Antigen Expression Research Articles

Enrichment of oligodendrocyte precursor phenotypes in subsets of low-grade glioneuronal tumours.

Current histological classification of low-grade glioneuronal tumours does not adequately represent their underlying biology. The neural lineage(s) and differentiation stage(s) involved and the cell state(s) affected by the recurrent genomic alterations are unclear. Here, we describe dysregulated oligodendrocyte lineage developmental programmes in three low-grade glioneuronal tumour subtypes. Ten dysembryoplastic neuroepithelial tumours, four myxoid glioneuronal tumours and five rosette-forming glioneuronal tumours were collected. Besides a comprehensive characterization of clinical features, known diagnostic markers and genomic alterations, we used comprehensive immunohistochemical stainings to characterize activation of rat sarcoma/mitogen-activated protein kinase pathway, involvement of neuronal component, resemblance to glial lineages and differentiation blockage along the stages of oligodendrocyte lineage. The findings were further complemented by gene set enrichment analysis with transcriptome data of dysembryoplastic neuroepithelial tumours from the literature. Dysembryoplastic neuroepithelial tumours, myxoid glioneuronal tumours and rosette-forming glioneuronal tumours occur at different ages, with symptoms closely related to tumour location. Dysembryoplastic neuroepithelial tumours and myxoid glioneuronal tumours contain oligodendrocyte-like cells and neuronal component. Rosette-forming glioneuronal tumours contained regions of rosette-forming neurocytic and astrocytic features. Scattered neurons, identified by neuronal nuclei antigen and microtubule-associated protein-2 staining, were consistently observed in all dysembryoplastic neuroepithelial tumours and myxoid glioneuronal tumours examined, but only in one rosette-forming glioneuronal tumour. Pervasive neurofilament-positive axons were observed only in dysembryoplastic neuroepithelial tumour and myxoid glioneuronal tumour samples. Alterations in B-Raf proto-oncogene, serine/threonine kinase, fibroblast growth factor receptor 1, fibroblast growth factor receptor 3 and platelet-derived growth factor receptor alpha occurred in a mutually exclusive manner, coinciding with strong staining of phospho-p44/42 mitogen-activated protein kinase and low apoptotic signal. All dysembryoplastic neuroepithelial tumours, myxoid glioneuronal tumours and the neurocytic regions of rosette-forming glioneuronal tumours showed strong expression of neuron-glia antigen 2, platelet-derived growth factor receptor alpha (markers of oligodendrocyte precursor cells) and neurite outgrowth inhibitor-A (a marker of developing oligodendrocytes), but lacked the expression of oligodendrocyte markers ectonucleotide pyrophosphatase/phosphodiesterase family member 6 and myelin basic protein. Notably, transcriptomes of dysembryoplastic neuroepithelial tumours were enriched in oligodendrocyte precursor cell signature, but not in signatures of neural stem cells, myelinating oligodendrocytes and astrocytes. Dysembryoplastic neuroepithelial tumour, myxoid glioneuronal tumour and rosette-forming glioneuronal tumour resemble oligodendrocyte precursor cells, and their enrichment of oligodendrocyte precursor cell phenotypes is closely associated with the recurrent mutations in rat sarcoma/mitogen-activated protein kinase pathway.

Electrospun photothermal tellurium nanosheet-loaded polycaprolactone/gelatin nanofibers to promote wound healing

Low-cost and biodegradable photothermal wound dressings with remarkable therapeutic effects are highly desirable for next-generation wound healing. Herein, we report an efficient photothermal wound dressing mat made of tellurium nanosheet (TeNS)-loaded electrospun polycaprolactone/gelatin (PCL/GEL) nanofibers. The TeNS-loaded PCL/GEL nanofibrous architectures showed antibacterial efficacy against Escherichia coli and Staphylococcus aureus of 87.68% and 94.57%, respectively. Under near-infrared (NIR) light illumination, they can facilitate cell proliferation as revealed by in vitro scratch assay. The results from in vivo skin wounds combined with tissue staining experiments further showed that the TeNS-loaded dressing could substantially promote wound healing under photothermal conditions. Using immunohistochemical analysis, we found that the TeNS-loaded PCL/GEL nanofibers [Formula: see text] NIR group have a high expression of specific antigens in epidermal growth factor (EGF) ([Formula: see text]) and endothelial cell adhesion molecule-31 (CD31, [Formula: see text]), verifying that the nanofibrous mat can stimulate EGF generation and microvessel proliferation. Furthermore, the PCL/GEL/Te[Formula: see text]NIR group has the lowest expression in endothelial cell adhesion molecule-68 (CD68, [Formula: see text]), suggesting that the nanofibrous mats have a high anti-inflammatory efficiency. Our work sheds light on the development of novel nonanti-inflammatory wound dressings via photothermal sterilization and the promotion of cell growth using two-dimensional (2D) nanosheets.

Targeted Drug Delivery by MMAE Farnesyl-Bioconjugated Multivalent Chemically Self-Assembled Nanorings Induces Potent Receptor-Dependent Immunogenic Cell Death.

