Peptide chemical modification is a valuable technique for improving peptide stability and bioactivity, particularly in drug discovery applications. Here, we report the development of a novel linker junction strategy using strain-promoted azide-alkyne cycloaddition (SPAAC) that enables the efficient formation of branched puromycin linkers with an average yield of 97%. This approach represents an improvement over traditional Michael-Addition methods, which typically yield ~15%-20%. The high yield of the SPAAC reaction and the near absence of by-products make the linkage by click reaction easy to purify. We demonstrate the utility of our linker design by successfully performing cDNA display and chemical modification strategies such as bicyclization of peptides. Our study demonstrates the functionality of the cDNA display system with the newly incorporated junction. In addition, the successful introduction of peptide bicyclization via tris-bromomethyl-benzene (TBMB) in cDNA display serves as a proof-of-concept for complex chemical modifications. Furthermore, the position of puromycin, which disrupts protein biosynthesis, has been determined. This approach offers novel insights into the discovery of chemically modified peptides and has the potential to accelerate the development of peptide-based therapeutics and diagnostics.

Synthetic multimeric peptide ligands of the natural cytotoxicity receptor, NKp30, on natural killer cells were developed in this study. A divergent solid phase peptide synthesis strategy was optimized for the conjugation of multiple peptide ligands, based on the parent TVPLN and related permutation sequences, into branched and hyperbranched peptides for structure-activity relationship studies. According to CD spectroscopy in aqueous trifluoroethanol, the parent TVPLN peptide transitioned from a β-turn (monomer and dimer) to an extended β-sheet and a helix-type conformation in its branched (trimer) and hyperbranched (tetramer) structures. The multimeric peptides were predicted to expand the binding interface to NKp30 according to molecular modeling and docking predictions. Flow cytometry revealed greater binding activity of the trimer and tetramer ligands onto NKp30-coated beads and the natural killer cells, while being displaced by the native B7H6 ligand in competitive binding studies on the NKp30-coated beads and reducing anti-NKp30 binding on the natural killer cells, suggesting some receptor-specific binding activity. Immunostimulatory activity assays showed little secretion of TNF-α and IFN-γ from the natural killer cells following peptide treatment, with the TVPLN monomer and dimer remaining superior to the rest. Select changes in sequence compositions and the ligated multimeric peptide display had an inhibitory effect on natural killer cell activation. This result was likely due to changes in bound vs unbound multimeric peptide structures that deviated from the bioactive β-turn of the TVPLN monomer and dimer for direct peptide engagement at the NKp30 active site. Nonetheless, the novel multimeric peptides showed improved cell binding activity relative to their linear counterparts and were nontoxic at lower (10 μM) doses, making them safe and effective for structure-activity studies for the discovery of novel peptide ligands of natural killer cells.

Tyrosine kinases control a wide range of cell signaling pathways that are central to human physiology, and they are dysregulated in a variety of human diseases, most notably cancers. Our understanding of tyrosine kinase biology hinges upon a clear delineation of their protein substrates. Thus, much effort has been invested into defining the substrate specificities of tyrosine kinases, which is partly driven by recognition of the amino acid sequences surrounding the phospho-acceptor tyrosine residues. Numerous methods have been developed to profile tyrosine kinase sequence recognition, and these approaches have collectively demonstrated that different tyrosine kinases have distinct substrate sequence preferences. Here, we describe one such method that combines bacterial peptide display and deep sequencing to study tyrosine kinase substrate preferences. Our approach enables rapid measurement of relative phosphorylation efficiencies for thousands of peptides simultaneously. The genetically-encoded nature of the peptide libraries used with this method allows for facile and cheap construction of libraries tailored to answer a variety questions. Notably, our approach is compatible with genetic code expansion via Amber codon suppression, which allows for the construction and screening of libraries containing non-canonical amino acids. Importantly, results from this assay correlate strongly with quantitative measurements of enzyme kinetics, they corroborate previously reported tyrosine kinase substrate preferences, and they can reveal new insights into tyrosine kinase substrate specificity.

