Search Results (10)

Chimeric Antigen Receptor T-cell (CAR-T) therapy has emerged as a transformative approach for cancer treatment, demonstrating remarkable success in patients with relapsed and refractory hematological malignancies. However, challenges persist in optimizing CAR-T cell production and improving therapeutic outcomes. One of the major hurdles is the efficiency of retroviral or lentiviral transduction during CAR-T cell manufacturing. Additionally, the heterogeneity of T-cell populations isolated from patients can impact CAR-T cell effectiveness and persistence in vivo. This article explores a novel strategy to address these challenges by focusing on serum-free medium and additive optimization. We propose a unique approach that incorporates the culturing of T cells in Nutri-T medium, along with 24 h of exposure to combined low concentrations of BX795 and rosuvastatin, to enhance the transduction efficacy and functionality of CAR-T cells. The results presented here provide promising insights into the potential of this strategy to produce more effective CAR-T cells for immunotherapy, ultimately advancing the field and benefiting cancer patients worldwide. Full article

Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common types of primary cutaneous T-cell lymphoma (CTCL). Proliferating cell nuclear antigen (PCNA) is expressed on the cell surface of cancer cells (csPCNA), but not on normal cells. It functions as an immune checkpoint ligand by interacting with natural killer (NK) cells through the NK inhibitory receptor NKp44, leading to the inhibition of NK cytotoxicity. A monoclonal antibody (mAb14) was established to detect csPCNA on cancer cells and block their interaction with NKp44. In this study, three CTCL cell lines and peripheral blood mononuclear cells (PBMCs) from patients with SS and healthy donors were analyzed for csPCNA using mAb14, compared to monoclonal antibody PC10, against nuclear PCNA (nPCNA). The following assays were used: immunostaining, imaging flow cytometry, flow cytometry, cell sorting, cell cycle analysis, ELISA, and the NK-cell cytotoxic assay. mAb14 successfully detected PCNA on the membrane and in the cytoplasm of viable CTCL cell lines associated with the G2/M phase. In the Sézary PBMCs, csPCNA was expressed on lymphoma cells that had an atypical morphology and not on normal cells. Furthermore, it was not expressed on PBMCs from healthy donors. In the co-culture of peripheral blood NK (pNK) cells with CTCL lines, mAb14 increased the secretion of IFN-γ, indicating the reactivation of pNK activity. However, mAb14 did not enhance the cytotoxic activity of pNK cells against CTCL cell lines. The unique expression of csPCNA detected by mAb14 suggests that csPCNA and mAb14 may serve as a potential biomarker and tool, respectively, for detecting malignant cells in SS and possibly other CTCL variants. Full article

Lung cancer cells in the tumor microenvironment facilitate immune evasion that leads to failure of conventional chemotherapies, despite provisionally decided on the genetic diagnosis of patients in a clinical setup. The current study follows three lung cancer patients who underwent “personalized” chemotherapeutic intervention. Patient-derived xenografts (PDXs) were subjected to tumor microarray and treatment screening with chemotherapies, either individually or in combination with the peptide R11-NLS-pep8; this peptide targets both membrane-associated and nuclear PCNA. Ex vivo, employing PDX-derived explants, it was found that combination with R11-NLS-pep8 stimulated antineoplastic effect of chemotherapies that were, although predicted based on the patient’s genetic mutation, inactive on their own. Furthermore, treatment in vivo of PDX-bearing mice showed an exactly similar trend in the result, corroborating the finding to be translated into clinical setup. Full article

Harnessing immune effector cells to benefit cancer patients is becoming more and more prevalent in recent years. However, the increasing number of different therapeutic approaches, such as chimeric antigen receptors and armored chimeric antigen receptors, requires constant adjustments of the transgene expression levels. We have previously demonstrated it is possible to achieve spatial and temporal control of transgene expression as well as tailoring the inducing agents using the Chimeric Antigen Receptor Tumor Induced Vector (CARTIV) platform. Here we describe the next level of customization in our promoter platform. We have tested the functionality of three different minimal promoters, representing three different promoters’ strengths, leading to varying levels of CAR expression and primary T cell function. This strategy shows yet another level of CARTIV gene regulation that can be easily integrated into existing CAR T systems. Full article

Multiple Myeloma (MM) is a devastating malignancy that evades immune destruction using multiple mechanisms. The NKp44 receptor interacts with PCNA (Proliferating Cell Nuclear Antigen) and may inhibit NK cells’ functions. Here we studied in vitro the expression and function of PCNA on MM cells. First, we show that PCNA is present on the cell membrane of five out of six MM cell lines, using novel anti-PCNA mAb developed to recognize membrane-associated PCNA. Next, we stained primary bone marrow (BM) mononuclear cells from MM patients and showed significant staining of membrane-associated PCNA in the fraction of CD38⁺CD138⁺ BM cells that contain the MM cells. Importantly, blocking of the membrane PCNA on MM cells enhanced the activity of NK cells, including IFN-γ-secretion and degranulation. Our results highlight the possible blocking of the NKp44-PCNA immune checkpoint by the mAb 14-25-9 antibody to enhance NK cell responses against MM, providing a novel treatment option. Full article

