A target discovery pipeline identified ILT3 as a target for immunotherapy of multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of plasma cells. To identify targets for MM immunotherapy, we develop an integrated pipeline based on mass spectrometry analysis of seven MM cell lines and RNA sequencing (RNA-seq) from 900+ patients. Starting from 4,000+ candidates, we identify the most highly expressed cell surface proteins. We annotate candidate protein expression in many healthy tissues and validate the expression of promising targets in 30+ patient samples with relapsed/refractory MM, as well as in primary healthy hematopoietic stem cells and T cells by flow cytometry. Six candidates (ILT3, SEMA4A, CCR1, LRRC8D, FCRL3, IL12RB1) and B cell maturation antigen (BCMA) present the most favorable profile in malignant and healthy cells. We develop a bispecific T cell engager targeting ILT3 that shows potent killing effects in vitro and decreased tumor burden and prolonged mice survival in vivo, suggesting therapeutic relevance. Our study uncovers MM-associated antigens that hold great promise for immune-based therapies of MM.

Loss of metabolic fitness drives tumor resistance after CAR-NK cell therapy and can be overcome by cytokine engineering.

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells is promising, with early-phase clinical studies showing encouraging responses. However, the transcriptional signatures that control the fate of CAR-NK cells after infusion and factors that influence tumor control remain poorly understood. We performed single-cell RNA sequencing and mass cytometry to study the heterogeneity of CAR-NK cells and their in vivo evolution after adoptive transfer, from the phase of tumor control to relapse. Using a preclinical model of noncurative lymphoma and samples from a responder and a nonresponder patient treated with CAR19/IL-15 NK cells, we observed the emergence of NK cell clusters with distinct patterns of activation, function, and metabolic signature associated with different phases of in vivo evolution and tumor control. Interaction with the highly metabolically active tumor resulted in loss of metabolic fitness in NK cells that could be partly overcome by incorporation of IL-15 in the CAR construct.

Activated B cells suppress T-cell function through metabolic competition.

BACKGROUND: B cells play a pivotal role in regulating the immune response. The induction of B cell-mediated immunosuppressive function requires B cell activating signals. However, the mechanisms by which activated B cells mediate T-cell suppression are not fully understood. METHODS: We investigated the potential contribution of metabolic activity of activated B cells to T-cell suppression by performing in vitro experiments and by analyzing clinical samples using mass cytometry and single-cell RNA sequencing. RESULTS: Here we show that following activation, B cells acquire an immunoregulatory phenotype and promote T-cell suppression by metabolic competition. Activated B cells induced hypoxia in T cells in a cell-cell contact dependent manner by consuming more oxygen via an increase in their oxidative phosphorylation (OXPHOS). Moreover, activated B cells deprived T cells of glucose and produced lactic acid through their high glycolytic activity. Activated B cells thus inhibited the mammalian target of rapamycin pathway in T cells, resulting in suppression of T-cell cytokine production and proliferation. Finally, we confirmed the presence of tumor-associated B cells with high glycolytic and OXPHOS activities in patients with melanoma, associated with poor response to immune checkpoint blockade therapy. CONCLUSIONS: We have revealed for the first time the immunomodulatory effects of the metabolic activity of activated B cells and their possible role in suppressing antitumor T-cell responses. These findings add novel insights into immunometabolism and have important implications for cancer immunotherapy.

KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape.

Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKTROG+) and NK cell hyporesponsiveness. This phenomenon could be offset by a dual-CAR system incorporating both an activating CAR against the cognate tumor antigen and an NK self-recognizing inhibitory CAR that transferred a 'don't kill me' signal to NK cells upon engagement with their TROG+ siblings. This system prevented trogocytic antigen-mediated fratricide, while sparing activating CAR signaling against the tumor antigen, and resulted in enhanced CAR-NK cell activity.

Natural killer cells in antitumour adoptive cell immunotherapy.

Natural killer (NK) cells comprise a unique population of innate lymphoid cells endowed with intrinsic abilities to identify and eliminate virally infected cells and tumour cells. Possessing multiple cytotoxicity mechanisms and the ability to modulate the immune response through cytokine production, NK cells play a pivotal role in anticancer immunity. This role was elucidated nearly two decades ago, when NK cells, used as immunotherapeutic agents, showed safety and efficacy in the treatment of patients with advanced-stage leukaemia. In recent years, following the paradigm-shifting successes of chimeric antigen receptor (CAR)-engineered adoptive T cell therapy and the advancement in technologies that can turn cells into powerful antitumour weapons, the interest in NK cells as a candidate for immunotherapy has grown exponentially. Strategies for the development of NK cell-based therapies focus on enhancing NK cell potency and persistence through co-stimulatory signalling, checkpoint inhibition and cytokine armouring, and aim to redirect NK cell specificity to the tumour through expression of CAR or the use of engager molecules. In the clinic, the first generation of NK cell therapies have delivered promising results, showing encouraging efficacy and remarkable safety, thus driving great enthusiasm for continued innovation. In this Review, we describe the various approaches to augment NK cell cytotoxicity and longevity, evaluate challenges and opportunities, and reflect on how lessons learned from the clinic will guide the design of next-generation NK cell products that will address the unique complexities of each cancer.