TP53 mutations are early truncal events across cancers 1,2 . These are perceived to encode tumour-specific neoantigens representing prime cytotoxic T lymphocyte (CTL) targets 3,4 . However, studies systematically examining the physical cell surface display of p53 peptides bound to major histocompatibility complex molecules (pMHC), their relative antigenicity, and resultant immunogenicity have yet to be conducted. Here, we develop an epitope discovery platform using p53-reconstituted lung cancer cells as well as various tumour cells as pMHC sources. Combining data-independent acquisition mass spectrometry (MS), nanoscale chromatography, and peptide detection based on probabilistic measure and three-dimensional ion visualization techniques allows attomole sensitivity identification of pMHCs. This approach excluded ∼97% of algorithm-based virtual p53 immunopeptidomes, highlighting that only a few p53 pMHCs can be presented by common human MHC (human leukocyte antigen, [HLA]) alleles. Strikingly, surface expressed neoantigens are restricted to the corresponding set of such limited self-p53 peptide arrays and unaffected by enhancing p53 proteasomal turnover. Further curtailment of MS-validated, high affinity p53 neoepitopes that are structurally deviant from self-pMHC occurs in established tumours due to immune selection against the antigen presenting MHC allele or by a novel mechanism involving p53 neoepitope destruction by endoplasmic reticulum aminopeptidase 1 (ERAP1). In contrast, given the extremely weak MHC affinity and resultant short-lived cell surface pMHC expression, the common p53 neoepitope R175H/HLA-A*02:01 escapes immune selection despite CTL with high quality T-cell receptors. Rigorous tumour-protective immunoediting makes effective truncal neoepitope targeting a challenge, requiring attentive MS analysis and functional vetting to focus protective cytolytic responses.

Dendritic cells (DCs) and B lymphocytes produce major histocompatibility complex class II molecules (MHCIIs) in large amounts to maximize the display of peptides and fulfill their antigen-presentation functions. The surface expression of MHCIIs in these cells is regulated via the ubiquitination of a single conserved lysine residue in the cytoplasmic tail of all known β-chains. This modification is carried out mainly by the MARCH1 E3 ubiquitin ligase. In MARCH1-deficient DCs, the lack of MHCII ubiquitination results in its excessive accumulation at the plasma membrane, disorganizing lipid rafts and tetraspanin webs. These membrane structures regulate numerous biological processes, allowing the interactions between signaling molecules, such as the B-cell receptor (BCR) and CD19. Nevertheless, the full impact of MARCH1 and the ubiquitin-dependent MHCII turnover on the development, activation, and functions of B cells remains to be explored. Here, we show that the absence of MHCII ubiquitination negatively affected the marginal zone (MZ) B-cell pool in mice. We provide evidence that this alteration of B-cell responses may, at least in part, be due to the proteotoxicity of MHCIIs on the CD81-containing tetraspanin web, which impacted the surface dynamics of CD19 and its capacity to activate the PI3K/Akt cascade during tonic BCR signaling. The reduced MZ B-cell pool impaired the immune response to a type 2 T-independent antigen. Interestingly, the germinal center (GC) response against a T-dependent antigen was also negatively affected. Altogether, our results demonstrate the importance of the ubiquitin-dependent control of MHCII proteostasis for B-cell functions.

Public health interventions involving improved water, sanitation and promotion of hygiene behaviors (WASH) plus nutrition, were implemented in Bangladesh in a large randomized controlled trial to assess impact on childhood enteric infection and diarrheal disease. Here, we evaluated magnitude and breadth of humoral responses to enteric pathogens in a subset of children among those received intervention (n=60) versus a control group (n=60) using an integrated method of bacterial display peptide library screening, next-generation sequencing and computational analysis to characterize individual antibody repertoires in serum collected at median ages of 3, 14 and 28 months. We determined high seroprevalence for enteric infections and show that antibody recognition of the putative epitopes and antigenic regions remained consistent over time. With mathematical models, we inferred waning of maternal immunity, and a subsequent immunity boost due to infection. Random peptide library screening has potential utility for rigorous analysis of antibody responses and identification of epitopes indicative of protective humoral immune responses.

