Antigen Clearance Research Articles

Exploring the reasons for liver cancer occurrence still after long-term nucleos(t)ide analogue therapy in patients with chronic hepatitis B

Chronic hepatitis B virus (HBV) infection is the main etiology of viral hepatitis, cirrhosis, and primary hepatocellular carcinoma and is also a major public health problem worldwide. With the progression of chronic infection, HBV DNA continuously integrates into host DNA and brings about integration and insertion mutations within host genes. In the process of repeated chronic inflammatory necrosis and compensatory regeneration, hepatocytes carrying HBV DNA integration have an advantage in proliferation and frequent clonal expansion formation that causes the accumulation of mutations over time and ultimately malignant transformation of hepatocytes. Although nucleos(t)ide analogs (NAs), currently the most widely used, can effectively inhibit viral replication, delay disease progression in patients with chronic hepatitis B, and significantly reduce the occurrence of end-stage liver disease, many patients still progress to liver cancer. Furthermore, even among clinically cured patients with hepatitis B surface antigen clearance, NAs have not significantly reduced the long-term occurrence of liver cancer, suggesting that their impact under previous treatment strategies for reducing the risk of liver cancer is limited. This could be due to antiviral treatment initiation at a time when patients already have numerous integrated cell proliferation colonies. Hence, NAs have not had any therapeutic impact on the expression of integrated HBV DNA fragments and viral proteins, such as hepatitis B surface antigen. In light of this, we suggest initiating antiviral treatment as early as possible to pursue clinical cure and ultimately reduce the risk of liver cancer in patients with chronic HBV infection.

Effect of dyslipidemia on HBsAg clearance in nucleos(t)ide analogues-experienced chronic hepatitis B patients treated with peginterferon alfa

BackgroundWhile previous reports have shown that hepatitis B virus (HBV) infection affects lipid metabolism and vice versa, the impact of dyslipidemia on the functional cure of HBV infection following peginterferon alfa (PegIFNα) therapy remains unknown. Hence, this study aimed to investigate the effect of dyslipidemia on hepatitis B surface antigen (HBsAg) clearance and develop a nomogram model for predicting patients for whom PegIFNα therapy is indicated.MethodsA total of 160 nucleos(t)ide analogues (NAs)- experienced chronic hepatitis B (CHB) patients treated with PegIFNα (180 µg/week) were enrolled in this study. The relationship between serum lipid and HBsAg clearance was analysed. Univariate and multivariate COX analyses were used to construct and plot the nomogram model. The area under the receiver operating characteristic curve (AUC) and calibration curve were used to evaluate the discrimination and calibration of the model, respectively.ResultsAfter 48 weeks of PegIFNα therapy, a total of 33 patients in the cohort achieved HBsAg clearance. Univariate and multivariate COX analyses indicated that dyslipidemia was significantly associated with HBsAg clearance and was an independent predictor of HBsAg clearance (HR = 0.243, P = 0.001). Kaplan-Meier survival analyses show that cumulative HBsAg clearance was significantly higher in the normolipidemic group than in the dyslipidemia group (log-rank test, P = 0.007). During the treatment, triglyceride showed an increasing trend, while the levels of total cholesterol, high-density lipoprotein, low-density lipoprotein, apolipoprotein A1 and apolipoprotein B decreased. Dyslipidemia and other indicators independently associated with HBsAg clearance were used to construct the nomogram model. The AUC of the model at 36-week and 48-week were 0.879 and 0.856, and the model demonstrated good discrimination and calibration.ConclusionDyslipidemia can affect the antiviral efficacy of PegIFNα in NAs-experienced CHB patients. Our findings suggest that the nomogram model constructed using serum lipid has good predictive power and may help physicians to identify the superior patients for PegIFNα therapy.

Differential T-cell profiles determined by Hepatitis B surface antigen decrease among people with Human Immunodeficiency Virus /Hepatitis B Virus coinfection on treatment

