Functional and structural investigation of a broadly neutralizing SARS-CoV-2 antibody.

Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance. Here, we isolated mAbs from donors with breakthrough infection with Omicron subvariants using a single-B cell screening platform. We identified a mAb, O5C2, which possesses broad-spectrum neutralization and antibody-dependent cell-mediated cytotoxic activities against SARS-CoV-2 variants, including EG.5.1. Single-particle analysis by cryo-electron microscopy revealed that O5C2 targeted an unusually large epitope within the receptor-binding domain of spike protein that overlapped with the angiotensin-converting enzyme 2 binding interface. Furthermore, O5C2 effectively protected against BA.5 Omicron infection in vivo by mediating changes in transcriptomes enriched in genes involved in apoptosis and interferon responses. Our findings provide insights into the development of pan-protective mAbs against SARS-CoV-2.

Multi-epitope protein production and its application in the diagnosis of opisthorchiasis.

BACKGROUND: Opisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis. METHODS: The Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique. RESULTS: The recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively. CONCLUSIONS: The high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.

Preparation and epitope analysis of monoclonal antibodies against African swine fever virus DP96R protein.

BACKGROUND: Many proteins of African swine fever virus (ASFV, such as p72, p54, p30, CD2v, K205R) have been successfully expressed and characterized. However, there are few reports on the DP96R protein of ASFV, which is the virulence protein of ASFV and plays an important role in the process of host infection and invasion of ASFV. RESULTS: Firstly, the prokaryotic expression vector of DP96R gene was constructed, the prokaryotic system was used to induce the expression of DP96R protein, and monoclonal antibody was prepared by immunizing mice. Four monoclonal cells of DP96R protein were obtained by three ELISA screening and two sub-cloning; the titer of ascites antibody was up to 1:500,000, and the monoclonal antibody could specifically recognize DP96R protein. Finally, the subtypes of the four strains of monoclonal antibodies were identified and the minimum epitopes recognized by them were determined. CONCLUSION: Monoclonal antibody against ASFV DP96R protein was successfully prepared and identified, which lays a foundation for further exploration of the structure and function of DP96R protein and ASFV diagnostic technology.

Predicting TCR sequences for unseen antigen epitopes using structural and sequence features.

T-cell receptor (TCR) recognition of antigens is fundamental to the adaptive immune response. With the expansion of experimental techniques, a substantial database of matched TCR-antigen pairs has emerged, presenting opportunities for computational prediction models. However, accurately forecasting the binding affinities of unseen antigen-TCR pairs remains a major challenge. Here, we present convolutional-self-attention TCR (CATCR), a novel framework tailored to enhance the prediction of epitope and TCR interactions. Our approach utilizes convolutional neural networks to extract peptide features from residue contact matrices, as generated by OpenFold, and a transformer to encode segment-based coded sequences. We introduce CATCR-D, a discriminator that can assess binding by analyzing the structural and sequence features of epitopes and CDR3-ß regions. Additionally, the framework comprises CATCR-G, a generative module designed for CDR3-ß sequences, which applies the pretrained encoder to deduce epitope characteristics and a transformer decoder for predicting matching CDR3-ß sequences. CATCR-D achieved an AUROC of 0.89 on previously unseen epitope-TCR pairs and outperformed four benchmark models by a margin of 17.4%. CATCR-G has demonstrated high precision, recall and F1 scores, surpassing 95% in bidirectional encoder representations from transformers score assessments. Our results indicate that CATCR is an effective tool for predicting unseen epitope-TCR interactions. Incorporating structural insights enhances our understanding of the general rules governing TCR-epitope recognition significantly. The ability to predict TCRs for novel epitopes using structural and sequence information is promising, and broadening the repository of experimental TCR-epitope data could further improve the precision of epitope-TCR binding predictions.

GNUV201, a novel human/mouse cross-reactive and low pH-selective anti-PD-1 monoclonal antibody for cancer immunotherapy.