Antibody-drug conjugates, nanoparticles, and liposomes have been used for anticancer drug delivery. The success of targeted killing of cancer cells relies heavily on the selectivity of the drug delivery systems. In most systems, antibodies or their fragments were used as targeting ligands. In this study, we have investigated the potential for protein-based octomeric chemically self-assembled nanorings (CSANs) to be used for anticancer drug delivery. The CSANs are composed of a DHFR-DHFR fusion protein incorporating an EGFR-targeting fibronectin and the anticancer drug MMAE conjugated through a C-terminal farnesyl azide. The anti-EGFR-MMAE CSANs were shown to undergo rapid internalization and have potent cytotoxicity to cancer cells across a 9000-fold difference in EGFR expression. In addition, anti-EGFR-MMAE CSANs were shown to induce immunological cell death. Thus, multivalent and modular CSANs are a potential alternative anticancer drug delivery platform with the capability of targeting tumor cells with heterogeneous antigen expression while activating the anticancer immune response.

Abstract PO1-26-05: Animal Study Compares CAR T cell Exhaustion & Ability to Kill Low Antigen expressing breast cancer cells among three CAR constructs including one with 1XX mutations

Abstract Background: CAR T cells for the treatment of solid tumor cancers has not yet achieved the same success as CAR T cells for treatment of blood cancers. Two of the hurdles that must be cleared for effective use of CAR T cells for solid tumor cancers are: 1) CAR T cell exhaustion; and 2) failure to recognize and kill low antigen expressing cancer cells. A promising approach to overcoming CAR T cell exhaustion, referred to as “1XX” was developed in the Sadelain Lab at MSKCC. Mutation of Tyrosines to Phenylalanine in ITAMs 2 and 3, of the CD3z signaling domain, restrict signaling to ITAM 1. This slowing down of signaling has been reported to be effective at eliminating or greatly reducing CAR T cell exhaustion in blood cancers [Feucht, et al 2019; Park et al 2022; Schoutrop, et al 2023]. Purpose: To evaluate the potential of the 1XX mutations to overcome CAR T cell exhaustion and inability to kill low antigen expressing cells in an animal model of breast cancer. Specifically, we tested the ability of three different CARs to eliminate human breast tumors that expressed variable levels of the target antigen, MUC1*, and their ability to suppress tumor recurrence over approximately 100 days. Methods: All three CARs were targeted to the tumor by the same antibody fragment, huMNC2, that recognizes MUC1*, which is the transmembrane cleavage product of MUC1 that functions as a potent growth factor receptor. The CARs are: 1) huMNC2-41BB-CD3z; 2) huMNC2-CD28-CD3z; and 3) huMNC2-CD28-1XX. Heterogeneous tumors expressing different levels of target antigen were made as follows. T47D breast cancer cells were engineered to express even more of the target, MUC1*, and were also engineered to fluoresce green. T47D wild type cells were engineered to fluoresce red. Heterogenous tumors consisting of 250,000 cells were implanted sub-cu into female NSG mice bearing 90-day estrogen pellets. The tumors comprised either 70% wild-type/30% overexpressing cells, or 85% wild-type/15% overexpressing cells or 92.5% wild-type/7.5% overexpressing cells. Tumor engraftment was verified by bioluminescence at Day 6 post implantation. There was a total of 150 animals, 5 animals per group. Animals were administered a single dose of CAR T cells into the tail vein on Day 7, wherein the Effector to Target ratio was either 10:1, 5:1 or 1:1. Tumor volume was measured weekly by Luciferase/Luciferin bioluminescence on an IVIS instrument. In addition, the red versus green fluorescence of the tumor was tracked periodically as an indicator of which cells, high or low antigen expressing, were being killed. Between Day 93 and Day 96, animals were sacrificed, cells were recovered from blood and spleen, recovered tumors were weighed, tumor cells dissociated and fluorescent images were captured to determine which cells escaped CAR T cell killing. Conclusions: The CAR with the 1XX mutations in CD3z, huMNC2-CD28-1XX, was much more effective at suppressing breast tumor recurrence than either CAR with wild-type CD3z. At sacrifice, significantly more CAR T cells were recovered from huMNC2-CD28-1XX than from huMNC2-41BB-CD3z or huMNC2-CD28-CD3z. At high dose, the CARs with wild-type CD3z effectively suppressed the high antigen expressing cells, but the recurrent tumors were essentially made up of the low antigen expressing cells that had escaped CAR T cell killing. At low CAR T cell dose, the CARs with wild-type CD3z appeared to become exhausted about 40 days post treatment, when tumors began to recur. Post sacrifice analysis of the recurrent tumors showed that they were made up of both high and low antigen expressing cells. huMNC2-CD28-1XX effectively killed both the high antigen and low antigen expressing cells as evidenced by the live fluorescent imaging and the post-sacrifice analysis of residual tumor. Citation Format: Cynthia Bamdad, Andrew Stewart, Benoit Smagghe, Mark Carter, Danica Walkley, Kevin Yi, Jac-Leen Nash, Michael Nash, Trevor Grant, Gregory Riley. Animal Study Compares CAR T cell Exhaustion & Ability to Kill Low Antigen expressing breast cancer cells among three CAR constructs including one with 1XX mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-05.

