Targeting Peptide Research Articles

Delivery of N-Cadherin Targeting Peptides to Vascular Tissues by Surface-Modified Polyurethane Nanoparticles via a Drug-Coated Balloon.

Restenosis remains a long-standing limitation to effectively maintain functional blood flow after percutaneous transluminal angioplasty (PTA). While the use of drug-coated balloons (DCBs) containing antiproliferative drugs has improved patient outcomes, limited tissue transfer and poor therapeutic targeting capabilities contribute to off-target cytotoxicity, precluding adequate endothelial repair. In this work, a DCB system was designed and tested to achieve defined arterial delivery of an antirestenosis therapeutic candidate, cadherin-2 (N-cadherin) mimetic peptides (NCad), shown to selectively inhibit smooth muscle cell migration in vitro and limit intimal thickening in early animal PTA models. To enable successful tissue transfer in the current work, a nanoparticle excipient system previously demonstrated to be an effective carrier of NCad in vitro was integrated with customized DCB coating methodologies designed to prevent therapeutic loss during delivery. DCB design took into consideration four components: (1) the angioplasty balloon; (2) a poly(ethylene oxide) (PEO) monolayer acting as a hydrophilic spacer between the balloon surface and the nanoparticles to assist with improved nanoparticle release; (3) surface-modified degradable polar hydrophobic ionic polyurethane (D-PHI) nanoparticles loaded with NCad to facilitate the transport of the therapeutic peptide into vascular tissue; and (4) a PEO sacrificial coating applied over the nanoparticle excipient layer to prevent premature losses during transit to the artery. The nanoparticle-DCB platform successfully delivered NCad to rat carotid tissue, with superior efficacy and increased permeation within the vessel wall compared with soluble NCad infusion alone. Nanoscale technologies in conjunction with enhanced DCB design properties hold promise in advancing the localized delivery of preventive restenosis therapies in vascular disease.

Synthesis of Sulfonated Peptides Using a Trifluoromethyltoluene-Protected Amino Acid.

A scalable, seven step synthesis is reported for a trifluoromethyl toluene protected sulfonated phenylalanine building block whose utility was demonstrated in the synthesis of four CXCR4-derived sulfonopeptides. When compared to a conventional trichloroethyl protected building block, overall yield was improved by up to 4-fold. We believe this building block will prove to be of significant value for the synthesis of a variety of peptide targets containing phenylalanine sulfonate, a bioisostere of tyrosine sulfate, enabling orthogonal protection strategies and improving synthetic efficiency and yield.

Rice bran peptides target lectin-like oxidized low-density lipoprotein receptor-1 to ameliorate atherosclerosis.

Food-derived multifunctional peptides offer numerous health benefits through different biochemical pathways. However, their impact on aging-related atherosclerotic cardiovascular disease (ASCVD), especially atherosclerosis, remains underexplored despite cardiovascular disease (CVD) being the leading cause of death globally. In this study, NHANES data and Mendelian randomization were used to analyze the association between lipid metabolism disorders, systemic immune responses, dietary inflammatory index, and ASCVD. The results showed that they were all positively correlated with ASCVD. A dietary intervention was used to induce a mouse model of atherosclerosis through a high-fat diet (HFD). Our findings demonstrate that rice bran peptide could mitigate the typical pathological features of atherosclerosis. Molecular docking analysis further predicted that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a key target of rice bran peptide. This prediction was validated through a two-cell model of endothelial cells and lox-1-interfered macrophages. Therefore, targeting LOX-1 with rice bran peptide inhibits the excessive uptake of oxidized LDL (ox-LDL) by macrophages, thereby hindering the mass production of foam cells, which is crucial in preventing the early onset of atherosclerosis.

Development of pH-responsive crosslinked peptides that form stable α-helices under weakly acidic conditions

Abstract α-Helical structures in protein surface are frequently related to protein–protein interactions that play important roles in signal transduction. We have developed various crosslinking agents that stabilize the α-helical structure of short peptides. In this article, we report pH-responsive crosslinked peptides that promote helical conformation of the peptides under weakly acidic conditions. We incorporated critical sequence of a synthetic estrogen receptor activity-regulating peptide into our pH-responsive crosslinked peptide. The pH control could provide total structural regulation of the target peptide.

Predictions to Increase Lasso Peptide Production in the Heterologous Host Streptomyces coelicolor M1152.