BackgroundSeveral studies have reported that combination antiretroviral therapy (cART) enhances the hepatitis B surface antigen (HBsAg) clearance rate in Human Immunodeficiency Virus-1/Hepatitis B Virus (HIV/HBV) coinfected patients, yet the associated immunological characteristics remain unclear.MethodsGlobal and specific immune phenotypic profiles were examined in 48 patients with HIV/HBV coinfection before cART and at 1-year, and 3-year after cART using flow cytometry. In addition, 61 patients with HBV monoinfection were included for comparison.ResultsHBsAg response (sAg-R) was defined as > 0.5 log decrease within six months of cART initiation, and 16 patients achieved it. Patients with sAg-R (the sAg-R group) exhibited distinct immune phenotypes compared to those of HBsAg-retained patients (the sAg-NR group). Notably, patients with sAg-R had lower CD4+ T cell counts and a higher number of HBcAg-specific T cells. Further, the sAg-R group exhibited upregulation of HLA-DR, Ki67, and PD-1 in CD4+ T cells and heightened HLA-DR and T-bet in CD8+ T cells. However, the sAg-R group had fewer TEMRA cells but more TEM and Th17 cells than those in the sAg-NR group. Expression of various markers, including HLA-DR+CD4+, Ki67+CD4+, PD-1+CD4+, CD38+CD8+, HLA-DR+CD8+, TIM-3+CD8+, HBV-specific CD4+ T cell secreting IFN-γ and IL-2, and specific CD8+ T cell secreting IFN-γ and IL-2, correlated with HBsAg decrease.ConclusionThe decline in HBsAg levels during cART in HIV/HBV coinfection involves significant alterations in CD4+ and CD8+ T cells phenotypes, offering a novel perspective on a functional HBV cure.

Slow clearance of histidine-rich protein-2 in Gabonese with uncomplicated malaria.

Detecting Plasmodium falciparum, the parasite responsible for the severest form of malaria, typically involves microscopy, polymerase chain reaction (PCR), or rapid diagnostic tests (RDTs) targeting the histidine-rich protein 2 or 3 (HRP2/3). While microscopy and PCR quickly turn negative after the infection is cleared, HRP2 remains detectable for a prolonged period. The exact duration of HRP2 persistence had not been well defined. Our study in Gabon tracked HRP2 levels over 4 weeks, resulting in a new model for antigen clearance. We discovered that a two-compartment model accurately predicts HRP2 levels, revealing an initial rapid reduction followed by a much slower elimination phase that can take several weeks. These findings are crucial for interpreting RDT results, as lingering HRP2 can lead to false positives, impacting malaria diagnosis and treatment decisions.

Management and outcomes of histoplasmosis in youth with inflammatory bowel disease in an endemic area.

Patients with inflammatory bowel disease (IBD) prescribed immunosuppressive therapies including antitumor necrosis factor (aTNF) therapies are at increased risk of histoplasmosis. We aim to evaluate the presentation, management, and outcomes of youth with IBD and concurrent histoplasmosis. Single center, retrospective review of youth with IBD diagnosed with histoplasmosis from January 12, 2007 to January 1, 2022. Management and outcomes were followed for up to 2 years after diagnosis. Nineteen patients (10 male, median age 16 years, range 8-22) with IBD were diagnosed with histoplasmosis: disseminated (N = 15/19; 79%), pulmonary (N = 3/19; 16%), lymph node (N = 1/19; 5%). At the time of histoplasmosis diagnosis, patients were predominantly receiving aTNF therapy (N = 17/19; 89%, median duration 21.9 months (interquartile range 8.5-52.0). Thirteen (13/19, 68%) patients required hospitalization and 2/19 (11%) required intensive care. All achieved antigen clearance with no recurrences. At the time of histoplasmosis diagnosis, aTNF was stopped in 15/17 (88%) patients and the following IBD therapies were initiated: 5-aminosalicylates (N = 4/19; 21%), 6-mercaptopurine (N = 3/19; 16%), enteral therapy (N = 2/19; 11%), and vedolizumab (N = 2/19; 11%); 6 of 19 (32%) received no IBD therapy and 2 of 19 (11%) patients continued aTNF. During follow-up, 6 of 19 (32%) patients had an emergency department (ED) visit and/or hospitalization for symptoms attributed to active IBD, all of whom had discontinued aTNF; one patient required colectomy. Severe histoplasmosis infection in youth with IBD was rare. IBD treatment was modified by reducing immunosuppression. Histoplasmosis outcomes were favorable, but multiple patients required hospitalization or ED visits for IBD symptoms. The optimal approach to managing IBD during histoplasmosis treatment is challenging and requires further study.

