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CAR natural killer cell therapy: Natural killer cell activation and expansion
Abstract
Currently, chimeric antigen receptor (CAR) T-cell therapy is an effective treatment method of hematological malignancies. However, T-lymphocyte-based immunotherapy has certain limitations for the scope of application of this approach. A promising alternative is CAR therapy based on natural killer (NK) cells, since it does not require detailed donor selection according to the human leukocyte antigen system; NK cells have a unique mechanism for recognizing and destroying tumor cells. In addition, NK cells do not cause severe toxic reactions when infused. The creation of a CAR NK product is a complex task includes cell culturing, using genetic engineering methods, and quality control testing of the resulting biomedical cell product (BMCP). For proliferation and effector function enhancement, NK cells require the presence of interleukins, feeder cells or their components, and immune system activators in the nutrient medium. This review focuses on various approaches to the activation and expansion of natural killer cells during cultivation, and also addresses the issues of the advantages and disadvantages of the chosen therapy and the regulatory aspects of creating a full-fledged BMCP.
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There is a pressing need for allogeneic chimeric antigen receptor (CAR)-immune cell therapies that are safe, effective and affordable. We conducted a phase 1/2 trial of cord blood-derived natural killer (NK) cells expressing anti-CD19 chimeric antigen receptor and interleukin-15 (CAR19/IL-15) in 37 patients with CD19⁺ B cell malignancies. The primary objectives were safety and efficacy, defined as day 30 overall response (OR). Secondary objectives included day 100 response, progression-free survival, overall survival and CAR19/IL-15 NK cell persistence. No notable toxicities such as cytokine release syndrome, neurotoxicity or graft-versus-host disease were observed. The day 30 and day 100 OR rates were 48.6% for both. The 1-year overall survival and progression-free survival were 68% and 32%, respectively. Patients who achieved OR had higher levels and longer persistence of CAR-NK cells. Receiving CAR-NK cells from a cord blood unit (CBU) with nucleated red blood cells ≤ 8 × 10⁷ and a collection-to-cryopreservation time ≤ 24 h was the most significant predictor for superior outcome. NK cells from these optimal CBUs were highly functional and enriched in effector-related genes. In contrast, NK cells from suboptimal CBUs had upregulation of inflammation, hypoxia and cellular stress programs. Finally, using multiple mouse models, we confirmed the superior antitumor activity of CAR/IL-15 NK cells from optimal CBUs in vivo. These findings uncover new features of CAR-NK cell biology and underscore the importance of donor selection for allogeneic cell therapies. ClinicalTrials.gov identifier: NCT03056339.
Natural killer (NK) cells, a unique component of the innate immune system, are inherent killers of stressed and transformed cells. Based on their potent capacity to kill cancer cells and good tolerance of healthy cells, NK cells have been successfully employed in adoptive cell therapy to treat cancer patients. In recent years, the clinical success of chimeric antigen receptor (CAR)-T cells has proven the vast potential of gene-manipulated immune cells as the main force to fight cancer. Following the lessons learned from mature gene-transfer technologies and advanced strategies in CAR-T therapy, NK cells have been rapidly explored as a promising candidate for CAR-based therapy. An exponentially growing number of studies have employed multiple sources of CAR-NK cells to target a wide range of cancer-related antigens, showing remarkable outcomes and encouraging safety profiles. Clinical trials of CAR-NK cells have also shown their impressive therapeutic efficacy in the treatment of hematological tumors, but CAR-NK cell therapy for solid tumors is still in the initial stages. In this review, we present the favorable profile of NK cells as a potential platform for CAR-based engineering and then summarize the outcomes and strategies of CAR-NK therapies in up-to-date preclinical and clinical investigations. Finally, we evaluate the challenges remaining in CAR-NK therapy and describe existing strategies that can assist us in devising future prospective solutions.
The immune system provides full protection for the body by specifically identifying ‘self’ and removing ‘others’; thus protecting the body from diseases. The immune system includes innate immunity and adaptive immunity, which jointly coordinate the antitumor immune response. T cells, natural killer (NK) cells and tumor-associated macrophages (TAMs) are the main tumor-killing immune cells active in three antitumor immune cycle. Cancer immunotherapy focusses on activating and strengthening immune response or eliminating suppression from tumor cells in each step of the cancer-immunity cycle; thus, it strengthens the body’s immunity against tumors. In this review, the antitumor immune cycles of T cells, natural killer (NK) cells and tumor-associated macrophages (TAMs) are discussed. Co-stimulatory and co-inhibitory molecules in the three activity cycles and the development of drugs and delivery systems targeting these molecules are emphasized, and the current state of the art of drug delivery systems for cancer immunotherapy are summarized.
Acute Myeloid Leukemia (AML) is a complex disease with rapid progression and poor/unsatisfactory outcomes. In the past few years, the focus has been on developing newer therapies for AML; however, relapse remains a significant problem. Natural Killer cells have strong anti-tumor potential against AML. This NK-mediated cytotoxicity is often restricted by cellular defects caused by disease-associated mechanisms, which can lead to disease progression. A stark feature of AML is the low/no expression of the cognate HLA ligands for the activating KIR receptors, due to which these tumor cells evade NK-mediated lysis. Recently, different Natural Killer cell therapies have been implicated in treating AML, such as the adoptive NK cell transfer, Chimeric antigen receptor-modified NK (CAR-NK) cell therapy, antibodies, cytokine, and drug treatment. However, the data available is scarce, and the outcomes vary between different transplant settings and different types of leukemia. Moreover, remission achieved by some of these therapies is only for a short time. In this mini-review, we will discuss the role of NK cell defects in AML progression, particularly the expression of different cell surface markers, the available NK cell therapies, and the results from various preclinical and clinical trials.
Natural killer (NK) cells play a crucial role in early immune defenses against transformed cells and are used in the therapeutic management of cancer. However, it is difficult to sufficiently obtain high purity activated NK cells for clinical application. The function of NK cells is dependent on the balance of activating and inhibitory signals. Strong and diverse stimuli are required to increase the function of NK cells. Radiotherapy modulates the expression of various immunomodulatory molecules that recruit and activate NK cells. NK cell-mediated antibody-dependent cellular cytotoxicity is one of the most potent cytotoxic effects of NK cells against target cancer cells. To generate activated and irradiated autologous peripheral blood mononuclear cells (PBMCs), cytokine and monoclonal antibody stimulation followed by ionizing radiation was performed in the present study. The expanded NK cells were cultured for 21 days using activated/irradiated autologous PBMCs. Colorectal cancer cells (SW480 and HT-29) were used to analyze the expression of NK group 2D ligands and EGFR by radiation. The cytotoxicity of radiation plus NK cell-based targeted therapy against colorectal cancer cell lines was analyzed using flow cytometry. Activated and irradiated PBMCs exhibited significantly increased expression of various activating ligands that stimulated NK cells. In total, >10,000-fold high-purity activated NK cells were obtained, with negligible T-cell contamination. To confirm the antitumor activity of the NK cells expanded by this method, the expanded NK cells were treated with cetuximab, radiotherapy, or a combination of cetuximab and radiotherapy in the presence of human colorectal cancer cells. Expanded NK cells were effective at targeting human colorectal cancer cells, particularly when combined with cetuximab and radiotherapy. Thus, in the present study, a novel method for high-purity activated NK cell expansion was developed using activated and irradiated PBMCs. In addition, combined radiotherapy and antibody-based immunotherapy with expanded NK cells may be an effective strategy to enhance the efficiency of treatment against colorectal cancer.
Simple Summary
Innate immune natural killer (NK) cells are capable of killing metastatic cancer cells without activation by antigen-presenting cells beforehand. The cytotoxic effects and immune regulation of NK cells are precisely controlled by an energetic balance of signals produced by a group of activating and inhibitory receptors expressed on NK cells, while metastatic tumor cells with multiple strategies escape immune cells attack. This review focuses on the critical function of NK cells in metastasis and recently developed objectively effective NK cell-based immunotherapies.
