June 26, 2022

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NK cells: NK cells in tumor immunity

NK cells: NK cells in tumor immunity

NK cells: NK cells in tumor immunity.   NK cell therapy is a promising field of clinical research, with good safety and preliminary efficacy for some cancer patients.


Human natural killer cells (NK) account for 15% of all circulating lymphocytes. NK cells were discovered in the 1970s and are mainly related to killing infected microorganisms and malignantly transformed allogeneic and autologous cells. NK cells exhibit anti-tumor cytotoxicity without prior sensitization and production of cytokines and chemokines that regulate various immune responses.

The field of tumor immunotherapy based on NK cells has reached an exciting juncture. Although these therapies have not achieved the same level of clinical success as adoptive T cell therapy, the early encouraging results have made NK cell therapy more and more enthusiastic in developing its potential. Let’s review the NK cells in tumor immunotherapy together.


NK cell biology

NK cells are the first subtype of innate lymphocytes (ILC) to be identified, which can produce a variety of effector functions on virus-infected and/or transformed cells, mainly cell killing and production of pro-inflammatory cytokines. NK cells and other ILC family members (type 1 ILC (ILC1s), ILC2s and ILC3s) are derived from the same lymphoid progenitor cells as B cells and T cells. The cytotoxic activity of NK cells makes them the most functionally similar to CD8+ T cells, and the cytokine production patterns of the ILC1, ILC2, and ILC3 populations correspond to the TH1, TH2, and TH17 subpopulations of CD4+ T cells, respectively.

The two most typical subgroups of NK cells are the CD56brightCD16- and CD56dimCD16+ populations. The number of CD56bright cells in peripheral blood is relatively small (90% of circulating NK cells are CD56dim), while the NK cells in tissues are mainly CD56bright. CD56bright NK cells are powerful cytokine producers, and unless stimulated by pro-inflammatory cytokines such as IL-15, their cytotoxicity is weak. In contrast, CD56dim NK cell population can mediate the continuous killing of infected and malignant cells, mainly through exocytosis of pre-assembled cytolytic particles containing granzyme B and perforin in the immune synapse, and finally induce target cells Apoptosis.

NK cells: NK cells in tumor immunity


Activating and inhibitory receptors of NK cells

Unlike B cells and T cells, NK cells do not express somatically rearranged antigen receptors, but a random combination of activated receptors and inhibitory receptors. The balance of stimulation signals and inhibitory signals through these different receptors produces a response or tolerance to target cells. MHC-I (major histocompatibility complex class I) antigen-specific inhibitory receptors can closely regulate NK cell-mediated cytotoxicity and lymphokine production.

The inhibitory signal of MHC-I specific receptor is essential for hematopoietic cells to avoid the destruction of NK cells. This concept is called “lost self” and was originally proposed by Ljunggren and Karre. This MHC-I recognition inhibitory receptor forms three families of NK cell surface receptors, namely KIRs (killer cell immunoglobulin-like receptors), LIRs (leukocyte immunoglobulin-like receptors) and NKG2A (natural killer) Cell Group 2 A).

KIRs are members of the immunoglobulin superfamily and are type I transmembrane molecules that recognize classic human leukocyte antigens A, B, and C (HLA-Ia). LIRs, also known as ILTs (immunoglobulin-like transcripts), form a second set of receptors, in addition to HLA Ia, they mainly recognize non-classical HLA-G (Ib) molecules. LIRs and KIRs belong to the same Ig superfamily. NKG2A is a member of the NKG2 family, including A, B, C, D, E, F, and H, and dimerizes with CD94 to form the NKG2A/CD94 receptor. It belongs to the C-type lectin family of receptors and recognizes non-classical HLA-EⅠ molecules as its ligands.

The killing effect of NK cells requires not only the detection of MHC-I molecules on transformed cells through inhibitory receptors, but also the activation of NK cells through activating receptors. Natural cytotoxicity receptors (NCR) are a group of activating receptors on the surface of natural killer cells, including NKp46, NKp30 and NKp44. These receptors, as well as NKG2D and DNAM-1 (DNAX helper molecule-1) recognize ligands expressed on the surface of virally infected or malignantly transformed cells.