BACKGROUND: Several PD-1 antibodies approved as anti-cancer therapies work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. These PD-1 antibodies lack inter-species cross-reactivity, necessitating surrogate antibodies for preclinical studies, which may limit the predictability and translatability of the studies. RESULTS: To overcome this limitation, we have developed an inter-species cross-reactive PD-1 antibody, GNUV201, by utilizing an enhanced diversity mouse platform (SHINE MOUSE™). GNUV201 equally binds to human PD-1 and mouse PD-1, equally inhibits the binding of human PD-1/PD-L1 and mouse PD-1/PD-L1, and effectively suppresses tumor growth in syngeneic mouse models. The epitope of GNUV201 mapped to the "FG loop" of hPD-1, distinct from those of Keytruda® ("C'D loop") and Opdivo® (N-term). Notably, the structural feature where the protruding epitope loop fits into GNUV201's binding pocket supports the enhanced binding affinity due to slower dissociation (8.7 times slower than Keytruda®). Furthermore, GNUV201 shows a stronger binding affinity at pH 6.0 (5.6 times strong than at pH 7.4), which mimics the hypoxic and acidic tumor microenvironment (TME). This phenomenon is not observed with marketed antibodies (Keytruda®, Opdivo®), implying that GNUV201 achieves more selective binding to and better occupancy on PD-1 in the TME. CONCLUSIONS: In summary, GNUV201 exhibited enhanced affinity for PD-1 with slow dissociation and preferential binding in TME-mimicking low pH. Human/monkey/mouse inter-species cross-reactivity of GNUV201 could enable more predictable and translatable efficacy and toxicity preclinical studies. These results suggest that GNUV201 could be an ideal antibody candidate for anti-cancer drug development.

Tumor Antigens beyond the Human Exome.

With the advent of immunotherapeutics, a new era in the combat against cancer has begun. Particularly promising are neo-epitope-targeted therapies as the expression of neo-antigens is tumor-specific. In turn, this allows the selective targeting and killing of cancer cells whilst healthy cells remain largely unaffected. So far, many advances have been made in the development of treatment options which are tailored to the individual neo-epitope repertoire. The next big step is the achievement of efficacious "off-the-shelf" immunotherapies. For this, shared neo-epitopes propose an optimal target. Given the tremendous potential, a thorough understanding of the underlying mechanisms which lead to the formation of neo-antigens is of fundamental importance. Here, we review the various processes which result in the formation of neo-epitopes. Broadly, the origin of neo-epitopes can be categorized into three groups: canonical, noncanonical, and viral neo-epitopes. For the canonical neo-antigens that arise in direct consequence of somatic mutations, we summarize past and recent findings. Beyond that, our main focus is put on the discussion of noncanonical and viral neo-epitopes as we believe that targeting those provides an encouraging perspective to shape the future of cancer immunotherapeutics.

Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin.

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

A novel chimeric vaccine containing multiple epitopes for simulating robust immune activation against Klebsiella pneumoniae.

BACKGROUND: Due to antibiotic resistance, the Klebsiella genus is linked to morbidity and death, necessitating the development of a universally protective vaccine against Klebsiella pathogens. METHODS: Core sequence analysis prioritized non-redundant host molecules and expected lipid bilayer peptides from fully sequenced Klebsiella genomes. These proteins were refined to identify epitopes, examining their immunogenicity, toxicity, solubility, and interaction with MHC alleles. Epitopes were linked to CPG ODN C274 via EAAAK, HEYGAEALERAG, and GGGS linkers to enhance immunological responses. The vaccine's tertiary structure was modelled and docked with MHC-I and MHC-II. RESULTS: Fifty-five proteins were recognized in the Vaxign collection as having remarkable features. Twenty-three proteins with potential pathogenicity were then identified. Eight options for vaccines emerged after the immunogenicity of proteins was examined. The best antigens were three proteins: MrkD, Iron-regulated lipid membrane polypeptides, and RmpA. These compounds were selected for their sensitivity. The structural protein sequences of K. pneumoniae were utilized to identify seven CTL epitopes, seven HTL epitopes, and seven LBL epitopes, respectively. The produced immunization displayed a stable contact with the receptors, based on molecular dynamic simulations lasting 250 nanoseconds. Intermolecular binding free energies also indicated the dominance of the van der Waals and electrostatic energies. CONCLUSION: In summary, the results of this study might help scientists develop a novel vaccine to prevent K. pneumoniae infections.

