Autologous patient-derived exhausted nano T-cells exploit tumor immune evasion to engage an effective cancer therapy.

BACKGROUND: Active targeting by surface-modified nanoplatforms enables a more precise and elevated accumulation of nanoparticles within the tumor, thereby enhancing drug delivery and efficacy for a successful cancer treatment. However, surface functionalization involves complex procedures that increase costs and timelines, presenting challenges for clinical implementation. Biomimetic nanoparticles (BNPs) have emerged as unique drug delivery platforms that overcome the limitations of actively targeted nanoparticles. Nevertheless, BNPs coated with unmodified cells show reduced functionalities such as specific tumor targeting, decreasing the therapeutic efficacy. Those challenges can be overcome by engineering non-patient-derived cells for BNP coating, but these are complex and cost-effective approaches that hinder their wider clinical application. Here we present an immune-driven strategy to improve nanotherapeutic delivery to tumors. Our unique perspective harnesses T-cell exhaustion and tumor immune evasion to develop a groundbreaking new class of BNPs crafted from exhausted T-cells (NExT) of triple-negative breast cancer (TNBC) patients by specific culture methods without sophisticated engineering. METHODS: NExT were generated by coating PLGA (poly(lactic-co-glycolic acid)) nanoparticles with TNBC-derived T-cells exhausted in vitro by acute activation. Physicochemical characterization of NExT was made by dynamic light scattering, electrophoretic light scattering and transmission electron microscopy, and preservation and orientation of immune checkpoint receptors by flow cytometry. The efficacy of chemotherapy-loaded NExT was assessed in TNBC cell lines in vitro. In vivo toxicity was made in CD1 mice. Biodistribution and therapeutic activity of NExT were determined in cell-line- and autologous patient-derived xenografts in immunodeficient mice. RESULTS: We report a cost-effective approach with a good performance that provides NExT naturally endowed with immune checkpoint receptors (PD1, LAG3, TIM3), augmenting specific tumor targeting by engaging cognate ligands, enhancing the therapeutic efficacy of chemotherapy, and disrupting the PD1/PDL1 axis in an immunotherapy-like way. Autologous patient-derived NExT revealed exceptional intratumor accumulation, heightened chemotherapeutic index and efficiency, and targeted the tumor stroma in a PDL1+ patient-derived xenograft model of triple-negative breast cancer. CONCLUSIONS: These advantages underline the potential of autologous patient-derived NExT to revolutionize tailored adoptive cancer nanotherapy and chemoimmunotherapy, which endorses their widespread clinical application of autologous patient-derived NExT.

Study of the Influence of the Wastewater Matrix in the Adsorption of Three Pharmaceuticals by Powdered Activated Carbon.

The use of powdered activated carbon (PAC) as an absorbent has become a promising option to upgrade wastewater treatment plants (WWTPs) that were not designed to remove pharmaceuticals. However, PAC adsorption mechanisms are not yet fully understood, especially with regard to the nature of the wastewater. In this study, we tested the adsorption of three pharmaceuticals, namely diclofenac, sulfamethoxazole and trimethoprim, onto PAC under four different water matrices: ultra-pure water, humic acid solution, effluent and mixed liquor from a real WWTP. The adsorption affinity was defined primarily by the pharmaceutical physicochemical properties (charge and hydrophobicity), with better results obtained for trimethoprim, followed by diclofenac and sulfamethoxazole. In ultra-pure water, the results show that all pharmaceuticals followed pseudo-second order kinetics, and they were limited by a boundary layer effect on the surface of the adsorbent. Depending on the water matrix and compound, the PAC capacity and the adsorption process varied accordingly. The higher adsorption capacity was observed for diclofenac and sulfamethoxazole in humic acid solution (Langmuir isotherm, R2 > 0.98), whereas better results were obtained for trimethoprim in the WWTP effluent. Adsorption in mixed liquor (Freundlich isotherm, R2 > 0.94) was limited, presumably due to its complex nature and the presence of suspended solids.

Challenges on optical printing of colloidal nanoparticles.