Association of hepatitis B core antibody level and hepatitis B surface antigen clearance in HBeAg-negative patients with chronic hepatitis B

ABSTRACT Predicting hepatitis B surface antigen (HBsAg) clearance is important for chronic hepatitis B (CHB) patients receiving pegylated interferon-alfa (Peg-IFN) therapy. We aimed to determine the predictive value of serum hepatitis B core antibody (anti-HBc) for HBsAg clearance. A total of 189 HBeAg-negative CHB patients who received Peg-IFN based therapy were retrospectively included and classified into two groups: nucleos(t)ide analogues (NAs) add-on Peg-IFN group (add-on group, n = 94) and Peg-IFN combined with NAs or Peg-IFN monotherapy group (combination or monotherapy group, n = 95). After 48 weeks of treatment, 27.5% (52/189) and 15.9% (30/189) of patients achieved HBsAg clearance and seroconversion, respectively. Patients in the combination or monotherapy group tended to achieve relatively higher HBsAg clearance (31.6% vs. 23.4%, p = 0.208) and seroconversion (21.1% vs. 10.6%, p = 0.050) rates than those in the add-on group. In combination or monotherapy group, anti-HBc levels at week 12 were lower in patients with HBsAg clearance (9.0 S/CO vs. 9.9 S/CO, p < 0.001) and seroconversion (8.8 S/CO vs. 9.8 S/CO, p < 0.001) than those without. Anti-HBc level at week 12 was an independent predictor of HBsAg clearance and seroconversion. Patients with lower anti-HBc levels at week 12 showed a more significant decline in HBsAg levels during treatment. Combination of anti-HBc at week 12 and baseline HBsAg could identify over 70% of patients who achieved HBsAg clearance after 48 weeks of treatment. In addition to HBsAg, anti-HBc level could be used as a promising marker for selecting HBeAg-negative CHB patients who are more likely to respond to Peg-IFN-based therapy.

Ciliary Function, Antigen Stasis and Asthma.

The prevalence of asthma exceeds 3% of the population. Asthma is observed to be more common in children following severe viral lower respiratory illnesses that affect ciliary function, but mechanisms linking ciliary function to asthma pathogenesis have been obscure. Recent data regarding primary ciliary dyskinesia (PCD) may help us to understand the association. Here, I will review what is known about the relationship between ciliary function and asthma. PCD is caused by pathologic variants in over 50 different genes that affect the structure and function of motile cilia. At the cellular level, a characteristic feature shared by most PCD patients is that antigens and other particles are not cleared from the epithelial surface. Poor antigen clearance results in pro-oxidant pathway activation and airway epithelial damage and may predispose PCD patients to DUOX1- and IL33-mediated asthma. Secondary ciliary dysfunction, such as that caused by viruses or by smoking, can also contribute to asthma development. Moreover, variants in genes that affect the function of cilia can be associated with poor lung function, even in the absence of PCD, and with increased asthma severity. The role of antigen stasis on the surface of dysfunctional airway cilia in the pathophysiology of asthma is a novel area for research, because specific airway clearance techniques and other therapeutic interventions, such as antioxidants, could be of value in preventing the development of asthma.

PPARβ/δ-orchestrated metabolic reprogramming supports the formation and maintenance of memory CD8+ T cells.

The formation of memory T cells is a fundamental feature of adaptative immunity, allowing the establishment of long-term protection against pathogens. Although emerging evidence suggests that metabolic reprogramming is crucial for memory T cell differentiation and survival, the underlying mechanisms that drive metabolic rewiring in memory T cells remain unclear. Here, we found that up-regulation of the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) instructs the metabolic reprogramming that occurs during the establishment of central memory CD8+ T cells. PPARβ/δ-regulated changes included suppression of aerobic glycolysis and enhancement of oxidative metabolism and fatty acid oxidation. Mechanistically, exposure to interleukin-15 and expression of T cell factor 1 facilitated activation of the PPARβ/δ pathway, counteracting apoptosis induced by antigen clearance and metabolic stress. Together, our findings indicate that PPARβ/δ is a master metabolic regulator orchestrating a metabolic switch that may be favorable for T cell longevity.

In silico design of multi-epitope vaccines against the hantaviruses by integrated structural vaccinology and molecular modeling approaches.

