Epitope Recognition Research Articles

Bdellovibrio bacteriovorus uses chimeric fibre proteins to recognize and invade a broad range of bacterial hosts

Predatory bacteria, like the model endoperiplasmic bacterium Bdellovibrio bacteriovorus, show several adaptations relevant to their requirements for locating, entering and killing other bacteria. The mechanisms underlying prey recognition and handling remain obscure. Here we use complementary genetic, microscopic and structural methods to address this deficit. During invasion, the B. bacteriovorus protein CpoB concentrates into a vesicular compartment that is deposited into the prey periplasm. Proteomic and structural analyses of vesicle contents reveal several fibre-like proteins, which we name the mosaic adhesive trimer (MAT) superfamily, and show localization on the predator surface before prey encounter. These dynamic proteins indicate a variety of binding capabilities, and we confirm that one MAT member shows specificity for surface glycans from a particular prey. Our study shows that the B. bacteriovorus MAT protein repertoire enables a broad means for the recognition and handling of diverse prey epitopes encountered during bacterial predation and invasion.

Pomab a Novel Monoclonal Type-I Antiphospholipid Antibody, Reduces Thrombin Generation By Selectively Binding to the Open Form of Prothrombin

Background. Anti-prothrombin (anti-PT) antibodies are a type of antiphospholipid antibodies frequently found in antiphospholipid syndrome (APS) patients. Previously, we identified two types of anti-PT antibodies, one binding to epitopes in kringle-1 that are hidden in the closed form but available in the open form (Type-I), and another one in which the epitopes are located in fragment-1 but accessible in both forms (Type-II). Our understanding of their functional characteristics remains limited. Goal. To identify Type-I monoclonal antibodies for mechanistic studies. Methods. Type-I anti-prothrombin antibodies were generated by immunizing mice with fragment-1 and producing monoclonal antibodies using hybridoma technology. Binding mechanisms and epitope recognition for one representative Type-I antibody, POmAb, were elucidated using surface plasmon resonance (SPR), single-molecule FRET (smFRET), and cryo-electron microscopy (cryo-EM). Functional evaluations were performed in human plasma and with purified components. In vivo hemostatic activity was assessed in a laser injury model of the cremasteric arteriole followed by intravital microscopy. Results. Type-I antibodies recognizing kringle-1 were obtained from immunization experiments. One of them, POmAb (Prothrombin Open monoclonal Antibody), was selected for in-depth mechanistic and structural studies. SPR studies showed that POmAb binds to the open form of prothrombin with low nanomolar affinity, and binding was impaired by the mutations S91A and Y93A in kringle-1. smFRET provided evidence of conformational selection, where POmAb preferentially selects the open conformation of prothrombin, thereby shifting the equilibrium towards the open form. Cryo-EM studies of the POmAb-prothrombin complex revealed an extended binding interface in kringle-1 which overlaps with the region occupied by the serine protease domain in the closed form, explaining why POmAb binds to the open form. In human plasma, POmAb prolonged the activated partial thromboplastin time and Russel's viper venom time, though the effect was modest. Further investigations using purified proteins and antibody fragments confirmed that POmAb reduces thrombin generation, although it does not completely abolish it, as the inhibition of thrombin generation occurs through a non-competitive mechanism with prothrombinase. In vivo studies corroborated in vitro findings, further proving reduced fibrin accumulation at the site of injury. Conclusions. We discovered POmAb, a Type-I antiphospholipid antibody. We demonstrated that it selectively binds to the open form of prothrombin, resulting in reduced thrombin generation. POmAb represents a novel tool for studying the role of Type-I antibodies in APS and advances the provocative new idea of conformational trapping of prothrombin as a novel class of anticoagulants.

