Conventional Antigen Research Articles

Diffuse Peritoneal Carcinomatosis of Prostate Cancer Unveiled by [89Zr]Zr-PSMA-617 PET/CT

Abstract We report an interesting case of a 64-year-old man with a history of radical prostatectomy for prostate cancer. The patient presented with steady increasing prostate-specific antigen levels, but with negative findings on previous multiple conventional prostate-specific membrane antigen (PSMA) PET/CT (with [68Ga]Ga-PSMA-11) and [18F]FDG PET/CT. A recently introduced PSMA tracer using long-lived 89Zr (half-life 3.3 days), [89Zr]Zr-PSMA-617, was administered in attempt to localize potential local recurrence or metastasis. [89Zr]Zr-PSMA-617 PET/CT clearly revealed diffuse peritoneal carcinomatosis and local recurrence of the prostate cancer, which were unidentified on previous conventional PET/CT scans. This case highly demonstrates the potential of PET imaging with the novel PSMA tracer [89Zr]Zr-PSMA-617.

Ebola Virus Infection of Flt3-Dependent, Conventional Dendritic Cells and Antigen Cross-presentation Leads to High Levels of T-Cell Activation.

Previous studies have described that Ebola virus (EBOV) infection of human monocyte-derived dendritic cells (moDCs) inhibits dendritic cell (DC) maturation, resulting in poor T-cell activation. However, it is unknown how other DC subsets distinct from moDCs respond to EBOV infection. To better understand how DCs initiate T-cell activation during EBOV infection, we assessed the response of conventional mouse DCs (cDCs) to EBOV infection utilizing a recombinant EBOV expressing the model antigen ovalbumin. In contrast to moDCs, mouse cDC2s and cDC1s were poorly infected with EBOV but were highly activated. DCs were able to prime CD8 T cells via cross-presentation of antigens obtained from cell debris of EBOV-infected cells. EBOV infection further enhanced DC cross-presentation. Our findings indicate that EBOV infection of cDCs results in activation rather than inhibition, leading to high levels of T-cell activation. With that we propose a mechanistic explanation for the excess T-cell activation observed in human Ebola virus disease.

Enhanced Lung Cancer Detection Using a Combined Ratio of Antigen-Autoantibody Immune Complexes against CYFRA 21-1 and p53.

The early detection of lung cancer (LC) improves patient outcomes, but current methods have limitations. Autoantibodies against tumor-associated antigens have potential as early biomarkers. This study evaluated the 9G testTM Cancer/Lung, measuring circulating complexes of two antigen-autoantibody immune complexes (AIC) against their respective free antigens (CYFRA 21-1 and p53) for LC diagnosis. We analyzed 100 LC patients and 119 healthy controls using the 9G testTM Cancer/Lung, quantifying the levels of AICs (CYFRA 21-1-Anti-CYFRA 21-1 autoantibody immune complex (CIC) and p53-Anti-p53 autoantibody immune complex (PIC)), free antigens (CYFRA 21-1 and p53), and ratios of AICs/antigens (LC index). The levels of the CICs and PICs were significantly elevated in LC compared to the controls (p < 0.0062 and p < 0.0026), while free antigens showed no significant difference. The CIC/CYFRA 21-1 and PIC/p53 ratios were also significantly higher in LC (all, p < 0.0001). The LC index, when combining both ratios, exhibited the best diagnostic performance with an area under the curve (AUC) of 0.945, exceeding individual CICs, PICs, and free antigens (AUCs ≤ 0.887). At a cut-off of 3.60, the LC index achieved 81% sensitivity and 95% specificity for LC diagnosis. It detected early-stage (Stage I-II) LC with 87.5% sensitivity, exceeding its performance in advanced stages (72.7%). The LC index showed no significant differences based on age, gender, smoking status (former, current, or never smoker), or pack years smoked. The LC index demonstrates promising potential for early LC diagnosis, exceeding conventional free antigen markers.

