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Therapies targeting the PD-1/PD-L1 pathway have transformed head and neck squamous cell carcinoma (HNSCC) treatment. However, predicting the response to anti-PD-1 therapy remains a clinical challenge. This study evaluated the functional binding of PD-1 ligands in 29 HNSCC patients and compared it to the standard PD-L1 Combined Positive Score (CPS). The assessment of PD-1 ligands' functionality advances the current ability to predict the response of HNSCC patients to anti-PD-1 therapy.
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BACKGROUND: The SARS-CoV-2 pandemic led to unprecedented testing demands, causing major testing delays globally. One strategy used for increasing testing capacity was pooled-testing, using a two-stage technique first introduced during WWII. However, such traditional pooled testing was used in practice only when positivity rates were below 2%. METHODS: Here we report the development, validation and clinical application of P-BEST - a single-stage pooled-testing strategy that was approved for clinical use in Israel. RESULTS: P-BEST is clinically validated using 3636 side-by-side tests and is able to correctly detect all positive samples and accurately estimate their Ct value. Following regulatory approval by the Israeli Ministry of Health, P-BEST was used in 2021 to clinically test 837,138 samples using 270,095 PCR tests - a 3.1fold reduction in the number of tests. This period includes the Alpha and Delta waves, when positivity rates exceeded 10%, rendering traditional pooling non-practical. We also describe a tablet-based solution that allows performing manual single-stage pooling in settings where liquid dispensing robots are not available. CONCLUSIONS: Our data provides a proof-of-concept for large-scale clinical implementation of single-stage pooled-testing for continuous surveillance of multiple pathogens with reduced test costs, and as an important tool for increasing testing efficiency during pandemic outbreaks.
Testing samples for SARS-CoV-2 is usually done on one sample at a time. However, the unprecedented demand for testing during the COVID-19 pandemic led to the adoption of pooled testing strategies, where samples are combined before being tested. This strategy requires two rounds: first, each pool of samples is tested, and then a second testing round is performed on individual samples from positive pools. We developed and implemented a pooling method for SARS-CoV-2 that requires a single round of testing, thus enabling the shorter turnaround times required during a pandemic. The method was approved for clinical use in Israel and was used to successfully test 837,138 clinical samples using fewer than a third of the tests usually required. Our study provides a blueprint for rapid implementation of efficient high-throughput testing in future pandemics.
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We investigated the role of ligand clustering and density in the activation of natural killer (NK) cells. To that end, we designed reductionist arrays of nanopatterned ligands arranged with different cluster geometries and densities and probed their effects on NK cell activation. We used these arrays as an artificial microenvironment for the stimulation of NK cells and studied the effect of the array geometry on the NK cell immune response. We found that ligand density significantly regulated NK cell activation while ligand clustering had an impact only at a specific density threshold. We also rationalized these findings by introducing a theoretical membrane fluctuation model that considers biomechanical feedback between ligand-receptor bonds and the cell membrane. These findings provide important insight into NK cell mechanobiology, which is fundamentally important and essential for designing immunotherapeutic strategies targeting cancer.
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Membrana Celular , Células Asesinas Naturales , Células Asesinas Naturales/inmunología , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Ligandos , Activación de Linfocitos , Fenómenos Biomecánicos , Modelos BiológicosRESUMEN
PURPOSE: In the pursuit of creating personalized and more effective treatment strategies for lung cancer patients, Patient-Derived Xenografts (PDXs) have been introduced as preclinical platforms that can recapitulate the specific patient's tumor in an in vivo model. We investigated how well PDX models can preserve the tumor's clinical and molecular characteristics across different generations. METHODS: A Non-Small Cell Lung Cancer (NSCLC) PDX model was established in NSG-SGM3 mice and clinical and preclinical factors were assessed throughout subsequent passages. Our cohort consisted of 40 NSCLC patients, which were used to create 20 patient-specific PDX models in NSG-SGM3 mice. Histopathological staining and Whole Exome Sequencing (WES) analysis were preformed to understand tumor heterogeneity throughout serial passages. RESULTS: The main factors that contributed to the growth of the engrafted PDX in mice were a higher grade or stage of disease, in contrast to the long duration of chemotherapy treatment, which was negatively correlated with PDX propagation. Successful PDX growth was also linked to poorer prognosis and overall survival, while growth pattern variability was affected by the tumor aggressiveness, primarily affecting the first passage. Pathology analysis showed preservation of the histological type and grade; however, WES analysis revealed genomic instability in advanced passages, leading to the inconsistencies in clinically relevant alterations between the PDXs and biopsies. CONCLUSIONS: Our study highlights the impact of multiple clinical and preclinical factors on the engraftment success, growth kinetics, and tumor stability of patient-specific NSCLC PDXs, and underscores the importance of considering these factors when guiding and evaluating prolonged personalized treatment studies for NSCLC patients in these models, as well as signaling the imperative for additional investigations to determine the full clinical potential of this technique.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Medicina de Precisión , Ensayos Antitumor por Modelo de Xenoinjerto , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/terapia , Animales , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Ratones , Medicina de Precisión/métodos , Femenino , Masculino , Persona de Mediana Edad , Anciano , Secuenciación del Exoma , Modelos Animales de EnfermedadRESUMEN
