target cells.

Peptide Phage Display: Molecular Principles and Biomedical Applications

Phage display (PD) is a technology based on the presentation of functional exogenous peptides on the capsid surface of bacteriophages. PD is performed by introducing a DNA sequence of interest at a specific position within a functional viral gene. In addition, peptide phage libraries are powerful tools for expressing a wide range of random peptides and for specific peptide screening. Specifically, PD applications include the analysis of binding and interactions between proteins, the identification of bioactive peptides that bind to receptors, the identification of disease-associated antigens, and the identification of cell-specific peptides. Since its emergence, PD technology has revolutionized several fields in the biological sciences, such as oncology, cell biology, and pharmacology, the innumerable applications for which will be described throughout this review.

Mapping Mimotopes for House Dust Mite Allergen Der f 7 Using a Specific Monoclonal Antibody.

The dust mite Dermatophagoides farinae is a common worldwide cause of indoor allergies induced by its proteins, including the mid-tier allergen Der f 7. To identify conformational epitopes in Der f 7 using mimotope mapping and computational modelling. Here, we used standard hybridoma technology to generate 3 new monoclonal antibodies against Der f 7 and performed mimotope mapping by probing a random peptide phage display library. Computational tools, including Minox and the DiscoTope-2.0 Server were used to assess the structure and potential position of antigenic residues within Der f 7. Thirteen mimotopes sharing the common sequence --XX[LST]P[-E][LI]MLPLR[-S]- were identified. Further, computationally-predicted conformational epitopes were found at residues 1-7, 10, 27, 76-81, 92, and 130-133 of Der f 7, and the key amino acids for these epitopes were deduced to be 2P, 3I, 10E, 27E, 78E, 79E, 81I, 130S, and 132E based on the common mimotope sequence. We identified Der f 7 peptide mimotopes that may model binding sites for blocking antibodies. These may guide the development of immunotherapy for individuals with hypersensitivity to Der f 7.

Swainsonine, an alpha-mannosidase inhibitor, may worsen cervical cancer progression through the increase in myeloid derived suppressor cells population.

Cervical cancer, caused by high oncogenic risk Human Papillomavirus (HPV) infection, continues to be a public health problem, mainly in developing countries. Using peptide phage display as a tool to identify potential molecular targets in HPV associated tumors, we identified α-mannosidase, among other enriched sequences. This enzyme is expressed in both tumor and inflammatory compartment of the tumor microenvironment. Several studies in experimental models have shown that its inhibition by swainsonine (SW) led to inhibition of tumor growth and metastasis directly and indirectly, through activation of macrophages and NK cells, promoting anti-tumor activity. Therefore, the aim of this work was to test if swainsonine treatment could modulate anti-tumor immune responses and therefore interfere in HPV associated tumor growth. Validation of our biopanning results showed that cervical tumors, both tumor cells and leukocytes, expressed α-mannosidase. Ex vivo experiments with tumor associated macrophages showed that SW could partially modulate macrophage phenotype, decreasing CCL2 secretion and impairing IL-10 and IL-6 upregulation, which prompted us to proceed to in vivo tests. However, in vivo, SW treatment increased tumor growth. Investigation of the mechanisms leading to this result showed that SW treatment significantly induced the accumulation of myeloid derived suppressor cells in the spleen of tumor bearing mice, which inhibited T cell activation. Our results suggested that SW contributes to cervical cancer progression by favoring proliferation and accumulation of myeloid cells in the spleen, thus exacerbating these tumors systemic effects on the immune system, therefore facilitating tumor growth.