Cell Cytotoxic Activity Research Articles

Combinatorial immunotherapy of anti-MCAM chimeric antigen receptor modified expanded natural killer cells and NKTR-255 against neuroblastoma

Pediatric patients with recurrent metastatic neuroblastoma (NB) have a dismal 5-year survival. Novel therapeutic approaches are urgently needed. The melanoma cell adhesion molecule (MCAM/CD146/MUC18) is expressed in a variety of pediatric solid tumors, including NB, and constitutes anovel target for immunotherapy. Here, we developed a chimeric antigen receptor (CAR) expressing natural killer (NK) cell-targeting MCAM by non-viral electroporation of CAR mRNA into exvivo expanded NK cells. Expression of anti-MCAM CAR significantly enhanced NK cell cytotoxic activity compared to mock NK cells against MCAMhigh but not MCAMlow/knockout NB cells invitro. Anti-MCAM-CAR-NK cell treatment significantly decreased tumor growth and prolonged animal survival in an NB xenograft mouse model. NKTR-255, a polymer-conjugated recombinant human interleukin-15 agonist, significantly stimulated NK cell proliferation and expansion and further enhanced the in vitro cytotoxic activity and invivo anti-tumor efficacyofanti-MCAM-CAR-NK cells against NB. Our preclinical studies demonstrate that exvivo expanded andmodified anti-MCAM-CAR-NK cells alone and/or incombination with NKTR-255 are promising novel alternative therapeutic approaches to targeting MCAMhigh malignant NB.

Development of BCMA-Targeted Bispecific Natural Killer Cell Engagers for Multiple Myeloma Treatment

Background: B-cell maturation antigen (BCMA)-targeted T cell-redirecting immunotherapies, including Chimeric Antigen Receptor (CAR) T-cell therapy and T-cell engagers have demonstrated remarkable success in treating relapsed/refractory (RR) multiple myeloma (MM), a malignancy of plasma cells. However, a significant challenge is the severe side effects associated with T-cell overactivation, leading to cytokine release syndrome and neurotoxicity in MM patients undergoing such therapies. Bispecific NK cell engagers (NKCEs) may offer a promising alternative by redirecting NK cell cytotoxic activity towards tumor cells without triggering cytokine release syndrome. Methods: In this study, we designed a series of BCMA × CD16 NKCEs that simultaneously engage BCMA and CD16 on MM and NK cells, respectively. We evaluated the functionality of these NKCEs in vitro with respect to their molecular design. Results: Our results indicate that the format design of NKCEs influences their functionalities, underscoring the importance of format selection in optimizing NKCE-based therapies for MM. This study provides valuable insights for developing next-generation NKCEs and advancing therapeutic strategies for MM and potentially other malignancies.

Generation, Characterization, and Preclinical Studies of a Novel NKG2A-Targeted Antibody BRY805 for Cancer Immunotherapy.

Immuno-oncology has revolutionized cancer treatment, with NKG2A emerging as a novel target for immunotherapy. The blockade of NKG2A using the immune checkpoint inhibitor (ICI) monalizumab has been shown to enhance the responses of both NK cells and CD8+ T cells. However, monalizumab has demonstrated limited efficacy in in vitro cytotoxic assays and clinical trials. In our study, we discovered and characterized a novel anti-NKG2A antibody, BRY805, which exhibits high specificity for the human CD94/NKG2A heterodimer complex and does not bind to the activating NKG2C receptor. In vitro cytotoxicity assays demonstrated that BRY805 effectively activated NK92 cells and primary NK cells, thereby enhancing the cytotoxic activity of effector cells against cancer cells overexpressing HLA-E, with significantly greater efficacy compared to monalizumab. Furthermore, BRY805 exhibited synergistic antitumor activity when combined with PD-L1 monoclonal antibodies. In a mouse xenograft model, BRY805 showed superior tumor control relative to monalizumab and demonstrated a favorable safety profile in non-human primate studies.

