1 INTRODUCTION

For decades, cancer treatments have been mainly centered around surgery, radiation therapy, and chemotherapy. However, over the past 5 years, cellular immunotherapy has increasingly become the fourth pillar of cancer therapy that shows encouraging curative potential in certain types of hematological neoplasms, such as B-cell lymphoma, leukemia, and multiple myeloma.¹ Cellular immunotherapy, as most prominently exemplified by the chimeric antigen receptor T (CAR-T) cell therapy, harnesses the body's immune system to specifically engage and eliminate cancer cells.^1-4 The goal of cancer immunotherapies is to enhance the patient's immune response to detect and clear cancerous cells, especially cells with accumulated mutations that allow them to evade the host immune system. To achieve this, immune cells are engineered to express specific receptors, such as chimeric antigen receptors (CARs). A typical CAR consists of a signal peptide for directing the correct position and topology of the engineered receptor, an extracellular antigen-specific single-chain variable fragment (scFv), a hinge region to provide stability and flexibility to access the target antigen, a transmembrane domain, as well as a 4-1BB costimulatory domain and a CD3ζ signaling domain to mediate T-cell activation, expansion, and persistence. Usually, this antigen is specific to cancer tissues or overexpressed in tumor cells when compared to normal tissues. For instance, among the six FDA-approved CAR-T cell therapies, four products (Kymriah, Yescarta, Tecartus, and Breyanzi) target the CD19 antigen overexpressed in leukemia or lymphoma cells to treat acute lymphoblastic leukemia (ALL) and relapsed or refractory diffuse B-cell lymphoma (DLBCL), whereas the remaining two products (Abecma and Carvykti) target the B-cell major antigen (BCMA) to treat multiple myeloma.^{1, 2, 5-10}

The introduction of a synthetic scFV into CAR enables engineered immune cells to recognize tumor cells in an MHC-unrestricted manner. Upon engagement with tumor antigen, CAR-T cells are activated to perform effector functions by activating several downstream signaling pathways, including the Fas/FasL axis, the perforin-granzyme axis, and cytokine release to sensitize the tumor microenvironment (TME). Recent CAR constructs consist of targeting domains and/or costimulatory domains that have been shown to improve cytotoxic function and induce rapid proliferation.¹ CAR-T cells have shown remarkable efficacy in patients with relapsed or refractory hematological malignancies.^1-4 However, their wide application is limited by several non-negligible side effects and/or inherent drawbacks, including graft-versus-host-disease (GvHD), cytokine release syndrome (CRS), potential “on-target off-tumor” cytotoxicity, and failure of efficient infiltration into solid tumors due to the immunosuppressive TME.^{11, 12} CAR-expressing NK (designated “CAR-NK”) cells, nevertheless, are capable of overcoming some of these roadblocks because of their advantages over CAR-T cells in killing malignant cells with fewer adverse side effects after transplantation.^{13, 14} Therefore, they can be exploited as an alternative therapeutic option.^{15, 16} Over the past 10 years, we have witnessed a steady increase of studies centered on CAR-NK cells (Figure 1 and Table 1).

NK cells are innate lymphocytes that possess substantial cytolytic activity against virus-infected cells and tumor cells.¹⁷ Human NK cells are characterized as CD3⁻CD56⁺ cells, and constitute 5%–15% of peripheral blood mononuclear cells (PBMCs).¹⁸ According to the different expression levels of CD56, NK cells can be further divided into two subtypes, CD56^dim and CD56^high NK cells.¹⁹ Unlike T cells and B cells, NK cells do not require prior antigens exposure and the engagement of antibodies to mediate antitumor immunity, making them ideal targets for cellular engineering to develop antitumor immunotherapies.²⁰ The effector responses and self-tolerance of NK cells mainly depend on the integration of a variety of activating receptors and inhibitory receptors.²¹ Major NK cell activation receptors include natural cytotoxicity receptors (NCRs, such as NKp30, NKp40, NKp44, and NKp46), NKG2D, NKG2C, and the Fc receptor CD16.²² In parallel, the inhibitory receptors provide self-tolerance and negative feedback signals. Inhibitory killer Ig-like receptors (KIRs) recognize class I human leukocyte antigen molecules (HLA-A, HLA-B, and HLA-C), whereas NKG2A can bind to HLA-E.^{23, 24} The recognition of tumor cells by NK cells is based on their unique ability to target and attack cells with low expression levels of MHC class I molecules on the cell surface. The natural antitumor effects of NK cells enable NK cell-based immunotherapeutic strategies. Not surprisingly, CAR-NK cell therapies have recently moved into the spotlight as an alternative cellular immunotherapeutic strategy and can be engineered to target diverse antigens. Unlike CAR-T cell therapy, CAR-NK cells can be derived from allogeneic donors and make it possible to produce “off-the-shelf” living therapeutics. Compared to CAR-T cells, which are mostly effective toward hematological malignancies, CAR-NK cells have shown equally encouraging therapeutic efficacy in solid tumors with enhanced infiltration into the TME.^{15, 25} Furthermore, CAR-NK cells can evade the resistant TME and achieve potent and persistent antitumor response. Given that engineered cells also possess the natural cytotoxic properties intrinsic to NK cells without the need for CARs (Figure 2), CAR-NK cells can mediate cytotoxic activity in both CAR-independent and CAR-dependent manners to maximize the tumor killing capability.²⁵