Perspectives in Immunotherapy: meeting report from the Immunotherapy Bridge, December 1st-2nd, 2021.

Over the past decade, immunotherapy has become an increasingly fundamental modality in the treatment of cancer. The positive impact of immune checkpoint inhibition, especially anti-programmed death (PD)-1/PD-ligand (L)1 blockade, in patients with different cancers has focused attention on the potential for other immunotherapeutic approaches. These include inhibitors of additional immune checkpoints, adoptive cell transfer (ACT), and therapeutic vaccines. Patients with advanced cancers who previously had limited treatment options available may now benefit from immunotherapies that can offer durable responses and improved survival outcomes. However, despite this, a significant proportion of patients fail to respond to immunotherapy, especially those with less immunoresponsive cancer types, and there remains a need for new treatment strategies.The virtual Immunotherapy Bridge (December 1st-2nd, 2021), organized by the Fondazione Melanoma Onlus, Naples, Italy in collaboration with the Society for Immunotherapy of Cancer addressed several areas of current research in immunotherapy, including lessons learned from cell therapies, drivers of immune response, and trends in immunotherapy across different cancers, and these are summarised here.

Generation of glucocorticoid-resistant SARS-CoV-2 T cells for adoptive cell therapy.

Adoptive cell therapy with virus-specific T cells has been used successfully to treat life-threatening viral infections, supporting application of this approach to coronavirus disease 2019 (COVID-19). We expand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observe that the choice of cytokines modulates the expansion, phenotype, and hierarchy of antigenic recognition by SARS-CoV-2 T cells. Culture with interleukin (IL)-2/4/7, but not under other cytokine-driven conditions, results in more than 1,000-fold expansion in SARS-CoV-2 T cells with a retained phenotype, function, and hierarchy of antigenic recognition compared with baseline (pre-expansion) samples. Expanded cytotoxic T lymphocytes (CTLs) are directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T cells cannot be expanded efficiently from the peripheral blood of non-exposed controls. Because corticosteroids are used for management of severe COVID-19, we propose an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

Avoiding Stops and Overcoming Roadblocks: Considerations for Improving Patient Access to CAR-Based Cell Therapies.

Adoptive cell therapy has significantly impacted the immuno-oncology landscape. The number of strategies currently in preclinical and clinical development is increasing at a rapid rate. Indeed, we are experiencing a transformative movement in cancer care as we shift toward highly personalized treatments designed to confront the specific challenges of each cancer. Advancements in genetic engineering methods and single-cell profiling technologies provide a level of understanding of the interactions between the immune system and cancer never before achieved. This knowledge, in turn, can be applied to the design and engineering of effective cancer-fighting treatments. As these promising new therapies progress toward clinical application, it becomes evident that we must develop robust methods for production and validation of cellular products to ensure consistency, safety, and efficacy, irrespective of cell type or indication. Herein, we provide an overview of the innovative approaches guiding the new generation of cell therapies and describe the benefits and challenges associated with emerging autologous and allogeneic platforms. Moreover, we discuss important considerations pertaining to process development, cost of goods, and manufacturing, and highlight their impact on the transfer of therapies from bench to bedside.

Glucuronoxylomannan in the Cryptococcus species capsule as a target for Chimeric Antigen Receptor T-cell therapy.

BACKGROUND AIMS: The genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: Cryptococcus gattii and Cryptococcus neoformans. To establish invasive human disease, inhaled cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infection's mortality rate ranges from 20% to 70%. Many HIV+/AIDS patients are affected by Cryptococcus infections, with 220,000 cases of cryptococcal meningitis reported worldwide in this population every year (C. neoformans infection statistics, via the Centers for Disease Control and Prevention, https://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/statistics.html). To escape from host immune cell attack, Cryptococcus covers itself in a sugar-based capsule composed primarily of glucuronoxylomannan (GXM). To evade phagocytosis, yeast cells increase to a >45-µm perimeter and become titan, or giant, cells. Cryptococci virulence is directly proportional to the percentage of titan/giant cells present during Cryptococcus infection. To combat cryptococcosis, the authors propose the redirection of CD8+ T cells to target the GXM in the capsule via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR). RESULTS: GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk in the extracellular domain, a CD28 transmembrane domain and CD28 and CD3-ς signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR+ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined with the use of brightfield microscopy. Large clusters of proliferating GXMR-CAR+ T cells were observed in GXM-treated cells, whereas no clusters were observed in control cells. Moreover, the interaction of GXM with GXMR-CAR+ T cells was detected via flow cytometry by using a GXM-specific antibody, and the recognition of GXM by GXMR-CAR T cells triggered the secretion of granzyme and interferon gamma (IFN-γ). The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by fluorescent microscopy, but no binding was detected in mock-transduced control T cells (NoDNA T cells). Moreover, lung tissue sections were stained with Gomori Methenamine Silver and evaluated by NanoZoomer (Hamamatsu), revealing a significantly lower number of titan cells, with perimeters ranging from 50 to 130 µm and giant cells >130 µm in the CAR T-cell treated group when compared with other groups. Therefore, the authors validated the study's hypothesis by the redirection of GXMR-CAR+ T cells to target GXM, which induces the secretion of cytotoxic granules and IFN-γ that will aid in the control of cryptococcosis CONCLUSIONS: Thus, these findings reveal that GXMR-CAR+ T cells can target C. neoformans. Future studies will be focused on determining the therapeutic efficacy of GXMR-CAR+ T cells in an animal model of cryptococcosis.

