Fas Receptor Research Articles

Combining Apoptotic Resistance and Cytokine Signaling to Improve Persistence and Anti-Tumor Activity of CAR Vδ2 T Cells In Vivo

Vγ9Vδ2 T cells (Vδ2 T cells hereafter) are an attractive platform for allogeneic chimeric antigen receptor (CAR)-modified cell therapy, as their invariant TCR minimizes their risk of graft-versus-host disease. However, the susceptibility of Vδ2 T cells to activation-induced cell death (AICD) and dependency on exogenous cytokine supplementation for prolonged persistence limit their in vivo efficacy. Addressing these limitations in would increase therapeutic potential of engineered Vδ2 T cells. Fas signaling is critical for AICD induction in Vδ2 T cells. To attenuate AICD, we generated a truncated Fas receptor lacking a signaling endodomain (tFas), to prevent downstream activation of apoptosis. Incubation with soluble FasL for 24h induced apoptosis in unmodified Vδ2 T cells and CD19.CAR-expressing Vδ2 T cells, while tFas-armed CD19.CAR-Vδ2 T cells resisted FasL-induced cell death. In extended (9-day) coculture experiments with CD19(+) NALM6 target cells at a 1:2 E:T ratio without exogenous cytokines, tFas-armed CD19.CAR-Vδ2 T cells produced enhanced anti-tumor control at early time points, compared to unarmed CD19.CAR-Vδ2 T cells which exhibited only transient activity, resulting in tumor regrowth. However, the lack of cytokine support limited long-term expansion of tFas/CD19.CAR-Vδ2 T cells resulting in tumor regrowth after 6 days of coculture. IL-18 signaling via MyD88 stimulates Vδ2 T cells, promoting their growth. Hence, we hypothesized combining IL-18/MyD88 signaling with AICD suppression would synergize to improve anti-tumor function of Vδ2 T cells. We generated a chimeric receptor consisting of the extracellular domain of Fas linked with intracellular MyD88 (Fas88) aiming to both prevent AICD and transmit MyD88 signaling upon engagement with FasL. Fas88-armed CD19.CAR-Vδ2 T cells resisted FasL-induced apoptosis similar to those armed with a tFas construct. Furthermore, in cocultures with NALM6 cells, Fas88/CD19.CAR-Vδ2 T cells resisted AICD and, in contrast to tFas/CD19.CAR-Vδ2 T cells, expanded over 9 days in the absence of exogenous cytokines and mediated a significantly superior tumor control. Next, we investigated the activity of armed CD19.CAR-Vδ2 T cells in a xenograft mouse model, where NALM6 cells (0.5 x 10 6 cells/mouse) were intravenously infused into NSG mice followed 5 days later by a single injection of Vδ2 T cells (4 x 10 6 cells/mouse). Both tumor and T cell populations were tracked via dual bioluminescent imaging. Unmodified CD19.CAR-Vδ2 T cells extended median survival from 18 days (control group) to 30 days but demonstrated limited persistence. tFas-armed CD19.CAR-Vδ2 T cells produced comparable tumor control, with a median survival of 31.5 days, suggesting that AICD suppression alone is insufficient for the long-term activity of CD19.CAR-Vδ2 T cells in vivo. In contrast, Fas88/CD19.CAR-Vδ2 T cells persisted and expanded in tumor-bearing mice improving the median survival to 47 days (p = 0.0478), indicating the ability of Fas88 to sustain functional persistence of CD19.CAR-Vδ2 T cells. This data shows that combining AICD suppression with MyD88 signaling improves the therapeutic potency of CAR-engineered Vδ2 T cells, demonstrating a means for exploiting Vδ2 T cells for allogeneic adoptive immunotherapy. Current work is focused on evaluating this approach for clinical translation.