In Silico Prediction of Plasmodium falciparum PfRipr Epitopes as Vaccine Candidates

Abstract Background The global malarial death toll in 2020 was ~627,000, 77% of which occurred in children <5 years of age. Plasmodium falciparum is the most lethal parasite of its genus but has evaded vaccine development due to its complex life cycle and redundant invasion mechanisms. Epitope-based vaccines hold significant promise due to their ability to target dominant and/or conserved regions in highly antigenically variable pathogens, including malaria. The recently characterized P. falciparum merozoite Rh5 interacting protein (PfRipr) is nonredundant, highly conserved, and essential for erythrocyte invasion, making it an ideal target for a bloodstage malaria vaccine. (Figure 1) We sought to analyze P. falciparum sequences from highly endemic areas using bioinformatic approaches that identify candidate PfRipr epitope-based bloodstage vaccine antigens for inclusion in a multiantigen vaccine. Figure 1: Model of binding and insertion of PRCR complex to erythrocyte basigin in merozoite invasion. Adapted from Scally et al. 2022. (https://doi.org/10.1038/s41564-022-01261-2 , https://creativecommons.org/licenses/by/4.0/) Methods Using two P. falciparum sequence datasets from Burkina Faso and Uganda, we assessed the immunogenic potential of PfRipr epitopes for T-cell receptor binding and B-cell recognition. T-cell receptor binding was predicted using NetMHCpan and NetMHCIIpan searching against MHC I and II alleles with high regional frequencies. Using an in-silico 3D model of PfRipr predicted via AlphaFold, tertiary structures of all PfRipr sample sequences were predicted via SWISS-MODEL then analyzed by ElliPro to identify linear and discontinuous B-cell epitopes. (Figure 2) Putative epitopes were filtered using binding thresholds, allele coverage, conservation, antigenicity, and allergenicity. (Figure 3) Figure 2: Protocol for B-cell epitope prediction using AlphaFold for template modeling, Swiss-Model for homology-based modeling of sample sequences, and ElliPro for continuous and discontinuous epitope prediction. Figure 3: Protocol for sorting T-cell epitopes using Average Allele Coverage (percentage of alleles in which the protein sequence was identified as an epitope) and B-cell epitopes using ElliPro Score (Protrusion Index representing surface accessibility of the epitope). Results The Burkina Faso PfRipr dataset predicted 1729 unique T-cell and 51 unique B-cell epitopes, and 28 T-cell and 4 B-cell epitopes remained post-filtration. Epitope prediction using the Ugandan dataset predicted 576 unique T-cell and 53 unique B-cell epitopes, and 57 T-cell and 7 B-cell epitopes remained post-filtration. Between the two datasets, there were 19 matching epitopes with 7 MHC I, 9 MHC II, 1 linear, and 2 discontinuous. (Tables 1-2, Figure 4) These epitopes were used to design a multi-epitope-based vaccine construct with a Flagellin adjuvant and peptide linkers to separate functional regions for immune processing. (Figure 5) Figure 4: 3D model of PfRipr with labeled B-cell epitopes. Predicted tertiary structure adapted from AlphaFold database. (https://doi.org/10.1038/s41586-021-03819-2, https://doi.org/10.1093/nar/gkab1061, https://creativecommons.org/licenses/by/4.0/) Figure 5: Schematic representation of hypothetical multi-epitope vaccine construct. FliC is added to the N-terminus to stimulate and amplify immune response. The EAAAK linker is used to separate the bifunctional fusion protein domains. AAY linkers are used to amplify MHC I and II epitopes and prevent junctional epitope binding. GPGPG linkers between B-cell epitopes function to minimize junctional epitopes and retain conformational-dependent immunogenicity. A six amino acid Histidine (6H) chain was added to the C-terminus to aid during lab purification processes. Conclusions Immunoinformatic tools employed in sequence identified candidate PfRipr epitopes used to design a bloodstage vaccine construct against P. falciparum. To validate their predicted immunogenicity, epitopes can be further investigated using in silico protein stabilization and docking simulations, in vitro methods such as HLA stabilization or T-cell activation assays, and in vivo methods using transgenic mouse models. The pipeline of immunoinformatic analyses formulated in this project can be further applied to a larger database of diverse P. falciparum sequences collected from other malaria endemic regions to develop vaccines effective against circulating strains.

In Vivo Treatment with Insulin-like Growth Factor 1 Reduces CCR5 Expression on Vaccine-Induced Activated CD4+ T-Cells.