Abstract B028: The Role of cDC1 Gene Signatures in gastric cancer prognosis and immune microenvironment modulation

Abstract Background and Aim: Gastric cancer (GC) continues to be a major global health burden, with a growing body of evidence highlighting the critical role of conventional type 1 dendritic cells (cDC1s) within the tumor microenvironment. This study aims to elucidate the prognostic significance of cDC1-associated genes—CD141, XCR1, CLEC9A, CADM1, and BTLA—in gastric cancer, as well as to explore the biological mechanisms that underpin their effects on patient outcomes. Materials and Methods: We conducted a comprehensive analysis of RNA- seq data from The Cancer Genome Atlas (TCGA-STAD) and Gene Expression Omnibus (GEO) datasets, focusing on the expression profiles of five cDC1-related genes. Patients were stratified into high and low expression groups based on a defined cDC1 gene signature. To assess the impact of these signatures, we utilized gene set variation analysis (GSVA) to examine relevant oncogenic pathways and performed Kaplan-Meier survival analysis along with Cox proportional hazards modeling to evaluate their prognostic value. Results: Our findings demonstrate that patients exhibiting a high cDC1-related gene signature had significantly worse overall and disease-free survival within the TCGA-STAD cohort. Further analysis revealed marked differences in CD8+ T cell infiltration and cytotoxic activity between the high and low cDC1 signature groups. Additionally, the high cDC1 signature group was strongly associated with upregulated expression of immune checkpoint proteins and the activation of oncogenic pathways, indicating a highly immunosuppressive tumor microenvironment. Conclusions: This study suggests that the cDC1-related gene signature may serve as a potent prognostic biomarker in gastric cancer, offering new insights into the complex interactions between the tumor and immune microenvironment. These results could pave the way for innovative therapeutic strategies that target the immune landscape in gastric cancer, potentially improving patient outcomes. Citation Format: Song-hee han. The Role of cDC1 Gene Signatures in gastric cancer prognosis and immune microenvironment modulation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B028.

Phenotypic and transcriptomic profiling of induced pluripotent stem cell (iPSC)-derived NK cells and their cytotoxicity against cancers

BackgroundAdoptive immunotherapy using natural killer (NK) cells has attracted considerable interest in numerous clinical trials targeting both hematological and solid tumors. Traditionally, NK cells are primarily derived from either peripheral blood (PB) or umbilical cord blood (UCB). However, these methods can lead to variability and heterogeneity within the NK cell population. In contrast, induced pluripotent stem cell (iPSC)-derived NK (iNK) cells provide a more controlled and uniform cellular population, suitable for large-scale clinical applications. This makes iNK cells a promising option for developing “off-the-shelf” immunotherapeutic products. Nevertheless, current NK cell differentiation protocols, which rely on embryoid body (EB) cultures, are labor-intensive and susceptible to unwanted heterogeneity during differentiation. Here, we developed a more efficient approach for generating iNK cells by employing a monolayer and feeder-free differentiation protocol, alongside optimized culture media.MethodsThe iNK cells were generated using a two-step in vitro monolayer feeder-free system following NK cell development. To evaluate their maturity, phenotypic analysis was performed using flow cytometry, comparing with PB-NK cells and the NK-92 cell line. Additionally, single-cell RNA sequencing was performed to examine their transcriptomic profiles. The cytotoxic activity of the iNK cells was evaluated by co-culturing with cholangiocarcinoma (CCA) and breast cancer (BCA) cell lines in both monolayer (2D) and tumor spheroid (3D) co-culture systems.ResultsWe successfully differentiated iPSCs into mesoderm (ME), hematopoietic stem/progenitor cells (HSPCs), and NK cells. The resulting iNK cells exhibited typical NK cell markers such as CD45, CD56, and CD16, and expressed key functional proteins, including both activating and inhibitory receptors. Single-cell RNA sequencing confirmed that the transcriptomic profile of our iNK cells closely resembles that of PB-NK cells. Importantly, our iNK cells demonstrated strong cytotoxic abilities against various CCA and BCA cell lines, surpassing the NK-92 cell line in both monolayer cultures and tumor spheroid cultures.ConclusionThis study highlights the potential of iPSCs as an effective alternative cell source for generating NK cells. Using a two-step in vitro monolayer feeder-free system, we successfully generated iNK cells that not only expressed key NK cell markers and their receptors but also displayed a transcriptomic profile closely resembling PB-NK cells. Furthermore, iNK cells exhibited cytotoxicity against CCA and BCA cell lines comparable to that of PB-NK cells. This approach could pave the way for off-the-shelf NK cell products, potentially enhancing the effectiveness of adoptive NK cell therapy.