Immunoinformatics design of a structural proteins driven multi-epitope candidate vaccine against different SARS-CoV-2 variants based on fynomer.

The ideal vaccines for combating diseases that may emerge in the future require more than simply inactivating a few pathogenic strains. This study aims to provide a peptide-based multi-epitope vaccine effective against various severe acute respiratory syndrome coronavirus 2 strains. To design the vaccine, a library of peptides from the spike, nucleocapsid, membrane, and envelope structural proteins of various strains was prepared. Then, the final vaccine structure was optimized using the fully protected epitopes and the fynomer scaffold. Using bioinformatics tools, the antigenicity, allergenicity, toxicity, physicochemical properties, population coverage, and secondary and three-dimensional structures of the vaccine candidate were evaluated. The bioinformatic analyses confirmed the high quality of the vaccine. According to further investigations, this structure is similar to native protein and there is a stable and strong interaction between vaccine and receptors. Based on molecular dynamics simulation, structural compactness and stability in binding were also observed. In addition, the immune simulation showed that the vaccine can stimulate immune responses similar to real conditions. Finally, codon optimization and in silico cloning confirmed efficient expression in Escherichia coli. In conclusion, the fynomer-based vaccine can be considered as a new style in designing and updating vaccines to protect against coronavirus disease.

Reinvestigation of the internal glycan rearrangement of Lewis a and blood group type H1 epitopes.

Protonated ions of fucose-containing oligosaccharides are prone to undergo internal glycan rearrangement which results in chimeric fragments that obfuscate mass-spectrometric analysis. Lack of accessible tools that would facilitate systematic analysis of glycans in the gas phase limits our understanding of this phenomenon. In this work, we use density functional theory modeling to interpret cryogenic IR spectra of Lewis a and blood group type H1 trisaccharides and to establish whether these trisaccharides undergo the rearrangement during gas-phase analysis. Structurally unconstrained search reveals that none of the parent ions constitute a thermodynamic global minimum. In contrast, predicted collision cross sections and anharmonic IR spectra provide a good match to available experimental data which allowed us to conclude that fucose migration does not occur in these antigens. By comparing the predicted structures with those obtained for Lewis x and blood group type H2 epitopes, we demonstrate that the availability of the mobile proton and a large difference in the relative stability of the parent ions and rearrangement products constitute the prerequisites for the rearrangement reaction.

Contemporary HIV-1 consensus Env with AI-assisted redesigned hypervariable loops promote antibody binding.

An effective HIV-1 vaccine must elicit broadly neutralizing antibodies (bnAbs) against highly diverse Envelope glycoproteins (Env). Since Env with the longest hypervariable (HV) loops is more resistant to the cognate bnAbs than Env with shorter HV loops, we redesigned hypervariable loops for updated Env consensus sequences of subtypes B and C and CRF01_AE. Using modeling with AlphaFold2, we reduced the length of V1, V2, and V5 HV loops while maintaining the integrity of the Env structure and glycan shield, and modified the V4 HV loop. Spacers are designed to limit strain-specific targeting. All updated Env are infectious as pseudoviruses. Preliminary structural characterization suggests that the modified HV loops have a limited impact on Env's conformation. Binding assays show improved binding to modified subtype B and CRF01_AE Env but not to subtype C Env. Neutralization assays show increases in sensitivity to bnAbs, although not always consistently across clades. Strikingly, the HV loop modification renders the resistant CRF01_AE Env sensitive to 10-1074 despite the absence of a glycan at N332.

In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model.

Oligoclonal mixtures of broadly-neutralizing antibodies can neutralize complex compositions of similar and dissimilar antigens, making them versatile tools for the treatment of e.g., infectious diseases and animal envenomations. However, these biotherapeutics are complicated to develop due to their complex nature. In this work, we describe the application of various strategies for the discovery of cross-neutralizing nanobodies against key toxins in coral snake venoms using phage display technology. We prepare two oligoclonal mixtures of nanobodies and demonstrate their ability to neutralize the lethality induced by two North American coral snake venoms in mice, while individual nanobodies fail to do so. We thus show that an oligoclonal mixture of nanobodies can neutralize the lethality of venoms where the clinical syndrome is caused by more than one toxin family in a murine challenge model. The approaches described may find utility for the development of advanced biotherapeutics against snakebite envenomation and other pathologies where multi-epitope targeting is beneficial.