Abstract PO1-18-04: Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968)

Abstract Background: Higher levels of stromal tumor-infiltrating lymphocytes (sTILs) in breast cancers are associated with increased likelihood of pathologic complete response (pCR) to chemotherapy and improved outcomes. DNA methyltransferase inhibitors (DNMTi) can augment immune responses to cancers by upregulating tumor antigen and MHC expression, decreasing numbers and activity of myeloid derived suppressor cells (MDSC), and increasing responsiveness of T lymphocytes. We have shown that the DNMTi decitabine augments the effectiveness of immunotherapy against murine triple negative breast cancer (TNBC) using murine 4T1 and E0771 mammary carcinoma models. Methods: In a single-arm phase 2 study, patients with HER2-negative breast cancer who were candidates for neoadjuvant chemotherapy (NCT) received decitabine (15 mg/m2 × 4 doses over 5 days) followed by 2 doses of pembrolizumab (pembro) (200 mg, 2 weeks apart) – collectively, window immunotherapy – prior to starting NCT. Two research biopsies were obtained: 1 prior to the window immunotherapy and 1 afterwards, prior to starting NCT. Biopsies were analyzed using established procedures to quantify sTILs and PD-L1 expression. Patients proceeded to NCT and tumor resection per standard of care and were followed up for immune-related adverse events (irAEs) and response. The primary endpoint was change in sTILs. Key secondary endpoints were occurrence of irAEs and pCR following neoadjuvant treatment. After the study was opened, reported results of a phase 3 trial (KEYNOTE 522) led to allowing patients with TNBC to receive additional pembro (200 mg q3w) concurrently with standard NCT and adjuvantly following resection (Cohort A2). Results: 46 patients (median age 54.5 yrs, range 28-72; 71.7% White, 28.3% Black; 100% female) were enrolled and treated on study. 21 patients had TNBC and did not receive neoadjuvant pembro concurrently with NCT nor adjuvant pembro (Cohort A), 7 patients had TNBC and did receive neoadjuvant and/or adjuvant pembro (Cohort A2), and 18 patients were ER+ or PR+ and received neither concurrent nor adjuvant pembro (Cohort B). Blood samples collected after decitabine administration before the first pembro dose showed a 59% decrease (P< 0.01) in monocytic MDSCs compared to baseline; decrease in granulocytic MDSCs was not statistically significant. 37 patients had paired biopsies adequate for sTIL evaluation with a mean change from 23.4% to 30.3% (absolute change 6.9%, P< 0.001). Cohorts A/A2 experienced an absolute sTIL increase of 7.4% (P< 0.01); Cohort B experienced absolute sTIL increase of 6.1% (P=0.01). PD-L1 expression in tumors (H-score using MoAb 22C3 clone) increased by 43% (P< 0.01) across all cohorts. 16 of the 39 patients (41.0%) who proceeded to resection achieved pCR (n=12 of 27 [44.4%] in Cohorts A/A2 and n=4 of 13 [30.8%] in Cohort B). The most frequently reported irAEs were adrenal insufficiency (n=5, 10.9%), maculopapular rash (n=3, 6.5%), and hypothyroidism (n=3, 6.5%). Conclusions: Treatment in the pre-neoadjuvant window with decitabine and pembro could potentially sensitize breast cancers to standard NCT by recruitment of TILs to the tumor tissue. The treatment was well -tolerated at the tested doses. Funding: financial support and drug (pembrolizumab) were provided by Merck Citation Format: Harry Bear, Xiaoyan Deng, Dipankar Bandyopadhyay, Michael Idowu, Maciej Kmieciak, Monique Williams, Giovanni Archer, Lindsey Gwaltney, Patrick Dillon, Daniel Flora, Daniel Stover, Andrew Poklepovic, Mary Hackney, Masey Ross, Hetal Vachhani, Raphael Louie, Kandace McGuire, Amelia Grover, Tasnim Rahman, Amber Hendrix. Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-18-04.