TMOD-02. PATIENT-DERIVED GLIOBLASTOMA ORGANOIDS AS REAL-TIME AVATARS FOR ASSESSING RESPONSES TO CLINICAL CAR-T CELL THERAPY

Abstract Patient-derived tumor organoids have been increasingly leveraged for disease modeling and preclinical studies, but rarely in real-time to aid with the interpretation of patient treatment responses in the clinical setting. Whether tumor organoids can be applied to mirror clinical activity in patients and even to predict responses for personalized medicine remains uncertain. We recently demonstrated promising early efficacy signals in a first-in-human, phase 1 study of dual-targeting chimeric antigen receptor T cells (EGFR-IL13Rα2 CAR-T cells) in patients with recurrent glioblastoma (clinical trial identifier: NCT05168423). Determination of meaningful clinical efficacy, in particular for CAR-T cell therapy, may take months to manifest. Given this challenge, real-time analysis of patient-derived glioblastoma organoids (GBOs) could help to provide early assessment of clinical efficacy and guide patient management. We analyzed six sets of GBOs generated on the same timeline as the production of patients’ CAR-T cell products. GBOs demonstrated the presence and retention of both targeted tumor-associated antigens EGFR and IL13Rα2 by immunohistochemistry. These GBOs were then treated with the same autologous CAR-T cell products received by patients in our phase 1 study in parallel with patient treatment. CAR-T cell treatment led to target antigen reduction assessed by immunohistochemistry and flow cytometry. Furthermore, we confirmed cytolysis of tumor cells in GBOs by cleaved caspase-3 immunostaining and by co-culture in the Axion Maestro Cellular Impedance platform. Importantly, the degree of cytolysis ex vivo significantly correlated with CAR-T cell engraftment detected in patients’ cerebrospinal fluid (CSF) for all six patients. Furthermore, cytotoxic activity of CAR-T cells as measured by TNFα, IL-2, and IFNγ release kinetics in GBOs mirrored cytokine levels in patient CSF samples over time. Our findings highlight a unique trial design and suggest GBOs as a valuable platform for real-time assessment of CAR-T cell bioactivity and for insights into immunotherapy efficacy.

Landscape of the Peripheral Immune Response Induced by Intraoperative Radiotherapy Combined with Surgery in Early Breast Cancer Patients.

A comprehensive analysis of the immune response triggered by intraoperative radiation therapy (IORT) remains incomplete. In this study, single-cell RNA sequencing and single-cell T cell receptor sequencing are conducted on peripheral blood mononuclear cells (PBMCs) from patient with early-stage breast cancer before and after IORT. Following IORT combined with surgery (defined as IORT+Surgery), PBMC counts remained stable, with increased proportions of T cells, mononuclear phagocytes, and plasma cells, and a reduction in neutrophil proportions. The cytotoxic score of CD8Teff_GZMK cells increased significantly post-IORT. Communication between CD8Teff_GZMK cells and other immune cells via MIF_CD74 and MIF_TNFRSF14 is decreased after IORT. cDCs showed an upregulation of the MCH II signaling pathway, while memory B cells exhibited enhanced activation of the B cell pathway. T cell clones expanded significantly after treatment. IORT+Surgery demonstrated the ability to partially suppress the anti-tumor effects of neutrophils. Flow cytometry analysis and co-culture experiments are utilized to delve deeper into the functional alterations in T cells. IORT+Surgery significantly enhanced T cell cytotoxic activity. Blockade of PD-1 of post-IORT PBMCs shows higher T-cell activity than that of pre-IORT PBMCs. This research highlights IORT's impact on immune cells, offering insights for targeting immune responses in breast cancer.

Kinetics of viral hemorrhagic septicemia virus (VHSV) and immune response in olive flounder (Paralichthys olivaceus) at altered temperature

Viral hemorrhagic septicemia virus (VHSV) is a pathogen that causes hemorrhagic septicemia in olive flounder at water temperatures below 15 °C, leading to symptoms such as abdominal swelling due to ascites and muscle hemorrhaging, and in severe cases, mortality. In this study, we investigated the proliferation of NCCs, the transcriptional analysis of CD4 and CD8, and the expression of pro-inflammatory cytokines (IL-1β, IL6, TNFα) and the cytokines (IL12, IL15, IFN-1β, IFNγ) involved in cytotoxic cell activation in the kidney of olive flounder during VHSV infection at suboptimal temperature (17 °C) and following a shift to optimal temperature (10 °C). Following viral infection, the population of NCCs and CD8 gene expression steadily increased. Meanwhile, IL12, IL15, and IFN-1β levels exhibited an early surge, reaching their peak within 1–2 days post-challenge at 17 °C. Notably, in olive flounder that were re-challenged with VHSV at 10 °C after an initial challenge at 17 °C, there was a rapid and robust expansion of NCCs and increased CD4 and CD8 gene expression, demonstrating the mounting of immune memory by NCCs and an efficient adaptive immune response from CD4+ and CD8+ T cells against VHSV infection.