Functional and antigenic characterization of SARS-CoV-2 spike fusion peptide by deep mutational scanning.

The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane fusion during virus entry and is part of a broadly neutralizing epitope. However, sequence determinants at the fusion peptide and its adjacent regions for pathogenicity and antigenicity remain elusive. In this study, we perform a series of deep mutational scanning (DMS) experiments on an S2 region spanning the fusion peptide of authentic SARS-CoV-2 in different cell lines and in the presence of broadly neutralizing antibodies. We identify mutations at residue 813 of the spike protein that reduced TMPRSS2-mediated entry with decreased virulence. In addition, we show that an F823Y mutation, present in bat betacoronavirus HKU9 spike protein, confers resistance to broadly neutralizing antibodies. Our findings provide mechanistic insights into SARS-CoV-2 pathogenicity and also highlight a potential challenge in developing broadly protective S2-based coronavirus vaccines.

Stabilized trimeric peptide immunogens of the complete HIV-1 gp41 N-heptad repeat and their use as HIV-1 vaccine candidates.

Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.

Assessing nanobody interaction with SARS-CoV-2 Nsp9.

The interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. A different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure.

Characterization of a novel format scFv×VHH single-chain biparatopic antibody against metal binding protein MtsA.

Biparatopic antibodies (bpAbs) are engineered antibodies that bind to multiple different epitopes within the same antigens. bpAbs comprise diverse formats, including fragment-based formats, and choosing the appropriate molecular format for a desired function against a target molecule is a challenging task. Moreover, optimizing the design of constructs requires selecting appropriate antibody modalities and adjusting linker length for individual bpAbs. Therefore, it is crucial to understand the characteristics of bpAbs at the molecular level. In this study, we first obtained single-chain variable fragments and camelid heavy-chain variable domains targeting distinct epitopes of the metal binding protein MtsA and then developed a novel format single-chain bpAb connecting these fragment antibodies with various linkers. The physicochemical properties, binding activities, complex formation states with antigen, and functions of the bpAb were analyzed using multiple approaches. Notably, we found that the assembly state of the complexes was controlled by a linker and that longer linkers tended to form more compact complexes. These observations provide detailed molecular information that should be considered in the design of bpAbs.

Structure-based epitope prediction and assessment of cross-reactivity of Myrmecia pilosula venom-specific IgE and recombinant Sol g proteins (Solenopsis geminata).

The global distribution of tropical fire ants (Solenopsis geminata) raises concerns about anaphylaxis and serious medical issues in numerous countries. This investigation focused on the cross-reactivity of allergen-specific IgE antibodies between S. geminata and Myrmecia pilosula (Jack Jumper ant) venom proteins due to the potential emergence of cross-reactive allergies in the future. Antibody epitope analysis unveiled one predominant conformational epitope on Sol g 1.1 (PI score of 0.989), followed by Sol g 2.2, Sol g 4.1, and Sol g 3.1. Additionally, Pilosulin 1 showed high allergenic potential (PI score of 0.94), with Pilosulin 5a (PI score of 0.797) leading in B-cell epitopes. The sequence analysis indicated that Sol g 2.2 and Sol g 4.1 pose a high risk of cross-reactivity with Pilosulins 4.1a and 5a. Furthermore, the cross-reactivity of recombinant Sol g proteins with M. pilosula-specific IgE antibodies from 41 patients revealed high cross-reactivity for r-Sol g 3.1 (58.53%) and r-Sol g 4.1 (43.90%), followed by r-Sol g 2.2 (26.82%), and r-Sol g 1.1 (9.75%). Therefore, this study demonstrates cross-reactivity (85.36%) between S. geminata and M. pilosula, highlighting the allergenic risk. Understanding these reactions is vital for the prevention of severe allergic reactions, especially in individuals with pre-existing Jumper Jack ant allergy, informing future management strategies.

Bilateral efforts to improve SERS detection efficiency of exosomes by Au/Na7PMo11O39 Combined with Phospholipid Epitope Imprinting.