Synthetic hemozoin as a nanocarrier for cross-presentation

It is known that conventional antigen presentation involves phagocytosis of antigens followed by its internalization in endocytic compartments and presentation of epitopes through MHC class II molecules for CD4 T cells. However, since 1976 a cross-presentation pathway has been studied, in which CD8 T cells are activated via MHC class I with antigens acquired through phagocytosis or endocytosis by dendritic cells (DCs). Among some important molecules involved in the cross-presentation, the C‐type lectin receptor of the Dectin‐1 cluster (CLECs), particularly the CLEC9A receptor, not only is expressed in dendritic cells but also presents a pivotal role in this context. In special, CLEC12A has been highlighted as a malaria pigment hemozoin (HZ) receptor. During Plasmodium infection, hemozoin crystals defend the parasite against heme toxicity within erythrocytes, as well as the released native HZ elicits pro-inflammatory responses and can induce cross-presentation. Particularly, this crystal can be synthesized from hematin anhydride and mimics the native form, and the gaps generated between the nanocrystal domains during its synthesis allow for substance coupling followed by its coating. Therefore, this study aimed to assess whether synthetic hemozoin (sHz) or hematin anhydride could be a nanocarrier and promote cross-presentation in dendritic cells. Firstly, it was verified that sHz can carry coated and coupled antigens, the compounds can associate to LAMP1-positive vesicles and decrease overall intracellular pH, which can potentially enhance the cross-presentation of ovalbumin and Leishmania infantum antigens. Thus, this study adds important data in the molecular intricacies of antigen presentation by showing not only the sHz immunomodulatory properties but also its potential applications as an antigen carrier.

Diagnosis of Clinically Suspected Amoebic Liver Abscess Using Conventional PCR and Antigen ELISA in Liver Aspirate and Serum

Diagnosis of Clinically Suspected Amoebic Liver Abscess Using Conventional PCR and Antigen ELISA in Liver Aspirate and Serum

Human erythroid progenitors express antigen presentation machinery.

Early-life immune exposures can profoundly impact lifelong health. However, functional mechanisms underlying fetal immune development remain incomplete. Erythrocytes are not typically considered active immune mediators, primarily because erythroid precursors discard their organelles as they mature, thus losing the ability to alter gene expression in response to stimuli. Erythroid progenitors and precursors circulate in human fetuses and neonates. Although there is limited evidence that erythroid precursors are immunomodulatory, our understanding of the underlying mechanisms remains inadequate. To define the immunobiological role of fetal and perinatal erythroid progenitors and precursors, we analyzed single cell RNA-sequencing data and found that transcriptomics support erythroid progenitors as putative immune mediators. Unexpectedly, we discovered that human erythroid progenitors constitutively express Major Histocompatibility Complex (MHC) class II antigen processing and presentation machinery, which are hallmarks of specialized antigen presenting immune cells. Furthermore, we demonstrate that erythroid progenitors internalize and cleave foreign proteins into peptide antigens. Unlike conventional antigen presenting cells, erythroid progenitors express atypical costimulatory molecules and immunoregulatory cytokines that direct the development of regulatory T cells, which are critical for establishing maternal-fetal tolerance. Expression of MHC II in definitive erythroid progenitors begins during the second trimester, coinciding with the appearance of mature T cells in the fetus, and is absent in primitive progenitors. Lastly, we demonstrate physical and molecular interaction potential of erythroid progenitors and T cells in the fetal liver. Our findings shed light on a unique orchestrator of fetal immunity and provide insight into the mechanisms by which erythroid cells contribute to host defense.

High-throughput light-induced immunoassay with milliwatt-level laser under one-minute optical antibody-coating on nanoparticle-imprinted substrate

The efficient detection of protein biomarkers is critical for public health. However, the sensitivity of conventional antigen test kits is relatively low for early diagnosis, and laboratory immunoassays require complex pretreatment processes overnight. If target nanomaterials could be remotely guided to the detection site, simpler and faster methods would be developed. Here, we reveal the mechanism of light-induced immunoassay that anti-spike-protein antibodies for SARS-CoV-2 were coated on our developed nanoparticle-imprinted plasmonic substrate (NPI-PS) over the submillimeter area within one minute and nanoparticles modified with spike proteins can be selectively detected within a few minutes at one or two orders of higher sensitivity via a two-step optical condensation using NPI-PS. NPI-PS exhibits high-performance optical condensation with high photothermal properties even under milliwatt-class nonresonant laser irradiation, enabling a wide range of quantitative measurements. These findings support an innovative strategy to mitigate pandemic threats and various diseases through the high-throughput detection of protein biomarkers.