Abstract
Metastatic tumors cause the most deaths in cancer patients. Treating metastasis remains the primary goal of current cancer research. Although the immune system prevents and kills the tumor cells, the function of the immune system in metastatic cancer has been unappreciated for decades because tumors are able to develop complex signaling pathways to suppress immune responses, leading them to escape detection and elimination. Studies showed NK cell-based therapies have many advantages and promise for fighting metastatic cancers. We here review the function of the immune system in tumor progression, specifically focusing on the ability of NK cells in antimetastasis, how metastatic tumors escape the NK cell attack, as well as the recent development of effective antimetastatic immunotherapies.
Purpose of Review
Chimeric antigen receptor (CAR) T cell therapy is an immunotherapy that has resulted in tremendous progress in the treatment of patients with B cell malignancies. However, the remarkable efficacy of therapy is not without significant safety concerns. Herein, we will review the unique and potentially life-threatening toxicities associated with CAR-T cell therapy and their association with treatment efficacy.
Recent Findings
Currently, CAR-T cell therapy is approved for the treatment of B cell relapsed or refractory leukemia and lymphoma, and most recently, multiple myeloma (MM). In these different diseases, it has led to excellent complete and overall response rates depending on the patient population and therapy. Despite promising efficacy, CAR-T cell therapy is associated with significant side effects; the two most notable toxicities are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The treatment of CAR-T-induced toxicity is supportive; however, as higher-grade adverse events occur, toxicity-directed therapy with tocilizumab, an IL-6 receptor antibody, and steroids is standard practice. Overall, a careful risk–benefit balance exists between the efficacy and toxicities of therapies. The challenge lies in the underlying pathophysiology of CAR-T-related toxicity which relies upon the activation of CAR-T cells.
Summary
Some degree of toxicity is expected to achieve an effective response to therapy, and certain aspects of treatment are also associated with toxicity. As progress is made in the investigation and approval of new CARs, novel toxicity-directed therapies and toxicity-limited constructs will be the focus of attention.
Multiple clinical trials exploring the potential of adoptive NK cell therapy for cancer have employed ex vivo expansion using feeder cells to obtain large numbers of NK cells. We have previously utilized the rhesus macaque model to clonally track the NK cell progeny of barcode-transduced CD34⁺ stem and progenitor cells after transplant. In this study, NK cells from barcoded rhesus macaques were used to study the changes in NK cell clonal patterns that occurred during ex vivo expansion, using culture protocols similar to those employed in clinical preparation of human NK cells including irradiated lymphoblastoid cell line (LCL) feeder cells or K562 cells expressing 4-1BBL and membrane-bound IL-21. NK expansion cultures resulted in the proliferation of clonally diverse NK cells, which at day 14 harvest contained greater than 50% of the starting barcode repertoire. Diversity as measured by Shannon index was maintained after culture. With both LCL and K562 feeders, proliferation of long-lived putative memory-like NK cell clones was observed, with these clones continuing to constitute a mean of 31% of the total repertoire of expanded cells. These experiments provide insight into the clonal makeup of expanded NK cell clinical products.
Hematological malignant neoplasms include more than a hundred different subtypes and account for about 4.8% of all neoplastic diseases in Russia. Despite significant advances in diagnosis and treatment, many of them remain incurable. In recent years, cell-based therapy appears to be a promising approach to the treatment of these incurable hematologic malignancies, showing striking results in various clinical trials. The most studied and advanced cell therapy is the therapy with T-lymphocytes modified with chimeric antigen receptors (CAR). However, although the US Food and Drug Administration has approved CAR T cells for the treatment of B-cell lymphoma and acute lymphoblastic leukemia, significant problems remain in terms of production, cost, and serious side effects. An alternative to the use of T cells can be the use of innate immune cells, in particular natural killer cells (NK), which have a high antitumor potential. Recent studies have shown the antitumor efficacy of a therapy that uses genetically modified natural killer cells CAR NK cells. The purpose of this review was to describe and systematize the experience of using CAR NK cells for the treatment of hematological neoplasms. The review presents the advantages and disadvantages of this method, as well as the problems that still have to be solved for its widespread introduction into clinical practice.
Interest in adoptive cell therapy for treating cancer is exploding due to early clinical successes of autologous chimeric antigen receptor (CAR) T lymphocytes. 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 (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), 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, CRS, or ICANS. NK cells can be obtained from blood, cord blood, or derived from HSPCs/iPSCs, and 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 cells 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.
Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.
The γδT cell subset of peripheral lymphocytes exhibits potent cancer antigen recognition independent of classical peptide MHC complexes, making it an attractive candidate for allogeneic cancer adoptive immunotherapy. The Vδ1-T cell receptor (TCR)-expressing subset of peripheral γδT cells has remained enigmatic compared to its more prevalent Vγ9Vδ2-TCR and αβ-TCR-expressing counterparts. It took until 2021 before a first patient was dosed with an allogeneic adoptive Vδ1 cell product despite pre-clinical promise for oncology indications stretching back to the 1980s. A contributing factor to the paucity of clinical progress with Vδ1 cells is the lack of robust, consistent and GMP-compatible expansion protocols. Herein we describe a reproducible one-step, clinically translatable protocol for Vδ1-γδT cell expansion from peripheral blood mononuclear cells (PBMCs), that is further compatible with high-efficiency gene engineering for immunotherapy purposes. Briefly, αβTCR- and CD56-depleted PBMC stimulation with known-in-the-art T cell stimulators, anti-CD3 mAb (clone: OKT-3) and IL-15, leads to robust Vδ1 cell expansion of high purity and innate-like anti-tumor efficacy. These Vδ1 cells can be virally transduced to express chimeric antigen receptors (CARs) using standard techniques, and the CAR-Vδ1 exhibit antigen-specific persistence, cytotoxicity and produce IFN-γ. Practicable, GMP-compatible engineered Vδ1 cell expansion methods will be crucial to the wide-spread clinical testing of these cells for oncology indications.
Natural killer (NK) cells are lymphocytes with a key role in the defense against viral infections and tumor cells. Although NK cells are classified as innate lymphoid cells (ILCs), under certain circumstances they exhibit adaptive and memory-like features. The latter may be achieved, among others, by a brief stimulation with interleukin (IL)-12, IL-15 and IL-18. These cytokine-induced memory-like (CIML) NK cells resemble the trained immunity observed in myeloid cells. CIML NK cells undergo transcriptional, epigenetic and metabolic reprogramming that, along with changes in the expression of cell surface receptors and components of cytotoxic granules, are responsible for their enhanced effector functions after a resting period. In addition, these memory-like NK cells persist for a long time, which make them a good candidate for cancer immunotherapy. Currently, several clinical trials are testing CIML NK cells infusions to treat tumors, mostly hematological malignancies. In relapse/refractory acute myeloid leukemia (AML), the adoptive transfer of CIML NK cells is safe and complete clinical remissions have been observed. In our review, we sought to summarize the current knowledge about the generation and molecular basis of NK cell memory-like responses and the up-to-date results from clinical trials with CIML NK cells.
Natural killer (NK) cells are unique innate immune cells and manifest rapid and potent cytotoxicity for cancer immunotherapy and pathogen removal without the requirement of prior sensitization or recognition of peptide antigens. Distinguish from the T lymphocyte-based cythotherapy with toxic side effects, chimeric antigen receptor-transduced NK (CAR-NK) cells are adequate to simultaneously improve efficacy and control adverse effects including acute cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). Moreover, considering the inherent properties of NK cells, the CAR-NK cells are “off-the-shelf” product satisfying the clinical demand for large-scale manufacture for cancer immunotherapy attribute to the cytotoxic effect via both NK cell receptor-dependent and CAR-dependent signaling cascades. In this review, we mainly focus on the latest updates of CAR-NK cell-based tactics, together with the opportunities and challenges for cancer immunotherapies, which represent the paradigm for boosting the immune system to enhance antitumor responses and ultimately eliminate malignancies. Collectively, we summarize and highlight the auspicious improvement in CAR-NK cells and will benefit the large-scale preclinical and clinical investigations in adoptive immunotherapy.
