Natural killer cells unleashed: Checkpoint receptor blockade and BiKE/TriKE utilization in NK-mediated anti-tumor immunotherapy

Natural killer (NK) cells have long been known to mediate anti-tumor responses without prior sensitization or recognition of specific tumor antigens. However, the tumor microenvironment can suppress NK cell function resulting in tumor escape and disease progression. Despite recent advances in cytokine therapy and NK cell adoptive transfer, tumor expression of ligands to NK – expressed checkpoint receptors can still suppress NK mediated tumor lysis. This review will explore many of the checkpoint receptors tumors utilize to manipulate the NK cell response as well as some of the current and upcoming pharmacological solutions to limit tumor suppression of NK cell function. Furthermore, we will discuss the potential to use these drugs in combinational therapies with novel antibody reagents such as bi- and tri-specific killer engagers (BiKEs and TriKEs) against tumor-specific antigens to enhance NK cell-mediated tumor rejection.     

A killer choice for cancer immunotherapy

The promise of cell-based immunotherapies for the treatment of cancer offers the potential of therapeutic synergy with chemo- and radiotherapies that may overcome current limitations leading to durable responses and prevention of recurrence. There is a wide array of cell-based immunotherapies that are either poised to enter cancer clinical trials or are in clinical trials, and many are showing some success. Yet within this field, there are clear obstacles that need to be overcome, including limited access across tissue barriers, development of antigen tolerance, and the immunosuppressive microenvironment of tumors. Through an understanding of immune cell signaling and trafficking, immune cell populations can be selected for adoptive transfer, and delivery strategies can be developed that circumvent these obstacles to effectively direct populations of cells with robust anti-tumor efficacy to the target. Within the realm of immune cell therapies, cytokine-induced killer (CIK) cells have demonstrated promising trafficking patterns, effective delivery of synergistic therapeutics, and stand-alone efficacy. Here, we discuss the next generation of CIK therapies and their application for the effective treatment of a wide variety of cancers.     

NK cell-based immunotherapy for malignant diseases

Natural killer (NK) cells play critical roles in host immunity against cancer. In response, cancers develop mechanisms to escape NK cell attack or induce defective NK cells. Current NK cell-based cancer immunotherapy aims to overcome NK cell paralysis using several approaches. One approach uses expanded allogeneic NK cells, which are not inhibited by self histocompatibility antigens like autologous NK cells, for adoptive cellular immunotherapy. Another adoptive transfer approach uses stable allogeneic NK cell lines, which is more practical for quality control and large-scale production. A third approach is genetic modification of fresh NK cells or NK cell lines to highly express cytokines, Fc receptors and/or chimeric tumor-antigen receptors. Therapeutic NK cells can be derived from various sources, including peripheral or cord blood cells, stem cells or even induced pluripotent stem cells (iPSCs), and a variety of stimulators can be used for large-scale production in laboratories or good manufacturing practice (GMP) facilities, including soluble growth factors, immobilized molecules or antibodies, and other cellular activators. A list of NK cell therapies to treat several types of cancer in clinical trials is reviewed here. Several different approaches to NK-based immunotherapy, such as tissue-specific NK cells, killer receptor-oriented NK cells and chemically treated NK cells, are discussed. A few new techniques or strategies to monitor NK cell therapy by non-invasive imaging, predetermine the efficiency of NK cell therapy by in vivo experiments and evaluate NK cell therapy approaches in clinical trials are also introduced.     

Improvement of adoptive cellular immunotherapy of human cancer using ex-vivo gene transfer

A variety of adoptive cellular strategies, aimed at boosting the immune system, have been tested in the management of metastatic diseases. Despite the drawbacks associated with ex vivo cell manipulation and upscaling, several such approaches have been assessed in the clinic. The use of lymphokine-activated killer (LAK) cells, auto-lymphocyte therapy (ALT) and tumor-infiltrating lymphocytes (TIL) have been the best studied and further trials are ongoing. Thus far, these approaches have not consistently shown benefit when compared to standard immune-based treatment with biologic response modifiers, notably, high-dose interleukin-2 (IL-2). More recently, it has been shown, in various animal models, that the ex vivo transfer of genes to cells of the immune system can have a dramatic impact on cancer immunotherapy. The application of gene transfer techniques to immunotherapy has animated the field of cell-based cancer therapy research. A wide variety of viral and non-viral gene transfer methods have been investigated in this context. Ex vivo strategies include gene delivery into tumor cells and into cellular components of the immune system, including cytotoxic T cells, NK, macrophages and dendritic cells (DC). Several of these approaches have already been translated into cancer therapy clinical trials. In this review, we focus on the rationale and types of ex vivo gene-based immunotherapy of cancer. Finally, the use of genetically modified DC for tumor vaccination and its prospects are discussed.     