Drug conjugates have emerged as promising tumor-targeted cytotoxics with an improved therapeutic index compared to classical chemotherapeutics. Although traditionally based on antibody ligands, high-throughput screening methods, such as peptide display and DNA-encoded chemical libraries, have enabled the isolation of ultra-high-affinity small ligands and the generation of drug conjugates with better tumor-targeting performance. This Perspective examines the history, major clinical milestones and future of drug conjugates for cancer treatment. We also discuss a new wave of combination modalities, linker strategies, and the development of conjugates based on large and small delivery vehicles.

Phage display is an ideal platform for selecting peptide hits and offers a diverse array of cyclic binders with high affinity. While many recently developed phage display platforms incorporate chemical strategies, the vast majority of these are detrimental to the phage life cycle due to cross-reactivity with the capsid protein. In contrast, enzyme catalysis, which combines high efficiency and biocompatibility, offers a promising approach for phage-based cyclic peptide display. However, enzyme-mediated cyclization approaches remain underexplored. Here, we present a tyrosinase-mediated phage display platform that enables one-step cyclization via o-quinone-cysteine coupling, which is a simple and efficient strategy that does not compromise phage infectivity. Importantly, the catalytic property of tyrosinase is highly selective and spatially constrained, allowing it to bypass native tyrosine residues in the phage and selectively recognize only the engineered tyrosine residues. Using this platform, we constructed a macrocyclic peptide library that facilitated the discovery of macrocyclic peptide inhibitors targeting therapeutically relevant proteins. Notably, the cyclic peptide ACI1 demonstrated potent inhibition of PIP4K2A kinase activity with an IC50 value of 0.93 ± 0.05 μM, while ACP1 effectively inhibited the dephosphorylation activity of PTP1B with an IC50 of 1.06 ± 0.25 μM. The generality and efficiency of this strategy highlight its potential as a valuable tool for the development of bioactive cyclic peptides.

The unique hierarchical core–shell structures of silica capsules contributing to delivery of active compounds have attracted broad interest. To overcome the limited cargo‐loading capacity of silica capsules but harness the silica mediated cargo protection, Escherichia coli is engineered to efficiently assemble submicrometer‐sized biopolyester particles (BPs) that densely display the positively charged RK1 peptide ((RKK)4G3Y), which mediates nucleation and growth of silica. The peptide‐coated BPs are tested as biomimetic template and treatment with silica precursors results in formation of BP core–silica shell structures. Electron microscopy reveals changes in BP surface morphology consistent with silica shell formation further confirmed through Fourier transform infrared spectroscopy. Thermogravimetric analysis shows 0.10 g of silica shell per gram of BPs is formed, with no impact on crystallinity as assessed by X‐ray diffraction. The payload of the lipophilic biopolyester core for the lipophilic model compound, curcumin, increases to an encapsulation efficiency of 23.6%. Silica coating of BPs does not cause cytotoxicity and BPs show efficient cellular uptake. This study presents an innovative and environmentally benign approach for efficiently assembling robust biopolyester core–silica shell structures. The nontoxic polymer core provides drug payload capacity, protected by a silica coating ideal for controlled drug delivery applications.

Discovery of bioactive peptides, including those acting to permeabilize and/or kill bacterial cells (antimicrobial peptides) has drawn extensive interest in recent years. However, current technologies for their identification are limited. To address these limitations, the Intracellular Release Peptide Display (IRPD) technology allowing the recombinant "display" of intracellular linear peptides was developed. IRPD uses the protease domain of the capsid protein from the Semliki Forest virus as a scaffold to express and liberate linear peptides intracellularly in Escherichia coli. IRPD is a universal platform that allows screening of millions of peptides and the discovery of bioactive peptides from direct target interactions and independent of the cell envelope barrier. Here, we identified peptides that cause increased bacterial cell envelope permeability and lysis. The most promising candidate, P38, effectively kills Gram-negative pathogens by disrupting the inner membrane without detectable resistance development. Thus, P38 constitutes an interesting hit peptide for further development.