While colloidal chemistry provides ways to obtain a great variety of nanoparticles with different shapes, sizes, material compositions, and surface functions, their controlled deposition and combination on arbitrary positions of substrates remain a considerable challenge. Over the last ten years, optical printing arose as a versatile method to achieve this purpose for different kinds of nanoparticles. In this article, we review the state of the art of optical printing of single nanoparticles and discuss its strengths, limitations, and future perspectives by focusing on four main challenges: printing accuracy, resolution, selectivity, and nanoparticle photostability.

Programme for Harmonization to the International Scale in Latin America for BCR-ABL1 quantification in CML patients: findings and recommendations.

Objectives The quantitation of BCR-ABL1 mRNA is mandatory for chronic myeloid leukemia (CML) patients, and RT-qPCR is the most extensively used method in testing laboratories worldwide. Nevertheless, substantial variation in RT-qPCR results makes inter-laboratory comparability hard. To facilitate inter-laboratory comparative assessment, an international scale (IS) for BCR-ABL1 was proposed. Methods The laboratory-specific conversion factor (CF) to the IS can be derived from the World Health Organization (WHO) genetic reference panel; however, this material is limited to the manufacturers to produce and calibrate secondary reference reagents. Therefore, we developed secondary reference calibrators, as lyophilized cellular material, aligned to the IS. Our purpose was both to re-evaluate the CF in 18 previously harmonized laboratories and to propagate the IS to new laboratories. Results Our field trial including 30 laboratories across Latin America showed that, after correction of raw BCR-ABL1/ABL1 ratios using CF, the relative mean bias was significantly reduced. We also performed a follow-up of participating laboratories by annually revalidating the process; our results support the need for continuous revalidation of CFs. All participating laboratories also received a calibrator to determine the limit of quantification (LOQ); 90% of them could reproducibly detect BCR-ABL1, indicating that these laboratories can report a consistent deep molecular response. In addition, aiming to investigate the variability of BCR-ABL1 measurements across different RNA inputs, we calculated PCR efficiency for each individual assay by using different amounts of RNA. Conclusions In conclusion, for the first time in Latin America, we have successfully organized a harmonization platform for BCR-ABL1 measurement that could be of immediate clinical benefit for monitoring the molecular response of patients in low-resource regions.

Positive cancer care in Peru: Patient and provider perspectives.

Peruvian women experience high mortality from reproductive cancers, partially due to suboptimal cancer care utilization and experiences. In this qualitative study, we examined factors contributing to positive cancer care experiences. Our sample included 11 cancer patients and 27 cancer providers who attended the First International Cancer Symposium survivorship conference in Lima, Peru in 2015. We conducted thematic analysis. Emergent themes revealed that, for patients, individualized empathic care by providers was an important facilitator to positive cancer care experiences. For providers, the ability to provide such care depended on provider norms and facility infrastructure to support such patient-centered practices.

"The disease is mine, the body is mine, I decide": Individual, interpersonal, and institutional barriers and facilitators among survivors of women's cancers in Andean countries.

Recent national cancer plans address high cancer mortality in Latin America, particularly in Andean countries. Little is known about which individual, interpersonal, and institutional facilitators and barriers persist, particularly from the perspective of cancer survivors. We conducted 15 semi-structured interviews with survivors of breast and cervical cancers during and after a Pan American Health Organization sponsored conference on women's cancers in Lima, Peru. We analyzed data using an inductive content analysis approach. Patients reported primarily psychosocial barriers and facilitators at individual, interpersonal, and institutional levels. Additionally, survivors provided recom-mendations to refine existing policy to improve the cancer care experience for patients.

Proton radiation effect on InAs avalanche photodiodes.

With increasing interest over the past decade in space-related remote sensing and communications using near-infrared (NIR) wavelengths, there is a need for radiation studies on NIR avalanche photodiodes (APDs), due to the high radiation environment in space. In this work, we present an experimental study of proton radiation effects on performance parameters of InAs APDs, whose sensitivity extends from visible light to ~3.5 µm. Three irradiation energies (10.0, 31.4, and 58.8 MeV) and four fluences (109 to 1011 p/cm2) were used. At the harshest irradiation condition (10.0 MeV energy and 1011 p/cm2 fluence) the APDs' avalanche gain and leakage current showed a measurable degradation. However, the responsivity of the APDs was unaffected under all conditions tested. The data reported in this article are available from the figshare digital repository (DOI: https://dx.doi.org/10.15131/shef. DATA: 4560562).