Clinical utility of natural killer cells in cancer therapy and transplantation

Natural killer (NK) cells recognize deranged cells that display stress receptors or loss of major histocompatibility complex (MHC) class I. During development, NK cells become “licensed” only after they encounter cognate human leukocyte antigen (HLA) class I, leading to the acquisition of effector function. NK cells can be exploited for cancer therapy in several ways. These include targeting with monoclonal antibodies alone or combined with ex vivo and in vivo NK cell activation to facilitate adoptive immunotherapy using donor-derived NK cell products to induce graft-vs-tumor effects. In the adoptive transfer setting, persistence and in vivo expansion requires lymphodepleting chemotherapy to prevent rejection and provide homeostatic cytokines (such as IL-15) that activate NK cells. IL-15 has the advantage of avoiding regulatory T-cell expansion. Clinical applications are currently being tested. To enhance in vivo expansion, IL-2 has been used at low doses. However, low dose administration also leads to the stimulation of regulatory T cells. Monoclonal antibodies and bispecific killer engagers (BiKEs) may enhance specificity by targeting CD16 on NK cells to tumor antigens. Inhibition of CD16 shedding may also promote enhanced cytotoxicity. Future strategies include exploiting favorable donor immunogenetics or ex vivo expansion of NK cells from blood, progenitors, or pluripotent cells. Comparative clinical trials are needed to test these approaches.     

Genetic approaches for antigen-selective cell therapy

Attempts to improve the efficacy of adoptive T-cell therapies have led to the development of innovative strategies that combine the high specificity of antibody molecules with the efficient trafficking properties and effector functions of immune cells. These antigen-selective cell therapies are designed to convert therapeutically important native antigens expressed on the cell surface (tumor associated antigens, viral envelope proteins) into recruitment points of effector functions, and address the goal of major histocompatibility complex- and exogenous cytokine-independent activation of mature effector T-cells. The most promising and best characterized antigen-selective strategy is based on the genetic manipulation of the recognition specificity of T-cells by grafting the recognition specificity of a monoclonal antibody onto a lymphocyte triggering molecule (TCR-associated polypeptides, Fc epsilon RI-gamma chain). Upon encountering specific antigen, cells harboring chimeric immune receptors (CIRs) are able to undergo specific stimulation and kill antigen bearing cells in both in vitro and in vivo model systems. Initial studies have focused on terminally differentiated effector cell-based protocols. However, recent data indicate that progenitor cell-based therapies allow the permanent generation of stable populations of CIR-expressing cells of multiple lineages, leading to long-term persistent systemic immunity. Emerging gene therapy strategies are based on the use of biespecic antibody fragments. The advantages of these biespecic antibody-mediated immune recruitment (BIR) approaches (trans-recruitment and multieffector activation) could complement conventional CIR-based immunotherapies. Although further scientific progress is required regarding the selection of the ideal effector cell/s and the definition of the optimal targeting and recruitment systems, clinical trials recently initiated in patients with advanced cancer and human immunodeficiency virus infection should help us to determine the real efficacy of these approaches. The relevance of these and other emerging concepts to cell-mediated immunotherapy is discussed.     

Chronically stimulated mouse invariant NKT cell lines have a preserved capacity to enhance protection against experimental tumor metastases

In pre-clinical models, CD1d restricted invariant Natural Killer T (iNKT) cells play a pivotal role in natural anti-tumor immune responses, mainly by trans-activating cells of both the innate and adaptive arms via swift and potent cytokine secretion. We have previously reported that patients with a severely reduced circulating iNKT cell pool have a poor clinical response to radio therapy of head and neck squamous cell carcinoma. Therefore, these patients might benefit from an immunotherapeutic approach aimed at the increase of circulating levels of iNKT cells. Furthermore, we have generated both human and mouse iNKT cell lines, and demonstrated that they had retained the capacity to release both Th1 and Th2 type cytokines even after long-term in vitro expansion using alpha-galactosylceramide (alphaGalCer) pulsed dendritic cells (DC). Here, we establish, in a pre-clinical tumor model that the large scale long lived polyclonal iNKT cell lines we generated have a preserved capacity to evoke an in vivo cytokine storm upon adoptive transfer, independently of supplemental alphaGalCer administration. This results in an augmented NK cell mediated protection against B16.F10 experimental lung metastases in vivo. These findings underscore the potential of autologous adoptive transfer of ex vivo expanded iNKT cells as a strategy to enhance immunotherapeutic modalities for the treatment of cancer patients.     