At the heart of the DNA/ALVAC/gp120/alum vaccine's efficacy in the absence of neutralizing antibodies is a delicate balance of pro- and anti-inflammatory immune responses that effectively decreases the risk of SIVmac251 acquisition in macaques. Vaccine efficacy is linked to antibodies recognizing the V2 helical conformation, DC-10 tolerogenic dendritic cells eliciting the clearance of apoptotic cells via efferocytosis, and CCR5 downregulation on vaccine-induced gut homing CD4+ cells. RAS activation is also linked to vaccine efficacy, which prompted the testing of IGF-1, a potent inducer of RAS activation with vaccination. We found that IGF-1 changed the hierarchy of V1/V2 epitope recognition and decreased both ADCC specific for helical V2 and efferocytosis. Remarkably, IGF-1 also reduced the expression of CCR5 on vaccine-induced CD4+ gut-homing T-cells, compensating for its negative effect on ADCC and efferocytosis and resulting in equivalent vaccine efficacy (71% with IGF-1 and 69% without).

A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells

Human epithelial growth factor receptor-2 (HER2) plays an oncogenic role in numerous tumors, including breast, gastric, and various other solid tumors. While anti-HER2 therapies are approved for the treatment of HER2-positive tumors, a necessity persists for creating novel HER2-targeted agents to resolve therapeutic resistance. Utilizing a synthetic nanobody library and affinity maturation, our study identified four anti-HER2 nanobodies that exhibited high affinity and specificity. These nanobodies recognized three distinct epitopes of HER2-ECD. Additionally, we constructed VHH-Fc and discovered that they facilitated superior internalization and showed moderate growth inhibition. Compared to the combination of trastuzumab and pertuzumab, the VHH-Fc combos or their combination with trastuzumab demonstrated greater or comparable antitumor activity in both ligand-independent and ligand-driven tumors. Most remarkably, A9B5-Fc, which targeted domain I of HER2-ECD, displayed significantly enhanced trastuzumab-synergistic antitumor efficacy compared to pertuzumab under trastuzumab-resistant conditions. Our findings offer anti-HER2 nanobodies with high affinity and non-overlapping epitope recognition. The novel nanobody-based HER2-targeted antibody, A9B5-Fc, binding to HER2-ECD I, mediates promising receptor internalization. It possesses the potential to serve as a potent synergistic partner with trastuzumab, contributing to overcoming acquired resistance.

Features and protective efficacy of human monoclonal antibodies targeting Mycobacterium tuberculosis arabinomannan.

A better understanding of the epitopes most relevant for antibody-mediated protection against tuberculosis (TB) remains a major knowledge gap. We have shown that human polyclonal IgG to the Mycobacterium tuberculosis (Mtb) surface glycan arabinomannan (AM) and related lipoarabinomannan (LAM) is protective against TB. To investigate the impact of AM epitope recognition and Fc-gamma receptor (FcgR)-binding on antibody functions against Mtb, we isolated a high-affinity human monoclonal antibody (mAb; P1AM25) to AM and show its binding to oligosaccharide (OS) motifs we previously found to be associated with in vitro functions of human polyclonal anti-AM IgG. Human IgG1 P1AM25, but not two other high-affinity human IgG1 anti-AM mAbs reactive with different AM OS motifs, enhanced Mtb phagocytosis by macrophages and reduced intracellular growth in an FcgR-dependent manner. P1AM25 in murine IgG2a, but neither murine IgG1 nor a non-FcgR-binding IgG, given intraperitoneally prior to and after aerosolized Mtb infection was protective in C57BL/6 mice. Moreover, we demonstrate the protective efficacy of human IgG1 P1AM25 in passive transfer with Mtb-infected FcgR-humanized mice. These data enhance our knowledge of the important interplay between both antibody epitope specificity and Fc effector functions in the defense against Mtb and could inform development strategies of vaccines against TB.

Site-oriented conjugation of poly(2-methacryloyloxyethyl phosphorylcholine) for enhanced brain delivery of antibody.

