Memory-like differentiation enhances NK cell responses against colorectal cancer.

Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB.

Avoiding a reproducibility crisis in regulatory toxicology-on the fundamental role of ring trials.

The ongoing transition from chemical hazard and risk assessment based on animal studies to assessment relying mostly on non-animal data, requires a multitude of novel experimental methods, and this means that guidance on the validation and standardisation of test methods intended for international applicability and acceptance, needs to be updated. These so-called new approach methodologies (NAMs) must be applicable to the chemical regulatory domain and provide reliable data which are relevant to hazard and risk assessment. Confidence in and use of NAMs will depend on their reliability and relevance, and both are thoroughly assessed by validation. Validation is, however, a time- and resource-demanding process. As updates on validation guidance are conducted, the valuable components must be kept: Reliable data are and will remain fundamental. In 2016, the scientific community was made aware of the general crisis in scientific reproducibility-validated methods must not fall into this. In this commentary, we emphasize the central importance of ring trials in the validation of experimental methods. Ring trials are sometimes considered to be a major hold-up with little value added to the validation. Here, we clarify that ring trials are indispensable to demonstrate the robustness and reproducibility of a new method. Further, that methods do fail in method transfer and ring trials due to different stumbling blocks, but these provide learnings to ensure the robustness of new methods. At the same time, we identify what it would take to perform ring trials more efficiently, and how ring trials fit into the much-needed update to the guidance on the validation of NAMs.

Bridging therapy with axicabtagene ciloleucel for large B-cell lymphoma: results from the US Lymphoma CAR-T Consortium.

ABSTRACT: During the manufacturing period of autologous chimeric antigen receptor (CAR) T-cell therapy, patients may experience a decline in their condition due to cancer progression. In this study, we investigated the impact of bridging therapy (BT) on the outcome of patients with relapsed/refractory large B-cell lymphoma who received antilymphoma treatment between leukapheresis and axicabtagene ciloleucel (axi-cel) infusion. We conducted our analysis using data from the multicenter US Lymphoma CAR-T Consortium, with a median follow-up of 33 months (range, 4.3-42.1). Out of the 298 patients who underwent leukapheresis, 275 patients received axi-cel. A total 52% of patients (n = 143) who received BT had a higher baseline risk profile than patients who did not receive BT, and these patients, as a group, had inferior outcomes compared with those who did not receive BT. However, after propensity score matching between the 2 groups, there were no statistically significant differences in overall response rate (77% vs 87%; P = .13), complete response rate (58% vs 70%; P = .1), progression-free survival (hazard ratio [HR], 1.25; P = .23), and overall survival (HR, 1.39; P=.09) between the BT group and the no-BT group, respectively. Analyzing the effects of BT in the whole cohort that underwent leukapheresis regardless of receiving axi-cel (intention-to-treat analysis) showed similar results. Radiation BT resulted in outcomes similar to those observed with nonradiation BT. Our findings suggest that BT may be safe without a significant impact on long-term survival for patients who require disease stabilization during the manufacturing period. Moreover, our results suggest that there is no clear advantage to using radiation-based BT over nonradiation-based BT.

Increased Cell Proliferation as a Key Event in Chemical Carcinogenesis: Application in an Integrated Approach for the Testing and Assessment of Non-Genotoxic Carcinogenesis.

In contrast to genotoxic carcinogens, there are currently no internationally agreed upon regulatory tools for identifying non-genotoxic carcinogens of human relevance. The rodent cancer bioassay is only used in certain regulatory sectors and is criticized for its limited predictive power for human cancer risk. Cancer is due to genetic errors occurring in single cells. The risk of cancer is higher when there is an increase in the number of errors per replication (genotoxic agents) or in the number of replications (cell proliferation-inducing agents). The default regulatory approach for genotoxic agents whereby no threshold is set is reasonably conservative. However, non-genotoxic carcinogens cannot be regulated in the same way since increased cell proliferation has a clear threshold. An integrated approach for the testing and assessment (IATA) of non-genotoxic carcinogens is under development at the OECD, considering learnings from the regulatory assessment of data-rich substances such as agrochemicals. The aim is to achieve an endorsed IATA that predicts human cancer better than the rodent cancer bioassay, using methodologies that equally or better protect human health and are superior from the view of animal welfare/efficiency. This paper describes the technical opportunities available to assess cell proliferation as the central gateway of an IATA for non-genotoxic carcinogenicity.