Production of specialized metabolites are restricted to the metabolic capabilities of the organisms. Genome-scale models (GEM)s are useful to study the whole metabolism and to find metabolic engineering targets to increase the yield of a target compound. In this work we use a modified model of Streptomyces coelicolor M145 to simulate the production of lagmysin A (LP4) and the novel lagmysin B (LP2) lasso peptide, in the heterologous host Streptomyces coelicolor M1152. Overexpression targets were identified using the flux scanning based on enforced objective flux (FSEOF) algorithm and flux variability analysis (FVA), considering growth in minimum and in complex medium. Thirteen reactions were found as candidate metabolic engineering targets for both lasso peptides considering both settings. We propose the overexpression of enzymes of the glycolysis pathway (GAPD, PGK, PGM and ENO) and leucine biosynthesis (IPPS, IPPMIb, IPPMIa, IPMD and OMCDC) to enhance the production of either lagmysin A or B.

Идентификация и предиктивный анализ аминокислотных паттернов, обуславливающих потенциальную антигиперурикемическую активность пептидов

Peptides offer a promising analogue to synthetic drugs in treating hyperuricemia. This article introduces reliable amino acid patterns that cause the inhibitory xanthine oxidase (CSR) activity of peptides. The research objective was to propose new antihyperuricemic peptides and prove their effectiveness by predictive analytics in silico. The study featured peptides with inhibitory xanthine oxidase activity. The authors developed a protocol for searching, identifying, and quantifying patterns of amino acid residues in target peptide sequences. The identified peptides were tested for physicochemical properties, pharmacokinetic profile, inhibitory xanthine oxidase activity, general and target biological activity, and toxicity. The research revealed amino acid patterns responsible for inhibiting the xanthine oxidase enzyme, as well as generated new peptide sequences. Forty-nine non-toxic peptides with different lengths of amino acid sequences demonstrated high antimicrobial and inhibitory potential against the targeted drugs used to treat hyperuricemia and type 2 diabetes mellitus. The peptides were low-molecular compounds of predominantly hydrophilic and hydrophobic nature, 4-7 amino acids long. They contained negatively charged amino acid residues of proline, tryptophan, and phenylalanine with an average molecular weight of 723 Da. The study offers an important insight into the molecular mechanisms of xanthine oxidase inhibition and opens up new prospects for developing novel antihyperuricemic peptide drugs.

Identification of Protein Hydrolysates from Sesame Meal and In Vivo Study of Their Gastric Mucosal Protective Effects.

This study aimed to investigate the protective effects and defense mechanisms of a sesame meal protein hydrolysate against ethanol-induced acute gastric mucosal injury in mice. The target peptides in the hydrolysate were identified by LC-MS/MS, the activity was predicted by PeptideRanker, and the KM mice were orally administered distilled water, a sesame peptide, and omeprazole for 24 consecutive days. Acute gastric mucosal injury was then induced in mice with 70% ethanol, except for the CK group. The sesame peptide significantly inhibited the over-accumulation of ALT, AST, MDA, TNF-α, IL-1β, and MPO, while promoting the reduction in GSH, T-AOC, GSSG, and EGF expression. In addition, a Western blotting analysis showed that sesame peptide significantly up-regulated the expression of HO-1 and NQO1 proteins in the Nrf2/Keap1 signaling pathway, and down-regulated Keap1 protein. The defense effect of a sesame peptide on gastric mucosa may be achieved by alleviating the overproduction of lipid peroxides and improving the antioxidant activity.

PEGylated Nanoliposomal Doxorubicin Conjugated with Specific TREM2 Peptides for Glioma-Targeting Therapy.