Hantaviruses are single-stranded RNA viruses belonging to the family Bunyaviridae that causes hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS) worldwide. Currently, there is no effective vaccination or therapy available for the treatment of hantavirus, hence there is a dire need for research to formulate therapeutics for the disease. Computational vaccine designing is currently a highly accurate, time and cost-effective approach for designing effective vaccines against different diseases. In the current study, we shortlisted highly antigenic proteins i.e., envelope, and nucleoprotein from the proteome of hantavirus and subjected to the selection of highly antigenic epitopes to design of next-generation multi-epitope vaccine constructs. A highly antigenic and stable adjuvant was attached to the immune epitopes (T-cell, B-cell, and HTL) to design Env-Vac, NP-Vac, and Com-Vac constructs, which exhibit stronger antigenic, non-allergenic, and favorable physiochemical properties. Moreover, the 3D structures were predicted and docking analysis revealed robust interactions with the human Toll-like receptor 3 (TLR3) to initiate the immune cascade. The total free energy calculated for Env-Vac, NP-Vac, and Com-Vac was -50.02 kcal/mol, -24.13 kcal/mol, and -62.30 kcal/mol, respectively. In silico cloning, results demonstrated a CAI value for the Env-Vac, NP-Vac, and Com-Vac of 0.957, 0.954, and 0.956, respectively, while their corresponding GC contents were 65.1%, 64.0%, and 63.6%. In addition, the immune simulation results from three doses of shots released significant levels of IgG, IgM, interleukins, and cytokines, as well as antigen clearance over time, after receiving the vaccine and two booster doses. Our vaccines against Hantavirus were found to be highly immunogenic, inducing a robust immune response that demands experimental validation for clinical usage.

Systematic review with network meta-analysis: sustained hepatitis B surface antigen clearance after pegylated interferon cessation.

The efficacy of different pegylated interferon (PEG-IFN) treatment strategies for achieving sustained hepatitis B surface antigen (HBsAg) clearance in chronic hepatitis B (CHB) remains controversial. This study assesses the efficacy of different PEG-IFN treatment regimens and factors influencing sustained HBsAg clearance after PEG-IFN discontinuation. PubMed , Embase , Web of Science , and the Cochrane Library databases were searched from inception to June 2023, regarding PEG-IFN therapy in CHB. Methodological quality was assessed using the Cochrane risk of bias tool. We explored sources of heterogeneity through univariate meta-regression. Frequentist network meta-analyses were used to compare the efficacy of different PEG-IFN treatment strategies. We analyzed 53 studies (including 9338 CHB patients). After PEG-IFN withdrawal, the annual rates of HBsAg clearance and seroconversion were 6.9% [95% confidence interval (CI), 5.10-9.31] and 4.7% (95% CI, 2.94-7.42). The pooled 1-, 3-, and 5-year sustained HBsAg clearance rates were 7.4%, 9.9%, and 13.0%, and the sustained HBsAg seroconversion rates were 6.6%, 4.7%, and 7.8%, respectively. HBsAg quantification, hepatitis B e antigen status, and PEG-IFN treatment protocols were major sources of heterogeneity. Baseline HBsAg quantification was significantly lower in patients with sustained HBsAg clearance versus those without ( P < 0.046). PEG-IFN combined with tenofovir has the highest probability of achieving HBsAg seroconversion (surface under the cumulative ranking of 81.9%). Sustained HBsAg clearance increased approximately linearly from years 1 to 5 after PEG-IFN discontinuation. Low baseline HBsAg quantification has a significant impact on sustained HBsAg clearance. PEG-IFN combined with tenofovir may be optimal in achieving sustained HBsAg seroconversion.

Serum sPD-1 and sPD-L1 as predictive biomarkers for HBsAg clearance in HBeAg-negative CHB patients undergoing IFN-based therapy.

For chronic hepatitis B (CHB) patients, there is still a need to improve hepatitis B surface antigen (HBsAg) clearance rates. This study aimed to assess the predictive effectiveness of soluble programmed cell death-1 (sPD-1) and soluble programmed cell death ligand-1 (sPD-L1) for HBsAg clearance in HBeAg-negative CHB patients undergoing peginterferon (Peg-IFN)-based antiviral treatment. This study encompassed 280 patients undergoing treatment with Peg-IFNα. Serum levels of sPD-1 and sPD-L1 were measured using ELISA kits at baseline, as well as at 12, 24 and 48 weeks. The primary endpoint of the study was the determination of HBsAg clearance at 48 weeks. Logistic regression analysis was employed to identify predictors of HBsAg clearance. The clearance group demonstrated significantly lower serum sPD-L1 levels compared to the non-clearance group. While both groups exhibited an increase in sPD-1 levels, only the clearance group showed a rise in sPD-L1 levels. Multivariate analysis identified sPD-L1 increase at 24 weeks, and HBsAg decline at 24 weeks as predictors for HBsAg clearance at 48 weeks. The combined use of these indicators showed a predictive performance for HBsAg clearance with an AUROC of 0.907 (95% CI: 0.861-0.953, p < 0.001). The study revealed an inverse relationship between the trends of sPD-1/sPD-L1 and HBsAg clearance during combined IFN and NAs treatment. Moreover, the magnitude of HBsAg reduction and sPD-L1 increase emerged as significant predictors for HBsAg clearance.