Abstract PO3-13-11: Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies

Abstract Emerging antibody drug conjugate (ADC) therapies targeting human trophoblast cell-surface antigen (TROP2) and human epidermal growth factor receptor 2 (HER2) are transforming the treatment landscape for breast cancer. Sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd) have gained approval for an overlapping set of "HER2-low" metastatic breast cancers, including hormone receptor (HR)-positive HER2 non-amplified and "triple-negative" subtypes. Nevertheless, the optimal selection of patients and treatment sequencing for these ADC therapies remains a clinical challenge. Clinical trial objective response rates to SG are approximately 30%, compared to 30-90% for T-DXd depending on HER2 expression levels. While both drugs are thought to be targeted therapies, the value of measuring the target and the best methods to do so are still not established. We believe that quantitative measurement of TROP2 and HER2 antigen expression levels could establish thresholds for responders, enabling more effective patient selection for ADC therapies. Here, we present a TROP2, high-sensitivity HER2, and cytokeratin (CK) quantitative immunofluorescence (QIF) multiplex assay. Using a ten-cell line standard array and proteomic mass spectrometry, we can convert tumor compartment QIF intensity to protein concentration in fmol/mm2 for tissue specimens. Anti-HER2, anti-TROP2 antibodies, and fluorescence detection systems were titrated and combined to maximize signal-to-noise ratio on our cell line standard array and breast cancer tissue microarrays (TMA). The multiplex assay was designed for automated slide stainers (Leica BOND Rx) and fluorescence slide scanning (Rarecyte CyteFinder II HT). We perform our analyses in QuPath using an image processing plugin developed for automated QIF/IHC analysis (Qymia). Reproducible TROP2 and HER2 QIF scoring (R² > 0.95) was achieved across multiple staining batches using serial sections of breast cancer TMAs and cell standard arrays. This assay has a TROP2 linear range between 0.63 - 9.17 fmol/mm2 (about 1 million TROP2 receptors/cell) and HER2 linear range between 0.09 - 0.565 fmol/mm2 (about 60,000 HER2 receptors/cell). We then applied this multiplex assay to two serial retrospective primary breast cancer cohorts from Yale University to quantitatively measure TROP2 and HER2 expression (338 clinical cases). We find a weak negative correlation between TROP2 and HER2 expression in our breast cancer cohorts (Pearson r = -0.14, p = 0.0097, n = 338). TROP2 expression levels were above the limit of detection (LOD) in 90.2% of cases, with 4.1% exceeding the limit of linearity (LOL), and a mean TROP2 expression of 4.05 fmol/mm2. For HER2, 67.2% of cases were above the LOD, with 7.1% exceeding the LOL, and a mean HER2 expression of 0.186 fmol/mm2. Both TROP2 and HER2 were below the LOD in 3.0% of cases, which we define as “negative”. We found 29.9% expressed TROP2 and were HER2-negative, and 6.8% expressed HER2 and were TROP2-negative. Our future studies will aim to quantitatively define expression thresholds for T-DXd and SG response with the goal to produce a clinical grade assay for ADC patient selection and determine the value of the assay to help select which ADC to give first. HER2 and TROP2 protein expression summary in Yale breast cancer cohort Table 1: Summary of HER2 and TROP2 protein expression levels in serial retrospective primary breast cancer cohort of 338 cases using our high-sensitivity HER2 and TROP2 multiplex immunofluorescent assay Citation Format: Charles Robbins, Mengni He, Revekka Khaimova, Katherine Bates, Nay Nwe Nyein Chan, Daniel Liebler, Regan Fulton, David Rimm. Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-11.

Abstract PO4-25-03: Comprehensive immune profiling reveals factors associated with neoadjuvant chemotherapy response in triple negative breast cancer

Abstract Background: KEYNOTE-522 has resulted in FDA approval of the immune checkpoint blocker pembrolizumab with neoadjuvant chemotherapy for patients with high-risk triple negative breast cancer (TNBC), given the remarkable improvement in pCR rate to 65% along with improvement in event free survival, while with chemotherapy alone, the pCR rate is 40-50%. Unfortunately, use of pembrolizumab is associated with several immune related adverse events (irAE), some of which can be life-threatening and debilitating, including cardiomyositis, encephalitis, and adrenal insufficiency, among others. Hence, there is an unmet need to identify biomarkers in the tumor microenvironment which could predict patients who may attain pCR with chemotherapy alone and be spared the side effects from the added PD-1 inhibition. Methods: Comprehensive immune profiling, including PD-L1 IHC and the expression of 395 immune genes by RNA-seq, was performed on 1:2 matched FFPE tumor samples from 23 stage 1-3 TNBC patients (2 stage 1, 6 stage 2, 15 stage 3). All patients were female with an average age of 48 years (range: 25-79 years). 14 patients were treated with neoadjuvant chemotherapy alone (NAC) and 9 were treated with neoadjuvant chemotherapy combined with the checkpoint inhibitor pembrolizumab (NAC+I). mRNA expression signatures of tumor inflammation (TIGS, weak/moderate/strong), cell proliferation (CP, poor/moderate/high), and cancer testis antigen expression burden (CTAB) were determined by RNA-sequencing. Statistical comparisons of quantitative biomarkers between groups were calculated using the Wilcoxon Rank-Sum test, overrepresentation analysis of categorical variables between groups was calculated by proportions test, and survival differences were quantified by Cox proportional hazards analysis. Pathological responses were documented as pathological complete response (pCR) vs. non-pCR. Results: Across the entire cohort, patients with a BMI < 30 had significantly higher PD-L1 expression (assessed by RNA) than those with a BMI ³ 30 [p=0.047]. While not a statistically significant association, the NAC+I group tended to have a higher pCR rate compared to the NAC group [44.4% vs. 28.6%, p=0.66]. Across the entire cohort, tumors with high CTA expression, designated as CTAB high, had a higher pCR rate than CTAB low tumors [54.5% vs 16.7%, p=0.089]. No significant difference in pCR rate between TIGS or CP groups was detected for the NAC group, but in the NAC+I group, highly proliferative tumors had a lower pCR rate than moderately proliferative tumors [0% vs. 80%, p=0.048]. The pCR and non-pCR groups for each treatment type also exhibited distinct gene expression profiles. Among the NAC group, underexpression of FOXP3 and overexpression of MAPK14 and CD44 were associated with pCR [p≤0.031], while underexpression of IFNB1, MAPK14, and CD44 was associated with non-pCR [p≤0.029]. Among the NAC+I group, overexpression of CXCR4, TNFR and CCL20 was associated with non-pCR [p≤0.046]. Interestingly, SDHA was significantly overexpressed in the non-pCR subset of patients in the NAC group and significantly underexpressed in the non-pCR subset of patients in the NAC+I group [p≤0.031]. Conclusions: The development of biomarkers of treatment response is essential to the integration of immunotherapy with chemotherapy as a combined cancer treatment. Our study profiled the immune context of both NAC and NAC+I and identified several key microenvironmental differences underlying divergent treatment response in both groups. A comprehensive understanding of these factors could potentially predict pCR to chemotherapy alone, enabling the avoidance of the unnecessary treatment of these tumors with immunotherapy. Citation Format: Robert Seager, Heidi Ko, Sarabjot Pabla, Maria-Fernanda Senosain, Pawel Kalinski, Erik Van Roey, Shuang Gao, Kyle Strickland, Rebecca Previs, Mary Nesline, Stephanie Hastings, Shengle Zhang, Jeffrey Conroy, Taylor Jensen, Marcia Eisenberg, Brian Caveney, Eric Severson, Shakti Ramkissoon, Shipra Gandhi. Comprehensive immune profiling reveals factors associated with neoadjuvant chemotherapy response in triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-03.