We introduce a novel approach for colloidal lithography based on the dry particle assembly into a dense monolayer on an elastomer, followed by mechanical transfer to a substrate of any material and curvature. This method can be implemented either manually or automatically and it produces large area patterns with the quality obtained by the state-of-the-art colloidal lithography at a very high throughput. We first demonstrated the fabrication of nanopatterns with a periodicity ranging between 200 nm and 2 µm. We then demonstrated two nanotechnological applications of this approach. The first one is antireflective structures, fabricated on silicon and sapphire, with different geometries including arrays of bumps and holes and adjusted for different spectral ranges. The second one is smart 3D nanostructures for mechanostimulation of T cells that are used for their effective proliferation, with potential application in cancer immunotherapy. This new approach unleashes the potential of bottom-up nanofabrication and paves the way for nanoscale devices and systems in numerous applications.
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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.
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T cells respond not only to biochemical stimuli transmitted through their activating, costimulatory, and inhibitory receptors but also to biophysical aspects of their environment, including the receptors' spatial arrangement. While these receptors form nanoclusters that can either colocalize or segregate, the roles of these colocalization and segregation remain unclear. Deciphering these roles requires a nanoscale platform with independent and simultaneous spatial control of multiple types of receptors. Herein, using a straightforward and modular fabrication process, we engineered a tunable nanoscale chip used as a platform for T cell stimulation, allowing spatial control over the clustering and segregation of activating, costimulatory, and inhibitory receptors. Using this platform, we showed that, upon blocked inhibition, cells became sensitive to changes in the nanoscale ligand configuration. The nanofabrication methodology described here opens a pathway to numerous studies, which will produce an important insight into the molecular mechanism of T cell activation. This insight is essential for the fundamental understanding of our immune system as well as for the rational design of future immunotherapies.
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The ex vivo activation and proliferation of cytotoxic T cells are critical steps in adoptive immunotherapy. Today, T cells are activated by stimulation with antibody-coated magnetic beads, traditionally used for cell separation. Yet, efficient and controlled activation and proliferation of T cells require new antibody-bearing materials, which, in particular, deliver mechanical and topographic cues sensed by T cells. Here, we demonstrate a new approach for the activation and proliferation of human cytotoxic T cells using an elastic microbrush coated with activating and costimulatory antibodies. We found that the microbrush topography affects the protrusion of the cell membrane and the elastic response to the forces applied by cells and can be optimized to yield the strongest activation of T cells. In particular, T cells stimulated by a microbrush showed a three-fold increase in degranulation and release of cytokines over T cells stimulated with state-of-the-art magnetic beads. Furthermore, the microbrush induced a T-cell proliferation of T cells that was more prolonged and yielded much higher cell doubling than that done by the state-of-the-art methods. Our study provides an essential insight into the physical mechanism of T-cell activation and proliferation and opens the floodgates for the design of novel stimulatory materials for T-cell-based immunotherapy.
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Señales (Psicología) , Linfocitos T Citotóxicos , Humanos , Inmunoterapia , Inmunoterapia Adoptiva/métodos , Activación de Linfocitos , Proliferación CelularRESUMEN
Accurate predictive biomarkers of response to immune checkpoint inhibitors (ICIs) are required for better stratifying patients with cancer to ICI treatments. Here, we present a new concept for a bioassay to predict the response to anti-PD1 therapies, which is based on measuring the binding functionality of PDL1 and PDL2 to their receptor, PD1. In detail, we developed a cell-based reporting system, called the immuno-checkpoint artificial reporter with overexpression of PD1 (IcAR-PD1) and evaluated the functionality of PDL1 and PDL2 binding in tumor cell lines, patient-derived xenografts, and fixed-tissue tumor samples obtained from patients with cancer. In a retrospective clinical study, we found that the functionality of PDL1 and PDL2 predicts response to anti-PD1 and that the functionality of PDL1 binding is a more effective predictor than PDL1 protein expression alone. Our findings suggest that assessing the functionality of ligand binding is superior to staining of protein expression for predicting response to ICIs.