Memory-like Differentiation, Tumor-Targeting mAbs, and Chimeric Antigen Receptors Enhance Natural Killer Cell Responses to Head and Neck Cancer.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell therapy-refractory cancers, but are frequently dysfunctional in patients with HNSCC. Strategies are needed to enhance NK cell responses against HNSCC. We hypothesized that memory-like (ML) NK cell differentiation, tumor targeting with cetuximab, and engineering with an anti-EphA2 (Erythropoietin-producing hepatocellular receptor A2) chimeric antigen receptor (CAR) enhance NK cell responses against HNSCC. EXPERIMENTAL DESIGN: We generated ML NK and conventional (c)NK cells from healthy donors, then evaluated their ability to produce IFNγ, TNF, degranulate, and kill HNSCC cell lines and primary HNSCC cells, alone or in combination with cetuximab, in vitro and in vivo using xenograft models. ML and cNK cells were engineered to express anti-EphA2 CAR-CD8A-41BB-CD3z, and functional responses were assessed in vitro against HNSCC cell lines and primary HNSCC tumor cells. RESULTS: Human ML NK cells displayed enhanced IFNγ and TNF production and both short- and long-term killing of HNSCC cell lines and primary targets, compared with cNK cells. These enhanced responses were further improved by cetuximab. Compared with controls, ML NK cells expressing anti-EphA2 CAR had increased IFNγ and cytotoxicity in response to EphA2+ cell lines and primary HNSCC targets. CONCLUSIONS: These preclinical findings demonstrate that ML differentiation alone or coupled with either cetuximab-directed targeting or EphA2 CAR engineering were effective against HNSCCs and provide the rationale for investigating these combination approaches in early phase clinical trials for patients with HNSCC.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

T-BET and EOMES sustain mature human NK cell identity and antitumor function.

Since the T-box transcription factors (TFs) T-BET and EOMES are necessary for initiation of NK cell development, their ongoing requirement for mature NK cell homeostasis, function, and molecular programming remains unclear. To address this, T-BET and EOMES were deleted in unexpanded primary human NK cells using CRISPR/Cas9. Deleting these TFs compromised in vivo antitumor response of human NK cells. Mechanistically, T-BET and EOMES were required for normal NK cell proliferation and persistence in vivo. NK cells lacking T-BET and EOMES also exhibited defective responses to cytokine stimulation. Single-cell RNA-Seq revealed a specific T-box transcriptional program in human NK cells, which was rapidly lost following T-BET and EOMES deletion. Further, T-BET- and EOMES-deleted CD56bright NK cells acquired an innate lymphoid cell precursor-like (ILCP-like) profile with increased expression of the ILC-3-associated TFs RORC and AHR, revealing a role for T-box TFs in maintaining mature NK cell phenotypes and an unexpected role of suppressing alternative ILC lineages. Our study reveals the critical importance of sustained EOMES and T-BET expression to orchestrate mature NK cell function and identity.

Development of a reference and proficiency chemical list for human steatosis endpoints in vitro.

The most prevalent liver disease in humans is non-alcoholic fatty liver disease, characterised by excessive hepatic fat accumulation, or steatosis. The western diet and a sedentary lifestyle are considered to be major influences, but chemical exposure may also play a role. Suspected environmental chemicals of concern include pesticides, plasticizers, metals, and perfluorinated compounds. Here we present a detailed literature analysis of chemicals that may (or may not) be implicated in lipid accumulation in the liver, to provide a basis for developing and optimizing human steatosis-relevant in vitro test methods. Independently collated and reviewed reference and proficiency chemicals are needed to assist in the test method development where an assay is intended to ultimately be taken forward for OECD Test Guideline development purposes. The selection criteria and considerations required for acceptance of proficiency chemical selection for OECD Test Guideline development. (i.e., structural diversity, range of activity including negatives, relevant chemical sectors, global restrictions, etc.) is described herein. Of 160 chemicals initially screened for inclusion, 36 were prioritized for detailed review. Based on the selection criteria and a weight-of-evidence basis, 18 chemicals (9 steatosis inducers, 9 negatives), including some environmental chemicals of concern, were ranked as high priority chemicals to assist in vitro human steatosis test method optimisation and proficiency testing, and inform potential subsequent test method (pre-)validation.

