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.

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NK cell-based immunotherapy for malignant diseases

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.

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Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

Immunotherapy is a rapidly expanding field of oncology aimed at targeting, not the tumor itself, but the immune system combating the cancerous lesion Of the many approaches currently under study to boost anti-tumor immune responses; modulation of immune co-receptors on lymphocytes in the tumor microenvironment has thus far proven to be the most effective Antibody blockade of the T cell co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) has become the first FDA approved immune checkpoint blockade; however, tumor infiltrating lymphocytes express a diverse array of additional stimulatory and inhibitory co-receptors, which can be targeted to boost tumor immunity Among these, the co-stimulatory receptor 4-1BB (CD137/TNFSF9) possesses an unequaled capacity for both activation and pro-inflammatory polarization of anti-tumor lymphocytes While functional studies of 4-1BB have focused on its prominent role in augmenting cytotoxic CD8 T cells, 4-1BB can also modulate the activity of CD4 T cells, B cells, natural killer cells, monocytes, macrophages, and dendritic cells 4-1BB's expression on both T cells and antigen presenting cells, coupled with its capacity to promote survival, expansion, and enhanced effector function of activated T cells, has made it an alluring target for tumor immunotherapy In contrast to immune checkpoint blocking antibodies, 4-1BB agonists can both potentiate anti-tumor and anti-viral immunity, while at the same time ameliorating autoimmune disease Despite this, 4-1BB agonists can trigger high grade liver inflammation which has slowed their clinical development In this review, we discuss how the underlying immunobiology of 4-1BB activation suggests the potential for therapeutically synergistic combination strategies in which immune adverse events can be minimized

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4-1BB Agonists: Multi-Potent Potentiators of Tumor Immunity.

Improving the therapeutic efficacy of T cells expressing a chimeric antigen receptor (CAR) represents an important goal in efforts to control B-cell malignancies. Recently an intrinsic strategy has been developed to modify the CAR itself to improve T-cell signaling. Here we report a second extrinsic approach based on altering the culture milieu to numerically expand CAR(+) T cells with a desired phenotype, for the addition of interleukin (IL)-21 to tissue culture improves CAR-dependent T-cell effector functions. We used electrotransfer of Sleeping Beauty system to introduce a CAR transposon and selectively propagate CAR(+) T cells on CD19(+) artificial antigen-presenting cells (aAPC). When IL-21 was present, there was preferential numeric expansion of CD19-specific T cells which lysed and produced IFN-γ in response to CD19. Populations of these numerically expanded CAR(+) T cells displayed an early memory surface phenotype characterized as CD62L(+)CD28(+) and a transcriptional profile of naive T cells. In contrast, T cells propagated with only exogenous IL-2 tended to result in an overgrowth of CD19-specific CD4(+) T cells. Furthermore, adoptive transfer of CAR(+) T cells cultured with IL-21 exhibited improved control of CD19(+) B-cell malignancy in mice. To provide coordinated signaling to propagate CAR(+) T cells, we developed a novel mutein of IL-21 bound to the cell surface of aAPC that replaced the need for soluble IL-21. Our findings show that IL-21 can provide an extrinsic reprogramming signal to generate desired CAR(+) T cells for effective immunotherapy.

https://cancerres.aacrjournals.org/content/canres/71/10/3516.full.pdf

Reprogramming CD19-Specific T Cells with IL-21 Signaling Can Improve Adoptive Immunotherapy of B-Lineage Malignancies

Natural killer (NK) cells are effective in combating infections and tumors, and as such are tempting for adoptive transfer therapy. However, they are not homogeneous but can be divided into three main subsets, including cytotoxic, tolerant and regulatory NK cells, with disparate phenotypes and functions in diverse tissues. The development and functions of such NK cells are controlled by various cytokines, such as fms-like tyrosine kinase 3 ligand (FL), kit ligand (KL), interleukin (IL)-3, IL-10, IL-12, IL-18, transforming growth factor (TGF)-β and common-γ chain family cytokines, which operate at different stages by regulating distinct signaling pathways. Nevertheless, the specific roles of each cytokine that regulates NK cell development or that shapes different NK cell functions remain unclear. In this review, we attempt to describe the characteristics of each cytokine and the existing protocols to expand NK cells using different combinations of cytokines and feeder cells. A comprehensive understanding of the role of cytokines in NK cell development and function will aid the generation of better efficacy for adoptive NK cell treatment.

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Developmental and Functional Control of Natural Killer Cells by Cytokines.

A large number of natural killer (NK) cells with high function are expected to generate especially in tumor adoptive immunotherapy. Here K562 cells were genetically modified to co-express major histocompatibility complex class I chain-related protein A (MICA), 4-1BB ligand, and IL-15, called K562-MICA-4-1BBL-IL-15. The modified K562 cells not only promoted activation, proliferation, and survival of NK cells, but also enhanced NK cell cytotoxicity. In long-term culture tests, K562-MICA-4-1BBL-IL-15 cells stimulated NK cell to expand mean 550 folds in 24-day culture and to cover from 14.8% of total peripheral blood monoclonal lymphocytes on day 1 to 86.7% on day 24. Prevalent NK cells after expansion enhanced the ability of killing targets and producing interferon gamma (IFN-γ), and kept high expression of activating receptors. The results indicated that K562-MICA-4-1BBL-IL-15 cells would be developed for expansion of NK cells ex vivo and may have important implications for clinical immunotherapy.

Ex vivo expansion of natural killer cells with high cytotoxicity by K562 cells modified to co-express major histocompatibility complex class I chain-related protein A, 4-1BB ligand, and interleukin-15.

Construction of a plasmid for co-expression of mouse membrane-bound form of IL-15 and RAE-1ε and its biological activity.

CD8(+) T cells play critical roles in immunosurveillance by killing malignant or virally infected cells. Interleukin 15 (IL-15) is a critical cytokine for promoting proliferation and the effector capacity of CD8(+) T cells, and has been used to support the growth of CD8(+) T cells in cellular therapies of neoplastic diseases. Recent studies have shown that IL-15, in synergy with other cytokines, such as IL-6, enhances the T-cell receptor (TCR)-independent proliferation and function of CD8(+) T cells. The aim of the present study was to investigate the role of BaF3-mb15-RAE cells in stimulating mouse CD8(+) T cells. BaF3 cells were cultured and B16F10 cells were grown in DMEM. MTT assay was used to detect the proliferation of CD8(+) T cells. Cells were analyzed using flow cytometry. The results showed that IL-15 synergistically acts with another T-cell stimulatory molecule, RAE1ɛ, to potently promote the proliferation of CD8(+) T cells, induce CD8(+) T-cell activation and enhance granzyme B and interferon-γ (IFN-γ) production in the absence of signaling via the TCR. Moreover, IL-15 in combination with RAE1ɛ resulted in a cooperative effect on CD8(+) T-cell-mediated cytotoxicity against B16F10 tumor cells. Thus, results of the present study showed that IL-15, in synergy with RAE1ɛ, enhances the TCR-independent effector function of CD8(+) T cells in vitro, which may be useful in the cellular immunotherapy of cancer.

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Xin-Yuan Pan

Papers

Ex vivo expansion of natural killer cells with high cytotoxicity by K562 cells modified to co-express major histocompatibility complex class I chain-related protein A, 4-1BB ligand, and interleukin-15.

Construction of a plasmid for co-expression of mouse membrane-bound form of IL-15 and RAE-1ε and its biological activity.

IL-15, in synergy with RAE-1ɛ, stimulates TCR-independent proliferation and activation of CD8(+) T cells.