Relevant Peptides Research Articles

Bacterial Quorum-Sensing Peptides as Immune Modulators Present in Systemic Circulation.

Quorum-sensing peptides (QSPs) are bacterial peptides traditionally considered only as inter-bacterial communication molecules. Recently, their involvement in microbiome-host interactions influencing host diseases such as cancer and sarcopenia were explored. However, it is still unknown to what extent these peptides have the potential to modulate the immune system. In this proof-of-concept study, we screened 89 QSPs for their potential to induce IL-6 and TNFα in murine splenocytes and J774 macrophages. Confirmatory experiments on the positive screening-hits were conducted using murine splenocytes and human PBMCs of different ages. Finally, to investigate the biological relevance of immunomodulatory QSPs, we analysed plasma in a human cohort for the presence of the immunomodulatory QSP Q010. To do this, we used a newly developed UHPLC-MS/MS method. Our findings indicated that specific QSPs activate immune cells in vitro, with Q007, Q010, Q017 and Q212 being the top four screening hits. Q007 and Q010 were affirmed in subsequent confirmatory experiments using murine splenocytes and human PBMCs. Finally, Q010 was detected in human plasma, demonstrating for the first time the presence of an immunomodulatory QSP in human circulation. In conclusion, our data are the first evidence indicating the potential of biologically relevant quorum-sensing peptides to modulate the immune system.

Cell density dependent restriction of N2O reductase in denitrifying Pseudomonas spp., a potential driver of N2O emissions in natural systems.

Denitrification is a major biological source and sink for the ozone-depleting greenhouse gas N2. Thus, the respiratory physiology of denitrifiers and the mechanisms determining their propensity for accumulation of N-oxides are of fundamental interest. Here, we report a pervasive positive correlation between cell density and N2O accumulation in Pseudomonas aeruginosa and P. fluorescens F113. We show that this was a result of quorum sensing by comparing the P. aeruginosa PAO1-UW wild type to a rhlI/lasI knockout mutant able to sense, but not synthesize the N-acyl-homoserine lactones (AHL) of the Rhl and Las circuits. Neither the transcription of nosZ (encoding N2O reductase, N2OR) nor the abundance of peptides of known relevance to denitrification could explain the restriction of N2O reduction in AHL-affected cultures. However, a protein shown to be involved in synthesis and repair of iron-sulphur (Fe-S) centers under NO stress, CyaY, was significantly downregulated in the AHL producing wild type. This hints to a possible route of N2OR-suppression via compromised Fe-S centers in the ancillary protein NosR. While the exact mechanism remains obscure, it appears that quorum sensing driven restriction of N2OR activity is common. Thus, given its ubiquity among prokaryotes, and the potential for cross-species and -strain effects, quorum sensing is plausibly a driver of N2O emissions in a range of systems.

Signal Peptide Efficiency: From High-Throughput Data to Prediction and Explanation.

The passage of proteins across biological membranes via the general secretory (Sec) pathway is a universally conserved process with critical functions in cell physiology and important industrial applications. Proteins are directed into the Sec pathway by a signal peptide at their N-terminus. Estimating the impact of physicochemical signal peptide features on protein secretion levels has not been achieved so far, partially due to the extreme sequence variability of signal peptides. To elucidate relevant features of the signal peptide sequence that influence secretion efficiency, an evaluation of ∼12,000 different designed signal peptides was performed using a novel miniaturized high-throughput assay. The results were used to train a machine learning model, and a post-hoc explanation of the model is provided. By describing each signal peptide with a selection of 156 physicochemical features, it is now possible to both quantify feature importance and predict the protein secretion levels directed by each signal peptide. Our analyses allow the detection and explanation of the relevant signal peptide features influencing the efficiency of protein secretion, generating a versatile tool for the de novo design and in silico evaluation of signal peptides.

Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase.

mRNA display is revolutionizing peptide drug discovery through its ability to quickly identify potent peptide binders of therapeutic protein targets. Methods to expand the chemical diversity of display libraries are continually needed to increase the likelihood of identifying clinically relevant peptide ligands. Orthogonal aminoacyl-tRNA synthetases (ORSs) have proven utility in cellular genetic code expansion, but are relatively underexplored for in vitro translation (IVT) and mRNA display. Herein, we demonstrate that the promiscuous ORS p-CNF-RS can incorporate noncanonical amino acids at amber codons in IVT, including the novel substrate p-cyanopyridylalanine (p-CNpyrA), to enable a pyridine-thiazoline (pyr-thn) macrocyclization in mRNA display. Pyr-thn-based selections against the deubiquitinase USP15 yielded a potent macrocyclic binder that exhibits good selectivity for USP15 and close homologues over other ubiquitin-specific proteases (USPs). Overall, this work exemplifies how promiscuous ORSs can both expand side chain diversity and provide structural novelty in mRNA display libraries through a heterocycle forming macrocyclization.

Biomarker Discovery by ORFeome Phage Display.

Phage display is an efficient and robust method for protein-protein interaction studies. Although it is mostly used for antibody generation, it can be also utilized for the discovery of immunogenic proteins that could be used as biomarkers. Through this technique, a genome or metagenome is fragmented and cloned into a phagemid vector. The resulting protein fragments from this genetic material are displayed on M13 phage surface, while the corresponding gene fragments are packaged. This packaging process uses the pIII deficient helperphage, called Hyperphage (M13KO7 ΔpIII), so open reading frames (ORFs) are enriched in these libraries, giving the name to this method: ORFeome phage display. After conducting a selection procedure, called "bio-panning," relevant immunogenic peptides or protein fragments are selected using purified antibodies or serum samples, and can be used as potential biomarkers. As ORFeome phage display is an in vitro method, only the DNA or cDNA of the species of interest is needed. Therefore, this approach is also suitable for organisms that are hard to cultivate, or metagenomic samples, for example. An additional advantage is that the biomarker discovery is not limited to surface proteins due to the presentation of virtually every kind of peptide or protein fragment encoded by the ORFeome on the phage surface. At last, the selected biomarkers can be the start for the development of diagnostic assays, vaccines, or protein interaction studies.

A Chemoenzymatic Approach to Investigate Cytochrome P450 Cross-Linking in Glycopeptide Antibiotic Biosynthesis.

Glycopeptide antibiotics (GPAs) are important and medically relevant peptide natural products. In the context of antimicrobial resistance (AMR), understanding and manipulating GPA biosynthesis is essential to discover new bioactive derivatives of these peptides. Among all the enzymatic steps in GPA biosynthesis, the most complex occurs during the maturation (cross-linking) of the peptide aglycone. This is achieved-while the peptide remains attached to the nonribosomal peptide synthetase (NRPS) machinery-through the action of a cytochrome P450 (CYP450 or Oxy)-mediated cyclization cascade. There is great interest in understanding the formation of the cross-links between the aromatic side chains in GPAs as this process leads to the cup-shaped aglycone, which is itself a requirement for antibiotic activity. In this regard, the use of in vitro experiments is crucial to study this process. To address the process of peptide cyclization during GPA biosynthesis, a series of peptide substrates and different Oxy enzymes are required. In this chapter, we describe a practical and efficient route for the synthesis of peptidyl-CoAs, the expression of proteins/enzymes involved in the in vitro cyclization assay, the loading of the PCP with peptidyl-CoAs, an optimized CYP450-mediated cyclization cascade and assay workup followed by mass spectrometry (MS) characterization. This in vitro assay affords high conversion tocyclic peptides and demonstrates the tolerance of the P450s for novel GPA precursor peptide substrates.