Real-world evidence of the safety and survival with CD19 CAR-T cell therapy for relapsed/refractory solid organ transplant-related PTLD.

The use of CD19 chimeric antigen receptor T-cell (CAR-T) therapy for relapsed/refractory solid organ transplantation (SOT)-related post-transplant lymphoproliferative disorder (PTLD) is not well studied. We conducted a multicentre, retrospective analysis of adults with relapsed/refractory SOT-associated PTLD. Among 22 relapsed/refractory SOT-PTLD patients, the pathology was monomorphic B cell. Prior SOTs included 14 kidney (64%), three liver (14%), two heart (9%), one intestinal (5%), one lung (5%), and one pancreas after kidney transplant (5%). The median time from SOT to PTLD diagnosis was 107 months. Pre-CAR-T bridging therapy was used in 55% of patients, and immunosuppression was stopped completely before CAR-T infusion in 64%. Eighteen (82%) patients experienced cytokine release syndrome: one (5%) each grade (G) 3 and G4. The immune effector cell-associated neurotoxicity syndrome was observed in 16 (73%) patients: six (27%) G3 and two (9%) G4. The overall response rate was 64% (55% complete response). Three patients (14%) experienced allograft rejection after CAR-T. The two-year progression-free survival and overall survival rates were 35% and 58%, respectively. Additionally, the achievement of CR post-CAR-T was strongly associated with survival. Collectively, the safety and efficacy of CD19 CAR-T therapy in relapsed/refractory SOT-related PTLD appeared similar to pivotal CAR-T data, including approximately one-third of patients achieving sustained remission.

OECD workshop consensus report: Ethical considerations with respect to human derived products, specifically human serum, in OECD test guidelines.

The ethical needs and concerns with use and sourcing of human materials, particularly serum, in OECD in vitro test guidelines were explored in a dedicated international workshop held in 2019. The health-related aspects of the donation procedure, including tissue screening, donor health, laboratory work health protection, permission from the donor for commercial use, payment of the donors and the potential for exploitation of low-income populations and data protection of the donors; supply, availability, and competition with clinical needs; traceability of the serum and auditability/GLP needs for the Test Guideline Programme, were examined. Here we provide the recommendations of the workshop with respect to the use of human serum, and potentially other human reagents, specifically with regard to test method development for OECD Test Guideline utility as part of the Mutual Acceptance of Data requirement across all OECD member countries. These include informed donor consent terminology, a checklist of human serum information requirements to be included with the Good Laboratory Practise report, and suitable sources for human serum to ensure waste supplies are used, that can no longer be used for medical purposes, ensuring no competition of supply for essential medical use.

The Cell Transformation Assay: A Historical Assessment of Current Knowledge of Applications in an Integrated Approach to Testing and Assessment for Non-Genotoxic Carcinogens.

The history of the development of the cell transformation assays (CTAs) is described, providing an overview of in vitro cell transformation from its origin to the new transcriptomic-based CTAs. Application of this knowledge is utilized to address how the different types of CTAs, variously addressing initiation and promotion, can be included on a mechanistic basis within the integrated approach to testing and assessment (IATA) for non-genotoxic carcinogens. Building upon assay assessments targeting the key events in the IATA, we identify how the different CTA models can appropriately fit, following preceding steps in the IATA. The preceding steps are the prescreening transcriptomic approaches, and assessment within the earlier key events of inflammation, immune disruption, mitotic signaling and cell injury. The CTA models address the later key events of (sustained) proliferation and change in morphology leading to tumor formation. The complementary key biomarkers with respect to the precursor key events and respective CTAs are mapped, providing a structured mechanistic approach to represent the complexity of the (non-genotoxic) carcinogenesis process, and specifically their capacity to identify non-genotoxic carcinogenic chemicals in a human relevant IATA.