Construction and analysis of a reliable five-gene prognostic signature for colon adenocarcinoma associated with the wild-type allelic state of the COL6A6 gene.

Tumors emerge by acquiring a number of mutations over time. The first mutation provides a selective growth advantage compared to adjacent epithelial cells, allowing the cell to create a clone that can outgrow the cells that surround it. Subsequent mutations determine the risk of the tumor progressing to metastatic cancer. Some secondary mutations may inhibit the aggressiveness of the tumor while still increasing the survival of the clone. Meaningful mutations in genes may provide a strong molecular foundation for developing novel therapeutic strategies for cancer. The somatic mutation and prognosis in colon adenocarcinoma (COAD) were analyzed. The copy number variation (CNV) and differentially expressed genes (DEGs) between the collagen type VI alpha 6 chain (COL6A6) mutation (COL6A6-MUT) and the COL6A6 wild-type (COL6A6-WT) subgroups were evaluated. The independent prognostic signatures based on COL6A6-allelic state were determined to construct a Cox model. The biological characteristics and the immune microenvironment between the two risk groups were compared. COL6A6 was found to be highly mutated in COAD at a frequency of 9%. Patients with COL6A6-MUT had a good overall survival (OS) compared to those with COL6A6-WT, who had a different CNV pattern. Significant differences in gene expression were established for 593 genes between the COL6A6-MUT and COL6A6-WT samples. Among them, MUC16, ASNSP1, PRR18, PEG10, and RPL26P8 were determined to be independent prognostic factors. The internally validated prognostic risk model, constructed using these five genes, demonstrated its value by revealing a significant difference in patient prognosis between the high-risk and low-risk groups. Specifically, patients in the high-risk group exhibited a considerably worse prognosis than did those in the low-risk group. The high-risk group had a significantly higher proportion of patients over 60 years of age and patients in stage III. Moreover, the tumor immune dysfunction and exclusion (TIDE) score and the expression of human leukocyte antigen (HLA) family genes were all higher in the high-risk group than that in the low-risk group. The allelic state of COL6A6 and the five associated DEGs were identified as novel biomarkers for the diagnosis and prognosis of COAD and may be therapeutic targets in COAD.

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

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

Chimeric hepatitis B surface antigen virus-like particles expressing the strep A epitope p*17 elicit a humoral immune response in mice

To optimize immunogenicity, bacterial epitopes in putative vaccine constructs can be presented to immune cells as multiple repeated structures on a defined nanoparticle. Virus-like particles (VLPs) are viral capsid proteins that self-assemble to form compact and highly ordered nanoparticles that are within the optimal size range for uptake by dendritic cells. VLPs mimic the live virus in size and form but contain no viral genetic material, are therefore noninfective and are the basis of safe and effective vaccines against hepatitis B virus (HBV) and human papillomavirus (HPV). Due to their particulate nature, molecular stability, and expression of high density and repetitive antigen displays, recombinant cell culture-derived VLPs are ideal platforms for the delivery of small molecules, including bacterial epitopes. We developed a putative vaccine by expressing a minimal epitope from the bacterium Streptococcus pyogenes (Strep A) on the surface of a recombinant VLP comprising multiple copies of HBV small envelope protein (HBsAg-S). Strep A is responsible for a wide spectrum of human infections and postinfectious diseases that disproportionately affect children and young adults living in resource-poor communities. No vaccine is currently available to offer sufficiently broad protection from the numerous and diverse strains of Strep A endemic in these at-risk populations. The Strep A antigen targeted by our vaccine construct is p*17, a cryptic epitope from a highly conserved region of the Strep A M-protein with demonstrated enhanced immunogenicity and broad protective potential against Strep A. To ensure surface expression and optimal immunogenicity, we expressed p*17 within the immunodominant “a” determinant of HBsAg-S. The recombinant VLPs (VLP-p*17) expressed in HEK293T cells spontaneously formed 22 nm particles and induced the production of high titers of p*17-specific IgG in BALB/c mice immunized with three 0.5 μg doses of VLP-p*17 formulated with adjuvant.