Antibody therapeutics are limited in treating brain diseases due to poor blood-brain barrier (BBB) penetration. We have discovered that poly 2-methacryloyloxyethyl phosphorylcholine (PMPC), a biocompatible polymer, effectively facilitates BBB penetration via receptor-mediated transcytosis and have developed a PMPC-shell-based platform for brain delivery of therapeutic antibodies, termed nanocapsule. Yet, the platform results in functional loss of antibodies due to epitope masking by the PMPC polymer network, which necessitates the incorporation of a targeting moiety and degradable crosslinker to enable on-site antibody release. In this study, we developed a novel platform based on site-oriented conjugation of PMPC to the antibody, allowing it to maintain key functionalities of the original antibody. With an optimized PMPC chain length, the PMPC-antibody conjugate exhibited enhanced brain delivery while retaining epitope recognition, cellular internalization, and antibody-dependent cellular phagocytic activity. This simple formula incorporates only the antibody and PMPC without requiring additional components, thereby addressing the issues of the nanocapsule platform and paving the way for PMPC-based brain delivery strategies for antibodies.

Determining epitope specificity of T-cell receptors with transformers.

T-cell receptors (TCRs) on T cells recognize and bind to epitopes presented by the major histocompatibility complex in case of an infection or cancer. However, the high diversity of TCRs, as well as their unique and complex binding mechanisms underlying epitope recognition, make it difficult to predict the binding between TCRs and epitopes. Here, we present the utility of transformers, a deep learning strategy that incorporates an attention mechanism that learns the informative features, and show that these models pre-trained on a large set of protein sequences outperform current strategies. We compared three pre-trained auto-encoder transformer models (ProtBERT, ProtAlbert, and ProtElectra) and one pre-trained auto-regressive transformer model (ProtXLNet) to predict the binding specificity of TCRs to 25 epitopes from the VDJdb database (human and murine). Two additional modifications were performed to incorporate gene usage of the TCRs in the four transformer models. Of all 12 transformer implementations (four models with three different modifications), a modified version of the ProtXLNet model could predict TCR-epitope pairs with the highest accuracy (weighted F1 score 0.55 simultaneously considering all 25 epitopes). The modification included additional features representing the gene names for the TCRs. We also showed that the basic implementation of transformers outperformed the previously available methods, i.e. TCRGP, TCRdist, and DeepTCR, developed for the same biological problem, especially for the hard-to-classify labels. We show that the proficiency of transformers in attention learning can be made operational in a complex biological setting like TCR binding prediction. Further ingenuity in utilizing the full potential of transformers, either through attention head visualization or introducing additional features, can extend T-cell research avenues. Data and code are available on https://github.com/InduKhatri/tcrformer.

Antibodies elicited by Plasmodium falciparum circumsporozoite proteins lacking sequentially deleted C-terminal amino acids reveal mouse strain and epitopes specific differences

Malaria affects ∼ ¼ billion people globally and requires the development of additional tools to aid in elimination efforts. The recently approved RTS,S/AS01 vaccine represents a positive step, however, the moderate efficacy necessitates the development of more efficacious vaccines. PfCSP is a key target antigen for pre-erythrocytic vaccines aimed at preventing Plasmodium falciparum malaria infections. Epitopes within the central repeat region and at the junction of the repeat and N-terminal domain are well documented as major protective B cell epitopes. On the other hand, a majority of antibodies against the epitopes in the C-terminal domain, have been shown to be non-protective against sporozoite challenge. The C-terminal domain, however, contains CD4+ and CD8+ T cell epitopes previously shown to be important for regulating immune responses. The present study was designed to further explore the immunomodulatory potential of the C-terminal domain using DNA vaccines encoding PfCSP with sequential C-terminal truncations following known T cell epitopes. Five DNA vaccines encoding different truncations of PfCSP within the C-terminal domain were administered via intramuscular route and in vivo electroporation for effective immunogenicity. Protection in mice was evaluated by challenge with transgenic P. berghei expressing PfCSP. In Balb/c mice, antibody responses and protective efficacy were both affected progressively with sequential deletion of C-terminal amino acid residues. Similar studies in C57Bl/6 mice revealed that immunizations with plasmids encoding truncated PfCSP showed partial protection from sporozoite challenge with no significant differences in antibody titers observed compared to full-length PfCSP DNA immunized mice. Further analysis revealed murine strain-specific differences in the recognition of specific epitopes.