PEGylated liposomes can deliver anti-cancer drugs to brain tumors, and achieve enhanced permeability and retention effects. Triggering receptor expressed on myeloid cells 2 (TREM2) is an excellent biomarker for precise therapyof glioma. The present study is aimed at designing PEGylatednanoliposomal doxorubicin (PLD) conjugated with peptides targeting TREM2 for glioma-targeting therapy. The specific peptides are designed with the Rosetta Peptiderive Protocol. Schrodinger's peptide-specific version of Glide is used for molecular docking.PLD modified with peptides (peptide-PLD) are engineered and prepared. Cell cycle, apoptosis, cell invasion and migration, cell viability, and colony-formation assays are performed to analyze glioma cell functions. The anti-tumor effects of peptide-PLD are validated in an intracranial U87-MG cells orthotopic glioma model. The targeting peptides HLRKLRKR and LRKLRLRL showed specific affinity for TREM2 and better cellular uptake in U87-MG cells. PLD with peptide modification demonstrated stable doxorubicin loading, small sizes (<60nm), and enrichment in the mouse brain. Peptide-PLD treatment inhibited the Akt/GSK3β/β-catenin pathway, thereby inhibiting cell invasion and migration, and colony-forming ability in U87-MG cells. The peptide modification of PLD achieved better suppression of glioma development than PLD.Overall, TREM2-targeting peptides are successfully designed, and peptide-PLD served as a potent drug delivery carrier for glioma-targeting therapy.

Active-Targeted ICG for Surgical Navigation and Fluorescence-Guided Laparoscopic Photothermal Ablation in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy, but there is limited improvement in its treatment. Near-infrared fluorescence (NIRF) imaging could potentially address the clinical challenges of PDAC. Indocyanine green (ICG) has been widely used in clinical practice; however, its short half-life and lack of active targeting greatly limit its application in pancreatic surgery. In this study, the active targeting peptide KTLLPTP (which actively recognizes PDAC cell surface overexpression Plectin-1) was modified to the ICG to create the novel contrast agent ICG-PTP, which actively targets PDAC cells. It was successfully applied to the NIRF imaging of the PDAC orthotopic mice model, achieving an improved tumor signal background ratio (T/N ratio) of 4.28, compared to 2.34 in the free ICG group. Next, Fluorescence-guided excision of subcutaneous/orthotopic PDAC using ICG-PTP was performed, accurately identifying the tumor margin and significantly facilitating resection efficiency. Finally, PDAC metastases were identified, and interventional photothermal ablation (iPTA) was performed under fluorescence laparoscope guidance. ICG-PTP exhibits good biosafety and clinical transitional potential. Thus, they can provide surgeons with efficient real-time tumor information and offer new treatment strategies for metastases. Accordingly, modification of probes for clinical use and adaptation studies of current equipment are the current focus.

The structural and functional analysis of mycobacteria cysteine desulfurase-loaded encapsulin

Encapsulin nanocompartments loaded with dedicated cargo proteins via unique targeting peptides, play a key role in stress resistance, iron storage and natural product biosynthesis. Mmp1 and cysteine desulfurase (Enc-CD) have been identified as the most abundant representatives of family 2 encapsulin systems. However, the molecular assembly, catalytic mechanism, and physiological functions of the Mmp1 encapsulin system have not been studied in detail. Here we isolate and characterize an Enc-CD-loaded Mmp1 encapsulin system from Mycobacterium smegmatis mc2155. The cryo-EM structure of the Mmp1 encapsulin and the crystal structure of the naked cargo Enc-CD have been determined. The structure shows that the Mmp1 protomer assembles two conformation models, the icosahedron (T = 1) and homodecamer, with the resolution of 2.60 Å and 2.69 Å. The Enc-CD at 2.10 Å resolution is dimeric and loaded into the Mmp1 (T = 1) encapsulin through the N-terminal long disordered region. Mmp1 encapsulin protects Enc-CD against oxidation as well as to maintain structural stability. These studies provide new insights into the mechanism by which Enc-CD-loaded encapsulin stores sulfur and provides a framework for discovery of new anti-mycobacterial therapeutics.

Ionic control of small GTPase HRas using calmodulin.

HRas is a small GTPase that plays physiologically important roles in various intracellular signal transduction processes, such as cell growth and proliferation. The structure and action mechanisms of HRas have been well-characterized, leading to widespread its use as a molecular switch in bionanomachines. calmodulin, a calcium ion-binding protein, acts as an ion-binding molecular switch and activates the target enzymes. We previously demonstrated that the fusion protein of HRas (M13-HRas) with the calmodulin target peptide M13 at the N-terminus of HRas exhibits reversible regulation of GTPase activity and the interaction between M13-HRas and the downstream signaling factor Raf by calcium ions with calmodulin. In this study, we prepared two new HRas fusion proteins with the M13 peptide at the C-terminus (HRas-M13) and both termini (M13-HRas-M13) of HRas and analyzed the calcium-dependent regulation of HRas function. M13-HRas-M13 more efficiently controlled GTPase, interaction with Raf, and the HRas regulator GEF by calcium ions with calmodulin.