Natural killer cell immunotherapies against cancer: checkpoint inhibitors and more

After many years of research, recent advances have shed new light on the role of the immune system in advanced-stage cancer. Various types of immune cells may be useful for therapeutic purposes, along with chemical molecules and engineered monoclonal antibodies. The immune effectors suitable for manipulation for adoptive transfer or drug targeting in vivo include natural killer (NK) cells. These cells are of particular interest because they are tightly regulated by an array of inhibitory and activating receptors, enabling them to kill tumor cells while sparing normal cells. New therapeutic antibodies blocking the interactions of inhibitory receptors (immune checkpoint inhibitors, ICI) with their ligands have been developed and can potentiate NK cell functions in vivo.     

Targeting the tumor microenvironment and T cell metabolism for effective cancer immunotherapy

The successful implementation of immunotherapies has provided new impetus in the fight against cancer. Antibody‐mediated blockade of immune checkpoint molecules PD‐1/PD‐L1 and CTLA‐4 has had a dramatic impact upon the treatment of previously intractable cancers such as malignant melanoma, while adoptive cell therapies using chimeric antigen receptor‐bearing T cells have proven highly efficacious in B cell leukemia. Furthermore, significant progress has been made in understanding the mechanisms by which tumors evade or become resistant to these immunotherapies. In this regard, approaches to broaden the applicability and enhance the efficacy of immunotherapies increasingly include modulation of tumor and immune cell metabolism. In this mini‐review, we highlight the most recent studies describing novel approaches and targets for the manipulation of the tumor microenvironment and T cell metabolism and describe how these approaches are being combined with current immunotherapies in preclinical studies.     

Prostate Cancer and Immunoproteome: Awakening and Reprogramming the Guardian Angels

Prostate cancer is a life-threatening molecular disorder that is undruggable to date because of stumbling blocks in the standardization of therapy. An emerging framework of research is addressing how pathways that are derailed during tumorigenesis are linked to immunological responses, which are instrumental in immunosurveillance of cancer. However, interestingly, cancer cells circumvent such immunosurveillance through development of poorly immunogenic tumor cell variants (immunoselection) and through subversion of the immunological nanomachinery (immunosubversion). Detailed mechanistic insights of molecular specificities that regulate natural killer (NK) cell function suggest that it might be promising to design NK cell-based immunotherapeutic interventions against prostate cancer. Here, we elucidate evidence for NK cell targeting of prostate cancer proteome and address critical questions that, in our view, need thoughtfulness for the development of successful NK cell-based therapies. This review also disproves our contemporary understanding of the versatile regulators of DNA damage repair (ATM, ATR) that trigger cell surface expression of NKG2D ligands and consequent elimination of the tumor cells by NK cells and other lymphocytes that express NK cell receptors. Substantial fraction of information has been generated that guarantees productive future for this technology as more optimized constructs, better trial designs, and improved platforms are being brought from benchtop to bedside.     

Novel immunotherapies for hematologic malignancies

The immune system is designed to discriminate between self and tumor tissue. Through genetic recombination, there is fundamentally no limit to the number of tumor antigens that immune cells can recognize. Yet, tumors use a variety of immunosuppressive mechanisms to evade immunity. Insight into how the immune system interacts with tumors is expanding rapidly and has accelerated the translation of immunotherapies into medical breakthroughs. Herein, we appraise novel strategies that exploit the patient's immune system to kill cancer. We review various forms of immune-based therapies, which have shown significant promise in patients with hematologic malignancies, including (i) conventional monoclonal therapies like rituximab; (ii) engineered monoclonal antibodies called bispecific T-cell engagers; (iii) monoclonal antibodies and pharmaceutical drugs that block inhibitory T-cell pathways (i.e. PD-1, CTLA-4, and IDO); and (iv) adoptive cell transfer therapy with T cells engineered to express chimeric antigen receptors or T-cell receptors. We also assess the idea of using these therapies in combination and conclude by suggesting multi-prong approaches to improve treatment outcomes and curative responses in patients.     

NK细胞在肿瘤免疫治疗中的进展

NK细胞是体内不同于T细胞和B细胞的一群淋巴细胞,由于其在机体抵抗病毒入侵及肿瘤防御方面发挥重要作用,现已成为肿瘤生物治疗的研究热点,以下将就NK细胞的来源、作用机制及目前NK细胞过继免疫治疗肿瘤的情况进行综述.     