Correlation between B-cell epitope profile and clinical features of anti-MDA5 antibody-positive dermatomyositis.

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive (MDA5+) dermatomyositis patients exhibit a variety of clinical features. We therefore investigated whether patterns of B-cell epitope recognition are linked to the clinical course of MDA5+ dermatomyositis. Our cross-sectional study used ELISA-based methods to determine the relationship between antibody recognition of overlapping 155 amino acid MDA5 subfragments and clinical features of 24 MDA5+ myositis patients. Correlations between clinical features and standardized anti-MDA5 subfragment antibody titres were assessed via Spearman's rank correlation coefficients. Twenty-four MDA5+ patients submitted serum samples within a median of 0 (interquartile range, 0-74) days from the initial clinic visit. In addition to typical dermatomyositis rashes, these patients exhibited muscle symptoms (n = 11), vascular dysfunction (n = 9) and interstitial lung disease (ILD) (n = 16). Female patients exhibited higher titres of antibodies recognizing fragment H (aa 905-1026) compared with male patients. Muscle involvement was associated with higher levels of anti-fragment F (aa 646-801) antibody. Conversely, patients with vascular abnormalities had higher anti-fragment B (aa 130-284) and E (aa 517-671) antibody titres than those without vascular dysfunction. Four patients died due to ILD progression and showed higher anti-fragment A (aa 1-155) antibody titres than the other 20 patients. Differences in the ratio of anti-fragment to anti-full-length MDA5 antibody titres were found for sex (H: anti-MDA5) and vascular dysfunction (anti-fragment B, E: anti-MDA5). Various clinical features of MDA5+ dermatomyositis correlated with levels of antibodies targeting selected subfragments of this autoantigen, providing a link between fragment-specific immune responses and disease course.

75P Possible significance of the proline at position 325 in the recognition of the MX35 epitope of the NaPi2b transporter by monoclonal antibodies

75P Possible significance of the proline at position 325 in the recognition of the MX35 epitope of the NaPi2b transporter by monoclonal antibodies

Determination of the epitopic peptides of fig mosaic virus and the single-chain variable fragment antibody by mass spectrometry

The study of antibody-antigen interactions, through epitope mapping, enhances our understanding of antibody neutralization and antigenic determinant recognition. Epitope mapping, employing monoclonal antibodies and mass spectrometry, has emerged as a rapid and precise method to investigate viral antigenic determinants. In this report, we propose an approach to improve the accuracy of epitopic peptide interaction rate recognition. To achieve this, we investigated the interaction between the nucleocapsid protein of fig mosaic virus (FMV-NP) and single-chain variable fragment antibodies (scFv-Ab). These scFv-Ab maintain high specificity similar to whole monoclonal antibodies, but they are smaller in size. We coupled this with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The experimental design involved using two different enzymes to digest FMV-NP separately. The resulting peptides were then incubated separately with the desired scFv-Ab at different incubation times and antibody concentrations. This allowed us to monitor the relative rate of epitopic peptide interaction with the antibody. The results demonstrated that, at a 1:1 ratio and after 2 h of interaction, the residues 122–136, 148–157, and 265–276 exhibited high-rate epitopic peptide binding, with reductions in peak intensity of 78%, 21%, and 22%, respectively. Conversely, the residues 250–264 showed low-rate binding, with a 15% reduction in peak intensity. This epitope mapping approach, utilizing scFv-Ab, two different enzymes, and various incubation times, offers a precise and dependable analysis for monitoring and recognizing the binding kinetics of antigenic determinants. Furthermore, this method can be applied to study any kind of antigens.

Structural basis of biased T cell recognition of immune-dominant epitope of SARS-CoV-2 spike protein

Structural basis of biased T cell recognition of immune-dominant epitope of SARS-CoV-2 spike protein

Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity.