Some co-receptors (2B4, NKp80, NTB-A and CD59) are also expressed, and they can only play a role in combination with other activating receptors. CD16 (or FcγRIII) is also an activating receptor, which is mainly expressed by CD56dim NK cell subsets, which is essential for the antibody-dependent cytotoxicity (ADCC) of IgG-coated target cells.



Anti-tumor effect mechanism of NK cells

NK cells have multiple functions and can limit the growth and spread of cancer cells. Under the guidance of TME innate and adaptive immune cells to produce pro-inflammatory chemokines, circulating NK cells can be recruited to the site of tumor occurrence. CXCR3–CXCR4, CX3CR1, and CCR3–CCR5 are the main chemotactic receptors differentially expressed by NK cell subsets, which respond to the chemokine gradient generated by the cancer immune response.

After entering TME, NK cells can kill cancer cells through a “lost self” mechanism. As mentioned earlier, the activation of NK cells is inhibited by inhibiting the binding of receptors to class I HLA (MHC I) molecules. However, many cancer cells down-regulate the expression of MHC I molecules to evade the detection of cytotoxic CD8+ T cells; therefore, due to the lack of MHC I-induced signal transduction by inhibiting receptors and the subsequent increase in activation signals, NK cells can recognize And cells that respond to this missing self-phenotype eventually lead to the lysis of the target cell. Therefore, NK cells have therapeutic potential when T cells cannot recognize cancer cells caused by down-regulation of MHC I.

NK cells: NK cells in tumor immunity

ADCC is another key mechanism for NK cell-mediated killing of cancer cells. This activity can be exploited by therapeutic monoclonal antibodies against tumor-associated antigens. In addition to directly inducing cytotoxicity, NK cells also respond to transformed cells by producing pro-inflammatory cytokines (including IFNγ and TNF). In addition to enhancing the cytotoxic CD8+ T cell response, these pleiotropic proteins have powerful anti-proliferation, anti-angiogenesis and pro-apoptotic effects on cancer cells.


Immunosuppression of NK cells in TME

Although NK cells are active in controlling tumor growth, they are sensitive to multiple immunosuppressive mechanisms, which are active in TME. Many soluble immunosuppressive molecules related to cancer have a negative impact on NK cell function, including IL-10, indoleamine 2,3-dioxygenase, prostaglandin E2 and macrophage migration inhibitory factor. Transforming growth factor β (TGF-β) is currently one of the most studied molecules with immunosuppressive effects on NK cells.

TGFβ can be produced by a variety of immune cell subpopulations in TME, including regulatory T (Treg) cells, myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAM), as well as their own cancer cells. This pleiotropic cytokine is known to down-regulate many aspects of NK cell function, including cytokine secretion, degranulation, metabolism, and mTOR signaling. Therefore, antagonizing transforming growth factor β is a potential strategy to improve the effect of immunotherapy. In fact, the dual-function “trap” for both transforming growth factor beta and immune checkpoint protein is being tested in several clinical trials (for example, NCT03631706).

Hypoxia is another important obstacle to the killing activity of NK cells in TME. Large solid tumors are not well vascularized, and therefore, usually contain a large amount of low oxygen concentration areas, in which the activity of NK cells is substantially (but not completely) disappeared. This situation leads to down-regulation of activated receptors and death receptors, granzyme B is degraded by autophagy, and cytokines secreted by NK cells are reduced. Various treatments aimed at overcoming hypoxia in TME, including hypoxia-induced prodrugs and HIF-1 targeted therapy, are under clinical development (for example, NCT01746979). In addition, the influencing factors that inhibit the activity of NK cells also include soluble ligands for NK cell activation receptors, IL-37 and CIS.

NK cells: NK cells in tumor immunity

Therefore, more and more studies have shown that effective methods can overcome the various defense effects of tumors on NK cells and the inhibitory effects of TME on these cells. These strategies may improve the effectiveness of NK cell therapy.