NK-92 cells are an off-the-shelf, cell-based immunotherapy currently in clinical trials for a variety of cancer types. As the most ‘NK-like’ cell line available, it is also an important research tool. To date, NK-92 cells have been cultivated in a costly and time-consumingly prepared specialized medium, complicating research with these cells. Here we show that NK-92 cells grow in the comparatively user-friendly RPMI medium supplemented with IL-2. We demonstrate that their metabolic activity and replication rates are even improved in RPMI. Furthermore, they can be grown in cell culture dishes and do not need to be expanded in ventilated flasks. We show that in RPMI the cells retain functional characteristics relating to receptor expression, IFN-γ secretion, and killing. Our findings will enable more researchers to work with and manipulate this cell line, hopefully leading to further discoveries and improved therapies.
Chimeric antigen receptors (CARs) are fusion proteins with an extracellular antigen recognition domain and numerous intracellular signaling domains that have been genetically modified. CAR-engineered T lymphocyte-based therapies have shown great success against blood cancers; however, potential fatal toxicity, such as in cytokine release syndrome, and high costs are some shortcomings that limit the clinical application of CAR-engineered T lymphocytes and remain to overcome. Natural killer (NK) cells are the focal point of current immunological research owing to their receptors that prove to be promising immunotherapeutic candidates for treating cancer. However, to date, manipulation of NK cells to treat malignancies has been moderately successful. Recent progress in the biology of NK cell receptors has greatly transformed our understanding of how NK cells recognize and kill tumor and infected cells. CAR-NK cells may serve as an alternative candidate for retargeting cancer because of their unique recognition mechanisms, powerful cytotoxic effects especially on cancer cells in both CAR-dependent and CAR-independent manners and clinical safety. Moreover, NK cells can serve as an ‘off-the-shelf product’ because NK cells from allogeneic sources can also be used in immunotherapies owing to their reduced risk of alloreactivity. Although ongoing fundamental research is in the beginning stages, this review provides an overview of recent developments implemented to design CAR constructs to stimulate NK activation and manipulate NK receptors for improving the efficiency of immunotherapy against cancer, summarizes the preclinical and clinical advances of CAR-NK cells against both hematological malignancies and solid tumors and confronts current challenges and obstacles of their applications. In addition, this review provides insights into prospective novel approaches that further enhance the efficiency of CAR-NK therapies and highlights potential questions that require to be addressed in the future.
Chimeric Antigen Receptors (CARs) have a unique facet of synthetic biology and offer a paradigm shift in personalized medicine as they can use and redirect the patient's immune cells to attack cancer cells. CAR-Natural Killer (NK) cells combine the targeted specificity of antigens with the subsequent intracellular signaling ability of the receptors to increase their anti-cancer functions. Importantly, CAR-NK cells can be utilized as universal cell-based therapy without requiring human leukocyte antigen (HLA) matching or earlier contact with tumor-associated antigens (TAAs). Indeed, CAR-NK cells can be adapted to recognize various antigens, hold higher proliferation capacity and in vivo persistence, show improved infiltration into the tumors, and ability to overcome the resistant tumor microenvironment leading to sustained cytotoxicity against tumors. Accumulating evidence from recent in vivo studies rendering CAR-NK cell anti-cancer competencies renewed attention in the context of cancer immunotherapy, since these redirected effector cells can be used in the development of the "off-the-shelf" anti-cancer immunotherapeutic products. In the current review, we focus on the therapeutic efficacy of CAR-NK cell therapies for treating various human malignancies, including hematological malignancies and solid tumors, and will discuss the recent findings in this regard, with a special focus on animal studies.
Natural killer (NK) cells are key innate immunity effectors that play a major role in malignant cell destruction. Based on expression patterns of CD16, CD56, CD57, and CD94, three distinct NK cell maturation stages have been described, which differ in terms of cytokine secretion, tissue migration, and the ability to kill target cells. Our study addressed NK cell maturation in bone marrow under three conditions: a normal developmental environment, during pre-leukemic state (myelodysplastic syndrome, MDS), and during leukemic transformation (acute myeloblastic leukemia, AML). In this study, we used a new tool to perform multicolor flow cytometry data analysis, based on principal component analysis, which allowed the unsupervised, accurate discrimination of immature, mature, and hypermature NK subpopulations. An impaired NK/T cell distribution was observed in the MDS bone marrow microenvironment compared with the normal and AML settings, and a phenotypic shift from the mature to the immature state was observed in NK cells under both the MDS and AML conditions. Furthermore, an impaired NK cell antitumor response, resulting in changes in NK cell receptor expression (CD159a, CD158a, CD158b, and CD158e1), was observed under MDS and AML conditions compared with the normal condition. The results of this study provide evidence for the failure of this arm of the immune response during the pathogenesis of myeloid malignancies. NK cell subpopulations display a heterogeneous and discordant dynamic on the spectrum between normal and pathological conditions. MDS does not appear to be a simple, intermediate stage but rather serves as a decisive step for the mounting of an efficient or ineffective immune response, leading to either the removal of the tumor cells or to malignancy.
Chimeric antigen receptor (CAR) technology has revolutionized cancer treatment, particularly in malignant hematological tumors. Currently, the BCMA-targeted second-generation CAR-T cells have showed impressive efficacy in the treatment of refractory/relapsed multiple myeloma (R/R MM), but up to 50% relapse remains to be addressed urgently. Here we constructed the BCMA-targeted fourth-generation CAR-T cells expressing IL-7 and CCL19 (i.e., BCMA-7 × 19 CAR-T cells), and demonstrated that BCMA-7 × 19 CAR-T cells exhibited superior expansion, differentiation, migration and cytotoxicity. Furthermore, we have been carrying out the first-in-human clinical trial for therapy of R/R MM by use of BCMA-7 × 19 CAR-T cells (ClinicalTrials.gov Identifier: NCT03778346), which preliminarily showed promising safety and efficacy in first two enrolled patients. The two patients achieved a CR and VGPR with Grade 1 cytokine release syndrome only 1 month after one dose of CAR-T cell infusion, and the responses lasted more than 12-month. Taken together, BCMA-7 × 19 CAR-T cells were safe and effective against refractory/relapsed multiple myeloma and thus warranted further clinical study.
Natural killer T (NKT) cells are a key component of innate immunity. Importantly, a growing body of evidence indicates that NKT cells play an integral role in various acute and chronic liver injuries. NKT cells participate in the progression of an injury through the secretion of cytokines, which promote neutrophil infiltration and enhance Fas ligand (FasL) and granzyme-mediated NKT cytotoxic activity. Therefore, examining the role of NKT cells in hepatic disease is critical for a comprehensive understanding of disease pathogenesis and may provide insight into novel approaches for treatment. For more than a century, mouse models that imitate the physiopathological conditions of human disease have served as a critical tool in biological and medical basic research, including studies of liver disease. Here, we review the role of NKT cells in various mouse models of hepatitis.
Natural killer (NK) cell is a specialized immune effector cell type that plays a critical role in immune activation against abnormal cells. Different from events required for T cell activation, NK cell activation is governed by the interaction of NK receptors with target cells, independent of antigen processing and presentation. Due to relatively unsophisticated cues for activation, NK cell has gained significant attention in the field of cancer immunotherapy. Many efforts are emerging for developing and engineering NK cell-based cancer immunotherapy. In this review, we provide our current understandings of NK cell biology, ongoing pre-clinical and clinical development of NK cell-based therapies and discuss the progress, challenges, and future perspectives.