CD30 is expressed on Hodgkin lymphomas (HL), many non-Hodgkin lymphomas (NHLs), and non-lymphoid malignancies in children and adults. Tumor expression, combined with restricted expression in healthy tissues, identifies CD30 as a promising immunotherapy target. An anti-CD30 antibody-drug conjugate (ADC) has been approved by the FDA for HL. While anti-CD30 ADCs and chimeric antigen receptors (CARs) have shown promise, their shortcomings and toxicities suggest that alternative treatments are needed. We developed novel anti-CD30 x anti-CD3 bispecific antibodies (biAbs) to coat activated patient T cells (ATCs) ex vivo prior to autologous re-infusions. Our goal is to harness the dual specificity of the biAb, the power of cellular therapy, and the safety of non-genetically modified autologous T cell infusions. We present a comprehensive characterization of the CD30 binding and tumor cell killing properties of these biAbs. Five unique murine monoclonal antibodies (mAbs) were generated against the extracellular domain of human CD30. Resultant anti-CD30 mAbs were purified and screened for binding specificity, affinity, and epitope recognition. Two lead mAb candidates with unique sequences and CD30 binding clusters that differ from the ADC in clinical use were identified. These mAbs were chemically conjugated with OKT3 (an anti-CD3 mAb). ATCs were armed and evaluated in vitro for binding, cytokine production, and cytotoxicity against tumor lines and then in vivo for tumor cell killing. Our lead mAb was subcloned to make a Master Cell Bank (MCB) and screened for binding against a library of human cell surface proteins. Only huCD30 was bound. These studies support a clinical trial in development employing ex vivo-loading of autologous T cells with this novel biAb.

A lateral flow immunochromatographic assay based on nanobody-oriented coupling strategy for aflatoxin B1 detection

Nonoriented coupling of nanobodies (Nb) to nanomaterials can considerably reduce epitope recognition or induce other dysfunctions, thereby restricting the application of Nb in field detection. Herein, using Avi-tag/streptavidin (Avi/SA)-oriented coupling strategy, we developed a lateral-flow immunochromatographic assay (LFIA) based on Nb-Avi/SA@quantum dots (QDs) probes (Nb-LFIA) for Aflatoxin B1 (AFB1) detection. Compared with nonoriented coupled Nb@QD probes, the detection sensitivity and stability of Nb-Avi/SA@QD probes were greatly retained. Furthermore, Nb showed higher tolerance to the extreme detection environments compared with monoclonal antibodies in traditional enzyme-linked immunosorbent assay and LFIA. After optimization, the limit of detection of Nb-LFIA was 0.095 ng mL−1, half-maximal inhibitory concentration was 0.85 ng mL−1, and the visual cut-off level was 1.25 ng mL−1. Nb-Avi/SA@gold nanoparticle probes were assessed for the wide applicability of the Avi/SA strategy, and they showed excellent performance (coefficient of determination = 0.999). Finally, the analysis performance of probes was assessed in oat samples, revealing a recovery rate of 88.8–116.7%, which was consistent with the results of high-pressure liquid chromatography coupled with tandem mass spectrometry. Altogether, our study provides a convenient and sensitive tool for AFB1 detection, and presents a viable path for efficient Nb application.

Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy

The Tcells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific Tcell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual Tcells to attack cancer in several ways simultaneously. Such "multipronged" Tcells exhibited superior recognition of cancer cells compared with conventional Tcell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.

The 2EF Antibody Targets a Unique N-Terminal Epitope of Trop-2 and Enhances the In Vivo Activity of the Cancer-Selective 2G10 Antibody.

Trop-2 proteolytic processing in cancer cells exposes epitopes that were specifically targeted by the 2G10 antibody. We sought additional recognition of Trop-2 within difficult-to-reach, densely packed tumor sites. Trop-2 deletion mutants were employed in immunization and screening procedures, and these led to the recognition of a novel epitope in the N-terminal region of Trop-2, by the 2EF antibody. The 2EF mAb was shown to bind Trop-2 at cell-cell junctions in MCF-7 breast cancer cells, and in deeply seated sites in prostate cancer, that were inaccessible to benchmark anti-Trop-2 antibodies. The 2EF antibody was shown to inhibit the growth of HT29 colon tumor cells in vitro, with the highest activity at high cell density. In vivo, 2EF showed anticancer activity against SKOv3 ovarian, Colo205, HT29, HCT116 colon and DU-145 prostate tumors, with the highest impact on densely packed tumor sites, whereby 2EF outcompeted benchmark anti-Trop-2 antibodies. Given the different recognition modes of Trop-2 by 2EF and 2G10, we hypothesized the effective interaction of the two mAb in vivo. The 2EF mAb was indeed demonstrated to enhance the activity of 2G10 against tumor xenotransplants, opening novel avenues for Trop-2-targeted therapy. We humanized 2EF by state-of-the-art CDR grafting/re-modeling, yielding the Hu2EF for therapy of Trop-2-expressing tumors in patients.