The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in-vivo neutralization of SARS-CoV-2 is not yet clear, and it is of high importance to delineate the role this process plays in antibody-mediated protection. Toward this aim, we have chosen two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, MD65 and BLN1 that target distinct domains of the spike (RBD and NTD, respectively). The Fc of these antibodies was engineered to include the triple mutation N297G/S298G/T299A that eliminates glycosylation and the binding to FcγR and to the complement system activator C1q. As expected, the virus neutralization activity (in-vitro) of the engineered antibodies was retained. To study the role of Fc-mediated functions, the protective activity of these antibodies was tested against lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice, when treatment was initiated either before or two days post-exposure. Antibody treatment with both Fc-variants similarly rescued the mice from death reduced viral load and prevented signs of morbidity. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in MD65 and BLN1 antibody-mediated protection, which should aid in the future design of effective antibody-based therapies. Full article

The new era of cancer treatments has made immune checkpoint inhibitors (ICIs) and emerging multikinase inhibitors (TKIs) the standards of care, thus drastically improving patient prognoses. Pembrolizumab is an anti-programmed cell death-1 antibody drug, and lenvatinib is a TKI with preferential antiangiogenic activity. We present, to our knowledge, the first reported series of cases consisting of patients with metastatic non–small cell lung cancer and malignant pleural mesothelioma who were treated with several types of chemotherapy combinations and ICIs followed by disease progression. They were subsequently treated with combined immunotherapy and TKI treatment, resulting in a near complete response within a very short time. Clinical responses were supported by in vitro testing of each patient’s lymphocytic response to pembrolizumab after pre-exposure of target cancer cells to lenvatinib. Full article

Over 50% of human papilloma positive head-and-neck cancer (HNC^HPV+) patients harbor genomic-alterations in PIK3CA, leading to hyperactivation of the phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) pathway. Nevertheless, despite PI3K pathway activation in HNC^HPV+ tumors, the anti-tumor activities of PI3K pathway inhibitors are moderate, mostly due to the emergence of resistance. Thus, for potent and long-term tumor management, drugs blocking resistance mechanisms should be combined with PI3K inhibitors. Here, we delineate the molecular mechanisms of the acquisition of resistance to two isoform-selective inhibitors of PI3K (isiPI3K), alpelisib (BYL719) and taselisib (GDC0032), in HNC^HPV+ cell lines. By comparing the transcriptional landscape of isiPI3K-sensitive tumor cells with that of their corresponding isiPI3K-acquired-resistant tumor cells, we found upregulation of insulin growth factor 2 (IGF2) in the resistant cells. Mechanistically, we show that upon isiPI3K treatment, isiPI3K-sensitive tumor cells upregulate the expression of IGF2 to induce cell proliferation via the activation of the IGF1 receptor (IGF1R). Stimulating tumor cells with recombinant IGF2 limited isiPI3K efficacy and released treated cells from S phase arrest. Knocking-down IGF2 with siRNA, or blocking IGF1R with AEW541, resulted in superior anti-tumor activity of isiPI3K in vitro and ex vivo. In vivo, the combination of isiPI3K and IGF1R inhibitor induced stable disease in mice bearing either tumors generated by the HNC^HPV+ UM-SCC47 cell line or HPV+ patient-derived xenografts. These findings indicate that IGF2 and the IGF2/IGF1R pathway may constitute new targets for combination therapies to enhance the efficacy of PI3K inhibitors for the treatment of HNC^HPV+. Full article

Activating alterations in PIK3CA, the gene coding for the catalytic subunit of phosphoinositide-3-kinase (PI3K), are prevalent in head and neck squamous cell carcinoma (HNSCC) and thought to be one of the main drivers of these tumors. However, early clinical trials on PI3K inhibitors (PI3Ki) have been disappointing due to the limited durability of the activity of these drugs. To investigate the resistance mechanisms to PI3Ki and attempt to overcome them, we conducted a molecular-based study using both HNSCC cell lines and patient-derived xenografts (PDXs). We sought to simulate and dissect the molecular pathways that come into play in PIK3CA-altered HNSCC treated with isoform-specific PI3Ki (BYL719, GDC0032). In vitro assays of cell viability and protein expression indicate that activation of the mTOR and cyclin D1 pathways is associated with resistance to PI3Ki. Specifically, in BYL719-resistant cells, BYL719 treatment did not induce pS6 and pRB inhibition as detected in BYL719-sensitive cells. By combining PI3Ki with either mammalian target of rapamycin complex 1 (mTORC1) or cyclin D1 kinase (CDK) 4/6 specific inhibitors (RAD001 and abemaciclib, respectively), we were able to overcome the acquired resistance. Furthermore, we found that PI3Ki and CDK 4/6 inhibitors have a synergistic anti-tumor effect when combined in human papillomavirus (HPV)-negative/PIK3CA-WT tumors. These findings provide a rationale for combining PI3Ki and CDK 4/6 inhibitors to enhance anti-tumor efficacy in HNSCC patients. Full article

Non-invasive detection and monitoring of lethal diseases, such as cancer, are considered as effective factors in treatment and survival. We describe a new disease diagnostic approach, denoted “reactomics”, based upon reactions between blood sera and an array of vesicles comprising different lipids and polydiacetylene (PDA), a chromatic polymer. We show that reactions between sera and such a lipid/PDA vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. The chromatic patterns were processed through machine-learning algorithms, and the bioinformatics analysis could distinguish both between cancer-bearing and healthy patients, respectively, as well between two types of cancers. Size-separation and enzymatic digestion experiments indicate that lipoproteins are the primary components in sera which react with the chromatic biomimetic vesicles. This colorimetric reactomics concept is highly generic, robust, and does not require a priori knowledge upon specific disease markers in sera. Therefore, it could be employed as complementary or alternative approach for disease diagnostics. Full article