Cell-based immunotherapy with cytokine-induced killer (CIK) cells: From preparation and testing to clinical application

ABSTRACT

Cell-based immunotherapy holds promise in the quest for the treatment of cancer, having potential synergy with surgery, chemotherapy and radiotherapy. As a novel approach for adoptive cell-based immunotherapy, cytokine-induced killer (CIK) cells have moved from the ‘bench to bedside’. CIK cells are a heterogeneous subset of ex-vitro expanded, polyclonal T-effector cells with both natural killer (NK) and T-cell properties, which present potent non-major histocompatibility complex-restricted cytotoxicity against a variety of tumor target cells. Initial clinical studies on CIK cell therapy have provided encouraging results and revealed synergistic antitumor effects when combined with standard therapeutic procedures. At the same time, issues such as inadequate quality control and quantity of CIK cells as well as exaggerated propaganda were continuously emerging. Thus, the Ministry of Health in China stopped CIK cell therapy in May 2016, which was a major setback for the innovation of CIK cell-based immunotherapy. Thus, it is very important to modify technical criteria to develop a standardized operation procedure (SOP) and standardized system for evaluating antitumor efficacy in a safe way.

KEYWORDS:

• cytokine-induced killer cells
• cytotoxicity
• contamination
• quality control
• tumor immunotherapy therapy

Introduction

Cytokine-induced killer (CIK) cell therapy, as a new option in the field of cancer immunotherapy, is a set of therapeutic strategies based on manipulating and co-opting a patient's or close relative's cells to treat cancer.^1,2 CIK cells are in-vitro-expanded T lymphocytes characterized by the presence of 2 main subsets. The first subset with a CD3⁺CD56⁺ phenotype can exert wide mainly MHC-unrestricted antitumor activity against both solid and hematologic malignancies.^3-5 The second subset with a CD3⁺CD56⁻ phenotype is more similar to conventional T lymphocytes.⁶ Issues favoring their facile clinical translation are the ready availability of large amounts of expanded CIK cells, their MHC-unrestricted tumor killing, simple to grow, potential effectiveness against many tumor types, and low cost. CIK cells can migrate to the tumor site after intravenous infusion since they express several chemokine receptors.⁷ CIK cells are only one among many immune cell-based therapies proposed for the treatment of cancer. Chimeric antigen receptor T (CART) cell therapy, another potential approach of immunotherapy, can recognize their target antigen via an interaction between the chimeric immuno-receptor and a cell surface ligand.⁸ However, CART cell therapy is currently limited to hematologic malignancies and not widely applied due to its very high cost.^1,9 Numerous clinical trials have been conducted using CIK cells for the treatment of both hematological and solid tumors. However, the main problem to the use of CIK cell immunotherapy is the lack of fine precision in use of the cells. Intriguing future perspectives and challenges are under investigation for synergy with other immunotherapy approaches, targeted therapies or even conventional chemotherapy.^10-13

The WHO and RECIST criteria established since years have been used to assess the effectiveness of CIK cell-based therapy; however, these criteria are not sufficient to fully describe all the important processes and cytotoxic agents. Since 2016, the number of institutions working on immunotherapy in China has considerably increased. However, some of these institutions lacked sufficient magnitude of CIK cells and standard quality control procedures. Furthermore, the exaggerated commercialization of the CIK cell-based immunotherapy indirectly caused the death of a patient suffering from synovial sarcoma, owing to which the Ministry of Health in China decided to stop CIK cell-based immunotherapy until an appropriate criterion is set-up. An effective and standardized informed consent procedure for CIK cell-based immunotherapy is expected to reduce the risk involved. Meanwhile, continuous quality improvement (CQI) will help to detect and prevent errors, thereby reducing harm to patients.¹⁴ Therefore, in the present study, the culture procedures for CIK cell-based immunotherapy, evaluation of cellular functions, and detection and management of contamination are presented. In addition, clinical examples involving cell-based tumor immunotherapy are discussed.

Growth and preparation of cells in Vitro

The brief preparation of CIK cells, as shown in Fig. 1A, is based on the first clinical trial using cell-based cancer immunotherapy.¹⁵ Although the procedures for CIK cell therapy lack quality control criteria, they have been relatively uniform, despite constant evolution in the past 30 y. The major procedures of CIK cell therapy followed on a large scale in Chinese cancer immunotherapy institutions are listed below and the corresponding operation is shown in Fig. 1B.^16-18

Figure 2. The concentration of viable cells. All the data were randomized collected from 50 tumor patients of different classification at different stages. And the samples would be counted at least 3 times.