[Guidelines for molecular monitoring of BCR-ABL1 in chronic myeloid leukemia patients by RT-qPCR]. / Recomendaciones metodológicas para el monitoreo molecular de BCR-ABL1 en pacientes con leucemia mieloide crónica por PCR cuantitativa en tiempo real.

Current clinical guidelines for managing chronic myeloid leukemia include molecular monitoring of BCR-ABL1 transcript quantitative reverse-transcription PCR. Despite the proven prognostic significance of molecular response, it is not widely appreciated that quantitative reverse-transcription PCR potentially produces highly variable data, which may affect the validity of results, making comparability between different laboratories difficult. Therefore, standardized reporting of BCR-ABL1 measurements is needed for optimal clinical management. An approach to achieve comparable BCR-ABL1 values is the use of an international reporting scale. Conversion to the international scale is achieved by the application of laboratory specific conversion factor that is obtained by using validated secondary reference calibrators. Moreover, with the aim to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate local laboratory results interpretation and reporting, we decide to prepare laboratory guidelines that will further facilitate interlaboratory comparative studies and independent quality-assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results, in particular for those most isolated laboratories, with not easy access to commercial kits or sample interchange programs.

Long-term follow-up of essential thrombocythemia patients treated with anagrelide: subgroup analysis according to JAK2/CALR/MPL mutational status.

BACKGROUND: Anagrelide represents a treatment option for essential thrombocythemia, although its place in therapy remains controversial. AIM: To assess the impact of mutational status in response rates and development of adverse events during long-term use of anagrelide. METHODS: We retrospectively evaluated 67 patients with essential thrombocythemia treated with anagrelide during 68 (4-176) months. RESULTS: Mutational frequencies were 46.3%, 28.3%, and 1.5% for JAK2V617F, CALR and MPL mutations. Anagrelide yielded a high rate of hematologic responses, which were complete in 49.25% and partial in 46.25%, without differences among molecular subsets. The rate of thrombosis during treatment was one per 100 patient-years, without excess bleeding. Anemia was the major adverse event, 30.3% at 5-yr follow-up, being more frequent in CALR(+) (P < 0.05). Myelofibrotic transformation developed in 14.9% (12.9%, 21%, and 12.5% in JAK2V617F(+), CALR(+), and triple-negative patients, respectively, P = NS) and those treated >60 months were at higher risk, OR (95% CI) 9.32 (1.1-78.5), P < 0.01, indicating the need for bone marrow monitoring during prolonged treatment. CONCLUSION: Although CALR(+) patients were at higher risk of developing anemia, anagrelide proved effective among all molecular subsets, indicating that mutational status does not seem to represent a major determinant of choice of cytoreductive treatment among essential thrombocythemia therapies.

A thorough analysis of the occurrence, removal and environmental risks of organic micropollutants in a full-scale hybrid membrane bioreactor fed by hospital wastewater.