Insight into the crystal structure analysis, vibrational studies, reactivities (MESP, HOMO-LUMO, NBO), and the anticancer activities of ruthenium diazide [Ru(POP)(PPh3)(N3)2] complex by molecular docking approach

This present work employs experimental and density functional theory (DFT) computations methods to investigate the structural, electronic, molecular interactions, and molecular docking of a ruthenium diazide [Ru(POP)(PPh3)(N3)2] complex with potential applications in apoptosis regulation. Crystallography analysis was conducted using Hirshfeld surface analysis which illustrated diverse intermolecular and intramolecular interactions, such as C H…O and CC….H respectively. Also, the following interactions and their percentage abundance existed in the complex O…H 1.5 %, C…H 19.9 %, H…H 68.8 %, C…N 0.2 % and N…H 9.9 %. With H…H interaction showcasing as dominant interaction in the complex. The molecular structure, vibrational analysis, and quantum chemical parameters were investigated within the framework of density functional theory at the ωB97XD/def2svp method. Molecular docking of the Ru-complex with receptor proteins involved in apoptosis regulation, including Death Receptor DR5, Estrogen Receptor Alpha (ERα), and Fas receptor, was performed. Results indicate potential interactions with these proteins, suggesting the Ru-complex's role in caspase-mediated apoptosis, particularly in colon and breast cancer cell lines. The investigation provides valuable insights into the reactivity and potential applications of the Ru-complex in cancer treatment.

Tumor necrosis factor superfamily 14 (TNFSF14/ LIGHT) attenuates neovascularization by reducing circulating endothelial progenitor cells and function

Abstract Background Tumor necrosis factor superfamily 14 (TNFSF14) is also known as LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) can bind to herpesvirus invasion mediator and lymphotoxin-β receptor to perform its biological activity. LIGHT signaling strengthens the synthesis of nitric oxide, reactive oxygen species, and cytokines and affects organ function. LIGHT also stimulates angiogenesis in tumors and induces synthesis of high endothelial venules, degrades extracellular matrix in thoracic aortic dissection, induces the expression of interleukin-8, cyclooxygenase-2, and cell adhesion molecules in endothelial cells. While LIGHT induces tissue inflammation, its effects on angiogenesis after tissue ischemia are unclear. Thus, we analyzed this effect in the current study. Methods C57BL/6 mice underwent hind limb ischemia surgery for model construction. Doppler ultrasound, immunohistochemical staining, and western blotting were employed to analyze the effects of LIGHT on angiogenesis. In addition, human endothelial progenitor cells (EPCs) were used for in vitro studies to analyze the possible mechanisms. Results In the mice, LIGHT injection inhibited angiogenesis in ischemic limbs. In vitro, LIGHT inhibited the expression of integrins and E-selectin; decreased migration and tube formation capabilities, mitochondrial respiration, and succinate dehydrogenase activity; and promoted senescence in EPCs. Western blotting revealed that the impairment of EPC function by LIGHT may be due to its effects on the normal operation of intracellular Akt signaling pathway and endothelial nitrite oxide synthase (eNOS) function. Conclusions LIGHT inhibits angiogenesis after tissue ischemia. LIGHT inhibition of intracellular Akt-eNOS signaling axis and mitochondrial respiration in EPCs impair cell function. These events are the possible causes of impaired angiogenesis.LIGHT decreases the recovery of ischemiaSenescence and dysfunction of EPC

Anti-Btla Antibody Tifcemalimab As a Single Agent or in Combination with Toripalimab in Relapsed/Refractory Lymphomas