Immune checkpoint suppression of NK cells

Research on ICI mainly focuses on the de-suppression of anti-tumor T cells. This treatment mode enhances the activity of NK cells and has also attracted considerable attention. For example, the humanized anti-NKG2A monoclonal antibody monalizumab is currently undergoing single-drug trials in a variety of cancer patients, or in combination with other immune checkpoint inhibitors. Preclinical data shows that this drug can significantly enhance NK cells and T cells. Cytotoxicity.

The clinical trial results of KIR antagonists were mixed. The KIR2D-specific monoclonal antibody IPH2101 (targeting KIR2DL1, KIR2DL2 and KIR2DL3) was suspended due to lack of efficacy in phase II clinical trials (NCT01248455). Another phase I trial of IPH2101 for AML patients showed that although the drug is well tolerated, it has no effect. Despite these dismal results, clinical trials of KIR inhibitors are not absolutely negative. In fact, the combined application of lirilumab and nivolumab produced good clinical activity (ORR 24%) in 29 patients with advanced head and neck squamous cell carcinoma (NCT01714739). These observations increase confidence in other ongoing clinical trials of anti-KIR monoclonal antibodies.

The T cell immune receptor (TIGIT) with Ig and ITIM domains is another inhibitory receptor expressed by NK cells and T cells, and its ligand is overexpressed on NK cells infiltrating human tumors. In some mouse models, TIGIT inhibition reverses NK cell failure and promotes NK cell-dependent anti-tumor immunity. Several active clinical trials have been designed to test the effectiveness and/or safety of anti-TIGIT drugs alone and in combination with other immune checkpoint inhibitors. (Such as NCT03119428, NCT04150965, NCT04047862, NCT04256421, NCT03563716 and NCT04294810)

T-cell immunoglobulin mucin receptor 3 (TIM-3) is another immune checkpoint protein with inhibitory properties. It is expressed by various white blood cells (including NK cells) and is compatible with known expression on human tumors. Body galectin-9 binding. Increased expression of TIM-3 on NK cells of patients with lung adenocarcinoma indicates a poor prognosis. Inhibition of TIM-3 of this NK cell in vitro enhances its cytotoxicity and IFNγ production. At present, some clinical trials are conducting TIM-3 inhibitor trials on various cancer patients (NCT0331142, NCT03066648, NCT03489343, NCT03680508, NCT03961971, NCT04370704, NCT02817633, NCT03099109, NCT03744468 and NCT04139902).

Inhibitory proteins are widely expressed in lymphocytes, including lymphocyte activation gene-3 (LAG-3). Data from mouse NK cell studies indicate that LAG-3 plays a positive role in mediating cytotoxicity to tumor cell lines. Encouraged by the preclinical effect of LAG-3 in inhibiting anti-tumor T cells, some clinical trials are testing the efficacy of this method on patients with various malignancies (including NCT01968109, NCT04150965, NCT04140500 and NCT03625323). Other immune checkpoints based on NK cells include CD96, Siglec-7/9, CD200R, CD47, CTLA-4, PD-1, and B7-H3. Combining these checkpoints for a coordinated anti-tumor response is the future to give full play to NK The direction of the cell killing tumor.


NK cell targeting adaptor

Various immune evasion mechanisms limit the degree of binding of NK cells to tumor cells in the body, which is a major obstacle to achieving extensive and effective NK cell therapy. In order to enhance the natural cytotoxicity of tumor-infiltrating NK cells, many research teams have been developing molecules that allow these cells to contact tumor cells in an antigen-specific manner. These molecules are usually bispecific or trispecific adapters composed of multiple antibodies (usually single-chain antibodies), allowing one domain to target NK cell activation receptors and the other to bind to specific tumor-associated antigens. For example, a trispecific killer adapter (TriKE) composed of two single-chain antibodies, one targeting CD16 on NK cells and the other targeting CD33 on AML cells, is connected by an IL-15 domain, which is designed to It enhances the survival and proliferation of NK cells.

This anti-CD16, IL-15, and anti-CD33 TriKE (GTB-3550) enhances several other important aspects of NK cell effector function. In preclinical studies, it has been observed to improve migration ability, increase continuous killing and shorten the first kill time. . It has demonstrated strong anti-tumor and pro-proliferation effects in a mouse xenograft model of human myelodysplastic syndrome. A phase I trial is currently underway, which involves relapsed and/or refractory AML or myelodysplastic Syndrome patients.