Background:
Interferon-γ (IFN-γ) plays a key role in activation of cellular immunity and subsequently, stimulation of antitumor immune-response. Based on its cytostatic, pro-apoptotic and antiproliferative functions, IFN-γ is considered potentially useful for adjuvant immunotherapy for different types of cancer. Moreover, it IFN-γ may inhibit angiogenesis in tumor tissue, induce regulatory T-cell apoptosis, and/or stimulate the activity of M1 proinflammatory macrophages to overcome tumor progression. However, the current understanding of the roles of IFN-γ in the tumor microenvironment (TME) may be misleading in terms of its clinical application.
Main body:
Some researchers believe it has anti-tumorigenic properties, while others suggest that it contributes to tumor growth and progression. In our recent work, we have shown that concentration of IFN-γ in the TME determines its function. Further, it was reported that tumors treated with low-dose IFN-γ acquired metastatic properties while those infused with high dose led to tumor regression. Pro-tumorigenic role may be described through IFN-γ signaling insensitivity, downregulation of major histocompatibility complexes, upregulation of indoleamine 2,3-dioxygenase, and checkpoint inhibitors such as programmed cell death ligand 1.
Conclusion:
Significant research efforts are required to decipher IFN-γ-dependent pro- and anti-tumorigenic effects. This review discusses the current knowledge concerning the roles of IFN-γ in the TME as a part of the complex immune response to cancer and highlights the importance of identifying IFN-γ responsive patients to improve their sensitivity to immuno-therapies.
Natural Killer (NK) cells and CD8⁺ cytotoxic T cells are two types of immune cells that can kill target cells through similar cytotoxic mechanisms. With the remarkable success of chimeric antigen receptor (CAR)-engineered T (CAR-T) cells for treating haematological malignancies, there is a rapid growing interest in developing CAR-engineered NK (CAR-NK) cells for cancer therapy. Compared to CAR-T cells, CAR-NK cells could offer some significant advantages, including: (1) better safety, such as a lack or minimal cytokine release syndrome and neurotoxicity in autologous setting and graft-versus-host disease in allogenic setting, (2) multiple mechanisms for activating cytotoxic activity, and (3) high feasibility for ‘off-the-shelf’ manufacturing. CAR-NK cells could be engineered to target diverse antigens, enhance proliferation and persistence in vivo, increase infiltration into solid tumours, overcome resistant tumour microenvironment, and ultimately achieve an effective anti-tumour response. In this review, we focus on recent progress in genetic engineering and clinical application of CAR-NK cells, and discuss current challenges and future promise of CAR-NK cells as a novel cellular immunotherapy in cancer.
Background:
Regarding to the increase of cancer deaths in recent years and disability of common therapies to eradicate cancers, as well as expansion of Natural Killer (NK) cell therapy, it seems so vital to find new useful therapies against cancers. Breast cancer is the second main cause of cancer death among women. As it is impossible for a majority of patients to receive NK cell therapy, an attempt was made to establish a low-cost and efficient method for expanding and activating NK cells against breast cancer cell line (MCF7).
Methods:
NK cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) applying either MACS based NK cell enrichment kit or antibodies and complement as cytotoxic method. Then, the NK cells were cultured in Stem Cell Growth Medium (SCGM) with feeder layer (irradiated PBMCs) along with PHA or OKT3. IL-2, IL-15 and IL-21 were used to expand NK cells and finally their cytotoxic activity was investigated by flow cytometry.
Results:
Highly pure NK cells were obtained and no significant difference between the two isolation methods was found. Using IL-2 plus IL-15, the number of NK cells increased up to100 fold after 16 days. No significant effect was observed after IL-21 treatment.
Conclusion:
Our data indicated that cytotoxicity method can be considered a low-cost alternative for NK cell isolation kits. It seems that culturing NK cells for 14 days in either PHA or OKT3 supplemented SCGM medium would be more effective than culturing for 16 days in the presence of IL-21.
Introduction
Acute myeloblastic leukaemia (AML) constitutes the second most common haematological malignancy in the paediatric population. Current treatment regimens are based on the administration of polychemotherapy, combining high doses of cytarabine with anthracyclines and topoisomerase inhibitors. Allogeneic haematopoietic stem cell transplantation (HSCT) is an option for high-risk patients with AML (and for intermediate-risk patients if a sibling donor is available). With this strategy, AML survival has increased substantially; however, it has remained stagnant at approximately 60%, with relapse being the principal culprit. The predominant role of the immune system and natural killer (NK) cells in controlling paediatric AML has gained importance within the context of HSCT. In this protocol, we propose incorporating this cell therapy as an adjuvant treatment through the infusion of activated and expanded haploidentical NK (NKAE) cells in paediatric patients with AML who are in cytological remission after completing consolidation therapy, and with no indication for HSCT.
Methods and analysis
Patients up to 30 years of age, diagnosed with AML, in their first cytological remission, who have completed both the induction and the consolidation phases of chemotherapy and do not meet the criteria for allogeneic HSCT are eligible. The patients will receive two doses of NKAE cells once a week, using a GMP K562-mbIL15-41BBL stimulus from a haploidentical donor and interleukin 2 subcutaneously. The patients will then be followed up for 36 months to assess the primary endpoint, which is the probability of relapse after NK cell infusion.
Ethics and dissemination
This clinical trial was approved by the Clinical Research Ethics Committee of La Paz University Hospital and The Spanish Agency of Medicines and Medical Devices. Findings will be disseminated through peer-reviewed publications, conference presentations and community reporting.
Trial registration number
EudraCT code: 2015-001901-15, ClinicalTrials.gov Identifier: NCT02763475 .
Background:
one of the promising approaches for treatment of some cancers is adoptive cell therapy using natural killer (NK) cells. Various methods have been investigated for ex vivo expansion of NK cells in large-scale, but most of them involved cancer or genetically modified cells as feeder layer and also some of them have the risk of T cell contamination and graft-versus-host disease (GVHD).
Method:
In this study, irradiated autologous peripheral blood mononuclear cells (PBMCs) as feeder layer with an anti-CD3 monoclonal antibody (mAb) were used. For activation and expansion of NK cells, human recombinant IL2 and IL15 were used. After co-culturing of expanded NK cells (eNKC) and isolated NK cells (iNKC) with hepatocellular carcinoma (HCC) cells, the viability of eNKC in compared to iNKC were analyzed by CCK-8 assay and degranulation of NK cells after co-culturing was assayed by measuring CD107a expression. Enzyme-Linked Immunosorbent Assay (ELISA) assay was used for the ability of NK cells to secretion of IFN-γ (interferon-γ) and TNF-α (Tumor Necrosis Factor-α) after co-culture with HCC cells. Real Time PCR analysis was used for expression of human Perforin and Granzyme B genes in the NK cells exposed to target HepG2 cells.
Result:
This method strongly expanded highly purified NK cells with powerful cytotoxicity against HCC cells. The expanded NK cells showed high level of expression of degranulation marker and human Perforin and Granzyme B genes, and also was secreted larger amounts of TNF-α and IFN-γ compared with fresh isolated NK cells.
Conclusion:
we proposed an effective method for expansion of cytotoxic NK cells using irradiated autologous PBMC as feeder layer for more successful transfer of allogeneic NK cell in immuno cell therapy of HCC.
NK cell adoptive therapy is a promising cancer therapeutic approach, but there are significant challenges that limiting its feasibility and clinical efficacy. One difficulty is the paucity of clinical grade manufacturing platforms to support the large scale expansion of highly active NK cells. We created an NK cell feeder cell line termed ‘NKF’ through overexpressing membrane bound IL-21 that is capable of inducing robust and sustained proliferation (>10,000-fold expansion at 5 weeks) of highly cytotoxic NK cells. The expanded NK cells exhibit increased cytotoxic function against a panel of blood cancer and solid tumor cells as compared to IL-2-activated non-expanded NK cells. The NKF-expanded NK cells also demonstrate efficacy in mouse models of human sarcoma and T cell leukemia. Mechanistic studies revealed that membrane-bound IL-21 leads to an activation of a STAT3/c-Myc pathway and increased NK cell metabolism with a shift towards aerobic glycolysis. The NKF feeder cell line is a promising new platform that enables the large scale proliferation of highly active NK cells in support of large scale third party NK cell clinical studies that have been recently intiatied. These results also provide mechanistic insights into how membrane-bound IL-21 regulates NK cell expansion.