Background: The B- and T-lymphocyte attenuator (BTLA) is an inhibitory receptor expressed on B, T and NK cells. Using Peripheral Blood Mononuclear Cell (PBMC) derived from cancer patients, co-blockade of the BTLA and PD-1 pathways improved antigen specific T cell response compared to either blockade alone. Tifcemalimab (JS004 or TAB004) is a humanized IgG4 monoclonal antibody with a hinge mutation (S228P) that binds BTLA and blocks its interaction with its ligand Herpesvirus Entry Mediator (HVEM). Tifcemalimab is the first BTLA antibody to enter the clinical development stage. Here, we report the safety and efficacy of tifcemalimab as a single agent or in combination with toripalimab (anti-PD-1) in patients with relapsed/refractory (R/R) lymphoma after long term follow-up. Methods: Eligible patients with R/R lymphoma were enrolled in this open-label, multicenter phase 1 study (NCT04477772). Tifcemalimab was administered as a monotherapy at escalating doses of 1, 3 and 10 mg/kg intravenously Q3W, followed by 3 mg/kg and 200 mg monotherapy dose expansion until disease progression or intolerable toxicity. During combination dose escalation, patients received ascending doses of tifcemalimab (100mg and 200mg) plus toripalimab (240mg). Dose-limiting toxicity (DLT) was evaluated by a safety monitoring committee. Study objectives included safety, pharmacokinetics, and efficacy. Results: By the cutoff date of July 19, 2023, a total of 71 patients were enrolled, including 25 in the monotherapy and 46 in the combination cohorts. The median follow-up time was 57 weeks. The median age was 38 (range 19-70) years with 50 (70%) male patients. The median prior line of therapy was 4 with 54 (76%) progressed upon prior anti-PD-1/L1 therapy. No DLT was observed in either monotherapy or combination dose escalation. Recommended phase 2 dose (RP2D) was determined to be 200 mg tifcemalimab as monotherapy or in combination with toripalimab. Sixty-seven (94%) patients experienced treatment emergent adverse events (TEAEs), 22 (31%) of whom experienced grade 3 or above TEAEs. The most common TEAEs were COVID-19 infection (30%), anemia (30%) and Infusion related reaction (27%). Four treatment-related adverse events led to the discontinuation of the study drug. Among 25 patients receiving monotherapy, 1 PR (follicular lymphoma) and 7 SD were observed with median progression-free survival (PFS) 2.1 months. Among 46 patients receiving combination therapy, 1 CR,16 PR (ORR 37.0%) and 20 SD (DCR 80.4%) were observed and estimated median PFS was 14.7 months. Thirty-four patients with Hodgkin's Lymphoma received RP2D of combination therapy and all of them had received previous chemotherapy and anti-PD-1 or anti-PD-L1 antibody, and 12 PR (ORR 35.3%) and 17 SD (DCR 85.3%) were observed with estimated median progression-free survival was 16.2 months. Conclusions: Tifcemalimab alone or in combination with toripalimab were well tolerated in all doses evaluated. The combination regimen showed favorable safety and durable efficacy in lymphoma patients with heavily pretreatment, providing evidence for future investigation.

Binding of herpesvirus entry mediator (HVEM) and HSV-1 gD affect reactivation but not latency levels.

Previously we reported that the HSV-1 latency associated transcript (LAT) specifically upregulates the cellular herpesvirus entry mediator (HVEM) but no other known HSV-1 receptors. HSV-1 glycoprotein D (gD) binds to HVEM but the effect of this interaction on latency-reactivation is not known. We found that the levels of latent viral genomes were not affected by the absence of gD binding to HVEM. However, reactivation of latent virus in trigeminal ganglia explant cultures was blocked in the absence of gD binding to HVEM. Neither differential HSV-1 replication and spread in the eye nor levels of latency influenced reactivation. Despite similar levels of latency, reactivation in the absence of gD binding to HVEM correlated with reduced T cell exhaustion. Our results indicate that HVEM-gD signaling plays a significant role in HSV-1 reactivation but not in ocular virus replication or levels of latency. The results presented here identify gD binding to HVEM as an important target that influences reactivation and survival of ganglion resident T cells but not levels of latency. This concept may also apply to other herpesviruses that engages HVEM.

Autoimmune lymphoproliferative syndrome (ALPS) due to a novel dominant negative germline mutation in the FAS gene.

Autoimmune lymphoproliferative syndrome (ALPS) due to a novel dominant negative germline mutation in the FAS gene.

MIF promotes Th17 cell differentiation in Hashimoto's thyroiditis by binding HVEM and activating NF-κB signaling pathway.

Hashimoto's thyroiditis is a typical thyroid autoimmune disease and Th17 cells are crucial in its development. In recent years, MIF (Macrophage Migration Inhibitory Factor) has been found to promote the secretion of IL-17A and the production and differentiation of Th17 cells. However, the specific mechanism of it remains unclear. Here, we found that the expression of MIF, IL-17A and HVEM (Herpes Virus Entry Mediator) were up-regulated in HT patients. The proportion of Th17 cells in peripheral blood mononuclear cells was positively correlated with the serum MIF protein level. We further found that the expression of HVEM and the phosphorylation level of NF-κB in peripheral blood mononuclear cells of HT patients were significantly increased. Therefore, we speculated that MIF promotes Th17 cell differentiation through HVEM and NF-κB signaling pathways. Further mechanism studies showed that MIF could directly bind to HVEM, and the stimulation of rhMIF in vitro could increase the expression of HVEM and activate NF-κB signaling pathways to promote Th17 cell differentiation. After blocking HVEM with HVEM antibody, the effect of MIF on Th17 cell differentiation disappeared. The results above show that the differentiation of Th17 cells is promoted by MIF combined with HVEM through NF-κB signaling pathways. Our research provides a new theory to the regulation mechanism of Th17 cell differentiation and gives hint to new potential therapeutic targets for HT.