Cost Analysis of Prostate Cancer Care Using a Biomarker-enhanced Diagnostic Strategy with Stockholm3

Background and objectiveBuilding on previous research demonstrating better prostate cancer (PC) diagnostics via a biomarker-enhanced approach, this study focuses on cost analysis of PC care using the Stockholm3 test. We assessed the economic impact in European health care systems using real-world evidence for diagnostic outcomes and relevant costs. MethodsWe evaluated two PC diagnostic strategies: (1) the conventional prostate-specific antigen (PSA) strategy with magnetic resonance imaging (MRI) and (2) PSA testing with a reflex to biomarkers at PSA ≥1.5 ng/ml in guiding decisions to perform MRI. Data from the Swedish National Prostate Cancer Register and Capio St. Göran Hospital provided real-world evidence, supplemented by health economic modeling. A comprehensive cost analysis was conducted using a Markov model for treatment pathways for four PC disease states and overall spending, for which costs from various European health care systems were used. A deterministic sensitivity analysis was performed across different cost and diagnostic scenarios. Key finding and limitationsThe average cost for the four disease states was €2 182 for benign disease, €10 023 for low-grade disease, €13 073 for intermediate- to high-grade localized or locally advance disease, and €271 210 for metastatic disease. The overall spending was €358 239 (7.7%) lower per 1000 men tested in the biomarker-enhanced strategy in comparison to the PSA strategy. The primary cost saving was attributed to lower treatment expenses for metastatic disease. Sensitivity analysis affirmed the robustness of the findings across various diagnostic and treatment scenarios. Conclusions and clinical implicationsBiomarker-enhanced diagnostic strategies may reduce health care costs for PC management and are likely to improve quality-adjusted life years in a scenario in which metastatic disease is reduced. Patient summaryWe explored different ways to detect prostate cancer more cost-effectively. We found that using a specific blood test, called Stockholm3, after a PSA (prostate-specific antigen) test to decide if an MRI scan (magnetic resonance imaging) is necessary could save money, mainly by identifying localized cancer earlier and reducing the need for expensive treatments for advanced cancer.

Biomolecule-regulation of fluorescent probe signaling: Homogeneous rapid portable protease sensing in serum

BackgroundAs is well documented, prostate cancer (PCa) being the second most prevalent cancer in men worldwide, emphasizing the importance of early diagnosis for prognosis. However, conventional prostate-specific antigen (PSA) testing lacks sufficient diagnostic efficiency due to its relatively low sensitivity and limited detection range. Mounting evidence suggests that matrix metalloproteinase 9 (MMP-9) expression increases with the aggressive behavior of PCa, highlighting the significance of detecting the serum level of MMP-9 in patients. Developing a non-immune rapid, portable MMP-9 detection strategy and investigating its representativeness of PCa serum markers hold considerable implications. ResultsHerein, our study developed a simple, homogeneous dual fluorescence and smartphone-assisted red-green-blue (RGB) visualization peptide sensor of MMP-9, utilizing cadmium telluride quantum dots (CdTe QDs) and calcein as signal reporters. The essence of our approach revolves around the proteolytic ability of MMP-9, exploiting the selective recognition of molecule-Cu2+ complexes with different molecular weights by CdTe QDs and calcein. Under optimized conditions, the limits of detection (LODs) for MMP-9 were 0.5 pg/mL and 6 pg/mL using fluorescence and RGB values readouts, respectively. Indeed, this strategy exhibited robust specificity and anti-interference ability. MMP-9 was quantified in 42 clinical serum samples via dual-fluorescence analysis, with 12 samples being visually identified with a smartphone. According to receiver operating characteristic curve (ROC) analysis, its sensitivity and specificity were 90 % and 100 %, respectively, with an area under curve (AUC) value of 0.903. Significance and noveltyOf note, the results of the aforementioned analysis were highly consistent with the serum level of PSA, clinical color Doppler flow imaging (CDFI), and histopathological results. Therefore, this simple, rapid, homogeneous fluorescence and visualization strategy can reliably measure MMP-9 levels and exhibit promising potential in point-of-care testing (POCT) applications for PCa patients.