Supramolecular Host:Guest Arrays Site-Selectively Recognize Peptide Phosphorylation and Kinase Activity.

A synergistic combination of cationic styrylpyridinium dyes and water-soluble deep cavitand hosts can recognize phosphorylated peptides with both site- and state-selectivity. Two mechanisms of interaction are dominant: either the cationic dye interacts with Trp residues in the peptide or the host:dye pair forms a heteroternary complex with the peptide, driven by both strong dye-peptide and cavitand-peptide binding (Kd values up to 4 μM). The presence of multiple recognition mechanisms results in varying fluorescence responses dependent on the phosphorylation state and position, eliminating the need for covalent modification of the peptide target. Differential sensing aided by machine learning algorithms permits full discrimination between differently positioned serine phosphorylations with a minimal 3-component array. The array is fully functional in the presence of protein kinase A (PKA) and its required cofactors and capable of site-selective monitoring of serine phosphorylation at the privileged PKA motif, in the presence of serine residues that do not undergo reaction, illustrating the potential of the system in kinase-based drug screening.

Targeting peptide antigens using a multiallelic MHC I-binding system.

Identifying highly specific T cell receptors (TCRs) or antibodies against epitopic peptides presented by class I major histocompatibility complex (MHC I) proteins remains a bottleneck in the development of targeted therapeutics. Here, we introduce targeted recognition of antigen-MHC complex reporter for MHC I (TRACeR-I), a generalizable platform for targeting peptides on polymorphic HLA-A*, HLA-B* and HLA-C* allotypes while overcoming the cross-reactivity challenges of TCRs. Our TRACeR-MHC I co-crystal structure reveals a unique antigen recognition mechanism, with TRACeR forming extensive contacts across the entire peptide length to confer single-residue specificity at the accessible positions. We demonstrate rapid screening of TRACeR-I against a panel of disease-relevant HLAs with peptides derived from human viruses (human immunodeficiency virus, Epstein-Barr virus and severe acute respiratory syndrome coronavirus 2), and oncoproteins (Kirsten rat sarcoma virus, paired-like homeobox 2b and New York esophageal squamous cell carcinoma 1). TRACeR-based bispecific T cell engagers and chimeric antigen receptor T cells exhibit on-target killing of tumor cells with high efficacy in the low nanomolar range. Our platform empowers the development of broadly applicable MHC I-targeting molecules for research, diagnostic and therapeutic applications.

Development of a Multiplexed LC-MS/MS Assay for the Quantitation of Podocyte Injury Biomarkers Nephrin, Podocalyxin, and Podocin in Human Urine.

CKD is frequently diagnosed only after a significant progression. GFR is the most common indicator of kidney function but is limited in detecting early CKD cases and distinguishing glomerular, tubular, and global CKD. Aiming to provide a glomeruli specific biomarker assay, we developed a peptide immunoaffinity targeted mass spectrometry method for the quantitation of three podocyte specific proteins in human urine: nephrin, podocalyxin, and podocin. Proteins in urine were precipitated, stable isotope labeled peptide standards incorporated, and digested with trypsin. Target peptides were enriched using an online antibody column prior to LC-MS/MS. The performance metrics for nephrin, podocalyxin, and podocin were evaluated: The lower limits of quantitation were 3.8, 22.0, and 5.4 pM, respectively. The intraplate relative error (RE) was within ±10.6%, ± 10.4%, and ±16.1%, and coefficient of variation (CV) was ≤27.2%, ≤ 14.1%, and ≤20.7% accordingly. The interplate RE was within ±7.0%, ± 3.8%, and ±3.0%, and CV was ≤17.2%, ≤ 12.1%, and ≤20.0% for the three analytes. The urinary nephrin, podocalyxin, and podocin concentrations in 60 healthy volunteers and 20 disease samples was measured, thereby establishing the basal levels of these protein and enabling future evaluation of their roles as noninvasive biomarkers of glomerular injury in the clinic.

Enhanced Molecular Imaging through a Versatile Peptide Nanofiber for Self-Assembly and Precise Recognition.