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Neoplasias , Humanos , Estudios Retrospectivos , Ligandos , Neoplasias/tratamiento farmacológicoRESUMEN
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.
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Antineoplásicos , Neoplasias Pulmonares , Humanos , Ratones , Animales , Sistemas de Liberación de Medicamentos , Péptidos/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Microambiente Tumoral , Modelos Animales de EnfermedadRESUMEN
Background: SARS-CoV-2 (COVID-19) elicits a T-cell antigen-mediated immune response of variable efficacy. To understand this variability, we explored transcriptomic expression of angiotensin-converting enzyme 2 (ACE2, the SARS-CoV-2 receptor) and of immunoregulatory genes in normal lung tissues from patients with non-small cell lung cancer (NSCLC). Methods: This study used the transcriptomic and the clinical data for NSCLC patients generated during the CHEMORES study [n = 123 primary resected (early-stage) NSCLC] and the WINTHER clinical trial (n = 32 metastatic NSCLC). Results: We identified patient subgroups with high and low ACE2 expression (p = 1.55 × 10-19) in normal lung tissue, presumed to be at higher and lower risk, respectively, of developing severe COVID-19 should they become infected. ACE2 transcript expression in normal lung tissues (but not in tumor tissue) of patients with NSCLC was higher in individuals with more advanced disease. High-ACE2 expressors had significantly higher levels of CD8+ cytotoxic T lymphocytes and natural killer cells but with presumably impaired function by high Thymocyte Selection-Associated High Mobility Group Box Protein TOX (TOX) expression. In addition, immune checkpoint-related molecules - PD-L1, CTLA-4, PD-1, and TIGIT - are more highly expressed in normal (but not tumor) lung tissues; these molecules might dampen immune response to either viruses or cancer. Importantly, however, high inducible T-cell co-stimulator (ICOS), which can amplify immune and cytokine reactivity, significantly correlated with high ACE2 expression in univariable analysis of normal lung (but not lung tumor tissue). Conclusions: We report a normal lung immune-tolerant state that may explain a potential comorbidity risk between two diseases - NSCLC and susceptibility to COVID-19 pneumonia. Further, a NSCLC patient subgroup has normal lung tissue expressing high ACE2 and high ICOS transcripts, the latter potentially promoting a hyperimmune response, and possibly leading to severe COVID-19 pulmonary compromise.
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Objectives: To evaluate the association between the achievements of medical students and whether they were admitted via the pre-medical track or the regular direct track. Methods: We performed a comparative retrospective data study using data from a three-year experimental cohort in a six-year medical school. We analyzed the academic achievements of all students admitted at one Israeli medical school between 2013-2015, either directly to the six-year program or via a pre-medical track. We compared averages of both yearly final grades and final medical examinations grades between the two groups. Descriptive statistics were calculated and differences between groups were evaluated using multivariate analysis. Results: Of the 324 students included in the study, 65 (20.1%) were enrolled in all three cohorts of the pre-medical track. Age and Gender distribution were nearly similar for both tracks. For the first two cohorts, the average final grades of year one of pre-medical students were significantly higher than those of regular direct track (F=(3,167) 6.10, p=0.001), but the opposite was true for the third cohort (F=(3,110)2.38, p=0.073). No further statistically significant differences were found neither between the groups in their final exams grades nor between choosing a MD/PhD optional track and admission pathway. Conclusions: Our results suggest promising achievements with the pre-medical admission pathway. This should encourage further discussion about the significant potential human resources lost by current admission processes and may question the effectiveness of six-year programs in medical schools.