CD72 is an inhibitory pattern recognition receptor that recognizes ribosomes and suppresses production of anti-ribosome autoantibody

B cell responses to nucleic acid-containing self-antigens that involve intracellular nucleic acid sensors play a crucial role in autoantibody production in SLE. CD72 is an inhibitory B cell co-receptor that down-regulates BCR signaling, and prevents the development of SLE. We previously showed that CD72 recognizes the RNA-containing self-antigen Sm/RNP, a target of SLE-specific autoantibodies, and induces B cell tolerance to Sm/RNP by specifically inhibiting B cell response to this self-antigen. Here, we address whether CD72 inhibits B cell response to ribosomes because the ribosome is an RNA-containing self-antigen and is a target of SLE-specific autoantibodies as well as Sm/RNP. We demonstrate that CD72 recognizes ribosomes as a ligand, and specifically inhibits BCR signaling induced by ribosomes. Although conventional protein antigens by themselves do not induce proliferation of specific B cells, ribosomes induce proliferation of B cells reactive to ribosomes in a manner dependent on RNA. This proliferative response is down-regulated by CD72. These results suggest that ribosomes activate B cells by inducing dual signaling through BCR and intracellular RNA sensors and that CD72 inhibits B cell response to ribosomes. Moreover, CD72−/− but not CD72+/+ mice spontaneously produce anti-ribosome autoantibodies. Taken together, CD72 induces B cell self-tolerance to ribosomes by recognizing ribosomes and inhibiting RNA-dependent B cell response to this self-antigen. CD72 appears to prevent development of SLE by inhibiting autoimmune B cell responses to multiple RNA-containing self-antigens. Because these self-antigens but not protein self-antigens induce RNA-dependent B cell activation, self-tolerance to RNA-containing self-antigens may require a distinct tolerance mechanism mediated by CD72.

Early induction of cytokine release syndrome by rapidly generated CAR T cells in preclinical models

Cytokine release syndrome (CRS) is a significant side-effect of conventional chimeric antigen receptor (CAR) T-cell therapy. To facilitate patient accessibility, short-term (st) CAR T cells, which are administered to patients only 24 h after vector exposure, are in focus of current investigations. Their impact on the incidence and severity of CRS has been poorly explored. Here, we evaluated CD19-specific stCAR T cells in preclinical models. In co-culture with tumor cells and monocytes, stCAR T cells exhibited anti-tumoral activity and potent release of CRS-related cytokines (IL-6, IFN-γ, TNF-α, GM-CSF, IL-2, IL-10). When administered to NSG-SGM3 mice, stCAR T cells, but not conventional CAR T cells, induced severe acute adverse events within 24 h, including hypothermia and weight loss, as well as high body scores, independent of the presence of tumor target cells. Human (IFN-γ, TNF-α, IL-2, IL-10) and murine (MCP-1, IL-6, G-CSF) cytokines, typical for severe CRS, were systemically elevated. Our data highlight potential safety risks of rapidly manufactured CAR T cells and suggest NSG-SGM3 mice as sensitive model for their preclinical safety evaluation.

RH genotypes and red cell alloimmunization rates in chronically transfused patients with sickle cell disease: A multisite study in the USA.

Red cell alloimmunization remains a challenge for individuals with sickle cell disease (SCD) and contributes to increased risk of hemolytic transfusion reactions and associated comorbidities. Despite prophylactic serological matching for ABO, Rh, and K, red cell alloimmunization persists, in part, due to a high frequency of variant RH alleles in patients with SCD and Black blood donors. We compared RH genotypes and rates of alloimmunization in 342 pediatric and young adult patients with SCD on chronic transfusion therapy exposed to >90,000 red cell units at five sites across the USA. Genotyping was performed with RHD and RHCE BeadChip arrays and targeted assays. Prevalence of overall and Rh-specific alloimmunization varied among institutions, ranging from 5% to 41% (p = .0035) and 5%-33% (p = .0002), respectively. RH genotyping demonstrated that 33% RHD and 57% RHCE alleles were variant in this cohort. Patients with RHCE alleles encoding partial e antigens had higher rates of anti-e identified than those encoding at least one conventional e antigen (p = .0007). There was no difference in anti-D, anti-C, or anti-E formation among patients with predicted partial or altered antigen expression compared to those with conventional antigens, suggesting that variant Rh on donor cells may also stimulate alloimmunization to these antigens. These results highlight variability in alloimmunization rates and suggest that a molecular approach to Rh antigen matching may be necessary for optimal prevention of alloimmunization given the high prevalence of variant RH alleles among both patients and Black donors.