Defect-Rich Metastable MoS2 Promotes Macrophage Reprogramming in Breast Cancer: A Clinical Perspective.

Tumor-associated macrophages (TAMs) play a crucial function in solid tumor antigen clearance and immune suppression. Notably, 2D transitional metal dichalcogenides (i.e., molybdenum disulfide (MoS2) nanozymes) with enzyme-like activity are demonstrated in animal models for cancer immunotherapy. However, in situ engineering of TAMs polarization through sufficient accumulation of free radical reactive oxygen species for immunotherapy in clinical samples remains a significant challenge. In this study, defect-rich metastable MoS2 nanozymes, i.e., 1T2H-MoS2, are designed via reduction and phase transformation in molten sodium as a guided treatment for human breast cancer. The as-prepared 1T2H-MoS2 exhibited enhanced peroxidase-like activity (≈12-fold enhancement) than that of commercial MoS2, which is attributed to the charge redistribution and electronic state induced by the abundance of S vacancies. The 1T2H-MoS2 nanozyme can function as an extracellular hydroxyl radical generator, efficiently repolarizing TAMs into the M1-like phenotype and directly killing cancer cells. Moreover, the clinical feasibility of 1T2H-MoS2 is demonstrated via ex vivo therapeutic responses in human breast cancer samples. The apoptosis rate of cancer cells is 3.4 times greater than that of cells treated with chemotherapeutic drugs (i.e., doxorubicin).

Selection and evaluation of single domain antibody against p19 subunit of IL-23 by phage display for potential use as an autoinflammatory therapeutic

IL-23 is a double-subunit cytokine that plays an important role in shaping the immune response. IL-23 was found to be associated with several autoinflammatory diseases by generating sustained inflammatory loops that lead to tissue damage. Antibody neutralization of IL-23 was proven to be effective in ameliorating associated diseases. However, antibodies as large proteins have limited tissue penetration and tend to elicit anti-drug antibodies. Additionally, anti-IL-23 antibodies target only one subunit of IL-23 leaving the other one unneutralized. Here, we attempted to isolate a recycling single domain antibody by phage display. One of IL-23 subunits, p19, was expressed in E. coli fused to Gamillus protein to stabilize the α-helix-only p19. To remove Gamillus binders, two biopanning methods were investigated, first, preselection with Gamillus and second, challenge with IL-23 then on the subsequent round challenge with p19-Gam. The isolation of calcium-dependent and pH-dependent recycling binders was performed with EDTA and citrate buffers respectively. Both methods of panning failed to isolate high-affinity and specific p19 recycling binders, while from the second panning method, a high affinity and specific p19 standard binder, namely H11, was successfully isolated. H11 significantly inhibited the gene expression of IL-17 and IL-22 in IL-23-challenged PBMCs indicating H11 specificity and neutralizing ability for IL-23. The new binder due to its small size can overcome antibodies limitations, also, it can be further engineered in the future for antigen clearance such as fusing it to cell penetrating peptides, granting H11 the ability to clear excess IL-23 and enhancing its potential therapeutic effect.

Subtractive proteomics-guided vaccine targets identification and designing of multi-epitopes vaccine for immune response instigation against Burkholderia pseudomallei

In this study, a methodical workflow using subtractive proteomics, vaccine designing, molecular simulation, and agent-based modeling approaches were used to annotate the whole proteome of Burkholderia pseudomallei (strain K96243) for vaccine designing. Among the total 5717 proteins in the whole proteome, 505 were observed to be essential for the pathogen's survival and pathogenesis predicted by the Database of Essential Genes. Among these, 23 vaccine targets were identified, of which fimbrial assembly chaperone (Q63UH5), Outer membrane protein (Q63UH1), and Hemolysin-like protein (Q63UE4) were selected for the subsequent analysis based on the systematic approaches. Using immunoinformatic approaches CTL (cytotoxic T lymphocytes), HTL (helper T lymphocytes), IFN-positive, and B cell epitopes were predicted for these targets. A total of 9 CTL epitopes were added using the GSS linker, 6 HTL epitopes using the GPGPG linker, and 6 B cell epitopes using the KK linker. An adjuvant was added for enhanced antigenicity, an HIV-TAT peptide for improved delivery, and a PADRE sequence was added to form a 466 amino acids long vaccine construct. The construct was classified as non-allergenic, highly antigenic, and experimentally feasible. Molecular docking results validated the robust interaction of MEVC with immune receptors such as TLR2/4. Furthermore, molecular simulation revealed stable dynamics and compact nature of the complexes. The binding free energy results further validated the robust binding. In silico cloning, results revealed GC contents of 50.73 % and a CIA value of 0.978 which shows proper downstream processing. Immune simulation results reported that after the three injections of the vaccine a robust secondary immune response, improved antigen clearance, and effective immune memory generation were observed highlighting its potential for effective and sustained immunity. Future directions should encompass experimental validations, animal model studies, and clinical trials to substantiate the vaccine's efficacy, safety, and immunogenicity.