The Role of FAS Receptor Methylation in Osteosarcoma Metastasis.

Osteosarcoma is the most frequent primary malignant bone tumor with an annual incidence of about 400 cases in the United States. Osteosarcoma primarily metastasizes to the lungs, where FAS ligand (FASL) is constitutively expressed. The interaction of FASL and its cell surface receptor, FAS, triggers apoptosis in normal cells; however, this function is altered in cancer cells. DNA methylation has previously been explored as a mechanism for altering FAS expression, but no variability was identified in the CpG island (CGI) overlapping the promoter. Analysis of an expanded region, including CGI shores and shelves, revealed high variability in the methylation of certain CpG sites that correlated significantly with FAS mRNA expression in a negative manner. Bisulfite sequencing revealed additional CpG sites, which were highly methylated in the metastatic LM7 cell line but unmethylated in its parental non-metastatic SaOS-2 cell line. Treatment with the demethylating agent, 5-azacytidine, resulted in a loss of methylation in CpG sites located within the FAS promoter and restored FAS protein expression in LM7 cells, resulting in reduced migration. Orthotopic implantation of 5-azacytidine treated LM7 cells into severe combined immunodeficient mice led to decreased lung metastases. These results suggest that DNA methylation of CGI shore sites may regulate FAS expression and constitute a potential target for osteosarcoma therapy, utilizing demethylating agents currently approved for the treatment of other cancers.

T Cells Expressing CAR Equipped With Extracellular Domain of BTLA Are Effective Against HVEM-over-expressing Melanoma Cell Lines.

Several chimeric antigen receptor (CAR) T cells have been used to treat melanoma but have not shown favorable results. This study investigated whether Herpes virus entry mediator (HVEM), which is overexpressed in melanoma, is a potential novel antigen for CAR T cell therapy. A CAR construct, composed of the BTLA extracellular domain for HVEM recognition (BTLA-28z), was developed and tested. Jurkat cells transduced with BTLA-28z exhibited enhanced IL-2 secretion when incubated with HVEM-over-expressing melanoma cells. KHYG-1 cells transduced with BTLA-28z also lysed melanoma cell lines. Using primary T cells, we generated CAR T cells targeting HVEM. BTLA-28z CAR T cells exhibited excellent lytic activities against melanoma cell lines. HVEM-targeting CAR T cells may be useful for the treatment of melanoma.

Bio-Assay-Guided Isolation of Fractions and Constituents with Antioxidant and Lipid-lowering Activity from Allium cepa.

Active fractions and constituents with antioxidant and lipid-lowering activities were investigated using bio-assay-guided isolation and identification. The data showed that the antioxidant fraction of A. cepa was AC50%, the main constituents of which were quercetin and isoquercitrin, by way of both ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) and bio-assay-guided purification and elucidation. Similarly, the lipid-lowering active fraction of A. cepa was AC30% with the main constituents of 3,4-dihydroxybenzoic acid and quercetin 3,4'-O-diglucoside. Also, bio-assay-guided isolation led to the isolation and identification of five known compounds with a purity of more than 98%, and quercetin was both the best free radical scavenger and lipid-lowering constituent. Moreover, the mechanism of the lipid-lowering effect of AC30% might be its reduction in mRNA expression levels of sterol regulatory element binding protein 2 (SREBP2) and FAS gene in lipid synthesis. Otherwise, reducing the mRNA expression level of lipid synthesis genes, including SREBP1, SREBP2, fatty acid synthetase (FASN), β-Hydroxy β-methylglutaryl-CoA (HMGCR), stearoyl CoA desaturase 1 (SCD1), and increasing the mRNA expression level of lipid decomposition gene, such as carnitine palmitoyl transferease-1 (CPT1), might be involved in the lipid-lowering activity of quercetin. This study suggested that Allium cepa might be used to prevent and treat oxidative stress and dislipidemia-related disorders, including NAFLD.

