Harnessing NK cells for cancer immunotherapy: immune checkpoint receptors and chimeric antigen receptors

Natural killer (NK) cells, key antitumor effectors of the innate immune system, are endowed with the unique ability to spontaneously eliminate cells undergoing a neoplastic transformation. Given their broad reactivity against diverse types of cancer and close association with cancer prognosis, NK cells have gained considerable attention as a promising therapeutic target for cancer immunotherapy. NK cell-based therapies have demonstrated favorable clinical efficacies in several hematological malignancies but limited success in solid tumors, thus highlighting the need to develop new therapeutic strategies to restore and optimize antitumor activity while preventing tumor immune escape. The current therapeutic modalities yielding encouraging results in clinical trials include the blockade of immune checkpoint receptors to overcome the immune-evasion mechanism used by tumors and the incorporation of tumor-directed chimeric antigen receptors to enhance NK cell antitumor specificity and activity. These observations, together with recent advances in the understanding of NK cell activation within the tumor microenvironment, will facilitate the optimal design of NK cell-based therapy against a broad range of cancers and, more desirably, refractory cancers.     

NK cells and cancer

In this review, we overview the main features and functions of NK cells, focusing on their role in cell-mediated immune response to tumor cells. In parallel, we discuss the information available in the field of NK cell receptors and offer a wide general overview of functional aspects of cell targeting and killing, focusing on the recent acknowledgments on the efficacy of NK cells after cytokine and mAb administration in cancer therapy. Since efficacy of NK cell-based immunotherapy has been proven in KIR-mismatch regimens or in TRAIL-dependent apoptosis, the ability to manipulate the balance of activating and inhibitory receptors on NK cells and of their cognate ligands, as well as the sensitivity of tumor cells to apoptosis, opens new perspectives for NK cell-based immunotherapy.     

Targeted cellular therapy with natural killer cells.

Natural killer (NK) cells are believed to be important contributors to a patient's immune armamentarium to fight cancer. However, cancer patients have reportedly defective NK cells and the malignant target frequently has developed mechanisms to escape detection of NK cells. Our research is aimed at overcoming this NK cell paralysis through three different approaches: instead of using autologous NK cells for adoptive immunotherapy, allogeneic NK cells are used that are not inhibited by self histocompatibility antigens. Further, NK cells, selected for a high affinity Fc receptor can, together with monoclonal antibodies, kill targets through ADCC irrespective of any inhibitory receptors. Finally, the genetic engineering of NK cells to express chimeric antigen receptors recognizing antigens on tumor target can overcome inhibitory mechanism and effectively lyse tumor cells.     

The therapeutic potential of immune cell-derived exosomes as an alternative to adoptive cell transfer

Adoptive cell transfer (ACT), a form of cell-based immunotherapy that eliminates cancer by restoring and strengthening the body’s immune system, has revolutionized cancer treatment. ACT entails intravenous transfer of either tumor-resident or peripheral blood-modified immune cells into cancer patients to mediate anti-tumor response. Although these immune cells control and eradicate cancer via enhanced cytotoxicity against specific tumor antigens, several side effects have been frequently reported in clinical trials. Recently, exosomes, potential cell-free therapeutics, have emerged as an alternative to cell-based immunotherapies, due to their higher stability under same storage condition, lower risk of GvHD and CRS, and higher resistance to immunosuppressive tumor microenvironment. Exosomes, which are nano-sized lipid vesicles, are secreted by living cells, including immune cells. Exosomes contain proteins, lipids, and nucleic acids, and the functional role of each exosome is determined by the specific cargo derived from parental cells. Exosomes derived from cytotoxic effectors including T cells and NK cells exert anti-tumor effects via proteins such as granzyme B and FasL. In this mini-review, we describe the current understanding of the ACT and immune cell-derived exosomes and discuss the limitations of ACT and the opportunities for immune cell-derived exosomes as immune therapies.     