Adoptive T-cell therapy of prostate cancer targeting the cancer stem cell antigen EpCAM

Background

Adoptive transfer of tumor infiltrating or circulating lymphocytes transduced with tumor antigen receptors has been examined in various clinical trials to treat human cancers. The tumor antigens targeted by transferred lymphocytes affects the efficacy of this therapeutic approach. Because cancer stem cells (CSCs) play an important role in tumor growth and metastasis, we hypothesized that adoptive transfer of T cells targeting a CSC antigen could result in dramatic anti-tumor effects.

Results

An EpCAM-specific chimeric antigen receptor (CAR) was constructed to transduce human peripheral blood lymphocytes (PBLs) and thereby enable them to target the CSC marker EpCAM. To investigate the therapeutic capabilities of PBLs expressing EpCAM-specific CARs, we used two different tumor models, PC3, the human prostate cancer cell line, which has low expression levels of EpCAM, and PC3M, a highly metastatic clone of PC3 that has high expression levels of EpCAM. We demonstrate that CAR-expressing PBLs can kill PC3M tumor cells in vitro and in vivo. Despite the low expression of EpCAM on PC3 cells, CAR-expressing PBLs significantly inhibited tumor growth and prolonged mouse survival in a PC3 metastasis model, probably by targeting the highly proliferative and metastatic population of cancer cells.

Conclusions

Our data demonstrate that PBLs expressing with EpCAM-specific CARs have significant anti-tumor activity against prostate cancer. Therefore, the adoptive transfer of T cells targeting EpCAM could have great potential as a cancer treatment.     

Harnessing innate and adaptive immunity for adoptive cell therapy of renal cell carcinoma

The development of immunotherapies for renal cell carcinoma (RCC) has been the subject of research for several decades. In addition to cytokine therapy, the benefit of various adoptive cell therapies has again come into focus in the past several years. Nevertheless, success in fighting this immunogenic tumor is still disappointing. RCC can attract a multitude of different effector cells of both the innate and adaptive immune system, including natural killer (NK) cells, γδ T cells, NK-like T cells, peptide-specific T cells, dendritic cells (DC), and regulatory T cells (Tregs). Based on intensive research on the biology and function of different immune cells, we now understand that individual cell types do not act in isolation but function within a complex network of intercellular interactions. These interactions play a pivotal role in the efficient activation and function of effector cells, which is a prerequisite for successful tumor elimination. This review provides a current overview of the diversity of effector cells having the capacity to recognize RCC. Aspects of the functions and anti-tumor properties that make them attractive candidates for adoptive cell therapies, as well as experience in clinical application are discussed. Improved knowledge of the biology of this immune network may help us to effectively harness various effector cells, placing us in a better position to develop new therapeutic strategies to successfully fight RCC.     

rhIL-2与阿霉素白蛋白磁微球靶向治疗肿瘤的协同作用

目的：观察重组人白细胞介素-2(rhIL-2)瘤内注射和阿霉素白蛋白磁微球(ADM-MAM)联合外磁场联合治疗H22荷瘤小鼠的协同作用，并探讨其抗肿瘤作用机制。方法：以荷瘤小鼠的瘤重为指标，观察药物的抗肿瘤活性。以乳酸脱氢酶释放法测NK细胞的杀伤活性。以MTT比色法测淋巴细胞的转化率。以流式细胞术检测肿瘤细胞的凋亡及p53、Fas和FasL的表达。用RT—PCR法测定IL—2及IL—12的表达。探讨抗肿瘤机制。结果：ADM—MAM靶向治疗与rhIL—2联用，可显著减小荷瘤小鼠的瘤重；提高NK细胞的杀伤活性和脾脏淋巴细胞的转化率；减小肿瘤细胞的增殖指数，上调肿瘤细胞p53、Fas和FasL的表达，以及增加脾脏淋巴细胞上IL—2及IL—12的表达。结论：ADM—MAM联合外磁场，具有显著增强抑瘤的作用。rhIL—2能增强ADM—MAM靶向治疗的抗肿瘤作用，其抗肿瘤协同作用主要是通过促进T细胞增殖，刺激NK细胞增长等提高机体的免疫功能而实现的。     

Dendritic cells in models of tumor immunity. Role of Flt3 ligand

Cancer immunotherapy might be based on the administration to cancer patients of dendritic cells (DC) 'pulsed' with tumor-specific antigens. An alternative approach is to directly expand and/or activate DC in vivo using the cytokine Flt3 Ligand (FL). In mice, FL can drive large expansion of both lymphoid-related DC that appears to selectively enhance Th1-like immune responses and myeloid-related DC that enhances a more mixed Th phenotype. Immunization of FL-treated mice with a protein antigen leads to increased production of antibodies specific for that protein as well as to antigen-specific helper T cell responses. Studies of mouse tumor models have demonstrated that FL administration leads to the generation of protective anti-tumor immune responses, these effects being mediated by CTL and/or NK cells. When, FL has only minor or short term effects, the anti-tumor response can be significantly amplified by adding other cytokines, known to act at the T cell level, such as CD40 ligand (CD40L) or 4-1BBL, a TNF family member. Thus, combination of FL + CD40L or FL + 4-1BBL have superior anti-tumor effects vs. either cytokine alone. In conclusion, cytokines offer a variety of novel approaches for the treatment of cancer, infectious or auto-immune diseases.     