Dynamics of The Γδtcr Repertoires During The Dedifferentiation Process and Pilot Implications for Immunotherapy of Thyroid Cancer.

γδ T cells are evolutionarily conserved T lymphocytes that manifest unique antitumor efficacy independent of tumor mutation burden (TMB) and conventional human leukocyte antigen (HLA) recognition. However, the dynamic changes in their T cell receptor (TCR) repertoire during cancer progression and treatment courses remain unclear. Here, a comprehensive characterization of γδTCR repertoires are performed in thyroid cancers with divergent differentiation states through cross-sectional studies. The findings revealed a significant correlation between the differentiation states and TCR repertoire diversity. Notably, highly expanded clones are prominently enriched in γδ T cell compartment of dedifferentiated patients. Moreover, by longitudinal investigations of the γδ T cell response to various antitumor therapies, it is found that the emergence and expansion of the Vδ2neg subset may be potentially associated with favorable clinical outcomes after post-radiotherapeutic immunotherapy. These findings are further validated at single-cell resolution in both advanced thyroid cancer patients and a murine model, underlining the importance of further investigations into the role of γδTCR in cancer immunity and therapeutic strategies.

Current Views about the Inflammatory Damage Triggered by Bacterial Superantigens and Experimental Attempts to Neutralize Superantigen-Mediated Toxic Effects with Natural and Biological Products.

Superantigens, i.e., staphylococcal enterotoxins and toxic shock syndrome toxin-1, interact with T cells in a different manner in comparison to conventional antigens. In fact, they activate a larger contingent of T lymphocytes, binding outside the peptide-binding groove of the major histocompatibility complex class II. Involvement of many T cells by superantigens leads to a massive release of pro-inflammatory cytokines, such as interleukin (IL)-1, IL-2, IL-6, tumor necrosis factor-alpha and interferon-gamma. Such a storm of mediators has been shown to account for tissue damage, multiorgan failure and shock. Besides conventional drugs and biotherapeutics, experiments with natural and biological products have been undertaken to attenuate the toxic effects exerted by superantigens. In this review, emphasis will be placed on polyphenols, probiotics, beta-glucans and antimicrobial peptides. In fact, these substances share a common functional denominator, since they skew the immune response toward an anti-inflammatory profile, thus mitigating the cytokine wave evoked by superantigens. However, clinical applications of these products are still scarce, and more trials are needed to validate their usefulness in humans.

Abstract C028: A phase 1/2 study investigating the safety and efficacy of autologous TAC T cells in subjects with unresectable, locally advanced or metastatic claudin 18.2+ solid tumors

Abstract CLDN18.2 is a tight junction protein found in differentiated gastric epithelial cells which can become abnormally expressed in a number of solid malignancies. This exposure profile illustrates the potential of CLDN18.2 as a candidate for targeted therapy. To date, there are no approved therapeutic agents directed against this target but promising data exist using monoclonal antibodies and conventional chimeric antigen receptor (CAR) T therapy. The T cell antigen coupler (TAC) technology is an approach to modifying T cells ex vivo, which allows recognition and cytotoxicity of tumor cells by co-opting the natural T cell receptor. TAC T cells demonstrate a safer profile than CAR T treatments. TAC01-CLDN18.2 is an autologous T-cell product comprising T cells expressing CLDN18.2 TAC. This is a first-in-human study (NCT05862324) to investigate the safety and preliminary anti-tumor activity of TAC01-CLDN18.2 in CLDN18.2+/HER2- solid tumors. Subjects will undergo leukapheresis and may receive bridging anticancer therapy as appropriate during cell manufacturing. Prior to TAC01-CLDN18.2 infusion, subjects will undergo low-intensity lymphodepletion chemotherapy. In phase I dose escalation, TAC01-CLDN18.2 will be administered at increasing doses (Cohorts 1-3) in adult subjects after ≥2 lines of therapy using the classic 3+3 dose escalation study design. Subjects with pancreatic ductal adenocarcinoma (PDAC) may have been treated with 1 line of prior therapy. CLDN18.2 expression levels will be determined centrally using a clinical trial assay validated across relevant indications. Dose limiting toxicities will be assessed up to 28 days from TAC01-CLDN18.2 infusion. A second dose may be administered, according to preidentified clinical and safety criteria. In Phase II, dose expansion groups will further evaluate the efficacy, safety, and pharmacokinetics of the optimal TAC01-CLDN18.2 dose, with the option of redosing. Indications will include gastric and esophageal adenocarcinoma (group A), PDAC (group B) and ovarian and non-small cell lung cancer (group C) in subjects after ≥2 and &amp;lt;4 lines of prior therapy (subjects with PDAC may have been exposed to 1 therapy). Definitions of eligible CLDN18.2 expression levels will be based on retrospective analysis of data from Phase 1 in association with clinical efficacy. A Simon 2-stage design will be used to enroll ≤57 subjects in Group A and ≤22 subjects in Group C. Group B will be exploratory and will enroll ≤10 subjects with an opportunity of cohort enrichment based on clinical efficacy data. Citation Format: Benjamin Schlechter, Ecaterina Dumbrava, Samuel Saibil, Martin Gutierez, Syma Iqbal, Davendra Sohal, Maria Apostolopoulou, Kara Moss, Deyaa Adib, Daniel Olson. A phase 1/2 study investigating the safety and efficacy of autologous TAC T cells in subjects with unresectable, locally advanced or metastatic claudin 18.2+ solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C028.

