NK cell therapy: Autologous and from cord blood

Cancer poses a serious threat to human health and stands as a leading cause of death worldwide. Statistics reveal approximately 10 million cancer-related deaths annually and over 19 million diagnosed cases in 2020. Breast cancer accounts for the highest percentage of cancer incidence in females at 11.7%, whereas lung cancer leads to males at 14.3% (1). Lung and breast cancers are also respectively the highest causes of mortality, accounting for 21.5% and 15.5% of cancer-related deaths. Alongside early detection and cancer prevention, novel cancer treatment methods are pivotal in enhancing treatment efficacy and improving the quality of life for cancer patients. Traditional treatments such as surgery, chemotherapy, and radiotherapy have been and are being used to treat various cancers. However, these treatments often lead to numerous side effects, such as oral mucositis, hepatorenal toxicity, hematopoietic suppression, and reduced patient quality of life, sometimes resulting in treatment cessation (2).

With the advancement in deep immune system research, immune cells have proven their significant role in cancer treatment and prevention, making immune therapy the fourth applied method in cancer treatment. Immune cell therapies developed on immune cell lines such as T lymphocytes, B lymphocytes, and Natural Killer (NK) cells have been derived from their role in the cancer immune system (3). NK cell therapy is one of the newest immune cell therapies under research and holds significant potential in immune cell therapy for cancer treatment. NK cells can be derived from cord blood or autologous sources.

NK NK Cells and NK Cell-Based Immunotherapies

NK cells are a type of white blood cell that, along with other cells like T cells, B cells, and macrophages, forms the innate immune system in humans (4). Originating from hematopoietic stem cells in the bone marrow, NK cells exist in the blood and some organs, such as the liver, uterus, and spleen. A distinguishing feature of NK cells from other cells is the absence of T cell receptors (TCR) expression and the presence of the CD56 marker. NK cell populations are divided into two types: CD56dim and CD56bright (5). Activating and inhibitory receptors present on the surface regulates the activity of NK cells. Signals received from these receptors when interacting with corresponding ligands on target cells enable NK cells to decide whether to eliminate or spare these cells or modulate the secretion of cytokines. The ligands are usually MHC-I molecules present on all body cells. In cancer cells, MHC-I molecules often exhibit imbalances, causing alterations in signals from inhibitory/activating receptors of NK cells, allowing NK cells to eliminate these cells (6).

The cytotoxic ability of NK cells is demonstrated through the secretion of perforin, a protein that creates pores in the target cell membrane, leading to cell membrane permeabilization, and granzymes, which activate caspases, causing cell death (7). NK cells can also directly eliminate target cells through the expression of FAS ligands and TNF-related apoptosis-inducing ligands (TRAIL) (6). Additionally, NK cells can release a range of cytokines and chemokines like IFN-γ, IL-10, CCL3, and CCL4, playing a vital role in promoting the activities of Lympho B and Lympho T cells to eliminate cancer cells (5). These biological characteristics of NK cells allow them to identify neighboring cells showing signs of cancer and eliminate them. Particularly, the absence of TCR receptors on their surface prevents NK cells from inducing graft-versus-host disease (GVHD). These unique NK cell characteristics, combined with in vitro cultivation methods to expand NK cell populations from various sources, either autologous or allogeneic, have created NK cell therapy in cancer treatment (Figure 1). Autologous NK cells mainly come from peripheral blood (PB-NK), while allogeneic NK cells can be obtained from cord blood (CB-NK), specialized NK cell lines, induced pluripotent stem cells (iPSC), and Car-NK (8).

Figure 1: NK Cell Therapy: A – Autologous NK Source from the Patient, B – Allogeneic Source from Healthy Donors, C – Commercial NK Source, Car-NK

Distinguishing between Autologous NK from Peripheral Blood and Cord Blood NK

In essence, NK cell therapy involves the isolation, cultivation, proliferation, and stimulation of NK cells before their infusion into the patient’s body (9). The major sources for NK cells in autologous therapy are the patient’s peripheral blood and bone marrow. In contrast, Cord Blood NK is isolated from the umbilical cord blood: the blood remaining in the cord and placenta after childbirth, which is then cryopreserved and stored in biobank.

In peripheral blood, PB-NK cells constitute about 10-15% of the total lymphocyte count. Of these, 90% are CD56dimCD16+ cells, referred to as mature NK cells, exhibiting high cytotoxicity due to full activation receptor expression on their surface, capable of secreting perforin and granzyme. The remaining 10% comprises CD56bright NK cells, capable of cytokine secretion (5). In Cord Blood NK therapy, NK cells constitute 25-30% of the total lymphocyte count, higher than in peripheral blood. The ratio of CD56dimCD16+ and CD56bright populations doesn’t significantly differ from peripheral blood (10). However, in cord blood, there are distinct NK cell populations differentiated by CD16, CD94, and CD117 markers, known as immature NK cells, exhibiting lower cytotoxicity compared to PB-NK (11).