Design, synthesis and evaluation of novel prostate-specific membrane antigen-targeted aryl [18F]fluorosulfate PET tracers

The expression of prostate-specific membrane antigen (PSMA) in prostate cancer is 100–1000 times higher than that in normal tissues, and it has shown great advantages in the diagnosis and treatment of prostate cancer. The combination of PSMA and PET imaging technology based on the principle of metabolic imaging can achieve high sensitivity and high specificity for diagnosis. Due to its suitable half-life (109 min) and good positron abundance (97%), as well as its cyclotron accelerated generation, 18F has the potential to be commercialize, which has attracted much attention. In this article, we synthesized a series of fluorosulfate PET tracers targeting PSMA. All four analogues have shown high affinity to PSMA (IC50 = 1.85–5.15 nM). After the radioisotope exchange labeling, [18F]L9 and [18F]L10 have PSMA specific cellular uptake (0.65 ± 0.04% AD and 1.19 ± 0.03% AD) and effectively accumulated in 22Rv1 xenograft mice model. This study demonstrates that PSMA-1007-based PSMA-targeted aryl [18F]fluorosulfate novel tracers have the potential for PET imaging in tumor tissues.

Comparison of Proliferating Cell Nuclear Antigen (PCNA) Expression among Nasal Polyp and Chronic Rhinosinusitis

Background and objectiveChronic rhinosinusitis is a common inflammatory disorder in sinonasal mucosa that could be developed with or without nasal polyps. Cellular proliferation is suggested as a possible mechanism of nasal polyp development. However, conducted studies in this context are limited. So, the present study's aim is the comparison of proliferating cell nuclear antigen (PCNA) expression in nasal polyps and chronic rhinosinusitis. Materials and methodsIn this cross-sectional study, 70 nasal polyp and 60 chronic rhinosinusitis samples from patients referred to Mostafa Khomeini Hospital, Tehran from 2017 to 2022 were immunohistochemically stained by PCNA marker. The percentage of PCNA nuclear expression was determined in two groups and its association with the type of pathological lesion and the patient's age and sex was analyzed by SPSS statistic software version 24 statistical software (IBM Statistics, USA). ResultsThe mean percentage expression of PCNA in nasal polyp and chronic rhinosinusitis samples was 16.55 ± 13.66 and 17.58 ± 12.68 respectively (ranging from 0 to 57 in both groups) however, there was no significant statistical difference between the two groups (p = 0.479). No relationship was found between PCNA expression with age and sex in none of the chronic rhinosinusitis and nasal polyp groups. ConclusionProliferative activity of the nasal epithelial cell is similar in chronic rhinosinusitis with and without nasal polyps and it is considered that the increase of epithelial cell proliferative activity probably has no role in nasal polyp development in patients with chronic rhinosinusitis.

Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human factor IX variant in mice and rhesus macaques

We investigated long-term human coagulation Factor IX expression of a novel variant when delivered into mice and rhesus macaques and compare transduction efficiencies using two different AAV capsids. In hemophilic mice injected with KP1-packaged rAAV expressing the hyperactive FIX variant specific activity plasma levels were 10-fold or 2-fold enhanced when compared to wild-type or Padua huFIX injected mice, respectively. In rhesus macaques AAV-LK03 capsid outperformed AAV-KP1 in terms of antigen expression and liver transduction. Two animals from each group showed sustained low-level huFIX expression at 3 months post-administration, while one animal from each group lost huFIX mRNA and protein expression over time despite comparable vector copies. We investigated if epigenetic differences in the vector episomes could explain this loss of transcription. Cut&Tag analysis revealed lower levels of activating histone marks in the two animals that lost expression. When comparing rAAV genome associated histone modifications in rhesus macaques to those in mice injected with the same vector, the activating histone marks were starkly reduced in macaque-derived episomes. Differential epigenetic marking of AAV genomes may explain different expression profiles in mice and rhesus macaques as well as the wide dose response variation observed in primates in both preclinical and human clinical trials.

Application of PLGA in Tumor Immunotherapy.

Biodegradable polymers have been extensively researched in the field of biomedicine. Polylactic-co-glycolic acid (PLGA), a biodegradable polymer material, has been widely used in drug delivery systems and has shown great potential in various medical fields, including vaccines, tissue engineering such as bone regeneration and wound healing, and 3D printing. Cancer, a group of diseases with high mortality rates worldwide, has recently garnered significant attention in the field of immune therapy research. In recent years, there has been growing interest in the delivery function of PLGA in tumor immunotherapy. In tumor immunotherapy, PLGA can serve as a carrier to load antigens on its surface, thereby enhancing the immune system's ability to attack tumor cells. Additionally, PLGA can be used to formulate tumor vaccines and immunoadjuvants, thereby enhancing the efficacy of tumor immunotherapy. PLGA nanoparticles (NPs) can also enhance the effectiveness of tumor immunotherapy by regulating the activity and differentiation of immune cells, and by improving the expression and presentation of tumor antigens. Furthermore, due to the diverse physical properties and surface modifications of PLGA, it has a wider range of potential applications in tumor immunotherapy through the loading of various types of drugs or other innovative substances. We aim to highlight the recent advances and challenges of plga in the field of oncology therapy to stimulate further research and development of innovative PLGA-based approaches, and more effective and personalized cancer therapies.