1,2,3‐Triazole‐Based New Aurones as Anticancer Agents with the Capability to Target Extracellular Digestive Enzymes

AbstractThis study involves the synthesis of a series of dimethyl substituted novel aurones, featuring 1,2,3‐triazole as an integral structure. All the newly synthesized compounds were thoroughly characterized using various spectroscopic tools and also subjected to computational analysis utilizing the DFT/B3LYP methodology, which involved the determination of frontier molecular orbital energy values and the computation of various quantum chemical parameters. Further their impact on cell viability and cytotoxic activity on the adenocarcinoma gastric cell line (AGS) was investigated using cell‐based MTT assay. Compounds 6d, 6o and 6p displayed significant cytotoxic activity, reducing cell viability to a greater extent with IC50 values of 9.74, 20.09, and 5.92 µM, respectively and even better than the standard chemotherapeutic drug leucovorin (IC50 = 30.8 µM). In addition, all the compounds were also screened for their extracellular enzymatic assay and through in vitro results compound 6n emerged as the efficient inhibitor of amylase (% inhibition = 51.92) and trypsin (% inhibition = 68.36), whereas an activation is observed for lipase (% activation = 269.48). In silico molecular docking was also conducted to assess the interactions between proteins and ligands, revealing the binding patterns of the synthesized compounds and the standard drug with receptor proteins.

Targeting PD-1+ T cells with small-format immunocytokines enhances IL-12 antitumor activity.

Immunostimulatory cytokines and immune checkpoint inhibitors hold promise as cancer therapeutics; however, their use is often limited by reduced efficacy and significant toxicity. In this study, we developed small-format immunocytokines (ICKs) based on interleukin-12 (IL-12) and blocking nanobodies targeting mouse and human PD-1 and PD-L1. Both PD-1 and PD-L1-targeted ICKs demonstrated similar in vitro performance, significantly increasing IL-12 tethering to immune cells and enhancing T cell cytotoxic activity compared to IL-12 alone. Antitumor efficacy of ICKs was evaluated by intratumoral delivery using self-amplifying RNA-based vectors or as recombinant proteins in mice. Despite effective PD-L1-mediated tumor anchoring and promising in vitro results, IL-12 antitumor activity was significantly enhanced only when specific targeting to intratumoral T cells was achieved via anti-PD-1 nanobody. This effect was also observed when the PD-1 specific ICK was delivered by electroporation of a DNA/RNA layered vector. Our findings suggest that targeting the appropriate type of cell within the tumor microenvironment could outperform tumor-anchoring strategies in the context of IL-12 therapy. Human versions of these ICKs were also developed, which showed to be active in human immune cells, opening an opportunity for clinical translation.

Melatonin mitigates vincristine-induced peripheral neuropathy by inhibiting TNF-α/astrocytes/microglial cells activation in the spinal cord of rats, while preserving vincristine's chemotherapeutic efficacy in lymphoma cells