Screening for prostate cancer in a city in Japan: age-specific prostate-specific antigen cutoff thresholds.

Older men have higher prostate-specific antigen levels than younger men. However, the current Japanese Urological Association guidelines recommend secondary screening at a cutoff value of 4.0ng/mL, even in older men. Here, we reexamined the cutoffs for older men using a prostate screening cohort in Japan and first performed an analysis to determine the indication cutoffs for detecting positive biopsies. Data from 68,566 prostate cancer screenings in the city in 2018 were combined with cancer registration data. The optimal prostate-specific antigen levels to predict prostate cancer in different age groups were calculated using receiver operating characteristic curves after determining whether a cancer was registered within one year of screening. At the conventional prostate-specific antigen threshold of 4.0ng/mL, the sensitivity, specificity, and negative predictive value were 94.9%, 91.7%, and 91.7%, respectively. The optimal prostate-specific antigen cutoff values for patients aged 50-59years, 60-69years, 70-79years, and over 80years were 3.900ng/mL, 4.014ng/mL, 4.080ng/mL, and 4.780ng/mL, respectively. The sensitivity and specificity of prostate cancer screening in the city were high, indicating a highly accurate screening. The prostate-specific antigen threshold was 4.78ng/mL in patients older than 80years. A higher prostate-specific antigen threshold may be useful in men over 80years of age to avoid excess biopsy and reduce costs. Our results suggest that the current Japanese method of using PSA 4.0ng/mL as a cutoff regardless of age may not be preferable for older men.

Novel SPEA Superantigen Peptide Agonists and Peptide Agonist-TGFαL3 Conjugate. In Vitro Study of Their Growth-Inhibitory Effects for Targeted Cancer Immunotherapy

Bacterial superantigens (SAgs) are effective T-cell stimulatory molecules that lead to massive cytokine production. Superantigens crosslink between MHC class II molecules on the Antigen Presenting Cells (APC) and TCR on T-cells. This enables them to activate up to 20% of resting T cells, whilst conventional antigen presentation results in the activation of 0.001–0.0001% of the T cell population. These biological properties of superantigens make them attractive for use in immunotherapy. Previous studies have established the effectiveness of superantigens as therapeutic agents. This, however, was achieved with severe side effects due to the high lethality of the native toxins. Our study aims to produce superantigen-based peptides with minimum or no lethality for safer cancer treatment. In previous work, we designed and synthesized twenty overlapping SPEA-based peptides and successfully mapped regions in SPEA superantigen, causing a vasodilatory response. We screened 20 overlapping SPEA-based peptides designed and synthesized to cover the whole SPEA molecule for T-cell activation and tumor-killing ability. In addition, we designed and synthesized tumor-targeted superantigen-based peptides by fusion of TGFαL3 either from the N′ or C′ terminal of selected SPEA-based peptides with an eight-amino acid flexible linker in between. Our study identified parts of SPEA capable of stimulating human T-cells and producing different cytokines. We also demonstrated that the SPEA-based peptide conjugate binds specifically to cancer cells and can kill this cancer. Peptides induce T-cell activation, and tumor killing might pave the way for safer tumor-targeted superantigens (TTS). We proposed the combination of our new superantigen-based peptide conjugates with other immunotherapy techniques for effective and safer cancer treatment.