Herpesvirus entry mediator as a potential biomarker in breast cancer compared with conventional cytotoxic T‑lymphocyte‑associated antigen 4.

Breast cancer (BC) is the most common cancer in women worldwide, with 2.3 million cases recorded in 2020. Despite improvements in cancer treatment, patients with BC still succumb to the disease, due to regional and distant metastases when diagnosed at later stages. Several immune checkpoint inhibitors have been approved for BC treatment, based on their expression and role in maintaining immunosurveillance against tumors. The present study aimed to evaluate the expression of 12 immune checkpoints in patients with BC, and assess their role as diagnostic and therapeutic markers. Expression levels were measured using reverse transcription-quantitative polymerase chain reaction. Among the 12 immune markers, herpesvirus entry mediator (HVEM) was found to be significantly upregulated in patients with malignant BC compared to non-malignant controls, with a relative fold change (FC) of 1.46 and P=0.012. A similar finding was observed for cytotoxic T-lymphocyte-associated antigen 4 (CTLA4; FC=1.47 and P=0.035). In addition, receiver operating characteristic curve analysis revealed that HVEM expression allowed significant differentiation between groups, with an area under the curve of 0.74 (P=0.013). Upregulation in both HVEM and CTLA4 was revealed to be significantly associated with the human epidermal growth factor receptor-2 (HER2)-enriched phenotype (FC=3.53, P=0.009 and FC=5.98, P=0.002, respectively), while only HVEM was significantly associated with the triple-negative phenotype (FC=2.07, P=0.016). Furthermore, HVEM was significantly higher in patients with grade III tumors (FC=1.88, P=0.025) and negative vascular invasion (FC=1.67, P=0.046) compared with non-malignant controls. Serum protein levels were assessed by multiplex immunoassay, and a significant increase in HVEM was detected in patients with malignant BC compared with that in non-malignant controls (P=0.035). These data indicated that HVEM may serve as a potential biomarker and target for immunotherapy, especially for certain types of BC.

Dual inhibition of BTLA and PD-1 can enhance therapeutic efficacy of paclitaxel on intraperitoneally disseminated tumors

BackgroundExpression of immune checkpoints in the tumor microenvironment is one mechanism underlying paclitaxel (PTX) chemoresistance. This study aimed to investigate whether the addition of checkpoint blockade to PTX can improve...

Independent antioxidant and anticancer properties of a novel thermostable lysozyme isolated from Bacillus paralicheniformis: in silico and in vitro studies.

In this study, we evaluated the independent anticancer properties of a novel heat-stable lysozyme derived from the thermophilic bacterium Bacillus paralicheniformis (BplzC) to identify potential alternative therapies to address the suboptimal outcomes of current cancer treatments. Using the String 10.5 database, an in-silico protein-protein interaction study predicted that BplzC was a strong functional partner of cytochrome c, indicating a potential role in cancer cell apoptosis. Further, the HDOCK server predicted that BplzC strongly bound to cell death receptors, such as cytokines FAS receptor, leading to activation of cytochrome c and subsequent apoptosis in the cancer cell line. In vitro assays demonstrated uniform apoptotic activity of BplzC against various cancer cell lines, while showing no apoptotic activity against normal non-cancer cell lines. And showing no apoptotic activity against normal non-cancer cell lines suggested a very specific mode of action and without any adverse side effects. Additionally, BplzC exhibited ROS scavenging activity and reducing ability comparable to ascorbic acid, and significantly accelerated HEK293 cell migration. Our findings suggest that BplzC has specific cytotoxic effects on cancer cells and may be a valuable natural source of antioxidants for future use in the nutritional and pharmaceutical sectors.