A large body of data shows that Natural Killer (NK) cells are immune effectors exerting a potent cytolytic activity against tumors and virus infected cells. The discovery and characterization of several inhibitory and activating receptors unveiled most of the mechanisms allowing NK cells to spare healthy cells while selectively attacking abnormal tissues. Nevertheless, the mechanisms ruling NK cell subset recirculation among the different compartments of human body have only lately started to be investigated. This is particularly true for pathological settings such as tumors or infected tissues but also for para-physiological condition like pregnant human uterine mucosa. It is becoming evident that the microenvironment associated to a particular clinical condition can deeply influence the migratory capabilities of NK cells. In this review we describe the main mechanisms and stimuli known to regulate the expression of chemokine receptors and other molecules involved in NK cell homing to either normal or pathological/inflamed tissues, including tumors or organs such as lung and liver. We will also discuss the role played by the chemokine/chemokine receptor axes in the orchestration of physiological events such as NK cell differentiation, lymphoid organ retention/egress and recruitment to decidua during pregnancy.
Natural killer (NK) cells are powerful immune effectors whose antitumor activity is regulated through a sophisticated network of activating and inhibitory receptors. As effectors of cancer immunotherapy, NK cells are attractive as they do not attack healthy self-tissues nor do they induce T cell-driven inflammatory cytokine storm, enabling their use as allogeneic adoptive cellular therapies. Clinical responses to adoptive NK-based immunotherapy have been thwarted, however, by the profound immunosuppression induced by the tumor microenvironment, particularly severe in the context of solid tumors. In addition, the short postinfusion persistence of NK cells in vivo has limited their clinical efficacy. Enhancing the antitumor immunity of NK cells through genetic engineering has been fueled by the promise that impaired cytotoxic functionality can be restored or augmented with the use of synthetic genetic approaches. Alongside expressing chimeric antigen receptors to overcome immune escape by cancer cells, enhance their recognition, and mediate their killing, NK cells have been genetically modified to enhance their persistence in vivo by the expression of cytokines such as IL-15, avoid functional and metabolic tumor microenvironment suppression, or improve their homing ability, enabling enhanced targeting of solid tumors. However, NK cells are notoriously adverse to endogenous gene uptake, resulting in low gene uptake and transgene expression with many vector systems. Though viral vectors have achieved the highest gene transfer efficiencies with NK cells, nonviral vectors and gene transfer approaches—electroporation, lipofection, nanoparticles, and trogocytosis—are emerging. And while the use of NK cell lines has achieved improved gene transfer efficiencies particularly with viral vectors, challenges with primary NK cells remain. Here, we discuss the genetic engineering of NK cells as they relate to NK immunobiology within the context of cancer immunotherapy, highlighting the most recent breakthroughs in viral vectors and nonviral approaches aimed at genetic reprogramming of NK cells for improved adoptive immunotherapy of cancer, and, finally, address their clinical status.
Natural killer (NK) cells are traditionally considered as innate cells, but recent studies suggest that NK cells can distinguish antigens, and that memory NK cells expand and protect against viral pathogens. Limited information is available about the mechanisms involved in memory-like NK cell expansion, and their role in bacterial infections and vaccine-induced protective immune responses. In the current study, using a mouse model of tuberculosis (TB) infection, we found that interferon-gamma producing CD3−NKp46+CD27+KLRG1+ memory-like NK cells develop during Bacille Calmette–Guérin vaccination, expand, and provide protection against challenge with Mycobacterium tuberculosis (M. tb). Using antibodies, short interfering RNA and gene-deleted mice, we found that expansion of memory-like NK cells depends on interleukin 21 (IL-21). NKp46+CD27+KLRG1+ NK cells expanded in healthy individuals with latent TB infection in an IL-21-dependent manner. Our study provides first evidence that memory-like NK cells survive long term, expansion depends on IL-21, and involved in vaccine-induced protective immunity against a bacterial pathogen.
Natural Killer (NK) cells are innate lymphoid cells (ILC) with cytotoxic and regulatory properties. Their functions are tightly regulated by an array of inhibitory and activating receptors, and their mechanisms of activation strongly differ from antigen recognition in the context of human leukocyte antigen presentation as needed for T-cell activation. NK cells thus offer unique opportunities for new and improved therapeutic manipulation, either in vivo or in vitro, in a variety of human diseases, including cancers. NK cell activity can possibly be modulated in vivo through direct or indirect actions exerted by small molecules or monoclonal antibodies. NK cells can also be adoptively transferred following more or less substantial modifications through cell and gene manufacturing, in order to empower them with new or improved functions and ensure their controlled persistence and activity in the recipient. In the present review, we will focus on the technological and regulatory challenges of NK cell manufacturing and discuss conditions in which these innovative cellular therapies can be brought to the clinic.
Objective:
Initial success with chimeric antigen receptor-modified T cell therapy for relapsed/refractory acute lymphoblastic leukemia is leading to expanded use through multicenter trials. Cytokine release syndrome, the most severe toxicity, presents a novel critical illness syndrome with limited data regarding diagnosis, prognosis, and therapy. We sought to characterize the timing, severity, and intensive care management of cytokine release syndrome after chimeric antigen receptor-modified T cell therapy.
Design:
Retrospective cohort study.
Setting:
Academic children's hospital.
Patients:
Thirty-nine subjects with relapsed/refractory acute lymphoblastic leukemia treated with chimeric antigen receptor-modified T cell therapy on a phase I/IIa clinical trial (ClinicalTrials.gov number NCT01626495).
Interventions:
All subjects received chimeric antigen receptor-modified T cell therapy. Thirteen subjects with cardiovascular dysfunction were treated with the interleukin-6 receptor antibody tocilizumab.
Measurements and main results:
Eighteen subjects (46%) developed grade 3-4 cytokine release syndrome, with prolonged fever (median, 6.5 d), hyperferritinemia (median peak ferritin, 60,214 ng/mL), and organ dysfunction. Fourteen (36%) developed cardiovascular dysfunction treated with vasoactive infusions a median of 5 days after T cell therapy. Six (15%) developed acute respiratory failure treated with invasive mechanical ventilation a median of 6 days after T cell therapy; five met criteria for acute respiratory distress syndrome. Encephalopathy, hepatic, and renal dysfunction manifested later than cardiovascular and respiratory dysfunction. Subjects had a median of 15 organ dysfunction days (interquartile range, 8-20). Treatment with tocilizumab in 13 subjects resulted in rapid defervescence (median, 4 hr) and clinical improvement.
Conclusions:
Grade 3-4 cytokine release syndrome occurred in 46% of patients following T cell therapy for relapsed/refractory acute lymphoblastic leukemia. Clinicians should be aware of expanding use of this breakthrough therapy and implications for critical care units in cancer centers.
Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenger RNA (mRNA) revealed not only cytokine-dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mammalian target of rapamycin (mTOR) signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15-stimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy.
Purpose:
Alternative strategies to EGFR blockage by mAbs is necessary in order to improve the efficacy of therapy in patients with locally advanced or metastatic pancreatic cancer. One such strategy includes the use of NK cells to clear cetuximab-coated tumor cells, as need for novel therapeutic approaches to enhance the efficacy of cetuximab is evident. We show that IL-21 enhances NK cell-mediated effector functions against cetuximab-coated pancreatic tumor cells irrespective of KRAS mutation status.
Experimental design:
NK cells from normal donors or donors with pancreatic cancer were used to assess ADCC, IFN-γ release, and T cell chemotaxis towards human pancreatic cancer cell lines. The in vivo efficacy of IL-21 in combination with cetuximab was evaluated in a subcutaneous and intraperitoneal model of pancreatic cancer.