Safety and clinical activity of autologous RNA chimeric antigen receptor T-cell therapy in myasthenia gravis (MG-001): a prospective, multicentre, open-label, non-randomised phase 1b/2a study

Chimeric antigen receptor (CAR) T cells are highly effective in treating haematological malignancies, but associated toxicities and the need for lymphodepletion limit their use in people with autoimmune disease. To explore the use of CAR T cells for the treatment of people with autoimmune disease, and to improve their safety, we engineered them with RNA (rCAR-T)-rather than the conventional DNA approach-to target B-cell maturation antigen (BCMA) expressed on plasma cells. To test the suitability of our approach, we used rCAR-T to treat individuals with myasthenia gravis, a prototypical autoantibody disease mediated partly by pathogenic plasma cells. MG-001 was a prospective, multicentre, open-label, phase 1b/2a study of Descartes-08, an autologous anti-BCMA rCAR-T therapy, in adults (ie, aged ≥18 years) with generalised myasthenia gravis and a Myasthenia Gravis Activities of Daily Living (MG-ADL) score of 6 or higher. The study was done at eight sites (ie, academic medical centres or community neurology clinics) in the USA. Lymphodepletion chemotherapy was not used. In part 1 (phase 1b), participants with Myasthenia Gravis Foundation of America (MGFA) disease class III-IV generalised myasthenia gravis received three ascending doses of Descartes-08 to determine a maximum tolerated dose. In part 2 (phase 2a), participants with generalised myasthenia gravis with MGFA disease class II-IV received six doses at the maximum tolerated dose in an outpatient setting. The primary objective was to establish safety and tolerability of Descartes-08; secondary objectives were to assess myasthenia gravis disease severity and biomarkers in participants who received Descartes-08. This trial is registered with clinicaltrials.gov, NCT04146051. We recruited 16 individuals for screening between Jan 7, 2020 and Aug 3, 2022. 14 participants were enrolled (n=3 in part 1, n=11 in part 2). Ten participants were women and four were men. Two individuals did not qualify due to low baseline MG-ADL score (n=1) or lack of generalised disease (n=1). Median follow-up in part 2 was 5 months (range 3-9 months). There was no dose-limiting toxicity, cytokine release syndrome, or neurotoxicity. Common adverse events were headache (six of 14 participants), nausea (five of 14), vomiting (three of 14), and fever (four of 14), which resolved within 24 h of infusion. Fevers were not associated with increased markers of cytokine release syndrome (IL-6, IL-2, and TNF). Mean improvements from baseline to week 12 were -6 (95% CI -9 to -3) for MG-ADL score, -7 (-11 to -3) for Quantitative Myasthenia Gravis score, -14 (-19 to -9) for Myasthenia Gravis Composite score, and -9 (-15 to -3) for Myasthenia Gravis Quality of Life 15-revised score. In this first study of an rCAR-T therapy in individuals with an autoimmune disease, Descartes-08 appeared to be safe and was well tolerated. Descartes-08 infusions were followed by clinically meaningful decreases on myasthenia gravis severity scales at up to 9 months of follow-up. rCAR-T therapy warrants further investigation as a potential new treatment approach for individuals with myasthenia gravis and other autoimmune diseases. Cartesian Therapeutics and National Institute of Neurological Disorders and Stroke of the National Institutes of Health.

Lymph node location and retention properties of DC‐SIGN engagement with the Immunoglobulin Oligomannoses of Follicular Lymphoma