Therapeutic gene modified cell based cancer vaccines

History of cancer immunotherapy lasts for more than 120 years. In 1891 William B. Coley injected bacteria into inoperable cancer (bone sarcoma) and observed tumor shrinkage. He is recognized as the "'"Father of Immunotherapy"'". Cancer immunotherapy is based on the ability of the immune system to recognize cancer cells and to affect their growth and expansion. Beside the fact that, tumor cells are genetically distinct from their normal counterparts, and should be recognized and eliminated by immune system, the tumor associated antigens (TAAs) are often poorly immunogenic due to immunoediting. This process allows tumor to evolve during continuous interactions with the host immune system, and eventually escape from immune surveillance. Furthermore, tumor microenvironment consists of immunosuppressive cells that release immunosuppressive factors including IL-6, IL-10, IDO, TGFβ or VEGF. Interactions between cancer and stroma cells create network of immunosuppressive pathways, while activation of immune defense is inhibited. A key to successful immunotherapy is to overcome the local immunosuppression within tumor microenvironment and activate mechanisms that lead to tumor eradication. There are two clinical approaches of immunotherapy: active and passive. Active immunotherapy involves stimulation of immune response to tumor associated antigens (TAAs), either non-specifically via immunomodulating agents or specifically employing cancer vaccines. This review presents the progress and breakthroughs in design, development and clinical application of selected cell-based tumor vaccines achieved due to the generation and development of gene transfer technologies.     

Characterizing the anti-tumor function of adoptively transferred NK cells in vivo

Natural killer (NK) cells represent a promising cell type to utilize for effective adoptive immunotherapy. However, little is known about the important cytolytic molecules and signaling pathways used by NK cells in the adoptive transfer setting. To address this issue, we developed a novel mouse model to investigate the trafficking and mechanism of action of these cells. We demonstrate that methylcholanthrene-induced RKIK sarcoma cells were susceptible to NK cell-mediated lysis in vitro and in vivo following adoptive transfer of NK cells in C57BL/6 RAG-2^−/−γc^−/− mice. Cytotoxic molecules perforin, granzymes B and M as well as the death ligand TRAIL and pro-inflammatory cytokine IFN-γ were found to be important in the anti-tumor effect mediated by adoptively transferred NK cells. Importantly, we demonstrate that adoptively transferred NK cells could traffic to the tumor site and persisted in vivo which correlated with the anti-tumor effect observed. Overall, the results of this study have important implications for enhancing NK cell-based immunotherapies.     

Adoptive transfer of gene-modified primary NK cells can specifically inhibit tumor progression in vivo

NK cells hold great potential for improving the immunotherapy of cancer. Nevertheless, tumor cells can effectively escape NK cell-mediated apoptosis through interaction of MHC molecules with NK cell inhibitory receptors. Thus, to harness NK cell effector function against tumors, we used Amaxa gene transfer technology to gene-modify primary mouse NK cells with a chimeric single-chain variable fragment (scFv) receptor specific for the human erbB2 tumor-associated Ag. The chimeric receptor was composed of the extracellular scFv anti-erbB2 Ab linked to the transmembrane and cytoplasmic CD28 and TCR-zeta signaling domains (scFv-CD28-zeta). In this study we demonstrated that mouse NK cells gene-modified with this chimera could specifically mediate enhanced killing of an erbB2(+) MHC class I(+) lymphoma in a perforin-dependent manner. Expression of the chimera did not interfere with NK cell-mediated cytotoxicity mediated by endogenous NK receptors. Furthermore, adoptive transfer of gene-modified NK cells significantly enhanced the survival of RAG mice bearing established i.p. RMA-erbB2(+) lymphoma. In summary, these data suggest that use of genetically modified NK cells could broaden the scope of cancer immunotherapy for patients.     

Rocaglamide enhances NK cell-mediated killing of non-small cell lung cancer cells by inhibiting autophagy

Targeting macroautophagy/autophagy is a novel strategy in cancer immunotherapy. In the present study, we showed that the natural product rocaglamide (RocA) enhanced natural killer (NK) cell-mediated lysis of non-small cell lung cancer (NSCLC) cells in vitro and tumor regression in vivo. Moreover, this effect was not related to the NK cell recognition of target cells or expressions of death receptors. Instead, RocA inhibited autophagy and restored the level of NK cell-derived GZMB (granzyme B) in NSCLC cells, therefore increasing their susceptibility to NK cell-mediated killing. In addition, we further identified that the target of RocA was ULK1 (unc-51 like autophagy activating kinase 1) that is required for autophagy initiation. Using firefly luciferase containing the 5´ untranslated region of ULK1, we found that RocA inhibited the protein translation of ULK1 in a sequence-specific manner. Taken together, RocA could block autophagic immune resistance to NK cell-mediated killing, and our data suggested that RocA was a promising therapeutic candidate in NK cell-based cancer immunotherapy.     