The Urban Wastewater Treatment Directive recent draft issued last October 2022 pays attention to contaminants of emerging concern including organic micropollutants (OMPs) and requires the removal of some of them at large urban wastewater treatment plants (WWTPs) calling for their upgrading. Many investigations to date have reported the occurrence of a vast group of OMPs in the influent and many technologies have been tested for their removal at a lab- or pilot-scale. Moreover, it is well-known that hospital wastewater (HWW) contains specific OMPs at high concentration and therefore its management and treatment deserves attention. In this study, a 1-year investigation was carried out at a full-scale membrane bioreactor (MBR) treating mainly HWW. To promote the removal of OMPs, powdered activated carbon (PAC) was added to the bioreactor at 0.1 g/L and 0.2 g/L which resulted in the MBR operating as a hybrid MBR. Its performance was tested for 232 target and 90 non-target OMPs, analyzed by UHPLC-QTOF-MS using a direct injection method. A new methodology was defined to select the key compounds in order to evaluate the performance of the treatments. It was based on their frequency, occurrence, persistence to removal, bioaccumulation and toxicity. Finally, an environmental risk assessment of the OMP residues was conducted by means of the risk quotient approach. The results indicate that PAC addition increased the removal of most of the key OMPs (e.g., sulfamethoxazole, diclofenac, lidocaine) and OMP classes (e.g., antibiotics, psychiatric drugs and stimulants) with the highest loads in the WWTP influent. The hybrid MBR also reduced the risk in the receiving water as the PAC dosage increased mainly for spiramycin, lorazepam, oleandomycin. Finally, uncertainties and issues related to the investigation being carried out at full-scale under real conditions are discussed.

Generating universal anti-CD19 CAR T cells with a defined memory phenotype by CRISPR/Cas9 editing and safety evaluation of the transcriptome.

Introduction: Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype. Methods: To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence. Results: In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments. Discussion: Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies. Conclusion: We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro. This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies.

Optical coherence tomography-based scanning system for shape determination, wall thickness mapping, and inner inspection of glass containers.

In this work we present a method that enables simultaneous measurement of shape and wall parameters of glass containers. The system is based on the optical coherence tomography technique, employing the spectral domain configuration. The data were obtained by measuring the spatial coordinates of a sequence of points in a predefined region of a sample that includes points on the surface and in the interior of the material. Dimensional parameters, thickness mapping, and tomography studies of the interior of the sample walls can be obtained from these measurements.

Global Surgeon Opinion on the Impact of Surgical Access When Using Endocutters Across Specialties.

Background: Despite design enhancements in endocutters, key challenges related to limited surgical access and space can impact stapling and, potentially, surgical outcomes. Objectives: This study aimed to develop consensus statements outlining the clinical value of precise articulation and greater anatomical access in minimally invasive surgery performed by bariatric, colorectal, and thoracic surgeons. Methods: Colorectal, bariatric, and thoracic surgeons from Japan, the United States, United Kingdom, and France participated in a 2-round modified Delphi panel. Round 1 included binary, Likert scale-type, multiple-response, and open-ended questions. These were converted to affirmative statements for round 2 if sufficient agreement was reached. Consensus was set at a predefined threshold of at least 90% of panelists across all surgical specialties and regions selecting the same option ("agree" or "disagree") for the affirmative statements. Results: Of the 49 statements in the round 2 questionnaire, panelists (n=135) reached consensus that (1) tissue slippage outside stapler jaws can occur due to limited access and space; (2) greater jaw aperture could help to manipulate thick or fragile tissue more easily; (3) articulation of an endocutter is clinically important in laparoscopic surgeries; (4) improved access to hard-to-reach targets and in limited space would improve safety; and (5) an endocutter with improved access through greater articulation would become common use. Discussion: By understanding user-specific challenges and needs from both specialty- and region-wide perspectives, endoscopic stapling devices can continue to be refined. In this study, improved articulation and greater jaw aperture were the key design features examined. Improved articulation and greater jaw aperture were key stapler design features identified in this study that may mitigate the risk of instrument clashes and intraoperative complications such as anastomotic leaks. Conclusions: This study gained insights into surgeons' perspective across a variety of specialties and from 3 distinct geographies. Participating surgeons reached consensus that an endocutter with greater jaw aperture and articulation may improve surgical access and has potential to improve surgical outcomes.

High cell-free DNA is associated with disease progression, inflammasome activation and elevated levels of inflammasome-related cytokine IL-18 in patients with myelofibrosis.

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver (JAK2/CALR/MPL) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1ß and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets.

Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells.

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 3' long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivator-free, all-one-one system capable of generating Dox-inducible ATMPs.

Removal of micropollutants using a membrane bioreactor coupled with powdered activated carbon - A statistical analysis approach.