Brexucabtagene Autoleucel for Relapsed or Refractory Mantle Cell Lymphoma in Standard-of-Care Practice: Results From the US Lymphoma CAR T Consortium.

PURPOSE: Brexucabtagene autoleucel (brexu-cel) is an autologous CD19-directed chimeric antigen receptor (CAR) T-cell therapy approved for relapsed/refractory mantle cell lymphoma (MCL). This therapy was approved on the basis of the single-arm phase II ZUMA-2 trial, which showed best overall and complete response rates of 91% and 68%, respectively. We report clinical outcomes with brexu-cel in the standard-of-care setting for the approved indication. PATIENTS AND METHODS: Patients who underwent leukapheresis between August 1, 2020 and December 31, 2021, at 16 US institutions, with an intent to manufacture commercial brexu-cel for relapsed/refractory MCL, were included. Patient data were collected for analyses of responses, outcomes, and toxicities as per standard guidelines. RESULTS: Of 189 patients who underwent leukapheresis, 168 (89%) received brexu-cel infusion. Of leukapheresed patients, 79% would not have met ZUMA-2 eligibility criteria. Best overall and complete response rates were 90% and 82%, respectively. At a median follow-up of 14.3 months after infusion, the estimates for 6- and 12-month progression-free survival (PFS) were 69% (95% CI, 61 to 75) and 59% (95% CI, 51 to 66), respectively. The nonrelapse mortality was 9.1% at 1 year, primarily because of infections. Grade 3 or higher cytokine release syndrome and neurotoxicity occurred in 8% and 32%, respectively. In univariable analysis, high-risk simplified MCL international prognostic index, high Ki-67, TP53 aberration, complex karyotype, and blastoid/pleomorphic variant were associated with shorter PFS after brexu-cel infusion. Patients with recent bendamustine exposure (within 24 months before leukapheresis) had shorter PFS and overall survival after leukapheresis in intention-to-treat univariable analysis. CONCLUSION: In the standard-of-care setting, the efficacy and toxicity of brexu-cel were consistent with those reported in the ZUMA-2 trial. Tumor-intrinsic features of MCL, and possibly recent bendamustine exposure, may be associated with inferior efficacy outcomes.

Allogeneic natural killer cell therapy.

Interest in adoptive cell therapy for treating cancer is exploding owing to early clinical successes of autologous chimeric antigen receptor (CAR) T lymphocyte therapy. However, limitations using T cells and autologous cell products are apparent as they (1) take weeks to generate, (2) utilize a 1:1 donor-to-patient model, (3) are expensive, and (4) are prone to heterogeneity and manufacturing failures. CAR T cells are also associated with significant toxicities, including cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, and prolonged cytopenias. To overcome these issues, natural killer (NK) cells are being explored as an alternative cell source for allogeneic cell therapies. NK cells have an inherent ability to recognize cancers, mediate immune functions of killing and communication, and do not induce graft-versus-host disease, cytokine release syndrome, or immune effector cell-associated neurotoxicity syndrome. NK cells can be obtained from blood or cord blood or be derived from hematopoietic stem and progenitor cells or induced pluripotent stem cells, and can be expanded and cryopreserved for off-the-shelf availability. The first wave of point-of-care NK cell therapies led to the current allogeneic NK cell products being investigated in clinical trials with promising preliminary results. Basic advances in NK cell biology and cellular engineering have led to new translational strategies to block inhibition, enhance and broaden target cell recognition, optimize functional persistence, and provide stealth from patients' immunity. This review details NK cell biology, as well as NK cell product manufacturing, engineering, and combination therapies explored in the clinic leading to the next generation of potent, off-the-shelf cellular therapies for blood cancers.

Analyses of Transcriptomics Cell Signalling for Pre-Screening Applications in the Integrated Approach for Testing and Assessment of Non-Genotoxic Carcinogens.