Briefly, heparinized peripheral blood (50 ml) is collected from the patients with tumors. The peripheral blood mononuclear cells (PBMCs) are isolated by Ficoll density gradient (Tianjin HY, China) centrifugation, followed by washing with 0.9% clinical-grade saline solution thrice. The cells are then incubated at 37°C for 24 h, under 5% CO₂, 95% relative humidity using X-VIVO 15 serum-free medium (Longza, Japan) containing 1000 U/ml IFN-γ (Peprotech, USA). After 24 h, anti-CD3 antibody (at a final concentration of 100 ng/ml, eBioscience, USA), 1 ng/ml IL-1α (Peprotech, USA) and 1000 U/ml recombinant human IL-2 (QuanGang, China) is added to the medium. After 5–7 d of culture, the final expansion levels are usually in the range of hundred fold and the cells are transferred into a clinical-grade cell culture bag. On days 7–10, cells are counted and 2.5 l of fresh medium containing 1000 U/ml rhIL-2 is added. On day 11, each of the products are sampled for sterility testing. Further, on day 14, autologous CIK cells are re-suspended in 200 ml normal saline and are confirmed to be free of bacterial, mycoplasma, or fungal contamination. The name and the admission number attached to the CIK cell bag are carefully checked before transfusion into the patient's body.

IFN-γ, added on day 0, plays its role through the activation of macrophages present in the culture, thus providing both contact-dependent and cytokine-mediated signals favoring the acquisition of a Th1 phenotype.^19-21

An initial mitogenic signal is provided by the anti-CD3 antibody (BD Biosciences, USA) and then sustained by the continuous presence of IL-1α and IL-2.^22,23

Testing of cells

Quantification of viable CIK cells

In modern biomedical research and in the bio-manufacturing industry, it is essential to routinely monitor the growth and proliferation of CIK cells by viable cell counting.²⁴ Every 2–4 days, the quality control personnel draws 3 ml of supernatant from the cell culture bag for cell counting to ensure the proper proliferation of CIK cells. All the samples are stained with 0.4% trypan blue (TB), and the cell size, concentration, and the viability of each sample is measured using the Countstar automated cell counter (Shanghai, China). At the end of the cultivation, the concentration of CIK cells should achieve 3–4 × 10⁶/ml (Fig. 2) and the viability should be > 92%.

Figure 1. The brief preparation of cytokine-induced killer (CIK) cells. (A) A brief description of CIK cells procedure in most clinical institutions on cell-based tumor immunotherapy. (B) The main preparation of CIK cells in lots of the immunotherapy institutions, which applied only about 50 ml whole blood cells instead of 100ml, and more monitoring measures were used during cultivation of the CIK cells. (NS, normal saline).

Composition of CIK cells

The CIK cell population obtained is mainly a CD3⁺ heterogeneous T-cell population with 2 predominant subsets, positive (CD3⁺CD56⁺) and negative (CD3⁺CD56⁻) for the simultaneous expression of CD56 membrane molecules, whereas the antitumor efficiency is mainly owing to the CD3⁺CD56⁺ cells.^25,26 The other cells, such as CD4⁺T cells, CD8⁺T cells, and T-regulatory lymphocytes (Treg cells) are mingled in the CIK cell population, which also influence the effect of immunotherapy.^27-30 On days 7, 10 and 14 of cultivation, the expressions of CD3, CD56, CD4, CD8, CD25, and CD127 molecules on the surface of CIK cells are detected by fluorescence-activated cell sorter (FACS, BD Bioscience, USA). The monoclonal antibodies used include anti- CD3+ to evaluate total T lymphocytes, CD3+/CD4+ to evaluate Helper T cells, CD3+/CD8+ to evaluate cytotoxic T cells (CTLs), CD3+/CD56+ to evaluate cytokine-induced killer cells (CIKs), CD3+/CD56- to evaluate natural killer (NK) cells, and CD4+/CD25+/CD127low/− to evaluate regulatory T cells (Tregs). Among them, the terminally differentiated CD3⁺CD56⁺ subset represents a key feature, a double T-cell and NK cell phenotype, which could exert a potent and widely MHC-unrestricted anti-tumor cytotoxicity. As a result, the percentage of CD3⁺CD56⁺ cells must exceed by 40%, with the proportion of CD8⁺T cells almost twice or more than the proportion of CD4⁺T cells. Simultaneously, the proportion of Tregs (CD4⁺CD25⁺CD127⁻) within the CIK cell culture should also be significantly decreased, which suggests that the immunosuppressive function of Tregs has been inhibited (Fig. 3). Higher amount of CD3⁺CD56⁺ cells detected in the supernatants of culture results in more benefits to the tumor patients, such as improved prognosis, reduced recurrence, and prolonged recurrence-free span.