Compared to engineered T-cells, adoptive NK cell-based therapy has several notable advantages. First, NK cells possess pleiotropic cytotoxicity effects, including the release of cytotoxic granules containing perforin and granzymes, secretion of cytokines, and active participation in antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by CD16 receptor.^{13, 26, 27} For example, activated NK cells can destroy cancer cells by direct cell cytotoxicity via the release of perforin and granzymes and/or by generating proinflammatory cytokines that further attract macrophages and dendritic cells into tumor sites.^27-29 Additionally, CAR-NK cells can be engineered with different costimulatory domains (such as 2B4) to enhance costimulatory specificity with regard to NK cell signaling, thereby leading to more rapid proliferation and higher antitumor efficacy.³⁰ Second, NK cells use similar killing mechanisms as CD8⁺ cytotoxic T-lymphocytes (CTLs) to target and destroy malignant or virus-infected cells yet possess different target recognition machinery. CTLs are activated by T-cell receptors (TCRs) as part of the adaptive immune response, whereas NK cells receive activating and inhibitory signals by their germline-encoded receptor repertoire.¹⁴ Because NK cells do not require human leukocyte antigen (HLA) compatibility or antigen recognition to mount an immune response, CAR-NK cells possess a reduced risk of GvHD from the allogeneic transfer of CAR-NK cells from donors.³¹ Third, CAR-NK cells possess a better safety profile, with CRS and neurotoxicity rarely observed in allogeneic CAR-NK cells in both preclinical studies and clinical trials. Activated NK cells typically secrete IFN-γ and GM-CSF,³² while activated CAR-T cells tend to elicit the production of copious amounts of IL-1α, IL-2, IL-6, TNF-α, IL-8, and IL-10, some of which are highly correlated with CRS.³³ These desirable features of CAR-NK cells could substantially mitigate side effects, reduce healthcare costs, and improve cancer patient outcomes.

2 CAR-NK CELL GENERATION

Autologous CAR-NK cell infusion is the safest approach for adoptive cell therapy because it relies on the patient's own blood as the cell source and obviates the need for immunosuppressive pretreatment of the patient. However, the reduced risk of alloreactivity inherent to NK cells allows CAR-NK cells to be generated from multiple allogeneic sources (Figure 3), including PBMCs, hematopoietic progenitor cells (HPCs), umbilical cord blood (UCB), NK-92 cell lines, and induced pluripotent stem cells (iPSCs).^{25, 34} This flexibility allows CAR-NK cells to be generated without the personalized, patient-specific production that CAR-T cell therapy often requires. Therefore, CAR-NK cells hold great promise to be manufactured as “off-the-shelf” products for on-demand and generic use in cancer patients with substantially reduced costs.

The gene encoding CAR is often introduced into peripheral blood-isolated (PB-NK) or stem cell-induced NK cells or NK-like cell lines via viral transduction using lentivirus or retrovirus, mRNA electroporation, or Sleeping Beauty transposon. CAR-expressing NK cells are then expanded in NK cell-specific expansion media with expensive cytokines for GMP-grade clinical application until adequate cell numbers are attained. PB-NK cells can be most easily obtained but have relatively low transduction efficiency and limited expansion capacity.³⁵ Stem cell-derived products offer a rich source of CAR-NK cells, and enable the generation of standardized generic therapies for any patient irrespective of the HLA haplotype. For instance, UCB stem cell-derived NK cells used in the first clinical trials of CAR-NK cell therapy have great proliferative competence. They can be expanded by 2000-fold ex vivo with over 90% purity for the functional NK cell population.^{36, 37} However, UCB-NK cells might suffer from condensed cytotoxicity due to their immature nature, and iPSC-derived NK cells are claimed to exhibit stronger anticancer activity than UCB-NK and remain as effective as PB-NK cells in several mouse models of cancer.^{38, 39}