SARS-CoV-2 RNA and Nucleocapsid Antigen Are Blood Biomarkers Associated With Severe Disease Outcomes That Improve in Response to Remdesivir.

Although antivirals remain important for the treatment COVID-19, methods to assess treatment efficacy are lacking. Here, we investigated the impact of remdesivir on viral dynamics and their contribution to understanding antiviral efficacy in the multicenter Adaptive COVID-19 Treatment Trial 1, which randomized patients to remdesivir or placebo. Longitudinal specimens collected during hospitalization from a substudy of 642 patients with COVID-19 were measured for viral RNA (upper respiratory tract and plasma), viral nucleocapsid antigen (serum), and host immunologic markers. Associations with clinical outcomes and response to therapy were assessed. Higher baseline plasma viral loads were associated with poorer clinical outcomes, and decreases in viral RNA and antigen in blood but not the upper respiratory tract correlated with enhanced benefit from remdesivir. The treatment effect of remdesivir was most pronounced in patients with elevated baseline nucleocapsid antigen levels: the recovery rate ratio was 1.95 (95% CI, 1.40-2.71) for levels >245 pg/mL vs 1.04 (95% CI, .76-1.42) for levels <245 pg/mL. Remdesivir also accelerated the rate of viral RNA and antigen clearance in blood, and patients whose blood levels decreased were more likely to recover and survive. Reductions in SARS-CoV-2 RNA and antigen levels in blood correlated with clinical benefit from antiviral therapy. NCT04280705 (ClinicalTrials.gov).

Prediction model for hepatitis B e antigen seroconversion in chronic hepatitis B with peginterferon-alfa treated based on a response-guided therapy strategy.

Models for predicting hepatitis B e antigen (HBeAg) seroconversion in patients with HBeAg-positive chronic hepatitis B (CHB) after nucleos(t)ide analog treatment are rare. To establish a simple scoring model based on a response-guided therapy (RGT) strategy for predicting HBeAg seroconversion and hepatitis B surface antigen (HBsAg) clearance. In this study, 75 previously treated patients with HBeAg-positive CHB underwent a 52-week peginterferon-alfa (PEG-IFNα) treatment and a 24-wk follow-up. Logistic regression analysis was used to assess parameters at baseline, week 12, and week 24 to predict HBeAg seroconversion at 24 wk post-treatment. The two best predictors at each time point were used to establish a prediction model for PEG-IFNα therapy efficacy. Parameters at each time point that met the corresponding optimal cutoff thresholds were scored as 1 or 0. The two most meaningful predictors were HBsAg ≤ 1000 IU/mL and HBeAg ≤ 3 S/CO at baseline, HBsAg ≤ 600 IU/mL and HBeAg ≤ 3 S/CO at week 12, and HBsAg ≤ 300 IU/mL and HBeAg ≤ 2 S/CO at week 24. With a total score of 0 vs 2 at baseline, week 12, and week 24, the response rates were 23.8%, 15.2%, and 11.1% vs 81.8%, 80.0%, and 82.4%, respectively, and the HBsAg clearance rates were 2.4%, 3.0%, and 0.0%, vs 54.5%, 40.0%, and 41.2%, respectively. We successfully established a predictive model and diagnosis-treatment process using the RGT strategy to predict HBeAg and HBsAg seroconversion in patients with HBeAg-positive CHB undergoing PEG-IFNα therapy.