Proof-of-Concept Study of Liposomes with a Set of SARS-CoV-2 Viral T-Cell Epitopes as a Vaccine

Potential nonameric epitopes of CD8+ T lymphocytes were selected from the composition of structural, accessory, and non-structural proteins of SARS-CoV-2 virus (13 peptides) and a 15-mer epitope of CD4+ T lymphocytes, from the S-protein, based on the analysis of publications on genome-wide immunoinformatic analysis of T-cell epitopes of the virus (Wuhan strain), as well as a number of clinical studies of immunodominant epitopes among patients recovering from COVID-19 disease. The peptides were synthesized and five compositions of 6–7 peptides were included in liposomes from egg phosphatidylcholine and cholesterol (~200 nm size) obtained by extrusion. After double subcutaneous immunization of conventional mice, activation of cellular immunity was assessed by the level of cytokine synthesis by splenocytes in vitro in response to stimulation with relevant peptide compositions. Liposomal formulation exhibiting the best result in terms of the formation of specific cellular immunity in response to vaccination was selected for further experiments. Evaluation of the protective efficacy of this formulation in an infectious mouse model showed a positive trend in the frequency of occurrence of hyaline-like membranes in the lumen of the alveoli, as well as a somewhat lower severity of microcirculatory disorders. The latter circumstance can potentially help reduce the severity of the disease and prevent its adverse outcomes. A method to produce liposome preparations with peptide compositions for long-term storage is under development.

Persulfate‐Mediated Oxidative C−H Functionalization of Glycinates to Access Diverse Unnatural Amino Acids

AbstractWe have developed an operationally simple and green approach to prepare robust α‐alkyl unnatural amino acids through the dual oxidative activation of glycine derivatives and alkylcarboxylic acids with persulfate as the sole oxidizing agent. This method allows for the utilization of naturally abundant alkylcarboxylic acids as radical sources without the assistance of catalysts. Notably, this method allows for the late‐stage decoration of biologically relevant carboxylic acids and peptides to construct diverse unnatural amino acids. In addition, the generality of the oxidative activation method enables a facile synthetic approach to α‐phosphonyl glycine derivatives by using phosphites and phosphine oxides.

Identification of putative orthologs of clinically relevant antimicrobial peptides in the equine ocular surface and amniotic membrane.

This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies. A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression. Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins. This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.

Impact of doubling peptide length on in vivo hydrogel stability and sustained drug release

Peptide-based hydrogels represent promising systems for the sustained release of different types of drugs, ranging from small molecules to biologicals. Aiming at subcutaneous injection, which is a desirable parenteral administration route, especially for biologicals, we herein focus on physically crosslinked systems possessing thixotropic behaviour. The purpose of this study was to evaluate the in vitro and in vivo properties of hydrogels based on the amphipathic hexapeptide H-FQFQFK-NH2, which served as the lead sequence. Upon doubling the length of this peptide, the dodecapeptide H-FQFQFKFQFQFK-NH2 gave a significant improvement in terms of in vivo stability of the hydrogel post-injection, as monitored by nuclear SPECT/CT imaging. This increased hydrogel stability also led to a more prolonged in vivo release of encapsulated peptide cargoes. Even though no direct link with the mechanical properties of the hydrogels before injection could be made, an important effect of the subcutaneous medium was noticed on the rheological properties of the hydrogels in post in vivo injection measurements. The results were validated in vivo for a therapeutically relevant analgesic peptide using the hot-plate test as an acute pain model. It was confirmed that elongation of the hydrogelator sequence induced more extended antinociceptive effects. Altogether, this simple structural modification of the hydrogelating peptide could provide a basis for reaching longer durations of action upon use of these soft biomaterials.

Applications of MALDI-MS/MS-Based Proteomics in Biomedical Research.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is one of the most widely used techniques in proteomics to achieve structural identification and characterization of proteins and peptides, including their variety of proteoforms due to post-translational modifications (PTMs) or protein–protein interactions (PPIs). MALDI-MS and MALDI tandem mass spectrometry (MS/MS) have been developed as analytical techniques to study small and large molecules, offering picomole to femtomole sensitivity and enabling the direct analysis of biological samples, such as biofluids, solid tissues, tissue/cell homogenates, and cell culture lysates, with a minimized procedure of sample preparation. In the last decades, structural identification of peptides and proteins achieved by MALDI-MS/MS helped researchers and clinicians to decipher molecular function, biological process, cellular component, and related pathways of the gene products as well as their involvement in pathogenesis of diseases. In this review, we highlight the applications of MALDI ionization source and tandem approaches for MS for analyzing biomedical relevant peptides and proteins. Furthermore, one of the most relevant applications of MALDI-MS/MS is to provide “molecular pictures”, which offer in situ information about molecular weight proteins without labeling of potential targets. Histology-directed MALDI-mass spectrometry imaging (MSI) uses MALDI-ToF/ToF or other MALDI tandem mass spectrometers for accurate sequence analysis of peptide biomarkers and biological active compounds directly in tissues, to assure complementary and essential spatial data compared with those obtained by LC-ESI-MS/MS technique.