The molecular mechanism of CD81 antibody inhibition of metastasis

Metastases are reduced in CD81KO mice. In addition, a unique anti-CD81 antibody, 5A6, inhibits metastasis in vivo and invasion and migration in vitro. Here, we probed the structural components of CD81 required for the antimetastatic activity induced by 5A6. We found that the removal of either cholesterol or the intracellular domains of CD81 did not affect inhibition by the antibody. We show that the uniqueness of 5A6 is due not to increased affinity but rather to its recognition of a specific epitope on the large extracellular loop of CD81. Finally, we present a number of CD81 membrane-associated partners that may play a role in mediating the 5A6 antimetastatic attributes, including integrins and transferrin receptors.

Final results: Dose escalation study of a personalized peptide-based neoantigen vaccine in patients with metastatic melanoma.

9551 Background: Despite the advances made with CPIs such as aPD-1/aPD-L1, aCTL4 and aLAG-3 there still is a significant unmet medical need for patients with metastatic cancers. As it has been shown that the effect of CPIs are mediated by tumor specific T cells, one strategy for increasing treatment success is to actively induce tumor specific T cells. Here, we report on a first-in-human clinical trial evaluating a personalized neoantigen vaccine (EVX-01) in patients with metastatic melanoma. Methods: Stage-IV metastatic melanoma patients were treated with standard aPD-1 in combination with a personalized tumor-specific neo-antigen vaccine. The vaccine consisted of multiple 15-27mer peptides comprising one or more patient specific neoantigens identified from tumor DNA and mRNA sequencing data by an integrated AI-platform; PIONEER and delivered together with the novel liposomal adjuvant CAF09b to potentiate immune responses. Results: Vaccines were designed and manufactured for each patient in less than 7 weeks. In total, 12 eligible patients received either 500ug (5 patients, reported in PMID: 35036074), 1000ug (3 patients) or 2000ug (4 patients) total peptide load keeping the peptide:CAF09b ratio constant. Four patients had stable disease on aPD-1 treatment for at least 4 months before enrolment in the study, whereas the remaining patients were treatment naïve. All patients received monotherapy with pembrolizumab (11) or nivolumab (1) during the vaccine manufacturing period. The personalized vaccine was shown to be safe and well tolerated with fatigue and injection site reactions being the most frequently reported events and only grade 1-2 events being related to the vaccine. Response was evaluated by RECIST 1.1, according to the investigator’s assessment with 8 patients (67%) having objective response (CR: n = 2, PR: n = 6), of which 1 patients had response initiation before addition of the vaccine. Importantly the vaccine induced neoantigen specific T cells in all patients analyzed. Both CD4+ and CD8+ T cells were detected, with the majority of responses being CD4+ T cell mediated. Interestingly, we report a significant correlation between the PIONEER prediction score and induced immunogenicity. Furthermore, the breadth of the neoantigen recognition seemed to be important for the clinical effect of the vaccine as responders demonstrated T cell recognition of more epitopes within the vaccine compared to non-responders. We found, in our cohort, that the neo-antigen scores effectively separated responders and non-responders, as opposed to the TMB (by FDA guidelines). Conclusions: aPD-1 treatment combined with a peptide-based personalized neo-antigen-based vaccine was shown to be feasible and safe with promising signs of efficacy, warranting further study. Further, the neo-antigen selection method appeared crucial for designing an efficacious vaccine. Clinical trial information: NCT03715985 .

Nature's Needle: Harnessing Bacterial Molecular Syringes

Nature's Needle: Harnessing Bacterial Molecular Syringes