Lactic acid bacteria (LAB) are promising mucosal vaccine vectors due to their safety, immunostimulatory properties, and the availability of genetic tools for certain strains. Bacterial flagellin has attracted attention as both a versatile scaffold for antigen surface display and a potent adjuvant via Toll-like receptor 5 (TLR5) activation. Despite these advantages, LAB-derived flagellin remains largely unexplored as an antigen display platform. In this study, we demonstrate the potential of flagellin (FliC2) from Ligilactobacillus agilis, one of the few flagellated LAB species, as a scaffold for the surface display of the HIV-1 membrane-proximal external region (MPER) epitope. Recombinant L. agilis strains were engineered to express FliC2 with the MPER epitope inserted at various positions within its hypervariable domain, identifying optimal sites for effective surface display. To enhance the adjuvant activity of FliC2, specific amino acid substitutions were introduced into the TLR5 recognition site, resulting in improved TLR5-stimulating activity in vitro. Immunization of mice with MPER-displaying L. agilis strains induced MPER-specific IgA and IgG responses, demonstrating the efficacy of the L. agilis flagellin-based display platform in eliciting both mucosal and systemic immune responses. This study is the first to demonstrate LAB-derived flagellin as an antigen display scaffold, highlighting L. agilis flagellin as a promising platform for mucosal vaccine development.IMPORTANCELactic acid bacteria (LAB) are promising delivery vehicles of active molecules, and surface display systems are gaining interest for efficiently displaying heterologous peptides and proteins. Flagellin, a TLR5 agonist, has been widely used as an adjuvant and an antigen scaffold, making it a potentially valuable platform for such systems. However, the potential of LAB-derived flagellin as a surface display scaffold remains largely unexplored. This study demonstrates that flagellin from Ligilactobacillus agilis, a flagellated LAB species, effectively functions as an antigen display platform, eliciting mucosal and systemic immune responses. Our findings highlight the feasibility of LAB-derived flagellin as a versatile tool for surface display of heterologous peptides, expanding its potential applications in vaccine development and mucosal immunotherapy.

A novel miniaturized filamentous phagemid as a gene delivery vehicle to target mammalian cells

Phage display technology, a molecular screening tool introduced in 1985, facilitates the display of peptides or proteins on phage surfaces to identify high-affinity ligands. Phage display has enabled the identification of peptides that enhance biomaterial biocompatibility, support stem cell proliferation and differentiation, and facilitate targeted drug delivery. Additionally, it has been utilized in bacterial detection, disease biomarker identification, and targeted therapies, including crossing the blood-brain barrier for improved cancer treatments. Despite challenges such as peptide stability and immunogenicity, ongoing innovations continue to enhance its therapeutic potential. This review discusses the potential of phage display technology for empowering healthcare through demonstrating its development in various biological fields in recent years.

Development of a pod pepper vein yellows virus-based expression vector for the production of heterologous protein or virus like particles in Nicotiana benthamiana.

Abstract 1941 Enhanced cellular and transdermal delivery of the modified chromatin using cell-penetrating peptide display

Abstract Multiple myeloma (MM) remains an incurable hematologic malignancy, and immunomodulatory imide drugs (IMiDs) have been used as an effective treatment option. A significant challenge with IMiDs such as pomalidomide, as with other treatment options, is the development of drug resistance. This has become a substantial impediment to extending patients' lives. Currently, no effective strategy exists for overcoming IMiD resistance in MM patients. Exploring alternative approaches that could improve the efficacy of pomalidomide might help myeloma patients live longer. Genetic alterations in cereblon (CRBN) contribute to more than one-third of clinical cases of pomalidomide resistance. This study aims to restore sensitivity to pomalidomide-resistant cells through mRNA-based wildtype CRBN restoration. First, we repeatedly exposed multiple myeloma cell lines (MM1.S) to pomalidomide at increasing doses, starting from 2 nM to 10 μM. Following six months of pomalidomide exposure, we generated MM1.S cells that are resistant to pomalidomide and confirmed the absence of CRBN with RNA sequencing and western blotting. Through in vitro transcription, we synthesized CRBN mRNA and electroporated it to the resistant cells. We found restoration of wildtype cereblon and subsequent pomalidomide sensitivity compared to cells without CRBN mRNA treatment. As electroporation is impractical beyond laboratory studies, we are designing a polymer-lipid hybrid nanoparticle to efficiently deliver the mRNA to the cells. Additionally, to ensure targeted delivery of the nanoparticle-coated CRBN mRNA to plasma cells, we applied phage display peptide library screens and identified a cyclic peptide with a high affinity for B-cell maturation antigen (BCMA), a protein known to be almost exclusively expressed by plasma and myeloma cells. This peptide is being validated for BCMA binding, and will be conjugated with the nanoparticle-coated CRBN mRNA for targeted delivery to the malignant plasma cells. Findings from this work will advance mRNA-based therapeutics for overcoming drug resistance caused by genetic alterations in target proteins. Citation Format: Joseph U. Ogbede, Michael S. Rogers, Robert J. D'Amato, Bruce R. Zetter. mRNA-based restoration of pomalidomide sensitivity in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4432.