Designing molecules for multivalent targeting of specific disease markers can enhance binding stability which is critical in molecular imaging and targeted therapy. Through rational molecular design, the nanostructures formed by self-assembly of targeting peptides are expected to achieve multivalent targeting by increasing the density of recognition ligands. However, the balance between targeting peptide self-assembly and molecular recognition remains elusive. In this study, we designed a targeting-peptide-based imaging probe system TAP which consist of the signal unit, the recognition motif, the assembly motif and a Pro-leverage. It is verified that TAP could specifically binds to PD-L1-positive tumor cells in a multivalent manner to produce biological effects, and could also be combined with imaging probes through unique self-assembly strategies. By the balance between the peptide self-assembly and targeting recognition, the specificity and stability can be improved while the accumulation capacity of the probes at the tumor site can be greatly enhanced compared with the conventional strategy, thus reducing side effects, providing an effective tool for diagnostic and therapeutic integration of tumors.

Deep mutational scanning-guided design of a high-affinity helix-loop-helix peptide targeting G-CSF receptor.

At present, mid-sized binding peptides have emerged as a new class of drug modalities. We have de novo designed a helix-loop-helix (HLH) peptide (MW: ∼4,500), constructed phage-displayed libraries, and screened the libraries against a variety of disease-related proteins to successfully obtain molecular-targeting HLH peptides. The next essential step in developing HLH peptides into therapeutics involves affinity engineering to optimize binding affinity and specificity. Here, we demonstrate deep mutational scanning to improve binding affinity over 1000-fold for an HLH peptide (P8-2KA; KD = 380nM) targeting granulocyte colony-stimulation factor receptor (G-CSFR). Site-saturation mutagenesis on the two helices was performed to produce a phage-displayed library that was screened against G-CSFR. The DNA sequences of mutants from the unselected and selected phage libraries were analyzed with next-generation sequencing. The enrichment ratio of each mutant was calculated from the sequencing data to identify beneficial mutations for G-CSFR binding. Grafting of the five beneficial mutations on P8-2KA dramatically increased the binding affinity (KD = 16nM), while cyclization of the HLH peptide with an intramolecular disulfide bond further increased binding affinity for G-CSFR (KD = 0.18nM). The combined strategy of phage-displayed library selection and deep mutational scanning-guided design generated high-affinity HLH peptides, emphasizing the potential of molecular-targeting HLH peptides as a new drug modality that serves as an alternative to antibodies.

In Silico-Designed G-Quadruplex Targeting Peptide Attenuates VEGF-A Expression, Preventing Angiogenesis in Cancer Cells.

Vascular endothelial growth factor-A (VEGF-A) is a growth factor and pluripotent cytokine that promotes angiogenesis in cancer cells, transitioning to an angiogenic phenotype. The binding of VEGF-A protein to VEGF receptors (VEGFR-1 and VEGFR-2) initiates a cascade of events that stimulates angiogenesis by facilitating the migration and enhancing the permeability of endothelial cells. The proximal promoter of the VEGF gene encompasses a 36-base pair region (from -85 to -50) that can form a stable G-quadruplex (G4) structure in specific conditions. The activity of the VEGF promoter is reliant on this structure. During cancer progression, the VEGF-A G4 succumbs to cellular pressure and fails to maintain a stable structure. This shifts the balance to form a duplex structure, increasing the transcription rate. Earlier research has tried to develop small-molecule ligands to target and stabilise G4, demonstrating the possibility of suppressing VEGF expression. However, they either lack specificity or toxic. Peptides, on the other hand, are significantly less studied as G4 binders. Here, we designed a peptide that successfully binds and stabilises the VEGF-A G4 while reducing its gene expression. This further alters the expression fate of the VEGF-A signalling cascade and blocks angiogenesis in cancer cells. We employed high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulation to elucidate the chemical details of G4-peptide interaction. In addition, we used qPCR and western blot techniques to investigate the expression pattern of the molecules implicated in the VEGF-A signalling cascade. The study explores the intricate relationship between peptides and quadruplex structures, revealing valuable insights that can improve the design of pharmacophores targeting the dynamic quadruplex structure. The results of our study are encouraging, opening possibilities for advancements in, the characterisation and optimisation of peptides as G-quadruplex ligands in view of their potential therapeutic uses.