With recent rapid advancement of methodological tools, mechanistic understanding of biological processes leading to carcinogenesis is expanding. New approach methodologies such as transcriptomics can inform on non-genotoxic mechanisms of chemical carcinogens and can be developed for regulatory applications. The Organisation for the Economic Cooperation and Development (OECD) expert group developing an Integrated Approach to the Testing and Assessment (IATA) of Non-Genotoxic Carcinogens (NGTxC) is reviewing the possible assays to be integrated therein. In this context, we review the application of transcriptomics approaches suitable for pre-screening gene expression changes associated with phenotypic alterations that underlie the carcinogenic processes for subsequent prioritisation of downstream test methods appropriate to specific key events of non-genotoxic carcinogenesis. Using case studies, we evaluate the potential of gene expression analyses especially in relation to breast cancer, to identify the most relevant approaches that could be utilised as (pre-) screening tools, for example Gene Set Enrichment Analysis (GSEA). We also consider how to address the challenges to integrate gene panels and transcriptomic assays into the IATA, highlighting the pivotal omics markers identified for assay measurement in the IATA key events of inflammation, immune response, mitogenic signalling and cell injury.

Use of New Approach Methodologies (NAMs) in regulatory decisions for chemical safety: Report from an EPAA Deep Dive Workshop.

New Approach Methodologies (NAMs) are considered to include any in vitro, in silico or chemistry-based method, as well as the strategies to implement them, that may provide information that could inform chemical safety assessment. Current chemical legislation in the European Union is limited in its acceptance of the widespread use of NAMs. The European Partnership for Alternative Approaches to Animal Testing (EPAA) therefore convened a 'Deep Dive Workshop' to explore the use of NAMs in chemical safety assessment, the aim of which was to support regulatory decisions, whilst intending to protect human health. The workshop recognised that NAMs are currently used in many industrial sectors, with some considered as fit for regulatory purpose. Moreover, the workshop identified key discussion points that can be addressed to increase the use and regulatory acceptance of NAMs. These are based on the changes needed in frameworks for regulatory requirements and the essential needs in education, training and greater stakeholder engagement as well the gaps in the scientific basis of NAMs.

Severity of Cytokine Release Syndrome Influences Outcome After Axicabtagene Ciloleucel for Large B cell Lymphoma: Results from the US Lymphoma CAR-T Consortium.

BACKGROUND: The majority of patients with large B-cell lymphoma treated with axicabtagene ciloleucel (axi-cel), an anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, develop cytokine release syndrome (CRS). Whether the lack of development of CRS with axi-cel is associated with inferior lymphoma outcomes is unknown. Additionally, relationship between CRS grade and lymphoma outcome is not well established. METHODS: The US Lymphoma CAR T Consortium includes seventeen US academic centers that contribute data independently of manufacturers. We analyzed the modified intent-to-treat population of 275 patients receiving axi-cel in two different ways: 1) Two group analysis comparing no CRS with any grade CRS; 2) Three group analysis comparing grade 0 CRS with grade 1 to 2 CRS, and grade 3-5 CRS. RESULTS: In this large multi-center observational cohort of 275 patients receiving axi-cel, 9% (n = 24) did not develop CRS, 84% (n = 232) developed grade 1-2 CRS, and 7% (n = 19) developed grade 3 to 5 CRS. Patients without CRS, compared with those having any grade CRS, had similar overall response rates (ORR), lower complete response (CR) rates and inferior progression free survival (PFS) with no statistically significant difference in overall survival (OS). Patients experiencing grade 1 to 2 CRS had superior CR rate and PFS, as compared to those without CRS or with grade 3 to 5 CRS. Grade 3 to 5 CRS was associated with a worse OS. CONCLUSION: Overall, durable responses were seen in patients that did not develop CRS, however grade 1 to 2 CRS was associated with better outcomes while those with grade 3 to 5 experienced the worse outcomes.

Candidate Proficiency Test Chemicals to Address Industrial Chemical Applicability Domains for in vitro Human Cytochrome P450 Enzyme Induction.