Equipped for Sexual Stings? Male-Specific Venom Peptides in Euscorpius italicus.

In the animal kingdom, intraspecific variation occurs, for example, between populations, different life stages, and sexes. For venomous animals, this can involve differences in their venom composition. In cases where venom is utilized in the context of mating, the differences in composition might be driven by sexual selection. In this regard, the genus Euscorpius is a promising group for further research, as some of these scorpions exhibit a distinct sexual dimorphism and are known to perform a sexual sting during mating. However, the venom composition of this genus remains largely unexplored. Here, we demonstrate that Euscorpius italicus exhibits a male-specific venom composition, and we identify a large fraction of the substances involved. The sex specificity of venom peptides was first determined by analyzing the presence/absence patterns of ion signals in MALDI-TOF mass spectra of venom samples from both sexes and juveniles. Subsequently, a proteo-transcriptomic analysis provided sequence information on the relevant venom peptides and their corresponding precursors. As a result, we show that several potential toxin precursors are down-regulated in male venom glands, possibly to reduce toxic effects caused to females during the sexual sting. We have identified the precursor of one of the most prominent male-specific venom peptides, which may be an ideal candidate for activity tests in future studies. In addition to the description of male-specific features in the venom of E. italicus, this study also includes a general survey of venom precursors in this species.

A thermodynamic investigation of amyloid precursor protein processing by human γ-secretase

Human γ-secretase cleaves the transmembrane domains (TMDs) of amyloid precursor protein (APP) into pathologically relevant amyloid-β peptides (Aβs). The detailed mechanisms of the unique endoproteolytic cleavage by the presenilin 1 domain (PS1) of γ-secretase are still poorly understood. Herein, we provide thermodynamic insights into how the α-helical APP TMD is processed by γ-secretase and elucidate the specificity of Aβ48/Aβ49 cleavage using unbiased molecular dynamics and bias-exchange metadynamics simulations. The thermodynamic data show that the unwinding of APP TMD is driven by water hydration in the intracellular pocket of PS1, and the scissile bond T32-L33 or L33-V34 of the APP TMD can slide down and up to interact with D257/D385 to achieve endoproteolysis. In the wild-type system, the L33-V34 scissile bond is more easily hijacked by D257/D385 than T32-L33, resulting in higher Aβ49 cleavage, while the T32N mutation on the APP TMD decreases the energy barrier of the sliding of the scissile bonds and increases the hydrogen bond occupancy for Aβ48 cleavage. In summary, the thermodynamic analysis elucidates possible mechanisms of APP TMD processing by PS1, which might facilitate rational drug design targeting γ-secretase.

Dicationic protic ionic liquids based on N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine

The interaction of N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine with carboxylic acids resulted in a new series of dicationic protic alkanolammonium ionic liquids (PAAILs). New PAAILs were characterized by NMR, FTIR spectroscopy, and thermal analysis. The conformation of N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediammonium cations and intermolecular interactions in salts of 1-hydroxy-2-naphthoic and oxalic acids were studied using X-ray diffraction supplemented by Hirshfeld surfaces analysis. Some pharmacokinetic parameters of the synthesized dicationic PAAILs have been predicted to evaluate their potential biomedical applications. In comparison with monocationic PAAILs, their antimicrobial and buffering activity in 68Ga-radiolabeling reactions of chelating agents and clinically relevant peptides were studied.