Vincristine (VCR), an anti-tubulin chemotherapy agent, is known to cause peripheral and central nerve damage, inducing severe chemotherapy-induced peripheral neuropathy (CIPN). Although melatonin has been recently recognized for its potential anti-neuropathic effects, its efficacy in countering VCR-induced neuropathy remains unclear. This study examines the neuroprotective potential of melatonin against VCR-induced neuropathy using a rat model. Neuropathic pain was induced through 10 VCR injections (0.1 mg/kg/day i.p.), administered in two five-day cycles with a two-day break. Melatonin treatment started two days before VCR administration and continued daily throughout the experiment. Rats were assigned to five groups: control, VCR, and three melatonin-treated groups receiving VCR with melatonin (5, 10, or 20 mg/kg/day i.p.). We assessed mechanical (von-Frey and Randall-Selitto tests) and thermal (hot-plate and tail-flick tests) hyperalgesia, motor coordination (rotarod test), and sciatic nerve conduction velocity (NCV). Changes in body weight, spinal cord histopathology (H&E), and proinflammatory markers (TNF-α, IL-1β, and IL-6), reactive astrocytes (GFAP) and microglial cells (IBA-1) were also assessed, as well as spinal cord degeneration (Nissl stain) and demyelination (LFB stain and MBP). Finally, the effect of melatonin on the cytotoxic activity of VCR against EL4 lymphoma cells was assessed using an MTT assay. Our results indicated that melatonin coadministration with VCR preserved spinal cord architecture, elevated nociceptive thresholds, improved motor coordination, enhanced NCV, and maintained normal body weight gain. Melatonin also reduced inflammation, decreased reactive astrocytes and microglia, and prevented neurodegeneration and demyelination in the spinal cord. Importantly, melatonin did not affect VCR's cytotoxic activity in cancer cells.

Three-dimensional silk fibroin scaffolded co-culture of human neuroblastoma and innate immune cells

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It accounts for 50 % of cancers diagnosed in infants less than 1 year old, and 10 % of all pediatric cancer deaths in the United States. High-risk patients have a less than 50 % 5-year survival rate with current treatment strategies. The complex tumor microenvironment of NB makes the development of treatment strategies for high-risk patients challenging. There is increasing evidence that intratumoral immune suppression plays an important role in the progression and invasion of NB tumors. Few three-dimensional (3D) cancer models include components of the innate immune system. This work develops a preclinical 3D NB-immune co-culture model using SK-N-AS NB cells, NK-92 natural killer cells, and THP-1 derived macrophages, co-cultured on porous 3D silk scaffolds to provide tumor architecture. Conditioned media and indirect co-culturing showed changes in SK-N-AS gene expression associated with immunoregulatory signaling, and changes in NK-92 gene expression that are associated with reduced cytotoxicity. This motivated the development of a 3D direct co-culture system in which NB cells were seeded prior to immune cells to allow incorporation and deposition of extracellular matrix within the construct. Immune cells were then incorporated into the model to achieve direct co-culture with SK-N-AS cells. Changes in THP-1 macrophage polarization toward a more M2-like phenotype were observed in 3D direct co-culture, as well as altered NK-92 cell protein secretion and cytotoxic activity. Preliminary testing of immunotherapeutics within the model was conducted on both NB-macrophage and NB-NK co-cultures, but the model demonstrated limited response to immunotherapeutics. This work lays the foundation for building high-throughput therapeutic screening models for the improved treatment NB and other solid tumors.

A recombinant fragment antigen-binding (Fab) of trastuzumab displays low cytotoxic profile in adult human cardiomyocytes: first evidence and the key implication of FcγRIIA receptor.

Fragment crystallizable gamma receptors (FcγRs) mediate various cellular responses with significant cardiovascular implications. They contribute to the anticancer activity of trastuzumab (TRZ), a recombinant humanized monoclonal antibody that interferes with human epidermal growth factor receptor 2 (HER2), thereby blocking its physiological function in cardiac cells. This is responsible for cardiac complications that hamper TRZ clinical application. In this study we investigated the involvement of FcγRs in the TRZ cardiotoxicity. We used a recombinant antigen-binding fragment (Fab) of TRZ (rFab-HER2) to examine whether the absence of the Fc region resulted in fewer cardiomyocyte toxicity while preserving TRZ's ability to inhibit HER2. When exposed to rFab-HER2, AC16 human adult ventricular cardiomyocytes were less vulnerable to damage and death, than to TRZ. Specifically, TRZ exhibited cytotoxicity at a lower concentration (150 µg/mL, corresponding to ~1 µM) compared to rFab-HER2 (250 µg/mL, corresponding to ~5 µM). Like TRZ, rFab-HER2 negatively modulated HER2 levels in cardiomyocyte (without inducing cytotoxic activity in BJ human fibroblast cells that either did not express or express very low levels of HER2) and inhibited the downstream ERK/AKT cascades. But rFab-HER2 did not alter cardiomyocyte mitochondrial dynamic balance, and affect apoptosis and inflammation, while it limited cytosolic and mitochondrial ROS indicators. On contrary, the Fc region (50-250 μg/mL) exerted direct cytotoxic action on cardiomyocytes (but not on human fibroblasts that lacked Fc receptors). TRZ (150 μg/mL) markedly upregulated the expression level of FcγRIIA (a FcγRs strongly involved in TRZ-induced antibody-dependent cellular toxicity) in cardiomyocytes, whereas the Fab fragment (150 μg/mL) had no effect. Our results demonstrate that Fc region plays an important pathogenic role in TRZ-induced cardiomyocyte toxicity. In addition, targeting FcγRIIA might contribute to the off-target effects of TRZ therapy.