Epitope topography of agonist antibodies to the checkpoint inhibitory receptor BTLA

B and T lymphocyte attenuator (BTLA) is an attractive target for a new class of therapeutics that attempt to rebalance the immune system by agonizing checkpoint inhibitory receptors (CIRs). Herpesvirus entry mediator (HVEM) binds BTLA in both trans- and cis-orientations. We report here the development and structural characterization of three humanized BTLA agonist antibodies, 22B3, 25F7, and 23C8. We determined the crystal structures of the antibody-BTLA complexes, showing that these antibodies bind distinct and non-overlapping epitopes of BTLA. While all three antibodies activate BTLA, 22B3 mimics HVEM binding to BTLA and shows the strongest agonistic activity in functional cell assays and in an imiquimod-induced mouse model of psoriasis. 22B3 is also capable of modulating HVEM signaling through the BTLA-HVEM cis-interaction. The data obtained from crystal structures, biochemical assays, and functional studies provide a mechanistic model of HVEM and BTLA organization on the cell surface and informed the discovery of a highly active BTLA agonist.

BTLA transcriptomic expression correlates with high levels of PD-1, PD-L2, and CTLA-4 checkpoints and with its ligand, HVEM, across diverse solid cancers.

e14641 Background: B and T lymphocyte attenuator (BTLA) is an immune checkpoint that is highly expressed on tumor-specific T cells and has been shown to be upregulated in several cancer types. The binding of BTLA to its ligand herpesvirus entry mediator (HVEM, also known as TNFRSF14) inhibits activation and proliferation of T lymphocytes, leading to decreased tumor killing. Early clinical trials of BTLA inhibitors are ongoing with preliminary results showing modest effects in advanced solid tumors. More recent clinical data suggests that combination checkpoint therapy with anti-BTLA and anti-PD-1 treatment provides greater benefit compared to monotherapy in patients with lymphoma. Methods: We analyzed RNA expression levels of 397 genes in various solid tumor types from 514 patients with various types of solid tumors seen at University of San Diego (UCSD), Moores Cancer Center. RNA expression was quantified at a CLIA-approved laboratory Omniseq (https://www.omniseq.com). Transcript abundance was normalized to internal housekeeping gene profiles and ranked (0-100 percentile) in a standardized manner to a reference population of 735 tumors spanning 35 histologies. We analyzed tumors with “High” (75-100 percentile rank) BTLA expression profiles. Odds ratio for high BTLA expression was calculated and multivariate analysis was utilized to determine significant correlations for multiple genes. Results: A total of 96 (18.7%) tumors had high BTLA (>75) expression across 32 different histologies. Cancers with the highest proportion of BTLA expression were small intestine (42%), breast (31%), pancreatic (29%), stomach (24%), neuroendocrine (20%), lung (20%), head and neck (17%) and colorectal (13%). There was significant association for BTLA and high PD-1 (p < 0.001), high CTLA-4 (p < 0.001), high PD-L2 (p = 0.05) and its ligand HVEM (p < 0.01). There was no significant association between BTLA expression and PD-L1, gender or stomach, neuroendocrine, head and neck, and lung histologies. Conclusions: High BTLA expression was found in a large number of tumor samples and significantly associated with other immune checkpoints with FDA-approved drugs. Furthermore, HVEM expression (ligand for BTLA) significantly correlates with high BTLA transcriptome levels. The BTLA/HVEM pathway has been associated with suppression of T cell-mediated anti-tumor activity and resistance to anti-PD-1 immunotherapy leading to poor clinical outcomes. The utilization of combination checkpoint therapy involving BTLA inhibitors and specific checkpoint blockade is currently being explored. Our results suggest that patient selection based on the RNA expression level of immune markers matched to corresponding immunotherapy agent, rather than a population-based approach, may be ideal for optimizing treatment response.

Investigation into aberrant B cell repertoire in myeloma on humoral immunity and patient survival.