Abstract 52: CRISPR-mediated knock-out of A20 lowers the activation threshold and induces metabolic rewiring of CAR-T cells

Abstract Background: Common failures of chimeric antigen receptor (CAR)-T cell therapy regimens stem from immune escape of target antigen low tumors. Strategies to improve CAR-T cell therapy efficacy include enhancing intrinsic effector function while preventing T cell exhaustion. The ubiquitin-modifying enzyme A20, is a key negative regulator of the NF-kB pathway downstream of T cell receptor activation in T cells. Dysfunctional CD8 T cells in tumors have high expression of A20, and deletion of A20 enhances antitumor activity of CD8 T cells in murine tumor models. We tested whether CRISPR-mediated knock-out (KO) of tumor necrosis factor α-induced protein 3 (TNFAIP3), the gene for A20, increases the effector function and alters the metabolic state of CAR-T cells. Results: A20 deletion led to upregulation of NF-κB target genes and resulted in higher levels of activation marker expression and effector cytokine secretion compared to unarmored CAR-T upon incubation with target cells. In addition, A20 KO CAR-T cells maintained prolonged expression of activation markers upon antigen clearance in the absence of enhanced differentiation, and were more resistant to dysfunction in a repeat challenge serial kill assay. In vivo, A20 deficient CAR-T exhibited increased anti-tumor efficacy against low, medium, and high target expressing tumor models. Consistent with NF-κB upregulation, A20 KO cells significantly upregulated key glycolytic intermediates suggesting that amino acid anabolism was fueled by glycolysis and the pentose phosphate pathway. Moreover, the preferential increase of glycolysis over oxidative phosphorylation confirmed that A20 KO CAR-T cells were skewed towards an effector-like phenotype. Notably, glutathione and its cysteine-glycine bi-products were upregulated upon activation to a higher level in A20 KO CAR-T suggesting enhanced resistance to oxidative stress, in line with their prolonged persistence. Conclusions: CRISPR-mediated knock-out of A20 lowers the activation threshold of CAR-T cells producing enhanced in vitro and in vivo anti-tumor efficacy particularly against tumors with very low levels of target expression. A20 KO induces metabolic rewiring of CAR-T cells by promoting glycolysis and amino acid anabolism to sustain their superior effector functions. This intrinsic armoring strategy can be applied to any CAR-T in any indication to increase tumor control and reduce immune escape. Citation Format: Nina Jia Chu, Carina Nava-Barbero, Ashley Merlino, Ping-Hsien Lee, David Baker, Ben Taylor, Gordon Moody, Letizia Giardino. CRISPR-mediated knock-out of A20 lowers the activation threshold and induces metabolic rewiring of CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 52.

Abstract 2720: Engineering sweeping antibodies for the clearance of tumor-derived, pro-tumorigenic cytokines

Abstract Soluble, tumor-derived, pro-tumorigenic (STP) cytokines have been shown to contribute to cancer cell proliferation and drug resistance. But therapeutic targeting of STP cytokines by monoclonal antibodies is limited by high concentrations and persistent production of antigen. We hypothesize that the ability of sweeping antibodies (swAbs) to actively clear soluble antigens will overcome these limitations. SwAbs differ from conventional antibodies via (1) pH-dependent binding of antigen, and (2) increased binding to the neonatal Fc receptor (FcRn). SwAbs facilitate clearance of soluble antigens by releasing antigen in the low pH environment of FcRn-expressing cells. SwAbs are then recycled via FcRn for repeated clearance of antigen.We are developing swAbs against four STP cytokines contributing to cancer cell proliferation, survival, migration or intra-tumoral angiogenesis: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and interleukin 16 (IL-16). We selected four existing neutralizing antibodies against these cytokines with available crystal structures and generated a second antibody targeting interleukin 16 with high affinity (clone 3B6; KD = 13 nM) de novo using phage display. We are using bio-layer interferometry and custom recycling/clearance assays for antibody characterization and pre-clinical validation. To introduce pH dependency into the four antibodies with crystal structures, we performed targeted histidine mutagenesis of residues at the interface with the respective target antigens. For the de novo-generated interleukin-16 antibody, we performed an unbiased histidine scanning approach by mutating residues across all six complementarity-determining regions (CDRs), which resulted in slight increases in pH sensitivity. We then performed a second round of mutagenesis of the most pH-sensitive 3B6 variants by replacing additional residues considered developability liabilities with histidine residues. We found that this approach significantly increased expression and affinity at pH 7 of the resulting 3B6 variants but only modestly increased pH sensitivity. In contrast, replacing a single residue in the heavy CDR1 in clone 14.1, a pre-clinical anti-IL-16 antibody, resulted in substantially increased pH sensitivity (KD(pH=7) = 2 nM; KD(pH&amp;lt;6) = n.b.). We are currently performing pH-sensitivity screenings for the three additional STP-specific antibodies. Our findings indicate that the ability to introduce pH sensitivity is largely dependent on the parental antibody and that some antibodies may not be amenable to this process. In addition, we show that histidine mutagenesis can be an effective approach to enhance expressibility and binding, while also increasing pH sensitivity. This project will answer the question whether anti-STP cytokine swAbs are an effective approach to treat patients with cancer. Citation Format: Jillian M. Baker, Nevil J. Singh, Tim Luetkens. Engineering sweeping antibodies for the clearance of tumor-derived, pro-tumorigenic cytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2720.