PI3Kδ activation, IL-6 overexpression, and CD37 loss cause resistance to naratuximab emtansine in lymphomas

AbstractCD37-directed antibody and cellular-based approaches have shown preclinical and promising early clinical activity. Naratuximab emtansine (Debio 1562; IMGN529) is an antibody-drug conjugate (ADC) incorporating an anti-CD37 monoclonal antibody conjugated to the maytansinoid DM1 as payload, with activity as a single agent and in combination with rituximab in patients with lymphoma. We studied naratuximab emtansine and its free payload in 54 lymphoma models, correlated its activity with CD37 expression, characterized 2 resistance mechanisms, and identified combination partners providing synergy. The activity, primarily cytotoxic, was more potent in B- than T-cell lymphoma cell lines. After prolonged exposure to the ADC, 1 diffuse large B-cell lymphoma (DLBCL) cell line developed resistance to the ADC due to the CD37 gene biallelic loss. After CD37 loss, we also observed upregulation of interleukin-6 (IL-6) and related transcripts. Recombinant IL-6 led to resistance. Anti-IL-6 antibody tocilizumab improved the ADC’s cytotoxic activity in CD37+ cells. In a second model, resistance was sustained by a PIK3CD activating mutation, with increased sensitivity to PI3Kδ inhibition and a functional dependence switch from MCL1 to BCL2. Adding idelalisib or venetoclax overcame resistance in the resistant derivative and improved cytotoxic activity in the parental cells. In conclusion, targeting B-cell lymphoma with the naratuximab emtansine showed vigorous antitumor activity as a single agent, which was also observed in models bearing genetic lesions associated with inferior outcomes, such as Myc Proto-Oncogene (MYC) translocations and TP53 inactivation or R-CHOP (rituximab, cyclophosphamide, doxorubicin, Oncovin [vincristine], and prednisone) resistance. Resistant DLBCL models identified active combinations of naratuximab emtansine with drugs targeting IL-6, PI3Kδ, and BCL2.

Synergistic Effect of Silver Nanoparticles with Antibiotics for Eradication of Pathogenic Biofilms.

The increase in nosocomial multidrug resistance and biofilm-forming bacterial infections led to the search for new alternative antimicrobial strategies other than traditional antibiotics. Silver nanoparticles (AgNP) could be a viable treatment due to their wide range of functions, rapid lethality, and minimal resistance potential. The primary aim of this study is to prepare silver nanoparticles and explore their antibacterial activity against biofilms. AgNPs with specific physicochemical properties such as size, shape, and surface chemistry were prepared using a chemical reduction technique, and then characterized by DLS, SEM, and FTIR. The activity of AgNPs was tested alone and in combination with some antibiotics against MDR Gram-negative and Gram-positive planktonic bacterial cells and their biofilms. Finally, mammalian cell cytotoxicity and hemolytic activity were tested using VERO and human erythrocytes. The findings of this study illustrate the success of the chemical reduction method in preparing AgNPs. Results showed that AgNPs have MIC values against planktonic organisms ranging from 0.0625 to 0.125 mg/mL, with the greatest potency against gram-negative bacteria. It also effectively destroyed biofilm-forming cells, with minimal biofilm eradication concentrations (MBEC) ranging from 0.125 to 0.25 mg/ml. AgNPs also had lower toxicity profiles for the MTT test when compared to hemolysis to erythrocytes. Synergistic effect was found between AgNPs and certain antibiotics, where the MIC was dramatically reduced, down to less than 0.00195 mg/ml in some cases. The present findings encourage the development of alternative therapies with high efficacy and low toxicity.

Sustained antiviral response against in vitro HIV-1 infection in peripheral blood mononuclear cells from people with chronic myeloid leukemia treated with ponatinib.