Results:
NK cell lysis of cetuximab-coated wild-type and mutant KRAS pancreatic cancer cell lines was significantly higher following NK cell IL-21 treatment. In response to cetuximab-coated pancreatic tumor cells, IL-21 treated NK cells secreted significantly higher levels of IFN-γ and chemokines, increased chemotaxis of T cells, and enhanced NK cell signal transduction via activation of ERK and STAT1. Treatment of mice bearing subcutaneous or intraperitoneal EGFR-positive pancreatic tumor xenografts with mIL-21 and cetuximab led to significant inhibition of tumor growth, a result further enhanced by the addition of gemcitabine.
Conclusions:
These results suggest that cetuximab treatment in combination with IL-21 adjuvant therapy in patients with EGFR-positive pancreatic cancer results in significant NK cell activation, irrespective of KRAS mutation status, and may be a potential therapeutic strategy.
Natural killer (NK) cells are increasingly used in clinical studies in order to treat patients with various malignancies. The following review summarizes platform lectures and 2013–2015 consortium meetings on manufacturing and clinical use of NK cells in Europe and United States. A broad overview of recent pre-clinical and clinical results in NK cell therapies is provided based on unstimulated, cytokine-activated, as well as genetically engineered NK cells using chimeric antigen receptors. Differences in donor selection, manufacturing and quality control of NK cells for cancer immunotherapies are described and basic recommendations are outlined for harmonization in future NK cell studies.
Natural Killer (NK) cells yield promise in therapy of hematologic malignancies. The clinical experience with adoptively transferred allogeneic NK cells over past two decades has revealed safety and minimal risk of CRS or ICANS. Unlike T cells which have to be genetically altered to avoid graft vs host disease (GVHD), HLA mismatched NK cells can be infused without GVHD risk. This makes them ideal for the development of off-the-shelf products. In this review we focus on NK biology relevant to the cancer therapy, the trajectory of NK therapeutics for leukemia, lymphoma, and myeloma; and advantages of the NK cell platform. We will also discuss novel methods to enhance NK cell targeting, persistence, and function in the tumor microenvironment. The future of NK cell therapy depends on novel strategies to realize these qualities.
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. This review gives an overview of natural killer (NK) cell-based immunotherapies. The authors describe the various engineering strategies used to increase NK cell cytotoxicity and persistence, as well as the current challenges and opportunities for the future design of next-generation NK cell therapies.
Background
Acute myeloid leukemia (AML) accounts for approximately 20% of pediatric leukemia cases, of which 30% of patients relapse. The antileukemia properties of natural killer (NK) cells and their safety profile have been reported in AML therapy.
Procedure
We proposed a phase II, open, prospective, multicenter, non-randomized clinical trial for the adoptive infusion of haploidentical K562-mb15-41BBL-activated and expanded NK (NKAE) cells as a consolidation strategy for children with favorable and intermediate-risk AML who were in their first complete remission after chemotherapy (NCT02763475). Prior to the NKAE cell infusion, patients underwent a lymphodepleting regimen. After the NKAE cell infusion, patients were administered low doses (1×10⁶/IU/m²) of subcutaneous interleukin-2. The primary study endpoint was AML relapse-free survival. We needed to include 35 patients to demonstrate a 50% reduction in relapses.
Results
Seven patients (median age, 7.4 years; range, 0.78–15.98) were administered 13 infusions of NKAE cells, with a median of 36.44×10⁶ cells/kg (range, 6.92×10⁶–193.2×10⁶ cells/kg). We observed chimerism in 4 patients (median chimerism, 0.065%; range, 0.05–0.27%). After a median follow-up of 33 moths, 6 (85.7%) patients remained in complete remission. The 3-year overall survival was 83.3% (95% CI 68.1–98.5), and the cumulative 3-year relapse rate was 28.6% (95% CI 11.5–45.7).
The study was terminated early due to low patient recruitment.
Conclusions
This study emphasizes the difficulties in recruiting patients for cell therapy trials, though NKAE cell infusion is safe and feasible. However, we cannot state any conclusions on the efficacy due to the small number of included patients and insufficient biological markers.
Multiple lines of evidence indicate that CAR-T cell based therapy and oncolytic virotherapy display robust performance in both immunocompetent and immunodeficient mouse models. Rare, yet highly successful attempts to combine these therapeutic platforms have also been reported. Interestingly, both approaches have shown pronounced efficacy in human trials, albeit these were limited to just a handful of malignancies. Specifically, CD19-specific CAR-T cell products (Kymriah and Yescarta) have been highly effective against B cell lymphomas and leukemias, whereas administering oncolytic viruses resulted in pronounced responses in melanoma (Imlygic and Rigvir) and nasopharyngeal carcinoma (Oncorine) patients. It is well established that efficacy of virotherapy as a standalone approach is largely restricted by the pre-existing and mounting immune response against viral antigens, and requires a relatively functional immune system, which is not typical for cancer patients, with the current antitumor therapy schemes. On the other hand, the most important challenges faced by the current CAR-T cell therapy formats include the lack of targetable tumor-specific surface antigens, tumor cell heterogeneity, and immunosuppressive tumor microenvironment, not to mention the unacceptably high costs. Remarkably, combining the two approaches may help address their individual bottlenecks. Namely, local acute inflammatory reaction induced by the viral infection may reverse tumor-associated immunosuppression and lead to more efficient homing and penetration of CAR-expressing lymphocytes into the tumor stroma; combined viral and CAR-mediated cytotoxicity may ensure the production of immunogenic cell debris and efficient presentation of tumor neoantigens, and potently recruit the patient's own bystander immune cells to attack cancer cells. Thus, testing the combinations of CAR-based and virolytic approaches in the clinical setting appears both logical and highly promising.
Background:
Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical efficacy in B-cell cancers. However, CAR T cells can induce substantial toxic effects, and the manufacture of the cells is complex. Natural killer (NK) cells that have been modified to express an anti-CD19 CAR have the potential to overcome these limitations.
Methods:
In this phase 1 and 2 trial, we administered HLA-mismatched anti-CD19 CAR-NK cells derived from cord blood to 11 patients with relapsed or refractory CD19-positive cancers (non-Hodgkin's lymphoma or chronic lymphocytic leukemia [CLL]). NK cells were transduced with a retroviral vector expressing genes that encode anti-CD19 CAR, interleukin-15, and inducible caspase 9 as a safety switch. The cells were expanded ex vivo and administered in a single infusion at one of three doses (1×105, 1×106, or 1×107 CAR-NK cells per kilogram of body weight) after lymphodepleting chemotherapy.
Results:
The administration of CAR-NK cells was not associated with the development of cytokine release syndrome, neurotoxicity, or graft-versus-host disease, and there was no increase in the levels of inflammatory cytokines, including interleukin-6, over baseline. The maximum tolerated dose was not reached. Of the 11 patients who were treated, 8 (73%) had a response; of these patients, 7 (4 with lymphoma and 3 with CLL) had a complete remission, and 1 had remission of the Richter's transformation component but had persistent CLL. Responses were rapid and seen within 30 days after infusion at all dose levels. The infused CAR-NK cells expanded and persisted at low levels for at least 12 months.
Conclusions:
Among 11 patients with relapsed or refractory CD19-positive cancers, a majority had a response to treatment with CAR-NK cells without the development of major toxic effects. (Funded by the M.D. Anderson Cancer Center CLL and Lymphoma Moonshot and the National Institutes of Health; ClinicalTrials.gov number, NCT03056339.).