Figure 3. Immunological assessment of T-cell subgroup. The percentage of CD3⁺CD4⁺ cells, CD3⁺CD8⁺cells and CD3⁺CD56⁺cells were evaluated using 2-color FACS. Treg(CD3⁺CD56⁺CD127⁻) cells were evaluated using 3-color FACS. Expression of CD25 and CD127 after gating on CD4⁺. The representative scatter plots were demonstrated here.

Cytotoxicity of CIK cells against tumor cells

The CD3⁺CD56⁺ CIK cells derived from CD3⁺CD56⁻ precursor T cells exhibit the highest cytotoxicity against target tumor cells.³¹ The antitumor mechanisms of CIK cells include 2 signaling pathways: first, the binding of NK-cell receptors to their ligands.^32,33 and second, the induction of tumor cell apoptosis by Fas ligand via the Fas signaling pathway.³⁴ Therefore, on day 14 of cultivation, the cytotoxicity of CIK cells collected are estimated by co-culturing with K562, a CIK cell-sensitive target cell line, at effector/target (E/T) ratios of 1:1, 10:1, 20:1 and 40:1. The tumor-lysis efficiency is determined by using CCK-8 assay kits (Dojindo, Japan), and the major killing molecules, such as NKG2D, NK1.1, perforin, FasL, and granzyme B are monitored by FACS. The total cytolysis activity during cell culture has been used as a unique marker for the success of autologous CIK cell generation.

Cytokine secretion by CIK cells

Besides directly killing the tumor cells, CIK cells secrete numerous cytokines that induce antitumor immune responses in patients. Cytokines production by CIK cells is fast and efficient, leading to effective function of CIK cells. IFN-γ and TNF-α are the main cytokines produced by CIK cells,^35,36 which are involved in regulating innate and adaptive immunities. The other positive regulatory cytokines that could be detected in tumor immunotherapy laboratories are IL-2 and IL-4.^37-39 The negative regulatory cytokines detected are usually IL-10 and TGF-β.^40,41 CIK cells can be used as a vehicle for delivering biological cytokines directly to the tumor microenvironment. In addition, these cytokines participate in rebuilding the patient's immune defense following tumor excision, chemotherapy, or radiotherapy.⁴²

The aging of CIK cells

Aging in CIK cells is characterized by a decrease in genome integrity due to immune dysfunction and commensal dysbiosis. Telomeres are nucleoprotein structures located at the end of eukaryotic chromosomes. Overwhelming evidence has indicated that telomere shortening induces the instability of chromosomes, which could also contribute to exponential increases in genomic instability of adult cells in response to replicative aging.^43-45 Therefore, it is necessary to study aging during CIK cell proliferation and self-renewal by monitoring the activity or magnitude of telomeres on day 0, 7, and day 14.^46-49 Although there is no significant evidence to suggest the correlation between the efficiency of tumor immunotherapy and the age of CIK cells, understanding of this correlation is important for the effective development of CIK cell-based immunotherapy.

Microbiological contamination

Safety is the main prerequisite for the adoptive infusion of CIK cells in patients with malignant cancers.⁵⁰ During CIK cell transfusion, various agents may be transmitted. However, in the past 20 years, such a case was not reported in any immunotherapy laboratory owing to strict supervision and appropriate and prompt disposal strategies. Microbiological contamination such as bacteria, viruses, or endotoxins may enter during blood collection from a patient or during CIK cell cultivation. Before blood collection, the patient is subjected to a series of blood tests to screen for HIV, Hepatitis A/B/C virus, or Treponema pallidum (Syphilis). If the results of any of these tests are positive, all the processes involved in the patient's CIK cell extraction and cultivation are conducted in an isolated culture environment by specifically trained personnel. In addition, on day 11 of cell cultivation, the culture supernatants of all the CIK cells from different patients are collected for screening for bacteria, viruses, and endotoxins separately.^51-53

Autologous CIK cell transfusion

The autologous CIK cells are administered via intravenous infusion within a period of 30 min. The infusion of these cells is well-tolerated and does not cause severe adverse events such as fever, myalgia, flu-like symptoms, and fatigue.⁵⁴ In general, patients receive 4–8 cycles of CIK cell infusion in a year and if the situation remains stable, more cycles of CIK maintenance treatment are given using the protocol mentioned above. Conversely, the CIK therapy is stopped if the patients do not respond well.