Detection of cancer-related exosomes in body fluids has become a revolutionary strategy for early cancer diagnosis and prognosis prediction. We have developed a two-step targeting detection method, termed PS-MIPs-NELISA SERS, for rapid and highly sensitive exosomes detection. In the first step, a phospholipid polar site imprinting strategy was employed using magnetic PS-MIPs (phospholipids-molecularly imprinted polymers) to selectively isolate and enrich all exosomes from urine samples. In the second step, a nanozyme-linked immunosorbent assay (NELISA) technique was utilized. We constructed Au/Na7PMo11O39 nanoparticles (NPs) with both surface-enhanced Raman scattering (SERS) property and peroxidase catalytic activity, followed by the immobilization of CD9 antibodies on the surface of Au/Na7PMo11O39 NPs. The Au/Na7PMo11O39-CD9 antibody complexes were then used to recognize CD9 proteins on the surface of exosomes enriched by magnetic PS-MIPs. Lastly, the high sensitivity detection of exosomes was achieved indirectly via the SERS activity and peroxidase-like activity of Au/Na7PMo11O39 NPs. The quantity of exosomes in urine samples from pancreatic cancer patients obtained by the PS-MIPs-NELISA SERS technique showed a linear relationship with the SERS intensity in the range of 6.21 × 107-2.81 × 108 particles/mL, with a limit of detection (LOD) of 5.82 × 107 particles/mL. The SERS signal intensity of exosomes in urine samples from pancreatic cancer patients was higher than that of healthy volunteers. This bidirectional MIPs-NELISA-SERS approach enables noninvasive, highly sensitive, and rapid detection of cancer, facilitating the monitoring of disease progression during treatment and opening up a new avenue for rapid early cancer screening.

Short-time ozone treatment promotes protease-mediated destruction of B cell allergen epitopes by altering the structural characteristics of whey protein.

A novel processing method combining short-time ozone pretreatment with hydrolysis has been developed to reduce whey protein allergenicity. The results showed that ozone treatment altered the whey protein spatial structure, initially increasing the surface hydrophobicity index, and then decreasing due to polymer formation as the time increased. Under the optimized conditions of alkaline protease-mediated hydrolysis, a 10-second pre-exposure to ozone significantly promoted the reduction in the IgE binding capacity of whey protein without compromising the hydrolysis efficiency. Compared with whey protein, the degranulation of KU812 cells stimulated by this hydrolysate decreased by 20.54%, 17.99%, and 22.80% for IL-6, ß-hexosaminidase, and histamine, respectively. In vitro simulated gastrointestinal digestion confirmed increased digestibility and reduced allergenicity. Peptidomics identification revealed that short-time ozonation exposed allergen epitopes, allowing alkaline protease to target these epitopes more effectively, particularly those associated with α-lactalbumin. These findings suggest the promising application of this processing method in mitigating the allergenicity of whey protein.

Enhancing antitumor response by efficiently generating large-scale TCR-T cells targeting a single epitope across multiple cancer antigens.

The need to contrive interventions to curb the rise in cancer incidence and mortality is critical for improving patients' prognoses. Adoptive cell therapy is challenged with quality large-scale production, heightening its production cost. Several cancer types have been associated with the expression of highly-immunogenic CTAG1 and CTAG2 antigens, which share common epitopes. Targeting two antigens on the same cancer could improve the antitumor response of TCR-T cells. In this study, we exploited an efficient way to generate large-fold quality TCR-T cells and also demonstrated that the common epitopes of CTAG1 and CTAG2 antigens provide an avenue for improved cancer-killing via dual-antigen-epitope targeting. Our study revealed that xeno/sera-free medium could expand TCR-T cells to over 500-fold, posing as a better replacement for FBS-supplemented media. Human AB serum was also shown to be a good alternative in the absence of xeno/sera-free media. Furthermore, TCR-T cells stimulated with beads-coated T-activator showed a better effector function than soluble T-activator stimulated TCR-T cells. Additionally, TCR-T cells that target multiple antigens in the same cancer yield better anticancer activity than those targeting a single antigen. This showed that targeting multiple antigens with a common epitope may enhance the antitumor response efficacy of T cell therapies.