Deciphering the conformational landscape of few selected aromatic noncoded amino acids (NCAAs) for applications in rational design of peptide therapeutics

Amino acids are the essential building blocks of both synthetic and natural peptides, which are crucial for biological functions and also important as biological probes for mapping the complex protein–protein interactions (PPIs) in both prokaryotic and eukaryotic systems. Mapping the PPIs through the chemical biology approach provides pharmacologically relevant peptides, which can have agonistic or antagonistic effects on the targeted biological systems. It is evidenced that ≥ 60 peptide-based drugs have been approved by the US-FDA so far, and the number will improve further in the foreseeable future, as ≥ 140 peptides are currently in clinical trials. However, natural peptides often require fine-tuning of their pharmacological properties by strategically replacing the αL-amino acids of the peptides with non-coded amino acids (NCAA), for which codons are absent in the genetic code for biosynthesis of proteins, prior to their applications as therapeutics. Considering the diverse repertoire of the NCAAs, the conformational space of many NCAAs is yet to be explored systematically in the context of the rational design of therapeutic peptides. The current study deciphers the conformational landscape of a few such Cα-substituted aromatic NCAAs (Ing: 2-indanyl-l-Glycine; Bpa: 4-benzoyl-l-phenylalanine; Aic: 2-aminoindane-2-carboxylic acid) both in the context of tripeptides and model synthetic peptide sequences, using alanine (Ala) and proline (Pro) as the reference. The combined data obtained from the computational and biophysical studies indicate the general success of this approach, which can be exploited further to rationally design optimized peptide sequences of unusual architecture with potent antimicrobial, antiviral, gluco-regulatory, immunomodulatory, and anti-inflammatory activities. Supplementary InformationThe online version contains supplementary material available at 10.1007/s00726-022-03175-z.

213-OR: Antidiabetic Potential of Novel, Long-Acting Amylin Analogue ZP8396 in ZDF Rats

Amylin improves glucose homeostasis through at least three distinct mechanisms of action, including prevention of the postprandial rise in plasma glucagon, slowing of gastric emptying, and increased satiety, leading to decreased caloric intake and potential weight loss. Clinical efficacy has been demonstrated with short-acting amylin analogue pramlintide. ZP8396, currently in phase 1 clinical testing, is a long-acting, amylin analogue designed to improve solubility, stability and allow for co-formulation with other relevant peptides. We previously reported that ZP8396 provides body weight reduction in DIO rats alone and in combination with semaglutide, a once-weekly GLP-1 agonist (Skarbaliene et al, Obesity Week 2021) . Here we investigated the antidiabetes potential of ZP8396 in Zucker Diabetic Fatty (ZDF) rats. The antidiabetes potential after a single subcutaneous (s.c.) injection of ZP8396 (nmol/kg) or vehicle was measured in ZDF rats (aged 12-13 weeks, n=10) , an animal model of type 2 diabetes, and continuously monitored for 168 h after dosing.ZP8396 induced a significant body weight loss in ZDF rats vs. vehicle group. A significant decrease in body weight was observed in rats treated with ZP8396 from initiation of the dosing and up to 96 h post-dose. In addition, single s.c. injection of ZP8396 showed a significant reduction in blood glucose levels at 24 h post-dose, reaching a 20 % difference in blood glucose levels vs. vehicle group (14,97±0,39 and 18,60±1,18 mmol/L, respectively; p <0.05) . Also, ZP8396 significantly reduced plasma glucagon levels, reaching a 55% difference in ZP8396-treated diabetic rats vs. vehicle group at 24 h post-dose (p <0.05) . In conclusion, in diabetic rat ZP8396 improved glycaemic status, supporting that long-acting amylin analogues may provide future valuable therapeutic options to manage diabetes. Further preclinical characterization of the antidiabetes effects of long-term treatment with ZP8396 is ongoing. Disclosure J.Skarbaliene: Employee; Zealand Pharma A/S, Stock/Shareholder; Zealand Pharma A/S. T.Baader-pagler: Employee; Boehringer Ingelheim International GmbH. P.Eriksson: Employee; Zealand Pharma A/S.

Solid-Phase Peptide Modification via Deaminative Photochemical Csp3 -Csp3 Bond Formation Using Katritzky Salts.