Characteristics and clinical treatment outcomes of chronic hepatitis B children with coexistence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg

BackgroundThe coexistence of hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (HBsAb) represents an uncommon serological pattern observed in patients with hepatitis B virus (HBV) infection, and its underlying mechanism and clinical significance have not been well established. The aim of this study was to investigate the association between this serological profile and clinical treatment outcomes in children with chronic hepatitis B (CHB).MethodsThis retrospective cohort study included 372 treatment-naïve CHB children from the Hunan Children’s Hospital. The participants were categorized into HBsAb-positive group and HBsAb-negative group. The associations between HBsAb positive status to clinical outcomes were assessed using Cox proportional hazard regression. Receiver operating characteristic curve was conducted to evaluate the prediction ability in HBsAg loss.ResultsThe coexistence of HBsAg and HBsAb accounted for 23.39% (87/372) of the participants. The crude incidence rates of HBsAg loss, hepatitis B e antigen (HBeAg) clearance, and HBV-DNA undetectability were higher in the HBsAb-positive group compared with the HBsAb-negative group (37.46 vs. 17.37, 49.51 vs. 28.66, 92.11 vs. 66.54 per 100 person-years, respectively, all P < 0.05). The Cox regression analysis revealed a significant association between this serological profile and an increased likelihood of HBsAg loss (HR = 1.78, P = 0.001), and HBeAg clearance (HR = 1.78, P = 0.001). In addition, a combination of HBsAb ≥ 0.84 log10 IU/L and age ≤ 5 years can help identify patients likely to achieve HBsAg loss after antiviral therapy, with an AUC of 0.71.ConclusionsChildren who are positive for both HBsAg and HBsAb demonstrate a higher probability of favorable outcomes after antiviral treatment. Thus, children with HBsAb-positive CHB should be actively treated to achieve functional cure.

Mechanism underlying severe deficiency of plasma ADAMTS-13 activity in immune thrombotic thrombocytopenic purpura

BackgroundImmune-mediated thrombotic thrombocytopenic purpura is caused by autoantibodies against ADAMTS-13, a plasma enzyme that cleaves von Willebrand factor. However, the mechanism resulting in severe deficiency of plasma ADAMTS-13 activity remains controversial. ObjectivesTo determine the mechanism of autoantibody-mediated severe deficiency of plasma ADAMTS13 activity in immune-mediated thrombotic thrombocytopenic purpura. MethodsFluorescence resonance energy transfer-VWF73 was used to determine plasma ADAMTS-13 activity. Enzyme-linked immunosorbent assay (ELISA) was used to determine anti–ADAMTS-13 immunoglobulin G. ELISA and capillary electrophoresis–based Western blotting were employed to assess plasma ADAMTS-13 antigen. ResultsWe showed that plasma ADAMTS-13 antigen levels varied substantially in the samples collected on admission despite all showing plasma ADAMTS-13 activity of <10 IU/dL (or <10% of normal level) using either ELISA or Western blotting. More severe deficiency of plasma ADAMTS-13 antigen (<10%) was detected in admission samples by ELISA than by capillary Western blotting. There was a significant but moderate correlation between plasma ADAMTS-13 activity and ADAMTS-13 antigen by either assay method, suggesting that severe deficiency of plasma ADAMTS-13 activity is not entirely associated with low levels of ADAMTS-13 antigen. ConclusionWe conclude that severe deficiency of plasma ADAMTS-13 activity primarily resulted from antibody-mediated inhibition, but the accelerated clearance of plasma ADAMTS-13 antigen via immune complexes may also contribute significantly to severe deficiency of plasma ADAMTS-13 activity in a subset of patients with acute immune-mediated thrombotic thrombocytopenic purpura.