Adoptive cell therapy: Living drugs against cancer.

Immunotherapy has made a profound impact in the treatment of cancer in the last decade. Insights from the study of the intricate relationships between immune cells and cancer have led to the clinical development of strategies that redirect the power of the immune system to target and eliminate cancer. Today, immunotherapy represents a rapidly expanding and transformative force comprising multiple modalities.

Generation of glucocorticoid resistant SARS-CoV-2 T-cells for adoptive cell therapy.

Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expanded SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observed that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions resulted in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs were directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells could not be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we developed an efficient strategy to inactivate the glucocorticoid receptor gene ( NR3C1 ) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

Rigor and Reproducibility of Cytometry Practices for Immuno-Oncology: A multifaceted challenge.

The rapid advancement of immunotherapy strategies has created a need for technologies that can reliably and reproducibly identify rare populations, detect subtle changes in modulatory signals, and assess antigenic expression patterns that are time-sensitive. Accomplishing these tasks requires careful planning and the employment of tools that provide greater sensitivity and specificity without demanding extensive time. Flow Cytometry has earned its place as a preferred analysis platform. This technology offers a flexible path to the interrogation of protein expression patterns and detection of functional properties in cell populations of interest. Mass Cytometry is a newcomer technology that has generated significant interest in the field. By incorporating mass spectrometry analysis to the traditional principles of flow cytometry, this innovative tool promises to significantly expand the ability to detect multiple proteins on a single cell. The use of these technologies in a manner that is consistent and reproducible through multiple sample sets demands careful attention to experiment design, reagent selection, and instrumentation. Whether applying flow or mass cytometry, reaching successful, reliable results involves many factors. Sample preparation, antibody titrations, and appropriate controls are major biological considerations that impact cytometric analysis. Additionally, instrument voltages, lasers, and run quality assessments are essential for ensuring comparability and reproducibility between analyses. In this article, we aim to discuss the critical aspects that impact flow cytometry, and will touch on important considerations for mass cytometry as well. Focusing on their relevance to immunotherapy studies, we will address the importance of appropriate sample processing and will discuss how selection of suitable panels, controls, and antibodies must follow a carefully designed plan. We will also comment on how educated use of instrumentation plays a significant role in the reliability and reproducibility of results.Through this work, we hope to contribute to the effort toward establishing higher standards for rigor and reproducibility of cytometry practices by researchers, operators, and general cytometry users employing cytometry-based assays in their work. © 2019 International Society for Advancement of Cytometry.

Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

Gene Correction of iPSCs from a Wiskott-Aldrich Syndrome Patient Normalizes the Lymphoid Developmental and Functional Defects.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by mutations in the gene encoding the WAS protein (WASp). Here, induced pluripotent stem cells (iPSCs) were derived from a WAS patient (WAS-iPSC) and the endogenous chromosomal WAS locus was targeted with a wtWAS-2A-eGFP transgene using zinc finger nucleases (ZFNs) to generate corrected WAS-iPSC (cWAS-iPSC). WASp and GFP were first expressed in the earliest CD34(+)CD43(+)CD45(-) hematopoietic precursor cells and later in all hematopoietic lineages examined. Whereas differentiation to non-lymphoid lineages was readily obtained from WAS-iPSCs, in vitro T lymphopoiesis from WAS-iPSC was deficient with few CD4(+)CD8(+) double-positive and mature CD3(+) T cells obtained. T cell differentiation was restored for cWAS-iPSCs. Similarly, defects in natural killer cell differentiation and function were restored on targeted correction of the WAS locus. These results demonstrate that the defects exhibited by WAS-iPSC-derived lymphoid cells were fully corrected and suggests the potential therapeutic use of gene-corrected WAS-iPSCs.