Background
Recent advances in T cell engineering have enabled clinical trials to evaluate the potential for adoptive transfer of T cells to target malignancy. A single treatment with engineered gene-modified T cells has the potential to generate potent and long-lasting anti-tumor immunity. We have reported initial clinical data on the use of T cells engineered to express a Chimeric Antigen Receptor (CAR) that targets CD19 (CTL019) in patients with advanced treatment-refractory CLL and pediatric ALL (Porter, et al NEJM 2011; Kalos et al. Sci Trans Med 2011, Grupp et al. NEJM 2013). The initial cohort of patients is now disease free between 1 and 3 years post-infusion. In this report we present data on the functional persistence, trafficking, and bioactivity of CTL019 cells from the initial cohort of CLL patients, a larger and more recently treated cohort of CLL patients, a cohort of pediatric ALL patients, as well as an initial cohort of adult ALL patients.
Methods
CAR engineering of patient T cells was accomplished by lentivirus transduction followed by in-vitro expansion using anti-CD3 and anti-CD28 antibody-coated beads. Persistence of gene-modified T cells was assessed by quantitative PCR. CAR19 surface expression was detected by flow. B cell aplasia was evaluated by multiparametric flow cytometry. Multiplex cytokine analyses were performed using LuminexTMtechnology.
Results
Detailed clinical outcomes for each patient cohort will be reported separately at this meeting (Porter, D.L.et al, Grupp S. et al). In summary, as of July 15, 2013: For CLL: A total of 24 adult patients with advanced relapsed and/or treatment-refractory CLL have been treated under two separate protocols. To date 5 patients have achieved ongoing CR, 7 patients PR and 12 patients were NR at the primary endpoint (within 3 months post infusion). For ALL (pediatric): A total of 14 pediatric patients with treatment refractory ALL have been treated and were evaluable under a single protocol. To date 8 patients have ongoing CR, 4 patients have relapsed including 2 with CD19-negative disease and 2 patients were NR. For ALL (Adult): A total of 3 adult patients with advanced relapsed and/or treatment-refractory ALL have been treated under a single protocol. All 3 patients have ongoing CR; one patient went into an allogeneic transplant while in CTL019-induced CR.
In all patients with CR, robust in vivo expansion of CTL019 cells was observed, as assessed by both molecular and flow cytometric analysis, followed by contraction and in all but one patient ongoing stable persistence of engineered cells, elimination of tumor B cells and ongoing B cell aplasia in blood and marrow at all evaluated time points (minimum 3 months, maximum ongoing at 35 months). In CR patients, peak marking exceeded 5% of total CD3+. Patients with PR demonstrated less robust in-vivo expansion accompanied by transient B cell elimination, while NR patients demonstrated minimal in-vivo expansion.
Clinical MRD analysis in patients was supplemented with Illumina-HiSeq/MiSeq-deep sequencing based molecular MRD analysis. These analyses demonstrate that CRs were accompanied with deep and sustained molecular remissions as assessed by the absence of the tumor specific clonotype defined in the enrollment samples.
All patients in CR experienced on-target cytokine release syndrome (CRS) and macrophage activation syndrome (MAS). Multiplex-cytokine analysis of serum samples from patients in CR demonstrated a broad pro-inflammatory signature with significant elevation in a subset of soluble immune modulators including IFN-g, IL-6, and IL2ra. In contrast, patients with NR did not have elevated serum cytokines. Elevation of cytokines coincided with expansion of CTL019 cells, elimination of B cells, and toxicity suggesting the potential for a cytokine-based diagnostic signature to monitor CTL019 treatment efficacy.
Conclusions
Adoptive transfer of CTL019 cells engineered to express CD137 and TCR-zeta signaling domains can result in in-vivo expansion, homing to marrow, and long-term functional persistence of engineered cells, accompanied by ongoing complete clinical responses and long-term B cell aplasia in a substantial fraction of patients with advanced, refractory and high risk CLL and relapsed refractory pre- B cell ALL. These results highlight the potential of CTL019 therapy to effectively target CD19-positive malignancy.
Disclosures
Kalos: Novartis corporation: CART19 technology, CART19 technology Patents & Royalties; Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other. Zheng:Novartis: Patents & Royalties. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Grupp:Novartis: Research Funding. June:Novartis: Patents & Royalties, Research Funding.
Natural Killer (NK) cells represent an exciting immunotherapeutic approach to treat cancer. We have shown that in vivo expansion and activation of donor NK cells supported by administration of IL-2 induces remission in patients with refractory AML. Recent clinical studies by our group have shown that IL-15 is superior to IL-2 to support NK cell persistence 14 days after adoptive transfer. However, only 36% of patients treated with 12 consecutive days of IL-15 had NK cell expansion to the level of ≥100 donor derived NK cells/µL blood compared to 10% in subjects treated with IL-2 (p=0.02). This leads us to conclude that we might not know the optimal route and interval to administer in vivo IL-15. We hypothesized that daily uninterrupted IL-15 dosing could lead to exhaustion or NK cellular stress. Therefore we designed an in vitro model system in which enriched NK cells are treated with three 3-day cycles of continuous IL-15 (IL-15cont) or were rested with a "gap" (skipping the middle cycle [IL-15gap]) before returning to the last cycle of IL-15. IL-15cont treatment yielded more proliferation and higher cell numbers compared to IL-15gap (4.8±0.44 vs. 1.9±0.26 million cells/ml, p < 0.0001) when cells were analyzed at the end of the three cycles (on day 9, where all in vitro measurements were taken). However, NK cell death, measured by flow cytometry, in the IL-15cont group was higher (18.9±2.2 vs 14.9±1.7 % cell death, p = 0.035) and this group also had an enrichment in genes involved in cell cycle checkpoint/ arrest, perhaps indicating more cellular stress in the IL-15cont. In an in vitro flow cytometric functional assay, the IL-15cont group had decreased activation when compared to the IL-15 gap group against K562 targets (43.6±2.1 vs 55.6±2.7 % CD107a [degranulation], p < 0.0001; 1.9±0.41 vs 7.1±0.93 % IFNg [inflammatory cytokine production], p = 0.0055). The decrease in NK cell activation correlated with a strong decrease in tumor target killing in an in vitro chromium release assay (Figure 1A) measuring killing of acute promyelocytic leukemia (HL-60) cell targets, in which the IL-15cont NK cells were potently outperformed by the IL-15gap cells (6.4±2.6 vs 51.5±4.8 % killing at 2.5:1 effector:target ratio, p < 0.0001). We used an in vivo xenogeneic model of AML, where conditioned NSG (NOD scid gamma) mice are engrafted with HL-60luc tumor targets 3 days prior to infusion with nothing, IL-15cont or IL-15gap human NK cells prepared within our 9 day culture system. Only the IL-15gap NK group mediated statistically significant tumor control when compared to tumor alone at two weeks following NK cell infusion (Figure 1B). To probe deeper into the functional defect we evaluated signaling after these treatments and noted decreased phosphorylation of several proteins in the IL-15cont group. These data led us to explore proteins involved in metabolism and we noted that CPT1A, a critical enzyme involved in fatty acid oxidation (FAO), was strongly increased in the IL-15gap treated NK cells (protein MFI of 15,759±2,603 [IL-15gap] vs 5,273±744 [IL-15cont], p = 0.009). Metabolic analysis using a Seahorse XFe24 analyzer showed an increased mitochondrial spare respiratory capacity (SRC) in the IL-15gap group, denoting better capability of the IL-15gap NK cells to respond to energetic demands (Figure 1C). In a separate experiment the groups were treated with etomoxir to inhibit CPT1A, and the SRC phenotype was reversed, with the IL-15gap group containing lower SRC than the IL-15cont group. To test these findings in a functional assay we repeated the IL-15cont treatment in combination with rapamycin, which can induce CPT1A through inhibition of mTORC1, and saw restoration of function to levels similar to IL-15gap (40.8±2.0 vs 49.3±2.9 % CD107a in the IL-15cont vs IL-15cont + rapamycin, p = 0.005; 2.4±0.47 vs 4.8±1.0 % IFNg in the IL-15cont vs IL-15cont + rapamycin, p = 0.03). These data indicate that NK cell functional exhaustion via continuous IL-15 signaling is mediated by a decrease in FAO. Intermittent IL-15 dosing or altering metabolism through other mechanisms may overcome this competition. These findings could impact ongoing clinical trials through simple alterations in dosing strategies in order to minimize NK cell exhaustion in the immunotherapeutic setting.