HIV-1 infection cannot be cured due to long-lived viral reservoirs formed by latently infected CD4+ T cells. "Shock and Kill" strategy has been considered to eliminate the viral reservoir and achieve a functional cure but the stimulation of cytotoxic immunity is necessary. Ponatinib is a tyrosine kinase inhibitor (TKI) clinically used against chronic myeloid leukemia (CML) that has demonstrated to be effective against HIV-1 infection in vitro. Several TKIs may induce a potent cytotoxic response against cancer cells that makes possible to discontinue treatment in people with CML who present long-term deep molecular response. In this longitudinal study, we analyzed the capacity of ponatinib to induce an antiviral response against HIV-1 infection in peripheral blood mononuclear cells (PBMCs) obtained from people with CML previously treated with imatinib for a median of 10years who changed to ponatinib for 12months to boost the anticancer response before discontinuing any TKI as part of the clinical trial NCT04043676. Participants were followed-up for an additional 12months in the absence of treatment. PBMCs were obtained at different time points and then infected in vitro with HIV-1. The rate of infection was determined by quantifying the intracellular levels of p24-gag in CD4+ T cells. The levels of p24-gag+ CD4+ T-cells were lower when these cells were obtained during and after treatment with ponatinib in comparison with those obtained during treatment with imatinib. Cytotoxicity of PBMCs against HIV-infected target cells was significantly higher during treatment with ponatinib than during treatment with imatinib, and it was maintained at least 12months after discontinuation. There was a significant negative correlation between the lower levels of p24-gag+ CD4+ T-cells and the higher cytotoxicity induced by PBMCs when cells were obtained during and after treatment with ponatinib. This cytotoxic immunity was mostly based on higher levels of Natural Killer and Tγδ cells seemingly boosted by ponatinib. In conclusion, transient treatment with immunomodulators like ponatinib along with ART could be explored to boost the antiviral activity of cytotoxic cells and contribute to the elimination of HIV-1 reservoir.

Flow cytometric analysis of the interaction of monoclonal antibodies to various epitopes of the HSP70 molecule with intracellular and membrane-associated forms of this protein

Currently, a large amount of data has demonstrated that many types of tumor cells, in contrast to their non-transformed forms, are characterized by the translocation of intracellular heat shock proteins 70 kDa (HSP70) to the surface of the plasma membrane. This made it possible to classify HSP70 exposed on the cell surface as a tumor-associated antigen and was the basis for searching the opportunities for the practical use of this phenomenon in clinical oncology. A significant argument in favor of the prospects of such studies was the discovered ability of HSP70, present on the surface of target cells, to enhance the cytotoxic activity of NK cells. In this regard, the work of many research groups is devoted to developing approaches to increasing the level of membrane-associated HSP70 in tumor tissues. At the same time, given the presence of such proteins on the surface of many types of tumor cells, the use of monoclonal antibodies that interact with HSP70 molecules for antitumor therapy can be considered as one of the promising approaches. It is well known that antibody preparations that selectively interact with cancer cells can be used for targeted antitumor immunotherapy. Previously, we obtained a panel of six B cell hybridomas producing monoclonal antibodies to the inducible and constitutive forms of human HSP70, directed to various epitopes of this molecule. It is significant that three varieties of hybridomas produced antibodies with specificity for binding sites on the C-terminal domain of the HSP70 molecule, and the second trio of monoclonal antibodies were specific to epitopes on the N-terminal domain of HSP70, while almost all known commercial antibodies interact only with C-terminal fragments of HSP70. In this work, we conducted a comparative study of the binding of the obtained antibodies to these proteins localized in different types of cells, both in the intracellular space and on the cell surface.The results obtained allow us to consider the identified varieties of monoclonal antibodies that most effectively recognize surface HSP70 as a promising basis for the creation of new drugs for antitumor immunotherapy.