Development of targeted antimicrobial peptides for Escherichia coli: Combining phage display and rational design for food safety application.

Escherichia coli surface display technology, which facilitates the stable display of target peptides and proteins on the bacterial surface through fusion with anchor proteins, has become a potent and versatile tool in biotechnology and biomedicine. The E.coli surface display strategy presents a unique alternative to classic intracellular and extracellular expression systems, facilitating the anchorage of target peptides and proteins on the cell surface for functional execution. This distinctive attribute also introduces a novel paradigm in the realm of biocatalysis, harnessing cells with surface-displayed enzymes to catalyze the conversion of substrates. This strategy effectively eliminates the requirement for enzyme purification, overcomes the limitations related to substrate transmembrane transport, improves enzyme activity and stability, and greatly reduces the cost of downstream product purification, thus making it widely used in biocatalysis. Here, we review recent advances in various surface display systems and surface display technology for biocatalytic applications. Additionally, we discuss the current limitations of this technology and several promising alternative display methods.

ABSTRACT Immunotherapy options for microsatellite stable (MSS) colorectal cancer are currently very limited. The lack of detectably unique or altered immunogens in the tumor microenvironment may be a factor. Radiation and chemotherapy may enhance immunotherapy by increasing cancer cell visibility through Major Histocompatibility Complex I (MHC I) expression. To investigate this, we treated MSS and microsatellite-instable (MSI) colon cancer cells with a topoisomerase inhibitor and analyzed MHC I-associated peptides. Treatment increased peptide numbers by 5% in RKO (MSI) cells and 83% in SW620 (MSS) cells, with 40–50% of peptides being exclusive to treatment. Additionally, clustering analysis revealed a set of peptides with uniquely conserved residues displayed only in treated MSS SW620 cells. Gene Ontology analysis of MHC I-displayed proteins revealed a treatment-induced increase in extracellular vesicle- and nuclear-derived proteins, alongside reduced cytosolic protein sampling. Overall, we present evidence for treatment-inducible differential display of peptides, some of which may affect interactions and functions of immune cells. Given the multitude of factors that modulate the effects of increased MHC I expression and associated peptides, further studies are needed to elucidate the pathophysiological implications of these changes.

Europium as one of the rare earth elements (REE) has outstanding properties in terms of its application for high-tech and renewable energy products. The high supply risk of REE, coupled with their low recovery rates from secondary sources, necessitates innovative recycling approaches. We introduce a phage display-based peptide biosorbent recycling technology that offers a cost-effective and environmentally friendly solution for recovering metal ions, supporting circular economy goals. In this study, we used phage surface display to screen for peptides with high affinity for europium (III) ions (Eu3+). Performing several independent biopanning experiments with the Ph.D.-12 Phage Display Peptide Library and different elution methods as well as combining them with next-generation sequencing, we identified eight peptides with moderate to good affinities for Eu3+ ions, verified by time-resolved laser fluorescence spectroscopy. The peptides EALTVNIKREME as well as DVHHVDGNDLQPFEGGGS and DSIHSDVTKDGRYPVEGGGS, the latter are variants of enriched dodecamers, proved to be the best candidates for future biosorption and selectivity studies. This study underscores the potential of phage surface display for peptide-based REE recovery, laying the foundation for selective recycling technologies from secondary raw materials.