Adaptive T cell immunotherapy in cancer

Impaired tumor-specific effector T cells contribute to tumor progression and unfavorable clinical outcomes. As a compensatory T cell-dependent cancer immunoediting strategy, adoptive T cell therapy(ACT) has achieved encouraging therapeutic results,and this strategy is now on the center stage of cancer treatment and research. ACT involves the ex vivo stimulation and expansion of tumor-infiltrating lymphocytes(TILs) with inherent tumor reactivity or T cells that have been genetically modified to express the cognate chimeric antigen receptor or T cell receptor(CAR/TCR), followed by the passive transfer of these cells into a lymphodepleted host. Primed T cells must provide highly efficient and long-lasting immune defense against transformed cells during ACT. Anin-depth understanding of the basic mechanisms of these living drugs can help us improve upon current strategies and design better next-generation T cell-based immunotherapies. From this perspective, we provide an overview of current developments in different ACT strategies, with a focus on frontier clinical trials that offer a proof of principle. Meanwhile, insights into the determinants of ACT are discussed, which will lead to more rational, potent and widespread applications in the future.     

T cell-mediated graft-versus-leukemia reactions after allogeneic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation represents the only curative approach for many hematological malignancies. During the last years the impact of the conditioning regimen has been re-assessed. With the advent of reduced-intensity conditioning the paradigm has changed from cytoreduction executed by high-dose radio-chemotherapy to immunological surveillance of leukemia by donor cells. Distinct subsets of T cells and NK cells contribute to graft-versus-leukemia reactions. So far, cytotoxic T lymphocytes are the mainstay of allogeneic immunotherapy. Here, we summarise the current knowledge of T cell-mediated graft-versus-leukemia reactions and present results from pre-clinical and clinical studies of T cell-based adoptive immunotherapy. We address the issues of feasibility and specificity of adoptive immunotransfer from a clinical point of view and discuss the prerequisites for successful clinical applications. Finally, the prospects for immunological research that have evolved with the increasing use of reduced-intensity conditioning and allogeneic stem cell transplantation are highlighted.     

Rocaglamide promotes the infiltration and antitumor immunity of NK cells by activating cGAS-STING signaling in non-small cell lung cancer

Background: Natural killer (NK) cell-based immunotherapy is clinically limited due to insufficient tumor infiltration in solid tumors. We have previously found that the natural product rocaglamide (RocA) can enhance NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells by inhibiting autophagy, and autophagic inhibition has been shown to increase NK cell tumor infiltration in melanoma. Therefore, we hypothesized that RocA could increase NK cell infiltration in NSCLC by autophagy inhibition.Methods: Flow cytometry, RNA-sequencing, real-time PCR, Western blotting analysis, and xenograft tumor model were utilized to assess the infiltration of NK cells and the underlying mechanism.Results: RocA significantly increased the infiltration of NK cells and the expressions of CCL5 and CXCL10 in NSCLC cells, which could not be reversed by the inhibitions of autophagy/ULK1, JNK and NF-κB. However, such up-regulation could be suppressed by the inhibitions of TKB1 and STING. Furthermore, RocA dramatically activated the cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) signaling pathway, and the inhibition/depletion of STING ablated the up-regulation of CCL5 and CXCL10, NK cell infiltration, and tumor regression induced by RocA. Besides, RocA damaged mitochondrial DNA (mtDNA) and promoted the cytoplasmic release of mtDNA. The mPTP inhibitor cyclosporin A could reverse RocA-induced cytoplasmic release of mtDNA.Conclusions: RocA could promote NK cell infiltration by activating cGAS-STING signaling via targeting mtDNA, but not by inhibiting autophagy. Taken together, our current findings suggested that RocA was a potent cGAS-STING agonist and had a promising potential in cancer immunotherapy, especially in NK cell-based immunotherapy.     

Artificial antigen-presenting cells: artificial solutions for real diseases

Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat a variety of life-threatening diseases. Unfortunately, current approaches for generating sufficient numbers of antigen-specific T cells lack the ability to serve as reproducible and economically viable methods. This has spurred the development of both cell- and non-cell-based artificial antigen-presenting cells to alleviate problems associated with peptide-loaded dendritic cells in current approaches to adoptive immunotherapy. Here, we review new strategies for the ex vivo generation of antigen-specific T cells and their clinical application. These new approaches have the potential to spearhead a new era of successful adoptive immunotherapy for cancer and infectious diseases.     

Challenges and prospects of immunotherapy as cancer treatment

The concept of cancer immunotherapy stems from the proposed function of the immune system, called immunosurveillance, to protect against growing tumors. Due to genetic aberrations, tumor cells display an altered repertoire of MHC-associated peptides that can lead to the activation of immune cells able to eliminate the transformed cells. In some instances, under the pressure of the immune system, both the tumor and its microenvironment are shaped and immune-resistant tumor variants are selected initiating the process of cancer immunoediting. This can impair not only host-generated immunosurveillance, but also attempts to harness the immune response for therapeutic purposes, namely immunotherapy. Rather than being an exhaustive review of the different approaches of cancer immunotherapy, the focus of this review is to provide the reader with future challenges of the field by proposing 'second generation' immunotherapy approaches that take into account immunosubversive mechanisms adopted by tumor cells. After an introduction on the process of immunosurveillance and immunoescape we will analyze why current immunotherapy approaches have not fulfilled their promise and will finish by summarizing what are the challenges for future approaches.     