Immunologic Factors Associated with Differential Response to Neoadjuvant Chemoimmunotherapy in Triple-Negative Breast Cancer.

Background: KEYNOTE-522 resulted in FDA approval of the immune checkpoint inhibitor pembrolizumab in combination with neoadjuvant chemotherapy for patients with early-stage, high-risk, triple-negative breast cancer (TNBC). Unfortunately, pembrolizumab is associated with several immune-related adverse events (irAEs). We aimed to identify potential tumor microenvironment (TME) biomarkers which could predict patients who may attain pathological complete response (pCR) with chemotherapy alone and be spared the use of anti-PD-1 immunotherapy. Methods: Comprehensive immune profiling, including RNA-seq gene expression assessment of 395 immune genes, was performed on matched FFPE tumor samples from 22 stage I-III TNBC patients (14 patients treated with neoadjuvant chemotherapy alone (NAC) and 8 treated with neoadjuvant chemotherapy combined with pembrolizumab (NAC+I)). Results: Differential gene expression analysis revealed that in the NAC group, IL12B and IL13 were both significantly associated with pCR. In the NAC+I group, LCK and TP63 were significantly associated with pCR. Patients in both treatment groups exhibiting pCR tended to have greater tumor inflammation than non-pCR patients. In the NAC+I group, patients with pCR tended to have greater cell proliferation and higher PD-L1 expression, while in the NAC group, patients with pCR tended to have lower cancer testis antigen expression. Additionally, the NAC+I group trended toward a lower relative dose intensity averaged across all chemotherapy drugs, suggesting that more dose reductions or treatment delays occurred in the NAC+I group than the NAC group. Conclusions: A comprehensive understanding of immunologic factors could potentially predict pCR to chemotherapy alone, enabling the avoidance of the unnecessary treatment of these patients with checkpoint inhibitors.

Obesity aggravates acute kidney injury resulting from ischemia and reperfusion in mice

In critically ill patients, overweight and obesity are associated with acute respiratory distress syndrome and acute kidney injury (AKI). However, the effect of obesity on ischemia–reperfusion injury (IRI)-induced AKI is unknown. We hypothesized that obesity would aggravate renal IRI in mice. We fed mice a standard or high-fat diet for eight weeks. The mice were divided into four groups and submitted to sham surgery or IRI: obese, normal, normal + IRI, obese, and obese + IRI. All studies were performed 48 h after the procedures. Serum glucose, cholesterol, and creatinine clearance did not differ among the groups. Survival and urinary osmolality were lower in the obese + IRI group than in the normal + IRI group, whereas urinary neutrophil gelatinase-associated lipocalin levels, tubular injury scores, and caspase 3 expression were higher. Proliferating cell nuclear antigen expression was highest in the obese + IRI group, as were the levels of oxidative stress (urinary levels of thiobarbituric acid-reactive substances and renal heme oxygenase-1 protein expression), whereas renal Klotho protein expression was lowest in that group. Expression of glutathione peroxidase 4 and peroxiredoxin 6, proteins that induce lipid peroxidation, a hallmark of ferroptosis, was lower in the obese + IRI group. Notably, among the mice not induced to AKI, macrophage infiltration was greater in the obese group. In conclusion, greater oxidative stress and ferroptosis might aggravate IRI in obese individuals, and Klotho could be a therapeutic target in those with AKI.

Single-cell RNA sequencing reveals the expansion of circulating tissue-homing B cell subsets in secondary acute dengue viral infection

The roles of antibodies secreted by subsets of B cells in dengue virus (DENV) infection have been extensively studied, yet, the contribution of tissue-homing B cells to antiviral immunity remains unclear. In this study, we performed a comprehensive analysis of B cell subpopulations in peripheral blood samples from DENV-infected patients using single-cell RNA-sequencing (scRNA-seq) datasets and flow cytometry. We showed that plasma cells (PCs) and plasmablasts (PBs) were the predominant B cell populations during the acute phase of secondary natural DENV infection, but not in convalescent phase nor in healthy controls. Interestingly, these cells expressed proliferation, adhesion, and tissue-homing genes, including SELPLG, a homing marker of the skin, the initial infected site of DENV. Flow cytometry analysis confirmed a significant upregulation of cell surface expression of a cutaneous lymphocyte-associated antigen (CLA) encoded by SELPLG in PCs and PBs, compared to naive and memory B cells from the same patients. The analysis of an independent single-cell B-cell receptor sequencing (scBCR-seq) dataset of DENV-infected patients revealed that the peripheral blood PCs and PBs exhibited the highest clonal expansion in secondary DENV infection compared to other B cell subsets. These clonally expanded cells also expressed the highest levels of tissue-homing genes, including SELPLG. In addition, by utilizing a public scRNA-seq dataset of SARS-CoV2 infection, we demonstrated the upregulation of several tissue-homing genes in PCs and PBs. Our study provides evidence for the potential roles of tissue-homing B cell subsets in the context of immune responses against viral infections in humans.