In preclinical studies, several other NKCE molecules have also been shown to have powerful anti-tumor effects. This new type of trifunctional NKCE molecule includes monoclonal antibody fragments targeting CD16, NKp46 and tumor-associated antigens CD19, CD20 or EGFR. The redirected optimized cell killing (ROCK) molecule includes a tetravalent bispecific adaptor molecule composed of two diabodies (scFv dimers). The ROCK molecule AFM13 targets lymphoma-related antigens CD30 and CD16a, the latter being the transmembrane form of CD16 expressed by NK cells. AFM13 is currently being tested in multiple phase I and/or phase II trials, including various solid and hematological malignancies (NCT02321592, NCT03192202, NCT01221571, NCT04101331, NCT02665650, and NCT04074746).

DF1001 is one of several drugs belonging to tri-specific NKCE (TriNKETs). The safety and effectiveness of this HER2-targeted TriNKETs are being studied in patients with various advanced HER2 solid tumors, including pembrolizumab, which binds to the anti-PD-1 antibody. Several other companies are also developing similar products and are expected to enter clinical trials in the near future.

NK cells: NK cells in tumor immunity


Adoptive NK cell therapy

Autologous NK cell infusion is the first major focus of adoptive NK cell therapy, because it is very convenient to use the patient’s own blood as a cell source, has low requirements for immunosuppressive therapy, and has a low risk of graft-versus-host disease (GvHD). Studies on this method have shown that the injected cells can expand in the body, but cannot produce an effective response to blood or solid tumors. This may be partly due to the inhibitory effect of the interaction between autologous NK cells and its own MHC I molecules. In addition, patients receiving infusion undergo a lot of pretreatment before cell collection and treatment, which may have a negative impact on the expansion and function of NK cells. These findings have prompted many research groups to switch from autologous NK cell therapy to allogeneic NK cell therapy.

Cord blood NK cells

Allogeneic peripheral blood NK cells are just one of many potential sources of therapeutic NK cells. NK cells account for about 10% of the total number of lymphocytes in the peripheral blood, and NK cells in the umbilical cord blood (UCB) account for 30% of the total number of lymphocytes; therefore, UCB is a reliable source of therapeutic effect NK cells. The therapeutic effect of UCBNK cells is currently being evaluated in some clinical trials (such as NCT01619761 and NCT02280525). UCB is also a rich source of hematopoietic progenitor cells (HPC), so it can be used as a substrate for in vitro differentiation of therapeutic NK cells with an ideal phenotype, including adaptive NK cells. This is an exciting new development in immunotherapy research and development. way.

NK cell line

Clonal NK cell lines, such as NK-92, KHYG-1 and YT cells, are alternative sources of allogeneic NK cells, and the NK-92 cell line has been extensively tested in clinical trials. However, these cells are aneuploid and therefore genetically unstable, which requires them to be irradiated before infusion. Irradiated NK-92 cells have been observed to kill tumor cells in cancer patients. The product is currently undergoing four clinical trials involving patients with a variety of malignancies, and is combined with anti-PD-L1 antibody avelumab plus tumor vaccine (NANT) or IL-15 superantigen.

Stem cell-derived NK cells

So far, most of the NK cell therapies used involve the use of peripheral blood NK cells, UCB NK cells or NK-92 cells, but each of the above cell sources has important disadvantages. In fact, due to issues related to costs, delays in blood collection, variability between donors, and leukocyte heterogeneity in blood donations, the focus of intense attention is to switch from traditional allogeneic cell sources to stem cell-derived NK cells. These cells can be used as a standardized “off-the-shelf” therapy for any patient.

Currently, NK cell products derived from stem cells from a variety of sources are being tested clinically, including NK cell products derived from UCB stem cells or induced pluripotent stem cells (IPSC).