8016 Background: B cells are critical to inhibiting tumor progression, as they generate tumor-reactive antibodies; promote anti-tumor killing capacity of NK cells and phagocytosis of macrophages/DCs; and enrich priming of T cells against the tumor. Multiple myeloma (MM) is a B-cell-derived malignancy, and its survival is partly driven by a deficiency in the immune response. Yet, the abnormalities of the B-cell-mediated humoral responses in MM are ill defined. Methods: We investigated B cell composition in bone marrow (BM) from patients with newly diagnosed MM (NDMM, N = 170), relapsed refractory MM (RRMM, N = 140) and healthy donors (HD, N = 21) by multi-color flow cytometry. Results: We observed that B1b cells was significantly reduced by 45% (p = 0.004) in NDMM patients compared to HD. B1b cells are important in generating long-lasting protective antibodies, particularly against vaccines, in a T cell independent fashion. Similarly, B2 cells, which can produce all antibody types, were significantly reduced in the periphery of NDMM and RRMM patients. Within the B2 cell population, germinal center (GC) matured B cells were significantly reduced in both BM and periphery of MM patients, as shown by significantly higher expression of Fas receptor. Because of deficiencies in the GC-maturation of these B cells, their ability to switch from IgM to IgG was also significantly decreased. On the other hand, the population of regulatory B cells (Bregs) were significantly elevated by 2.3-fold (p < 0.05) in the BM and periphery of NDMM and periphery of RRMM patients. However, the ability of Breg cells to produce suppressive cytokines, like IL-10, was significantly reduced (p = 0.03), indicating impaired suppressive functional capacity. We investigated whether this dysregulated B cell homeostasis impacts myeloma patient survival following treatment. We found that a higher proportion of Bregs and a lower percent of B1a in BM and periphery prior to treatment was associated with favorable progression-free survival (PFS) in both NDMM and RRMM patients. We hypothesized that reduction of B2 cells and their inability to have GC-based maturation could impair antibody generation against acute infections and tumor neoantigens. Therefore, we evaluated MM patients’ ability to produce antibodies against hepatitis B surface antigen-based vaccination. We observed that only 31% of MM patients (N = 32) respond to the vaccine compared to 90% of HD. Furthermore, MM patients’ antibody titers were significantly lower ( < 120 IU/L) compared to > 5000IU/L in HD. Conclusions: All together, these results show that in MM patients, vaccine-responsive populations of B cells are reduced, with fewer mature B cells with limited repertoires in GC to fight against acute infections, and with a high number of dysregulated regulatory B cells. These results indicate that compromised B cell functionality in MM patients could impact therapeutic outcome.

Evaluation of microsatellite instability status, a definitive predictive biomarker for immune checkpoint inhibitors (ICI), in underrepresented minorities (URM) with gastrointestinal (GI) cancers.

3538 Background: Mismatch repair deficiency (dMMR) results in MSI-H state and is the first tumor type-agnostic biomarker predictive of ICI response. Among GI cancers, MSI-H is most frequent in colorectal cancer (CRC 15%), gastroesophageal (GEC, 5%) and other (small bowel, hepatopancreatobiliary; 1%). For CRC, MSI-H can be attributed to germline mutation (Lynch syndrome, 3%) or somatic inactivation (sporadic, 12%) of foundational MMR genes. Studies evaluating ICI efficacy in dMMR cancers focus primarily on non-Hispanic White (NHW) patients (pts). We present prevalence, tumor genomic features, and outcomes in pts from a large cohort at Memorial Sloan Kettering (MSK). Methods: Retrospective analysis of MSI-H GI cancers from MSK-IMPACT database. Pts were grouped by self-reported race and ethnicity into 4 study arms: NHW, Asian, non-Hispanic Black (NHB), and Hispanic. Age, tumor type, tumor mutation burden (TMB), and MMR genes were analyzed. Overall survival (OS) estimated with Kaplan-Meier. Results: Of 776 pts with MSI-H GI cancers: 623 (80.3 %) NHW, 60 (7.7 %) Hispanic, 50 Asian (6.5 %), and 43 (5.5 %) NHB. CRC (76%), GEC (14%), other cancers (10%). We present initial evaluation of CRC and GEC: Median age, TMB, and most frequently altered MMR genes (MMR gene FA) are in table. Median OS (mOS) in NHW/URM by receipt of ICI in MSI-H CRC were 38.5m/25.3m (p 0.07) in no-ICI group, 34.2m/28.7m (p 0.64) in +ICI group; MSI-H GEC 43.4m/30m (p 0.44) in no-ICI group, 28.8m/26.7m in +ICI group. Conclusions: Number of URM MSI-H CRC/GEC pts is 7 to 15-fold less than NHW, with no such difference in % MSI-H/MSS between groups; reflecting significant undertesting in URM pts. In MSI-H CRC, median age (m-Age) at sequencing was younger in URM compared to NHW; pronounced in Asian and Hispanic patients, who were 10+ years younger than NHW. No such age difference seen in GEC. No difference in mOS detected between NHW and URM, however a non-significant trend towards worse mOS in URM was observed in the no-ICI group. Next steps include validation of clinico-genomics of MSI-H GI cancers in other large cohorts, including TEMPUS (N = 768) which is ongoing. [Table: see text]

Innovative retargeted oncolytic herpesvirus against nectin4-positive cancers.