Introduction: The oligomannose-type glycans occupying the surface immunoglobulin antigen-binding site (sIg-Mann) are a tumor-founding post-translational requirement of classic follicular lymphoma (FL). These are universally acquired and a clonal necessity during the entire natural history of FL from the early stages throughout transformation into EZB-DLBCL (Chiodin G, Blood 2021, Odabashian M, Blood 2020). The low-affinity interaction of sIg-Mann with its specific ligand DC-SIGN distinguishes from conventional high-affinity antigen:Ig protein interactions for promoting prolonged low-level growth and prosurvival signals via PI3K/AKT and not endocytosis (Linley A, Blood 2015). However, the location and function of DC-SIGN remains to be further elucidated. Here, we investigated the hypothesis that the specific DC-SIGN:Ig-Mann interaction functions to promote retention of FL cells in tissue niches maintaining selective survival advantage. Methods: Immunofluorescence imaging was used to determine DC-SIGN histological location in primary FL lymph nodes. In WSU-FSCCL cells, DC-SIGN effects on sIg redistribution were measured by dSTORM; adhesion to VCAM-1 in presence/absence of inhibitors was measured by flow cytometry. Results: Immunofluorescence of lymph nodes from FL patients revealed that DC-SIGN was expressed on interfollicular CD163+ macrophages and, remarkably, on CD23+ follicular dendritic cells (FDC), claiming an influence on FL cell retention and survival. dSTORM revealed that DC-SIGN induced less dense and more diffuse surface Ig clusters compared to anti-Ig, reflecting an immature sIg redistribution unable to promote endocytosis and death. However, DC-SIGN efficiently induced adhesion of sIg-Mann+ve lymphoma cells to VCAM-1, known to be expressed on both lymph node macrophages and FDC, and inhibited migration towards SDF-1 in vitro. Blocking of DC-SIGN carbohydrate-recognition domain completely abrogated DC-SIGN-induced adhesion. Although intracellular signals were significantly lower, DC-SIGN induced levels of adhesion similar to those induced by anti-Ig. Adhesion to VCAM-1 was observed at concentrations from 20 µg/ml down to 20 ng/ml, even when AKT and ERK phosphorylation was not detectable. Either proximal inhibition of the PI3K/AKT pathway, or distal inhibition of ARP2/3, formin, and Cdc42 for lamellipodium formation at the surface membrane, suppressed adhesion. Conclusions: These results reveal an important mechanism of the DC-SIGN:sIg-Mann interaction that fine-tunes signals for membrane adaptation towards tumor cell adhesion, without promotion of cell death. This possibly facilitates FL cell retention in the lymph node protected tumor niche where selective growth and survival signals are maintained. Interrupting this interaction will be a novel way towards tumour-specific targeted therapy in FL patients. The research was funded by: This research was funded by Blood Cancer UK (grant 18009), Cancer Research UK (ECRIN-M3 accelerator award C42023/A29370, program C2750/A23669, and BTERP project C36811/A29101). D.T. was funded by the Eyles Cancer Immunology PhD scholarship, G.C. was funded by the Eyles Cancer Immunology Fellowship and the Southampton Cancer Immunology Centre Pump-priming award 2021). Keywords: Indolent non-Hodgkin lymphoma, Microenvironment, Targeting the Tumor Microenvironment Conflicts of interests pertinent to the abstract. F. Forconi Employment or leadership position: University of Siena Consultant or advisory role: Beigene Honoraria: Abbvie, Janssen-cilag, Beigene, Astra-Zeneca Research funding: Cancer Research UK Educational grants: Beigene, Abbvie Other remuneration: BC Platform

Beads- and oil-free single molecule assay with immuno-rolling circle amplification for detection of SARS-CoV-2 from saliva

Digital enzyme linked immunosorbent assays (ELISA) can be used to detect various antigens such as spike (S) or nucleocapsid (N) proteins of SARS-CoV-2, with much higher sensitivity compared to that achievable using conventional antigen tests. However, the use of microbeads and oil for compartmentalization in these assays limits their user-friendliness and causes loss of assay information due to the loss of beads during the process. To improve the sensitivity of antigen test, here, we developed an oil- and bead-free single molecule counting assay, with rolling circle amplification (RCA) on a substrate. With RCA, the signal is localized at the captured region of an antigen, and the signal from a single antigen molecule can be visualized using the same immune-reaction procedures as in the conventional ELISA. Substrate-based single molecule assay was theoretically evaluated for kd value, and the concentration of capture and detection antibodies. As a feasibility test, biotin-conjugated primer and mouse IgG conjugates were detected even at femto-molar concentrations with this digital immuno-RCA. Using this method, we detected the N protein of SARS-CoV-2 with a limit of detection less than 1 pg/mL more than 100-fold improvement compared to the detection using conventional ELISA. Furthermore, testing of saliva samples from COVID-19 patients and healthy controls (n = 50) indicated the applicability of the proposed method for detection of SARS-CoV-2 with 99.5% specificity and 90.9% sensitivity.