The occurrence of micropollutants in wastewater is largely documented as well as the environmental risk posed by their residues in the aquatic environment. Many investigations have been carried out and plan to study and improve their removal efficiency in existing wastewater treatment plants. At the same time, efforts are being made to develop new technologies or upgrade existing ones to increase the removal of a selection of micropollutants. Due to the great variability in their chemical and physical properties, it would be advisable to find representative compounds or identify the factors which most influence the removal mechanisms under specific conditions. This study analyses the removal efficiencies of a great number of micropollutants in wastewater treated in a membrane bioreactor coupled with powdered activated carbon (PAC), which was the subject of a review article we have recently published. The main operational parameters (i.e. PAC dosage, PAC retention time and sludge retention time) and compound physico-chemical properties (i.e. octanol-water distribution coefficient, charge and molecular weight) were first selected on the basis of a dedicated screening step and then an attempt was carried out to clarify their influence on the removal of micropollutants from wastewater during its treatment. To this end, a statistical analysis, mainly based on exploratory methods (cluster analysis and principal component analysis) and regression analysis, was carried out to compare and discuss the different results published in the scientific literature included in the cited review article. It emerged, that, based on the collected dataset, micropollutant charge and LogDow seem to play the most important role in the removal mechanisms occurring in MBR coupled with PAC.

High-Quality SiO2/O-Terminated Diamond Interface: Band-Gap, Band-Offset and Interfacial Chemistry.

Silicon oxide atomic layer deposition synthesis development over the last few years has open the route to its use as a dielectric within diamond electronics. Its great band-gap makes it a promising material for the fabrication of diamond-metal-oxide field effects transistor gates. Having a sufficiently high barrier both for holes and electrons is mandatory to work in accumulation and inversion regimes without leakage currents, and no other oxide can fulfil this requisite due to the wide diamond band-gap. In this work, the heterojunction of atomic-layer-deposited silicon oxide and (100)-oriented p-type oxygen-terminated diamond is studied using scanning transmission electron microscopy in its energy loss spectroscopy mode and X-ray photoelectron spectroscopy. The amorphous phase of silicon oxide was successfully synthesized with a homogeneous band-gap of 9.4 eV. The interface between the oxide and diamond consisted mainly of single- and double-carbon-oxygen bonds with a low density of interface states and a straddling band setting with a 2.0 eV valence band-offset and 1.9 eV conduction band-offset.

The Economic Burden of Localized Prostate Cancer and Insights Derived from Cost-Effectiveness Studies of the Different Treatments.

Prostate cancer has huge health and societal impacts, and there is no clear consensus on the most effective and efficient treatment strategy for this disease, particularly for localized prostate cancer. We have reviewed the scientific literature describing the economic burden and cost-effectiveness of different treatment strategies for localized prostate cancer in OECD countries. We initially identified 315 articles, studying 13 of them in depth (those that met the inclusion criteria), comparing the social perspectives of cost, time period, geographical area, and severity. The economic burden arising from prostate cancer due to losses in productivity and increased caregiver load is noticeable, but clinical decision-making is carried out with more subjective variability than would be advisable. The direct cost of the intervention was the main driver for the treatment of less severe cases of prostate cancer, whereas for more severe cases, the most important determinant was the loss in productivity. Newer, more affordable radiotherapy strategies may play a crucial role in the future treatment of early prostate cancer. The interpretation of our results depends on conducting thorough sensitivity analyses. This approach may help better understand parameter uncertainty and the methodological choices discussed in health economics studies. Future results of ongoing clinical trials that are considering genetic characteristics in assessing treatment response of patients with localized prostate cancer may shed new light on important clinical and pharmacoeconomic decisions.

Physiological lentiviral vectors for the generation of improved CAR-T cells.

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms. However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%-50% of the treated patients. Most CAR-T cells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor α (EF1α)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-α) and interferon (IFN)-É£ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.

Efficacy and safety of universal (TCRKO) ARI-0001 CAR-T cells for the treatment of B-cell lymphoma.

Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CAR-T products delays treatment and precludes standardization. Allogeneic off-the-shelf CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation αCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.