Reprogramming adult cells into pluripotent stem cells to differentiate into NK cells and expand to produce final products is a unique feature of iPSC-based methods. This method standardizes starting materials and provides greater uniformity and reproducibility in the development and management of NK cell therapy. A clinical trial is currently recruiting patients with blood or solid cancer to test the safety of these ready-made iPSC-derived NK cells. In addition, some genetically modified iPSC-derived NK cell products are undergoing clinical investigations for various cancer patients. These products include iPSC-derived NK cells expressing a high-affinity CD16 variant that is also resistant to proteolytic lysis of ADM17.

CAR-NK cells

With the clinical success of CAR-T cell therapy, a lot of efforts have been applied to explore the efficacy of CAR-NK cell products and their potential advantages over similar T cells.

A CAR-NK cell product that expresses genes encoding anti-CD19 CAR and IL-15 and an inducible caspase-9 suicide switch. The suicide switch uses a small molecule dimerization agent to eradicate genetically engineered cells in the body. Data from preclinical studies of this product show that it effectively kills primary leukemia cells expressing CD19 in vitro. Due to the expression of IL-15, the survival time of NK cells is significantly prolonged, and the suicide switch is quickly eliminated after triggering the suicide switch. In patients with CD19+ B-cell lymphoma or CLL, the CR rate of this CAR-NK cell product was 64% (7/11), without any major adverse reactions (no CRS, neurotoxicity or GvHD).

The current clinical trials of CAR-NK cells are mainly focused on products derived from stem cells or progenitor cells. A large number of clinical trials of CAR NK-92 cells are ongoing, but the requirements for irradiation and subsequent durability issues are the potential limitations of the clinical efficacy of these products. A series of Phase I trials of CAR NK cells from different sources, including autologous peripheral blood NK cells, UCB NK cells, NK-92 cells, and IPSC, which are aimed at a variety of cancers, such as AML, all or other B cell malignancies , Non-small cell lung cancer (NSCLC), ovarian cancer or glioblastoma, currently active.

Adaptive NK cells

Adaptive NK cells are another emerging source of therapeutic NK cells. Adaptive NK cells are a naturally occurring cell population that expands in humans after HCMV infection or reactivation. The interaction between the HCMV UL40-derived peptide antigen-HLA-E complex and NKG2C on NK cells leads to the proliferation of NKG2ChiCD57+NK cells. NKG2ChiCD57+NK cells down-regulate certain proteins involved in intracellular signal transduction, including PLZF, SYK and FcεRIγ . Adaptive NK cells have some unique effector properties, including enhanced ADCC, enhanced cytokine response, and inherent resistance to the immunosuppressive effects of MDSCs and Treg cells. Currently, three clinical trials are evaluating the efficacy of these adaptive NK cells for various cancer patients.

CIML NK cells

Cytokine-induced memory-like (CIML) NK cells are another option for allogeneic cell therapy and have unique advantages compared with other types of NK cell products. CIML-NK cells are activated temporarily by IL-12, IL-15 and IL-18 in vitro to produce NK cells with enhanced cytokine response and activate receptor stimulation that lasts for several weeks to several months. Infusion of these CIML-NK cells into mice carrying AML xenografts can produce powerful anti-tumor effects and significantly extend survival time. In the phase I trial, CIML NK cells have good safety, expanded in 44% of evaluable AML patients (4/9) and induced remission.



Natural killer cells are a unique group of anti-tumor effector cells that have cytotoxicity, cytokine production and immune memory functions that are not restricted by MHC, making them a key role in the innate and adaptive immune response system. Some cancers are related to dysfunctional NK cells. Therefore, repairing such NK cells may be a potential option for anti-tumor immunotherapy. One method of this repair is to suppress immune checkpoints, which means that cancer cells can escape immune response by controlling inhibitory receptors on the surface of immune cells.

In addition, NK cell therapy is a promising field of clinical research, with good safety and preliminary efficacy for some cancer patients. In particular, several groups have independently confirmed that adoptive allogeneic NK cells have powerful anti-tumor activity in AML patients.

Finally, the use of NK cells and antibodies, immune cell adapters, CARs and other checkpoint inhibitors combined methods for testing, these methods are designed to regulate the suppression of checkpoints and enhance anti-tumor capabilities. The goal is to find a NK cell-based treatment strategy that is safe and has the exact efficacy required for a wide range of clinical applications.





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