Sequential immunization with chimeric hemagglutinin ΔNS1 attenuated influenza vaccines induces broad humoral and cellular immunity

Influenza viruses pose a threat to public health as evidenced by severe morbidity and mortality in humans on a yearly basis. Given the constant changes in the viral glycoproteins owing to antigenic drift, seasonal influenza vaccines need to be updated periodically and effectiveness often drops due to mismatches between vaccine and circulating strains. In addition, seasonal influenza vaccines are not protective against antigenically shifted influenza viruses with pandemic potential. Here, we have developed a highly immunogenic vaccination regimen based on live-attenuated influenza vaccines (LAIVs) comprised of an attenuated virus backbone lacking non-structural protein 1 (ΔNS1), the primary host interferon antagonist of influenza viruses, with chimeric hemagglutinins (cHA) composed of exotic avian head domains with a highly conserved stalk domain, to redirect the humoral response towards the HA stalk. In this study, we showed that cHA-LAIV vaccines induce robust serum and mucosal responses against group 1 stalk and confer antibody-dependent cell cytotoxicity activity. Mice that intranasally received cH8/1-ΔNS1 followed by a cH11/1-ΔNS1 heterologous booster had robust humoral responses for influenza A virus group 1 HAs and were protected from seasonal H1N1 influenza virus and heterologous highly pathogenic avian H5N1 lethal challenges. When compared with mice immunized with the standard of care or cold-adapted cHA-LAIV, cHA-ΔNS1 immunized mice had robust antigen-specific CD8+ T-cell responses which also correlated with markedly reduced lung pathology post-challenge. These observations support the development of a trivalent universal influenza vaccine for the protection against group 1 and group 2 influenza A viruses and influenza B viruses.

Abstract A036: The role of hypoxia in CD8+ T cell localization and function in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy characterized by poor response to existing therapeutic options. This resistance to treatment is partly due to its extensive inflammatory, desmoplastic stromal response, along with a hypoxic microenvironment. Although tumor hypoxia induces adaptive responses in both cancer cells and the surrounding stroma, most studies on the role of hypoxia in tumorigenesis have focused on cancer cell-intrinsic properties. The impact of hypoxia on stromal cells themselves and their interactions with cancer cells has remained largely unknown. We previously showed that inflammatory cancer-associated fibroblasts (iCAFs), which express high levels of cytokines and chemokines, are preferentially located in hypoxic regions of PDAC, and that hypoxia promotes an inflammatory fibroblast phenotype. The iCAF phenotype has been correlated with highly immunosuppressive features of PDAC. These findings and our preliminary observation that CD8+ T cells are largely excluded from hypoxic PDAC regions, raise the possibility that hypoxia suppresses the infiltration and activation of CD8+ T cells by modulating their interactions with inflammatory fibroblasts. By culturing CD8+ T cells under hypoxia with conditioned media from co-cultures of pancreatic cancer cells and fibroblasts, we have found that cancer cells and fibroblasts decrease CD8+ T cell proliferation and cytotoxic activity under low oxygen conditions. We are working to elucidate the mechanism by which hypoxia regulates CD8+ T cell infiltration and activation through cancer cell-fibroblast crosstalk. Citation Format: Ashley M. Mello, Tenzin Ngodup, Katelyn L. Donahue, Marina Pasca Di Magliano, Kyoung Eun Lee. The role of hypoxia in CD8+ T cell localization and function in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A036.

Preparation and characterization of chitosan nanoparticles with extracts of Rheum ribes, evaluation of biological activities of extracts and extract loaded nanoparticles

The biological activities of different parts of the Rheum ribes plant were evaluated comparatively. Extracts showing strong biological activity were identified and it was determined which of the extract-loaded nanoparticles showed stronger activity. Cytotoxic activity of R. ribes extracts was calculated on glial (C6) and fibroblast (NIH 3T3) cells using XTT assay. Spectrophotometry was used to evaluate the impact of these compounds on the enzyme activities of human carbonic anhydrase I and II (hCA I and hCA II). The findings showed that chitosan NPs with extracts loaded on them have a lower IC50 value and more cytotoxic activity in C6 cells than chitosan NPs with only extracts. R. ribes young shoots ultrasonic methanol extract (RYU) was shown to have the strongest antiproliferative efficacy against C6 cells. Results showed that RYU and ultrasonic methanol extract of R. ribes radix (RRU) were determined as the best carbonic anhydrase inhibitors. According to results of particle size, encapsulation efficiency, and release studies of chitosan NPs, it has been observed that they are suitable for application. At a concentration of 10 µg/mL, it was found that none of the R. ribes extracts exhibited cytotoxic action toward the NIH 3T3 cell line. According to results of particle size, encapsulation efficiency, and release studies of chitosan NPs, it has been observed that they are suitable for application. It was observed that none of the extracts of R. ribes at a concentration of 10 µg/mL showed cytotoxic activity in the NIH 3T3 cell line.