Clonal cell lines with NK cell-like properties, including NK-92, YT, and KHYG-1, could serve as alternative sources for CAR-NK cells, with NK-92 most extensively used in both preclinical and clinical applications. The NK92 cells are easier for genetic engineering and ex vivo expansion with a doubling time of approximately 24−36 h. However, because this cell line is derived from a 50-year-old white male diagnosed with rapidly progressive non-Hodgkin's lymphoma, NK92 cells have to be lethally irradiated before infusion back to the patient, which tends to limit the in vivo persistence of the engineered cells and impair durable clinical efficacy.^{40, 41} This obstacle has been partially tackled via the expression of a high-affinity CD16 variant (haNK) to boost the effector function.⁴² The clinical efficacy of this novel approach, along with the combined use of cancer vaccines or other agents, is yet to be revealed by ongoing clinical trials (NCT03387085, NCT03853317, NCT03387111, and NCT03586869).

In general, CAR-NK cells have safer profiles than clinically approved CAR-T cells, and elicit minimal CRS with decreased or negligible neurotoxicity. The decrease in neurotoxicity and CRS can be explained by the different cytokine production patterns between NK and T cells. CAR-NK cells mainly secrete IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas CAR-T cells predominantly secrete proinflammatory interleukins (e.g., IL-6) and TNF-α, which are highly associated with CRS and neurotoxicity.^{13, 34} Additionally, because of the limited lifespan of CAR-NK cells circulating within the body, CAR-NK cells have a reduced risk of on-target/off-target toxicity to normal tissues. The addition of tumor antigen-specific adapter molecules (AMs) and CAR immune cells that are specific for these AMs leads to a more targeted, precise, and temporally restricted immune response.⁴³ As a result, multiple antigens can be targeted simultaneously by using CAR-NK cells. Collectively, advances in gene manipulation and bioengineering techniques have resulted in CAR-NK cell therapy with strong antitumor activity and mostly nontoxic effects on normal tissues after infusion into patients.¹⁶

3 THERAPEUTIC EXPLORATION WITH CAR-NK CELL THERAPY

Following the huge clinical successes of CAR-T therapies, CAR-NK cell-based therapies have been increasingly tested and optimized in various mouse models of tumors, as well as in clinical trials (Table 1). However, due to the limited progress of clinical trials, biosafety and efficacy of CAR engineered NK cell therapies in human are yet to be rigorously examined in the clinical setting. We present herein some representative preclinical achievements by using CAR-NK cells derived from different cell sources.

4 OVERCOMING ROADBLOCKS IN CAR-NK CELL THERAPY

Despite all the aforementioned encouraging advances, several roadblocks have to be addressed to accelerate the clinical translation of NK cell-based therapy. Recent efforts have been predominantly directed to reprogram and modify NK cells to maximize their expansion and persistence, promote their trafficking or infiltration, and combat the immunosuppressive TME.

5 CONCLUSION

Compared to T cells, NK cells are important innate immune cells of the body, which can kill tumor cells without pre-sensitization. The accelerated research progress of NK cell-based therapy shows a promising efficacy on solid tumors. CAR-NK is expected to be one of the most anticipated engineered cell therapies following the recent success of CAR-T cell therapy. In addition to directly eradicating tumors expressing the target antigen, CAR-NK cells retain their natural cytotoxicity toward tumor cells with the loss of surface antigens, making it possible to overcome tumor antigen escape that is often encountered during CAR-T cell therapy. Because NK cells express CD16 (also known as FcγRIIIa) on their surface, they can interact with the Fc fragment of antibodies to trigger ADCC. CAR-NK cells can thus be further combined with existing mAb-based therapies to prevent tumor escape and boost the tumor-killing efficacy. These properties indicate that a wider range of target antigens for CAR-NK cells can be exploited in parallel. Creative immune-engineering and powerful genome editing techniques are likely to overcome many hurdles facing the expansion, persistence, trafficking, infiltration, and survival of CAR-NK cells. In aggregate, CAR-NK, as a relatively new addition to cellular immunotherapy, holds great promise to become a first-line strategy in cancer treatment and, therefore, has high translational values in the clinic to directly benefit cancer patients.

AUTHOR CONTRIBUTIONS

Yubin Zhou conceived the idea and supervised the work. Yubin Zhou, Siyao Liu, Kaycee Nguyen, and Dongyong Park drafted the manuscript with inputs and further editing from all other authors.

CONFLICT OF INTEREST

Professor Yubin Zhou is a member of Chronic Diseases and Translational Medicine editorial board and is not involved in the peer review process of this article. The remaining authors declare no conflict of interest.

ETHICS STATEMENT

None.