Immunotherapy of cancer: promise and reality

The notion that the immune system regulates cancer development is now well established. An overwhelming amount of data from animal models, together with compelling data from human patients, indicate that the immune system is instrumental in scanning and irradicating tumors. Analysis of individuals with congenital or acquired immunodeficiencies or patients undergoing immunosuppressive therapy has documented a highly elevated incidence of virally induced malignancies and cancers compared with immunocompetent individuals [1-3]. During the last decade, thanks to the breakthoughts in understanding the molecular mechanisms responsible for immune activation, the tumor antigen identification, the dendritic cell biology, the immunogenecity of tumors, the immune escape mechanisms, the host-tumor relationship, we are facing a new area of tumor immunotherapy. The basic advances were translated in therapeutical applications and have changed the view of immunotherapy from "a dream scenario" to a clinical fourth modality to cancer treatments. Multiple cancer trials using active immunization with vaccines or adoptive immunotherapy have been conducted with only very limited success. There are still a number of issues that still need to be resolved including a better understanding of immune escape mechanisms. Cancer vaccines continue to be evaluated and may lead to the emergence of clinically useful new treatments. A comprehensive approach to define the intricate molecular program initiated by tumor cells to resist to escape and the immune system of the host may help in breaking down the barriers to a more adapted cancer immunotherapy.     

A malignus melanoma immunterápiájának lehetoségei

Despite their well-documented immunogenicity, malignant melanomas belong to the most aggressive tumor types. A potential explanation for this is the suboptimal activation of tumor infiltrating T cells. In order to boost immune responses against tumors, a variety of treatment modalities have been tested in animal models and in clinical setting. Antigen-nonspecific approaches (e.g., IFN-alpha and IL-2), as well as active specific immunotherapeutical modalities based on the use of autologous or allogeneic tumor cell-save been investigated in clinical trials of melanoma. The identification of melanoma-associated antigens has opened new avenues in antigen-specific immunotherapy. A promising alternative for the delivery of different forms of melanoma antigens is the application of dendritic cells, the most potent antigen presenting cells capable of eliciting efficient T-cell response. Beside active immunotherapy, immune response against melanoma antigens could be increased through the adoptive transfer of tumor infiltrating lymphocytes or antigen specific T-cell clones. The most important conclusion that can be drawn from the results of published immunotherapy studies is that these modalities are able to induce durable complete tumor regressions,mostly with reasonable toxicity; however, generally only in a minority of patients. This points to the importance of appropriate patient selection, with regard to the expression of the targeted antigens and HLA molecules, as well as to the general immunocompetence of the patients. A crucial and still unsolved question is monitoring immune activation during treatment, although there are promising new tools that could prove useful in this respect. The presence of tumor-reactive CTL in the circulation or in the tumors does not guarantee an efficient immune response. It is important to assess if these T cells are in an activated and functional state. Finally, in several single target antigen-based clinical studies a therapy-induced immunoselection of antigen-negative clones, leading to disease progression, was observed. This could be overcome with the use of antigen cocktails or whole tumor approaches. A better understanding of the mechanisms of action of immunotherapeutical modalities may enhance the success rate of these strategies.     

Dendritic cell recovery post-lymphodepletion: a potential mechanism for anti-cancer adoptive T cell therapy and vaccination

Adoptive transfer of autologous tumor-reactive T cells holds promise as a cancer immunotherapy. In this approach, T cells are harvested from a tumor-bearing host, expanded in vitro and infused back to the same host. Conditioning of the recipient host with a lymphodepletion regimen of chemotherapy or radiotherapy before adoptive T cell transfer has been shown to substantially improve survival and anti-tumor responses of the transferred cells. These effects are further enhanced when the adoptive T cell transfer is followed by vaccination with tumor antigens in combination with a potent immune adjuvant. Although significant progress has been made toward an understanding of the reasons underlying the beneficial effects of lymphodepletion to T cell adoptive therapy, the precise mechanisms remain poorly understood. Recent studies, including ours, would indicate a more central role for antigen presenting cells, in particular dendritic cells. Unraveling the exact role of these important cells in mediation of the beneficial effects of lymphodepletion could provide novel pathways toward the rational design of more effective anti-cancer immunotherapy. This article focuses on how the frequency, phenotype, and functions of dendritic cells are altered during the lymphopenic and recovery phases post-induction of lymphodepletion, and how they affect the anti-tumor responses of adoptively transferred T cells.     