Research by Luevano and colleagues compared the perfoarin secretion, granzyme expression, FAS-L, and TRAIL surface molecule expression, which are characteristics of direct cytotoxicity between PB-NK and CB-NK cells. Both cell types were cultured in an IL-2-supplemented environment to proliferate and activate NK cells. Results showed that after seven days of cultivation, CD56dim-NK cell populations from cord blood exhibited about 30-40% lower expression of perforin, granzyme, and FAS-L compared to PB-CD56dim-NK. CD56bright-NK populations from both cord blood and peripheral blood showed similar expression levels (12). The reduced cytotoxicity in CB-NK cells might be due to lower expression of activating receptors KIRs, NKG2C, NKp46, and DNAM-1 compared to PB-NK (Figure 2). Conversely, CB-NK cells showed higher expression of inhibitory receptor NKG2A compared to PB-NK (12,13).

Figure 2. Stimulatory and Inhibitory Receptors on PB-NK and UC-NK Cells

NK cell therapy requires a substantial quantity of NK cells for treatment. To expedite the proliferation of NK cells and activate them into highly cytotoxic mature stages, researchers introduce cytokines and feeder cells during the cultivation process. The most used cytokine is IL-2, supplemented by IL-12, IL-15, IL-18, or IL-21 and Type I IFN. These cytokines interact with receptors on NK cells, stimulating NK cell division and the expression of stimulatory receptors (14). UC-NK cells require a fivefold higher IL-2 concentration for activation compared to PB-NK cells (14). Initially, this might be due to fewer IL-2 receptors and reduced STAT5 phosphorylation in UC-NK cells compared to NK cells. Moreover, after IL-2 activation, PB-NK cells exhibit higher expression of activation receptors like NKp44 and CD69 compared to UC-NK cells, indicating higher cytotoxicity of PB-NK cells when activated by IL-2 (12). According to Alnabhan’s research, UC-NK and PB-NK cells both respond to IL-15 and IL-18 activation. However, UC-NK cells respond more significantly to IL-15 than IL-2. When activated by IL-15 and IL-18, these cells exhibit enhanced cytotoxicity and increased cytokine production like IFN-gamma, with a role in anti-tumor activities (15).

Advantages and Disadvantages of Autologous NK Therapy

Until recently, 92% of clinical studies on NK cell usage originated from peripheral blood, with autologous NK cell therapy accounting for 13%. This raises concerns about the safety and treatment efficacy of using peripheral blood-derived NK therapy versus autologous sources (16). Regarding NK cell sources, the advantages of autologous NK therapy include easy mobilization, with up to 90% of mature NK cells in the PB-NK population, demonstrating high cytotoxicity (5). Furthermore, autologous sources don’t trigger graft-versus-host reactions upon infusion due to the inclusion of T lymphocytes during cultivation. Compared to allogeneic sources, autologous NK cells don’t require safety assessments for transmitted products, such as virus and mycoplasma tests, and don’t incur expenses for cord blood units, reducing costs. Most importantly, autologous PB-NK cells fully comply with legal and safety aspects compared to donor-derived NK cells, cord blood, and commercial NK cell lines (17). The effective proliferation capability of PB-NK cells surpasses that of other NK cell types. PB-NK cells respond better at low IL-2 concentrations compared to UC-NK cells, and their cytotoxicity is also higher when activated by IL-2 (18,12).

NK cells account for only 10% of the total lymphocyte count in peripheral blood results in a lower mobilization of NK cells compared to commercial NK cell lines. Additionally, the lower homogeneity of PB-NK cells, because of their inclusion of various cell types, poses challenges (18). Moreover, for cancer patients, mobilizing peripheral blood to isolate NK cells often requires significant time and treatments or surgeries, whereas alternative NK cell sources like UC-NK or allogeneic NK cells are readily available (17,10). In cancer patients, using NK cells directly for treatment may lead to minimal NK cell activity. The inhibition of tumor cell destruction could explain this based on decreased recognition of MHC-I complex on tumor cells because of KIR inhibition (19).

Rosenberg’s study using autologous PB-NK cells activated with IL-2 to treat 8 renal cancer patients with a dose of 4.7×1010 NK cells/kg showed NK cell persistence in patients’ bodies for several weeks to months. However, these NK cells did not significantly express the NKG2D activating receptor on their surface and lacked the ability to lyse cancer cells in vitro without re-stimulation by IL-2 (20). Furthermore, another limitation of using autologous NK cells is their susceptibility to damage and difficulty in recognizing previously treated cancer cells, significantly impacting the use of autologous NK cells in cancer immunotherapy (21). The limited function of autologous NK cells in cancer patients is mainly because of KIR mismatching (22). The compatibility of NK cells is driven by the mismatch between KIR receptors and their ligands expressed on the surface of target tumor cells during hematopoietic stem cell transplantation (HSCT), demonstrating potent anti-tumor activity and limiting graft-versus-host disease (GvHD) (23). Ruggeri’s study used haploidentical hematopoietic stem cell transplantation for leukemia patients with myeloid leukemia, focusing on KIR mismatch between donor and recipient NK cells. Results from 51 patients showed a very low relapse rate of only 3%, with a complete remission rate of up to 67% for critically ill patients.

Although the use of autologous NK cells in cancer treatment is still under research and evaluation, it has shown notable results. Autologous NK cell therapy remains quite safe and is being strongly applied to cancer prevention and recurrence reduction (9). Additionally, other allogeneic NK cell therapies, such as those from healthy donors, commercially available NK cell lines, cord blood, and pluripotent stem cells (iPSCs), are also being evaluated and clinically tested. The results and clinical trials contribute significantly to making NK cell therapy for cancer treatment and prevention more reliable and potentially becoming a more effective treatment trend compared to other traditional treatment methods.

References:

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