Nectin4 is a recently discovered tumor associated antigen expressed in cancers that constitute relevant unmet clinical needs, including the undruggable triple negative breast cancer, pancreatic ductal carcinoma, bladder/urothelial cancer, cervical cancer, lung carcinoma and melanoma. So far, only one nectin4-specific drug-Enfortumab Vedotin-has been approved and the clinical trials that test novel therapeutics are only five. Here we engineered R-421, an innovative retargeted onco-immunotherapeutic herpesvirus highly specific for nectin4 and unable to infect through the natural herpes receptors, nectin1 or herpesvirus entry mediator. In vitro, R-421 infected and killed human nectin4-positive malignant cells and spared normal cells, e.g., human fibroblasts. Importantly from a safety viewpoint, R-421 failed to infect malignant cells that do not harbor nectin4 gene amplification/overexpression, whose expression level was moderate-to-low. In essence, there was a net threshold value below which cells were spared from infection, irrespective of whether they were malignant or normal; the only cells that R-421 targeted were the malignant overexpressing ones. In vivo, R-421 decreased or abolished the growth of murine tumors made transgenic for human nectin4 and conferred sensitivity to immune checkpoint inhibitors in combination therapies. Its efficacy was augmented by the cyclophosphamide immunomodulator and decreased by depletion of CD8-positive lymphocytes, arguing that it was in part T cell-mediated. R-421 elicited in-situ vaccination that protected from distant challenge tumors. This study provides proof-of-principle specificity and efficacy data justifying nectin4-retargeted onco-immunotherapeutic herpesvirus as an innovative approach against a number of difficult-to-drug clinical indications.

Anti-HVEM mAb therapy improves antitumoral immunity both in vitro and in vivo, in a novel transgenic mouse model expressing human HVEM and BTLA molecules challenged with HVEM expressing tumors

BackgroundTumor necrosis factor superfamily member 14 (TNFRSF14)/herpes virus entry mediator (HVEM) is the ligand for B and T lymphocyte attenuator (BTLA) and CD160-negative immune co-signaling molecules as well as viral...

ALPS Diagnosis: Different Phenotype in Child and Mother

Autoimmune lymphoproliferative syndrome (ALPS) is a group of genetic disorders characterized by early-onset lymphoproliferation, autoimmune cytopenias, and susceptibility to malignancy. We present two cases: mother and son with different phenotype at onset.A 7-year-old child presented with anemia, thrombocytopenia and splenomegaly. In his family history: born from non-consanguineous parents, his mother had a splenectomy due to hemolytic anemia of unknown cause. In the child, initial differential diagnosis ruled out infection or malignancy. Bone marrow aspirate had erythroid hyperplasia, giant metamyelocytes and bands and low thrombopoyesis. His immunological evaluation had hypergammaglobulinemia, elevated TCRαβ+-DNT cells (10%). ALPS was suspected and he started Prednisolone and Mycophenolate.A mutation in FAS gene was identified (exon 9, c.785T > A, p.Ile262Asn). His mother, a 45-year-old female presented with a history of lymphadenopathy. In her past medical history, she had hemolytic anemia and splenomegaly, a splenectomy was performed and the anemia resolved. Initial evaluation was started because breast cancer was suspected. Lymph node biopsy was performed where malignancy was ruled out, but unspecific inflammatory infiltrate was found. An immunological evaluation was performed. The first that was identified was the history of his son with ALPS (not specified by any other physician). Due to her history, the lymphoproliferation and anemia Sanger sequencing of her sońs mutation was performed. It revealed the same mutation and treatment was started. FAS gene with 85% frequency is the main etiological cause of genetically diagnosed patients with ALPS phenotype. The factors that determine the penetrance of clinical ALPS have not been entirely determined. Penetrance appears to be relatively associated with the location and type of the mutation and even varies in individuals carrying the same genetic mutation.