Introduction of unnatural amino acids can significantly improve the binding affinity and stability of peptides. Commercial availability of such amino acids is limited, and their synthesis is a long and tedious process. We here describe a method that allows the functionalization of peptides directly on solid‐support by converting lysine residues to Katritzky salts, and subjecting them to a photochemical Giese reaction under mild reaction conditions. The method avoids the need for amino acid synthesis and instead offers a late‐stage modification route for rapid peptide diversification. While numerous modification approaches at the lysine amine have been described, this work provides the first example of deaminative functionalization of peptides at lysine. The two‐step protocol is compatible with various substrates, lysine analogues, resins, and all proteinogenic amino acids. Finally, by leveraging solid‐phase modification, this protocol facilitates the functionalization of longer peptides as was demonstrated using biologically relevant peptides of up to 15 amino acids.

Selective Labeling of Peptides with o-Carboranes via Manganese(I)-Catalyzed C-H Activation.

A robust method for the selective labeling of peptides via manganese(I) catalysis was devised to achieve the C‐2 alkenylation of tryptophan containing peptides with 1‐ethynyl‐o‐carboranes. The manganese‐catalyzed C−H activation was accomplished with high catalytic efficiency, and featured low toxicity, high functional group tolerance and excellent E‐stereoselectivity. This approach unravels a promising tool for the assembly of o‐carborane with structurally complex peptides of relevance to applications in boron neutron capture therapy.

Benefits of a Mixed-Mode Stationary Phase to Address the Challenging Purification of an Industrially Relevant Peptide: A Proof-of-Concept Study

Peptides are a class of biomolecules with a great potential from the therapeutic point of view, because of their unique biological properties. Industrially, the production stategies adopted produce both the target peptide and a series of impurities that must be removed. Preparative chromatography is the technique of choice for the large-scale purification of biomolecules, generally performed in reversed-phase mode, using hydrophobic adsorbents (e.g., C8 stationary phases). A promising and innovative alternative is represented by mixed-mode columns, which bear two different ligands on the particle surface, exploiting two different retention mechanisms to improve the separation. This work represents a proof-of-concept study focused on the comparison of a hydrophobic adsorbent and a mixed-mode one (bearing both hydrophobic groups and charged ones) for the purification of a crude peptide mixture. Thanks to more-favourable thermodynamics, it was found that, when collecting the whole peak excluding fractions of the peak tail, the mixed-mode column led to an increase in the recovery of roughly +15%, together with a slight improvement in purity at the same time, with respect to the traditional hydrophobic column. In addition, if the whole peak, including the tail, is collected, the performance of the two columns are similar in terms of purity and recovery, but the pepetide elutes as a narrower peak with the mixed mode. This leads to a collection pool showing a much-higher peptide concentration and to lower solvent volumes needed, which is a beneficial achievement when targeting more sustainable processes. These results are very advantageous from the industrial viewpoint, because they also involve a decrease in the peptide amount contained in the peak tail, which must be reprocessed again to satisfy purity requirements.

CAD v1.0: Cancer Antigens Database Platform for Cancer Antigen Algorithm Development and Information Exploration.

Cancer vaccines have gradually attracted attention for their tremendous preclinical and clinical performance. With the development of next-generation sequencing technologies and related algorithms, pipelines based on sequencing and machine learning methods have become mainstream in cancer antigen prediction; of particular focus are neoantigens, mutation peptides that only exist in tumor cells that lack central tolerance and have fewer side effects. The rapid prediction and filtering of neoantigen peptides are crucial to the development of neoantigen-based cancer vaccines. However, due to the lack of verified neoantigen datasets and insufficient research on the properties of neoantigens, neoantigen prediction algorithms still need to be improved. Here, we recruited verified cancer antigen peptides and collected as much relevant peptide information as possible. Then, we discussed the role of each dataset for algorithm improvement in cancer antigen research, especially neoantigen prediction. A platform, Cancer Antigens Database (CAD, http://cad.bio-it.cn/), was designed to facilitate users to perform a complete exploration of cancer antigens online.