Aggregation-Prone Antimicrobial Peptides Target Gram-negative Bacterial Nucleic Acids and Protein Synthesis

Aggregation of antimicrobial peptides (AMPs) enhances their efficacy by destabilising the bacterial cell wall, membrane, and cytosolic proteins. Developing aggregation-prone AMPs offers a promising strategy to combat antibiotic resistance, though predicting such AMPs and understanding bacterial responses remain challenging. Octopus bimaculoides, a cephalopod species, lacks known AMP gene families, yet its protein fragments were used to predict AMPs via artificial intelligence tools. Four peptides (Oct-P1, Oct-P2, Oct-P3, and Oct-P4) were identified based on their aggregation propensity. Among them, Oct-P2 reduced the viability of Escherichia coli and Staphylococcus aureus by up to 90%, confirmed by confocal laser scanning microscopy and scanning electron microscopy. It further aggregated plasmid DNA in vitro, and the presence of extracellular DNA reduced their antibacterial activity. With knockout mutants, it revealed that Oct-P2 was internalised into bacterial cells, possibly through membrane transport proteins, enhancing its antibacterial effect. Aggregation-induced emission assays and molecular dynamics simulations revealed that Oct-P2 aggregates with transcription promoter DNA, inhibiting transcription and translation in vitro. This dual-target mechanism not only highlights the potential of Oct-P2 as a lead template for new antimicrobial drug development, but also opens a new window for discovering AMPs from protein fragments against the upcoming challenge of bacterial infections. Statement of significanceA popular strategy for identifying antimicrobial peptides (AMPs) in specific genomes uses the conserved regions of AMP families, but this strategy has limitations in organisms lacking classical AMP gene families, such as Octopus. Fragments from non-antimicrobial proteins serve as a rich source for the identification of new AMPs. In this study, we used artificial intelligence tools to search for potential candidate AMP sequences from non-antimicrobial proteins in Octopus bimaculoides. The successful identification of aggregation-prone AMPs was shown to decrease bacterial viability, increase permeability, and reduce biomass. One candidate, Oct-P2, kills the gram-negative bacteria E. coli by aggregating with DNA and inhibiting transcription and translation, suggesting a new intracellular mechanism of AMP activity.

Structural Characterization of Mycobacterium tuberculosis Encapsulin in Complex with Dye-Decolorizing Peroxide.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the world's deadliest infectious disease. Mtb uses a variety of mechanisms to evade the human host's defenses and survive intracellularly. Mtb's oxidative stress response enables Mtb to survive within activated macrophages, an environment with reactive oxygen species and low pH. Dye-decolorizing peroxidase (DyP), an enzyme involved in Mtb's oxidative stress response, is encapsulated in a nanocompartment, encapsulin (Enc), and is important for Mtb's survival in macrophages. Encs are homologs of viral capsids and encapsulate cargo proteins of diverse function, including those involved in iron storage and stress responses. DyP contains a targeting peptide (TP) at its C-terminus that recognizes and binds to the interior of the Enc nanocompartment. Here, we present the crystal structure of the Mtb-Enc•DyP complex and compare it to cryogenic-electron microscopy (cryo-EM) Mtb-Enc structures. Investigation into the canonical pores formed at symmetrical interfaces reveals that the five-fold pore for the Mtb-Enc crystal structure is strikingly different from that observed in cryo-EM structures. We also observe DyP-TP electron density within the Mtb-Enc shell. Finally, investigation into crystallographic small-molecule binding sites gives insight into potential novel avenues by which substrates could enter Mtb-Enc to react with Mtb-DyP.

Level of Expression of MHCI-Presented Neoepitopes Influences Tumor Rejection by Neoantigen-Specific CD8+ T Cells.

Neoantigen-targeted therapy holds an array of benefits for cancer immunotherapy, but the identification of peptide targets with tumor rejection capacity remains a limitation. To better define the criteria dictating tumor rejection potential, we examined the capacity of high-magnitude T-cell responses induced toward several distinct neoantigen targets to regress MC38 tumors. Despite their demonstrated immunogenicity, vaccine-induced T-cell responses were unable to regress established MC38 tumors or prevent tumor engraftment in a prophylactic setting. Although unable to kill tumor cells, T cells showed robust killing capacity toward neoantigen peptide-loaded cells. Tumor-cell killing was rescued by saturation of target peptide-loaded MHCs on the cell surface. Overall, this study demonstrates a pivotal role for target protein expression levels in modulating the tumor rejection capacity of neoantigens. Thus, inclusion of this metric, in addition to immunogenicity analysis, may benefit antigen prediction techniques to ensure the full antitumor effect of cancer vaccines.