Disclosures
Cooley: Fate Therapeutics: Research Funding. Miller:Oxis Biotech: Consultancy, Other: SAB; Fate Therapeutics: Consultancy, Research Funding.
Extracellular adenosine is a key immunosuppressive metabolite that restricts activation of cytotoxic lymphocytes and impairs antitumor immune responses. Here, we show that engagement of A2A adenosine receptor (A2AR) acts as a checkpoint that limits the maturation of natural killer (NK) cells. Both global and NK-cell–specific conditional deletion of A2AR enhanced proportions of terminally mature NK cells at homeostasis, following reconstitution, and in the tumor microenvironment. Notably, A2AR-deficient, terminally mature NK cells retained proliferative capacity and exhibited heightened reconstitution in competitive transfer assays. Moreover, targeting A2AR specifically on NK cells also improved tumor control and delayed tumor initiation. Taken together, our results establish A2AR-mediated adenosine signaling as an intrinsic negative regulator of NK-cell maturation and antitumor immune responses. On the basis of these findings, we propose that administering A2AR antagonists concurrently with NK cell–based therapies may heighten therapeutic benefits by augmenting NK cell–mediated antitumor immunity.
Significance: Ablating adenosine signaling is found to promote natural killer cell maturation and antitumor immunity and reduce tumor growth. Cancer Res; 78(4); 1003–16. ©2017 AACR.
Chimeric antigen receptors (CARs) have been used to redirect the specificity of autologous T-cells against leukemia and lymphoma with promising clinical results.(1-3) Extending this approach to allogeneic T-cells is problematic as they carry a significant risk of graft-versus-host disease (GVHD). Natural killer (NK) cells are highly cytotoxic effectors, killing their targets in a non-antigen specific manner without causing GVHD. Cord blood (CB) offers an attractive, allogeneic, off-the-self source of NK cells for immunotherapy. We transduced CB-derived NK cells with a retroviral vector incorporating the genes for CAR-CD19, IL-15 and inducible caspase-9-based suicide gene (iC9), and demonstrated efficient killing of CD19-expressing cell lines and primary leukemia cells in vitro, with dramatic prolongation of survival in a xenograft Raji lymphoma murine model. IL-15 production by the transduced CB-NK cells critically improved their function. Moreover, iC9/CAR.19/IL-15 CB-NK cells were readily eliminated upon pharmacologic activation of the iC9 suicide gene. In conclusion, we have developed a novel approach to immunotherapy using engineered CB-derived NK cells which are easy to produce, exhibit striking efficacy and incorporate safety measures to limit toxicity. This approach should greatly improve the logistics of delivering this therapy to large numbers of patients, a major limitation to current CAR-T cell therapies.Leukemia accepted article preview online, 20 July 2017. doi:10.1038/leu.2017.226.
Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell antileukemia potential is unclear. Cytokine-induced memory-like NK cells differentiate after a brief preactivation with interleukin-12 (IL-12), IL-15, and IL-18 and exhibit enhanced responses to cytokine or activating receptor restimulation for weeks to months after preactivation. We hypothesized that memory-like NK cells exhibit enhanced antileukemia functionality. We demonstrated that human memory-like NK cells have enhanced interferon-g production and cytotoxicity against leukemia cell lines or primary human AML blasts in vitro. Usingmass cytometry, we found that memory-like NK cell functional responses were triggered against primary AML blasts, regardless of killer cell immunoglobulin-like receptor (KIR) to KIR-ligand interactions. In addition, multidimensional analyses identified distinct phenotypes of control and memory-like NK cells from the same individuals. Humanmemory-like NK cells xenografted into mice substantially reduced AML burden in vivo and improved overall survival. In the context of a first-in-human phase 1 clinical trial, adoptively transferred memory-like NK cells proliferated and expanded in AML patients and demonstrated robust responses against leukemia targets. Clinical responses were observed in five of nine evaluable patients, including four complete remissions. Thus, harnessing cytokine-induced memory-like NK cell responses represents a promising translational immunotherapy approach for patients with AML.
Natural killer (NK) cells are promising anti-tumor effector cells, but the generation of sufficient NK cell numbers for adoptive immunotherapy remains challenging. Therefore, we developed a method for highly efficient ex vivo expansion of human NK cells.
Ex vivo expansion of NK cells in medium containing IL-2 and irradiated clinical-grade feeder cells (EBV-LCL) induced a 22–fold NK cell expansion after one week that was significantly increased to 53–fold by IL-21. Repeated stimulation with irradiated EBV-LCL and IL-2 and addition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with 1011–fold NK cell expansion after six weeks. Compared to naive NK cells, expanded NK cells up-regulated TRAIL, NKG2D and DNAM-1, had superior cytotoxicity against tumor cell lines in vitro and produced more IFN-γ and TNF-α upon PMA/Iono stimulation. Most importantly, adoptive transfer of NK cells expanded using feeder cells, IL-2 and IL-21 led to significant inhibition of tumor growth in a melanoma xenograft mouse model, which was greater than with NK cells activated with IL-2 alone. Intriguingly, adoptively transferred NK cells maintained their enhanced production of IFN-γ and TNF-α upon ex vivo restimulation, although they rapidly lost their capacity to degranulate and mediate tumor cytotoxicity after the in vivo transfer.
In conclusion, we developed a protocol for ex vivo NK cell expansion that results in outstanding cell yields. The expanded NK cells possess potent anti-tumor activity in vitro and in vivo and could be utilized at high numbers for adoptive immunotherapy in the clinic.
Translating cellular therapy from the laboratory to the clinic is a complicated process that involves scale-up of procedures to generate clinically relevant cell numbers, adaptation to reagents and equipment that are qualified for human use, establishing parameters of safety for reagents and equipment that are not already qualified for human use, codifying these processes into standards of practice and rules of conduct, and obtaining approval from regulatory bodies based on those codified standards and rules. As the laws and regulations that apply to cellular therapy will vary by time and geography, this chapter reviews some common key principles for the manufacturing of NK cells for human use that will need to be considered within the constraints of local policies and regulations.
Background aims:
Umbilical cord blood transplantation (UCBT) is increasingly used to treat acute leukemias. UCB units are thawed and infused in their entirety at transplant, precluding later use as immunotherapy to prevent or treat leukemia relapse.
Methods:
We developed a device that selectively thaws only 1 mL of the UCB unit, leaving the remaining UCB unit cryopreserved for subsequent transplantation. We also show that large numbers of CD56(+) natural killer (NK) cells can be expanded from these 1-mL fractions of selectively accessed UCB. Immunomagnetic depletion of CD3(+) cells of the 1-mL fraction was performed, and the cells were subsequently stimulated with irradiated Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCLs) and set to culture in media containing interleukin (IL)-2.
Results:
When a 1:20 ratio of total nucleated cells to EBV-LCL feeder cells was used, day-21 and day-35 NK cell cultures initiated from 1 mL of UCB contained a median of 430 × 10(6) (range: 44-4321 × 10(6)) and 6092 × 10(6) (range: 165-20947 × 10(6)) CD3(-)CD56(+) NK cells. These cells expressed high levels of CD161, LFA-1, CD69, NKG2D, NKp30, NKp44, NKp80 and NKp46. UCB-derived NK cells were highly cytotoxic against K562 leukemia cells, although cytotoxicity was slightly lower than in expanded PBMC-derived NK cells.
Conclusions:
We have developed and optimized a strategy to selectively access a small fraction from cryopreserved UCB and show that large numbers of CD56(+) cells can be expanded from this selectively accessed fraction. This strategy presents a method to explore whether early adoptive transfer of NK cells expanded from the same UCB unit used for transplantation can prevent leukemic relapse and decrease graft-versus-host disease after UCBT.