Stimulating natural killer cells to protect against cancer: recent developments

Current cancer immunotherapies have begun to target cell types involved in innate immunity, such as natural killer (NK) cells that recognize and kill tumor cells. Recent advances in the study of NK cell biology have generated interest in manipulating these cells to generate anti-tumor responses. A rise in the number of activated NK cells has been shown to prevent and treat cancer in many preclinical models and is a positive clinical factor in human tumors. This article will focus on recent research on the ability of IL-15 and Toll-like receptor ligands to stimulate NK cell activity against cancer. The potential of these therapies, both alone and in conjunction with traditional and other vaccine platforms, will be reviewed. The current status of these therapies in clinical trials will also be discussed. Targeting these cell types in the context of human cancers may be an essential factor in future cancer treatments.     

Stimulating natural killer cells to protect against cancer: recent developments

Current cancer immunotherapies have begun to target cell types involved in innate immunity, such as natural killer (NK) cells that recognize and kill tumor cells. Recent advances in the study of NK cell biology have generated interest in manipulating these cells to generate anti-tumor responses. A rise in the number of activated NK cells has been shown to prevent and treat cancer in many preclinical models and is a positive clinical factor in human tumors. This article will focus on recent research on the ability of IL-15 and Toll-like receptor ligands to stimulate NK cell activity against cancer. The potential of these therapies, both alone and in conjunction with traditional and other vaccine platforms, will be reviewed. The current status of these therapies in clinical trials will also be discussed. Targeting these cell types in the context of human cancers may be an essential factor in future cancer treatments.     

Combining targeted therapy with immunotherapy. Can 1 + 1 equal more than 2?

Targeted therapies have induced high response rates and improved survival in patients with cancer. However, the long-term effectiveness of targeted therapies has been limited by the development of acquired resistance in the majority of patients. On the other hand, the modern immunotherapy strategies have been associated with durable responses but in limited number of patients. Accordingly, research efforts have been focused on examining the effects of combinations of targeted therapy and immunotherapy in several different histological subtypes of cancer. There has been accumulated evidence to suggest that targeted therapy can induce immune effects in the tumor cells, the host immune system, and the tumor microenvironment. Subsequently, clinical trials have been designed to examine the efficacy of combining immune checkpoint blockade or adoptive cell transfer with tyrosine kinase inhibitors, HER family blockade, anti-angiogenic agents, histone deacetylase inhibitors, and cancer stem cell inhibitors. To date, the combination of immunotherapy with targeted therapy has demonstrated potential as a cancer treatment strategy, but further optimizations are required and caution must be taken to avoid toxicity. The current review summarizes existing evidence and provides rationale supporting the use of combined targeted and immune-therapy approaches in patients with different types of cancer.     

Cell-based vaccination against melanoma – background, preliminary results, and perspective

Melanoma is the prototype of a tumor to which many forms of immunotherapy have been applied extensively over the past two decades. Melanoma vaccines (active specific immunotherapy) are designed to modulate the immune system and have subsequent anti-tumor effects with minimal toxicity. Previous attempts to produce melanoma vaccines include immunization with whole tumor cells/cell lysates admixed with nonspecific adjuvants. While these vaccines generate enhanced anti-tumor immunity in a subset of patients, some of whom survive for longer than historical controls, no clinical benefit has so far been demonstrated in a properly controlled phase III study. Genetic modifications of tumor cells to make them express cytokines afford new-generation melanoma vaccines, and generate long-lasting systemic antitumor immunity in animal models. Translation of these preclinical results primarily into melanoma patients with advanced diseases shows the potential to induce systemic antitumor immune responses and in some instances tumor regression with acceptably low toxicity. The efficacy of this novel vaccine approach would be expected to be higher when used in a postsurgical adjuvant setting when the tumor load is small. Other novel vaccine approaches such as dendritic cell-based therapy also hold promise for the treatment of melanoma. The clinical value of all these new approaches will eventually have to be established in prospectively randomized clinical studies.