Cytochrome P450 (CYP) enzymes play a key role in the metabolism of both xenobiotics and endogenous chemicals, and the activity of some CYP isoforms are susceptible to induction and/or inhibition by certain chemicals. As CYP induction/inhibition can bring about significant alterations in the level of in vivo exposure to CYP substrates and metabolites, CYP induction/inhibition data is needed for regulatory chemical toxicity hazard assessment. On the basis of available human in vivo pharmaceutical data, a draft Organisation for Economic Co-operation and Development Test Guideline (TG) for an in vitro CYP HepaRG test method that is capable of detecting the induction of four human CYPs (CYP1A1/1A2, 2B6, and 3A4), has been developed and validated for a set of pharmaceutical proficiency chemicals. However to support TG adoption, further validation data was requested to demonstrate the ability of the test method to also accurately detect CYP induction mediated by industrial and pesticidal chemicals, together with an indication on regulatory uses of the test method. As part of "GOLIATH", a European Union Horizon-2020 funded research project on metabolic disrupting chemical testing approaches, work is underway to generate supplemental validated data for an additional set of chemicals with sufficient diversity to allow for the approval of the guideline. Here we report on the process of proficiency chemical selection based on a targeted literature review, the selection criteria and considerations required for acceptance of proficiency chemical selection for OECD TG development (i.e. structural diversity, range of activity, relevant chemical sectors, global restrictions etc). The following 13 proposed proficiency chemicals were reviewed and selected as a suitable set for use in the additional validation experiments: tebuconazole, benfuracarb, atrazine, cypermethrin, chlorpyrifos, perfluorooctanoic acid, bisphenol A, N,N-diethyl-m-toluamide, benzo-[a]-pyrene, fludioxonil, malathion, triclosan, and caffeine. Illustrations of applications of the test method in relation to endocrine disruption and non-genotoxic carcinogenicity are provided.

Mechanistic Interrogation of Cell Transformation In Vitro: The Transformics Assay as an Exemplar of Oncotransformation.

The Transformics Assay is an in vitro test which combines the BALB/c 3T3 Cell Transformation Assay (CTA) with microarray transcriptomics. It has been shown to improve upon the mechanistic understanding of the CTA, helping to identify mechanisms of action leading to chemical-induced transformation thanks to RNA extractions in specific time points along the process of in vitro transformation. In this study, the lowest transforming concentration of the carcinogenic benzo(a)pyrene (B(a)P) has been tested in order to find molecular signatures of initial events relevant for oncotransformation. Application of Enrichment Analysis (Metacore) to the analyses of the results facilitated key biological interpretations. After 72 h of exposure, as a consequence of the molecular initiating event of aryl hydrocarbon receptor (AhR) activation, there is a cascade of cellular events and microenvironment modification, and the immune and inflammatory responses are the main processes involved in cell response. Furthermore, pathways and processes related to cell cycle regulation, cytoskeletal adhesion and remodeling processes, cell differentiation and transformation were observed.

Hematopoietic cell transplantation donor-derived memory-like NK cells functionally persist after transfer into patients with leukemia.

Natural killer (NK) cells are innate lymphoid cells that eliminate cancer cells, produce cytokines, and are being investigated as a nascent cellular immunotherapy. Impaired NK cell function, expansion, and persistence remain key challenges for optimal clinical translation. One promising strategy to overcome these challenges is cytokine-induced memory-like (ML) differentiation, whereby NK cells acquire enhanced antitumor function after stimulation with interleukin-12 (IL-12), IL-15, and IL-18. Here, reduced-intensity conditioning (RIC) for HLA-haploidentical hematopoietic cell transplantation (HCT) was augmented with same-donor ML NK cells on day +7 and 3 weeks of N-803 (IL-15 superagonist) to treat patients with relapsed/refractory acute myeloid leukemia (AML) in a clinical trial (NCT02782546). In 15 patients, donor ML NK cells were well tolerated, and 87% of patients achieved a composite complete response at day +28, which corresponded with clearing high-risk mutations, including TP53 variants. NK cells were the major blood lymphocytes for 2 months after HCT with 1104-fold expansion (over 1 to 2 weeks). Phenotypic and transcriptional analyses identified donor ML NK cells as distinct from conventional NK cells and showed that ML NK cells persisted for over 2 months. ML NK cells expressed CD16, CD57, and high granzyme B and perforin, along with a unique transcription factor profile. ML NK cells differentiated in patients had enhanced ex vivo function compared to conventional NK cells from both patients and healthy donors. Overall, same-donor ML NK cell therapy with 3 weeks of N-803 support safely augmented RIC haplo-HCT for AML.

Systemic IL-15 promotes allogeneic cell rejection in patients treated with natural killer cell adoptive therapy.

Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793.