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Logro , Estudiantes de Medicina , Estudios de Cohortes , Humanos , Estudios Retrospectivos , Facultades de MedicinaRESUMEN
PURPOSE: The prognosis of patients with non-small-cell lung cancer (NSCLC), traditionally determined by anatomic histology and TNM staging, neglects the biological features of the tumor that may be important in determining patient outcome and guiding therapeutic interventions. Identifying patients with NSCLC at increased risk of recurrence after curative-intent surgery remains an important unmet need so that known effective adjuvant treatments can be offered to those at highest risk of recurrence. METHODS: Relative gene expression level in the primary tumor and normal bronchial tissues was used to retrospectively assess their association with disease-free survival (DFS) in a cohort of 120 patients with NSCLC who underwent curative-intent surgery. RESULTS: Low versus high Digital Display Precision Predictor (DDPP) score (a measure of relative gene expression) was significantly associated with shorter DFS (highest recurrence risk; P = .006) in all patients and in patients with TNM stages 1-2 (P = .00051; n = 83). For patients with stages 1-2 and low DDPP score (n = 29), adjuvant chemotherapy was associated with improved DFS (P = .0041). High co-overexpression of CTLA-4, PD-L1, and ICOS in normal lung (28 of 120 patients) was also significantly associated with decreased DFS (P = .0013), suggesting an immune tolerance to tumor neoantigens in some patients. Patients with DDPP low and immunotolerant normal tissue had the shortest DFS (P = 2.12E-11). CONCLUSION: TNM stage, DDPP score, and immune competence status of normal lung are independent prognostic factors in multivariate analysis. Our findings open new avenues for prospective prognostic assessment and treatment assignment on the basis of transcriptomic profiling of tumor and normal lung tissue in patients with NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Antígeno B7-H1/análisis , Antígeno CTLA-4/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Pulmón/química , Neoplasias Pulmonares/tratamiento farmacológico , Estudios Prospectivos , Estudios Retrospectivos , TranscriptomaRESUMEN
The current Covid-19 pandemic has a profound impact on all aspects of our lives. Aside from contagion by aerosols, the presence of the SARS-CoV-2 is ubiquitous on surfaces that millions of people handle daily. Therefore, controlling this pandemic involves the reduction of potential infections via contaminated surfaces. We developed antiviral surfaces by preparing suspensions of copper and cupric oxide nanoparticles in two different polymer matrices, poly(methyl methacrylate) and polyepoxide. For total copper contents as low as 5%, the composite material showed remarkable antiviral properties against the HCoV-OC43 human coronavirus and against a model lentivirus and proved well-resistant to accelerated aging conditions. Importantly, we showed that the Cu/CuO mixture showed optimal performances. This product can be implemented to produce a simple and inexpensive coating with long-term antiviral properties and will open the way to developing surface coatings against a broad spectrum of pathogens including SARS-CoV-2.
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COVID-19 , Coronavirus Humano OC43 , Nanocompuestos , Antivirales , COVID-19/prevención & control , Cobre/farmacología , Humanos , Pandemias/prevención & control , SARS-CoV-2RESUMEN
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.
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Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Inmunoterapia Adoptiva , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T , Microambiente Tumoral/genéticaRESUMEN
Cancer immunotherapies are highly potent and are gaining wide clinical usage. However, severe side effects require focusing effector immune cell activities on the tumor microenvironment (TME). We recently developed a chimeric antigen receptor tumor-induced vector (CARTIV), a synthetic promoter activated by TME factors. To improve CARTIV functions including background, activation levels, and synergism, we screened a library of promoters with variations in key positions. Here, we present a screening method involving turning ON/OFF stimulating TNFα and IFNγ cytokines, followed by sequential cell sorting. Sequencing of enriched promoters identified seventeen candidates, which were cloned and whose activities were then validated, leading to the identification of two CARTIVs with lower background and higher induction. We further combined a third hypoxia element with the two-factor CARTIV, demonstrating additional modular improvement. Our study presents a method of fine-tuning synthetic promoters for desired immunotherapy needs.
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Inmunoterapia , Neoplasias , Microambiente Tumoral , Biblioteca de Genes , Humanos , Neoplasias/terapia , Regiones Promotoras GenéticasRESUMEN
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.
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Mieloma Múltiple , Línea Celular Tumoral , Humanos , Células Asesinas Naturales , Mieloma Múltiple/metabolismo , Receptor 2 Gatillante de la Citotoxidad Natural/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
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.
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Transduction of primary T cells has become prominent with the introduction of chimeric antigen receptor T-cell therapy. Although there are many protocols for the transduction of human T cells, it remains a challenge to transduce murine T cells. We present an optimized protocol for the retroviral transduction of murine CD4 T cells, which overcomes major challenges including large-scale production and long-term culturing of transduced cells. The optimized protocol combines high transduction efficiency with a low rate of cell death. For complete details on the use and execution of this protocol, please refer to Eremenko et al., 2019.
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Inmunoterapia Adoptiva/métodos , Transducción Genética/métodos , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citometría de Flujo/métodos , Vectores Genéticos/genética , Ratones , Retroviridae/genéticaRESUMEN
The blood-brain barrier acts as a major barrier for the entrance of most therapeutics into the brain, impeding treatment for neurological disorders. Intracerebroventricular (ICV) injection of T cells is a useful tool for cell therapy of neurological disorders including neurodegenerative and neuropsychiatric diseases and brain tumors. Here, we present an optimized ICV injection of T cells with improved injection efficiency at pathological sites within the brain parenchyma. We describe details of the surgical procedure and verification of injection via immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Fisher et al. (2014); Strominger et al., (2018); Mittal et al. (2019); Eremenko et al. (2019).