Correlation of immune fitness with response to teclistamab in relapsed/refractory multiple myeloma in the MajesTEC-1 study

AbstractTeclistamab, an off-the-shelf B-cell maturation antigen (BCMA) × CD3 bispecific antibody that mediates T-cell activation and subsequent lysis of BCMA-expressing myeloma cells, is approved for the treatment of patients with relapsed/refractory multiple myeloma (R/RMM). As a T-cell redirection therapy, clinical outcomes with teclistamab may be influenced by patient immune fitness and tumor antigen expression. We correlated tumor characteristics and baseline immune profiles with clinical response and disease burden in patients with R/RMM from the pivotal phase 1/2 MajesTEC-1 study, focusing on patients treated with 1.5 mg/kg of teclistamab (N = 165). Peripheral blood samples were collected at screening, and bone marrow samples were collected at screening and cycle 3. Better clinical outcomes to teclistamab correlated with higher baseline total T-cell counts in the periphery. In addition, responders (partial response or better) had a lower proportion of immunosuppressive regulatory T cells (Tregs), T cells expressing coinhibitory receptors (CD38, PD-1, and PD-1/TIM-3), and soluble BCMA and a T-cell profile suggestive of a more cytolytic potential, compared with nonresponders. Neither frequency of baseline bone marrow BCMA expression nor BCMA-receptor density was associated with clinical response to teclistamab. Improved progression-free survival was observed in patients with a lower frequency of T cells expressing exhaustion markers and immunosuppressive Tregs. Overall, response to teclistamab was associated with baseline immune fitness; nonresponders had immune profiles suggestive of immune suppression and T-cell dysfunction. These findings illustrate the importance of the contribution of the immune landscape to T-cell redirection therapy response. This trial was registered at www.ClinicalTrials.gov as #NCT03145181/NCT04557098.

The lack of HBsAg secretion does neither facilitate induction of antiviral T cell responses nor Hepatitis B Virus clearance in mice

Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts is regarded as a key contributor. HBsAg is expressed in HBV-infected hepatocytes and those carrying an HBV integration. Whether either HBsAg secretion or the high antigen amount expressed in the liver determines its immunomodulatory properties, however, remains unclear. We, therefore, developed a novel HBV animal model that allowed us to study the role of secreted HBsAg. We introduced a previously described HBs mutation, C65S, abolishing HBsAg secretion into a replication-competent 1.3-overlength HBV genome and used adeno-associated virus vectors to deliver it to the mouse liver. The AAV-HBV established a carrier state of wildtype and C65S mutant HBV, respectively. We investigated antiviral B- and T-cell immunity in the HBV-carrier mice after therapeutic vaccination. Moreover, we compared the effect of a lacking HBsAg secretion with that of an antiviral siRNA. While missing HBsAg secretion allowed for higher levels of detectable anti-HBs antibodies after therapeutic vaccination, it did neither affect antiviral T-cell responses nor intrahepatic HBV gene expression, irrespective of the starting level. A treatment with HBV siRNA restricting viral antigen expression within hepatocytes, however, improved the antiviral efficacy of therapeutic vaccination, irrespective of the ability of HBV to secrete HBsAg. Our data indicate that clearing HBsAg from blood cannot significantly impact HBV persistence or T-cell immunity. This indicates that a restriction of hepatic viral antigen expression will be required to break HBV immunotolerance.

Target Role of Monocytes as Key Cells of Innate Immunity in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic, systemic, and inflammatory autoimmune condition characterized by synovitis, pannus formation (with adjacent bone erosion), and joint destruction. In the perpetuation of RA, fibroblast-like synoviocytes (FLSs), macrophages, B cells, and CD4+ T-cells-specifically Th1 and Th17 cells-play crucial roles. Additionally, dendritic cells, neutrophils, mast cells, and monocytes contribute to the disease progression. Monocytes, circulating cells primarily derived from the bone marrow, participate in RA pathogenesis. Notably, CCR2 interacts with CCL2, and CX3CR1 (expressed by monocytes) cooperates with CX3CL1 (produced by FLSs), facilitating the migration involved in RA. Canonical "classical" monocytes predominantly acquire the phenotype of an "intermediate" subset, which differentially expresses proinflammatory cytokines (IL-1β, IL-6, and TNF) and surface markers (CD14, CD16, HLA-DR, TLRs, and β1- and β2-integrins). However, classical monocytes have greater potential to differentiate into osteoclasts, which contribute to bone resorption in the inflammatory milieu; in RA, Th17 cells stimulate FLSs to produce RANKL, triggering osteoclastogenesis. This review aims to explore the monocyte heterogeneity, plasticity, antigenic expression, and their differentiation into macrophages and osteoclasts. Additionally, we investigate the monocyte migration into the synovium and the role of their cytokines in RA.