Neuroendocrine regulation and tumor immunity

The morphogenetic events leading to the transendothelial passage of lymphoid and tumoral cells are analyzed in light of a very recent and global theory of intercellular communication designated as the Triune Information Network (TIN). The TIN system is based on the assumption that cell-cell interactions primarily occur through cell surface informations or topobiological procesess, whose mechanisms rely upon expression of adhesion molecules, and are regulated by an array of locally-borne (autocrine/paracrine signals and autonomic inputs) and distantly-borne (endocrine secretions) messages. The final aim of the TIN is to control homeostatic functions crucial for the organism survival, like morphogenesis. Knowledge of the TIN signals involved in lymphoid and tumoral cell intravasation might offer a new perspetive to study the mechanisms of tumor immunity. Recognition of tumor target cells by immune cytotoxic effectors, in fact, can be considered a notable case of TIN-mediated cell to cell interaction. In particular, Natural Killer (NK) cells play a role in the cell-mediated control of tumor growth and metastatic spreading. Cell targeting and killing are dependent on the different NK cell receptors and on the efficacy of NK cells after cytokine and monoclonal antibody administration in cancer therapy. Since efficacy of NK cell-based immunotheraphy has been proven in KIR-mismatch regimens or in TRAIL-dependent apoptosis, the ability to manipulate the balance of activating and inhibitory receptors on NK cells and of their cognate ligands as well as the sensitivity of tumor cells to apoptosis, opens new perspectives for NK cell based immunotherapy.     

Characterization of human natural killer cells for therapeutic use

As a part of the innate immune system, natural killer (NK) cells are cytotoxic lymphocytes that can exert cytotoxic activity against infected or transformed cells. Furthermore, due to their expression of a functional Fc receptor, they have also been eluded as a major effector fraction in antibody-dependent cellular cytotoxicity. These characteristics have led to multiple efforts to use them for adoptive immunotherapy against various malignancies.  There are now at least 70 clinical trials testing the safety and efficacy of NK cell products around the world in early-phase clinical trials. NK cells are also being tested in the context of tumor retargeting via chimeric antigen receptors, other genetic modification strategies, as well as tumor-specific activation strategies such as bispecific engagers with or without cytokine stimulations. One advantage of the use of NK cells for adoptive immunotherapy is their potential to overcome HLA barriers. This has led to a plethora of sources, such as cord blood hematopoietic stem cells and induced pluripotent stem cells, which can generate comparatively high cytotoxic NK cells to peripheral blood counterparts. However, the variety of the sources has led to a heterogeneity in the characterization of the final infusion product. Therefore, in this review, we will discuss a comparative assessment strategy, from characterization of NK cells at collection to final product release by various phenotypic and functional assays, in an effort to predict potency of the cellular product.     

mRNA-transfected dendritic cells: a promising strategy in immunotherapy

Dendritic cells play a central role in the initiation of the immune response as they are the only antigen-presenting cells able to prime naive T cells. This makes the dendritic cells the vector of choice to use as a cell-based vaccine in immunotherapy. Although there are several strategies to deliver antigen to dendritic cells, the ones transfected with mRNA coding for tumor or viral antigens are able to induce potent antigen specific T-cell responses directed against multiple epitopes. In this review, we report several advances made in the field of anti-tumoral and anti-HIV immunotherapy using mRNA-transfected dendritic cells-based approaches.     

Cytokine-driven regulation of NK cell functions in tumor immunity: role of the MICA-NKG2D system

Natural killer (NK) cells are critical players during tumor growth control in immunocompetent hosts. These cells also establish a cross-talk with dendritic cells (DCs) and promote a Th1-mediated immunity. NKG2D is a pivotal receptor that directs the tumoricidal activity of NK cells through the recognition of a group of ligands such as MICA widely expressed on different tumors. Here we will review the most important tumor immune escape mechanisms that compromise the functionality of NKG2D and its cognate ligands, including TGF-beta secretion, tumor shedding of soluble MICA, and additional mechanisms that compromise the tumoricidal activity of NKG2D-expressing cells. Such mechanisms may also dampen the cross-talk between NK cells and DCs during the anti-tumor immune responses. Recent knowledge may lead to innovative approaches to promote efficient NK cell-mediated anti-tumor immune responses.