Clinical use

A growing number of clinical trials have been performed and the outcome suggests that CIK therapy yields highly compelling clinical responses in several solid carcinomas, such as hepatocellular carcinoma or B-cell malignant lymphoma. The data of randomized clinical trials for CIK cell-based therapy were searched on PubMed and analyzed. Within the 68 matches found, 24 published papers were excluded due to the lack of clinical information and unrelated tumors and the clinical information from 44 papers^34,54-97 published on CIK cell therapy was analyzed (Table 1). The patients in the immunotherapy group received at least 4 cycles of transfusion of CIK-cells, with more than 1 × 10⁹ CIK cells transfused into patients within 1 h via the peripheral vein.

Table 1. A summarization of the Clinical information on the CIK cell-based cancer immunotherapy.

Reference numbers	Cancer disease	Stage of disease	Total patients(n)	Patients(n) treated with CIK cells	Combined with standard cancer therapy	Synergism with other immunotherapy approaches	Adverse reaction	Intended therapeutic benefit
^55-60	Non-small cell lung cancer	I∼IV	529	304	Intravenous chemotherapy	Dendritic cells(DCs) with CIK cells	—	The CIK cell treatment to improve the clinical outcomes of conventional chemotherapy.
^61	Lung cancer	I∼IV	364	209	—	—	—	The CIK cells were able to improve the immune functions of patients with lung cancer.
^62-64	Multiple myeloma	I∼III	152	78	Oral & Intravenous chemotherapy	DCs with CIK cells	—	The DC-CIK cell therapy improved the quality of life, clinical index and survival.
^65,66	Breast cancer	I∼III	288	148	Intravenous chemotherapy	DCs with CIK cells	Alleviated	The DC-CIK cell therapy improve the quality of life.
^34,67-73	Renal cancer	I∼IV	301	160	—	CIK cells transfected with the IL-2 gene or DCs with CIK cells	No adverse events with grade greater than 2 were reported	The different CIK cell-based immunotherapies would improve the immune status of patients.
^34,74,75	Colorectal cancer	I∼IV	418	388	Oral chemotherapy, radiotherapy surgery	CIK cells transfected with the IL-2 gene or DCs with CIK cells	—	The different CIK cell-based immunotherapies would reduce the recurrence rate and promote the survival time of patients.
^76,77	Gastric cancer	III/IV	208	106	Oral & Intravenous chemotherapy	—	—	The CIK cell therapy prolong DFS and improve OS.
^54,78-89	Hepatocellular carcinoma	I/II/III/IV	1203	650	TACE and RFA or after radical resection	Dendritic cells with CIK cells	—	The DC-CIK cell therapy were capable of reducing recurrence, prolonging the recurrence-free span, and attacking HBV, decrease the times of TACE or RFA.
^90	Brain cancer		20	20	Oral & Intravenous chemotherapy	RetroNectin activated CIKs	—	R-CIKs combined with conventional therapies could improve the prognosis of brain tumor patients
^34,91-93	Haematopoietic tumor	I∼IV	72	72	Oral & Intravenous chemotherapy	CIK cells transfected with the IL-2 gene or CIK cells alone	—	The CIK cell therapy could significantly enhanced immune functions and increasing absolute numbers of effector cells without side effects.
^94,95	Pancreatic cancer	IV	78	48	Oral chemotherapy	—	—	The CIK therapy was well tolerated in a second-line setting in patients with gemcitabine-refractory and advanced pancreatic cancer.
^96	Esophageal carcinoma	I∼IV	68	34	Radiotherapy	DCs with CIK cells	hepatic and renal dysfunction, fever, allergy, and bone marrow depression were assessed	The therapy could improve patients' quality of life and immune function, decrease bone marrow suppression, and lengthen survival time.
^97	Nasopharyngeal Carcinoma	—	222	112	Intravenous chemotherapy	—	—	The sequential CIK treatment may be effective in enhancing the therapeutic efficacy.

DFS, disease-free survival; OS: overall survival; TACE, transcatheter arterial chemoembolization; RFA, radiofrequency ablation.

The majority of tumor patients in these clinical trials were treated with CIK cells along with conventional therapy, to achieve a synergistic antitumor effect. Other studies suggest that the cytotoxicity of CIK cells can be enhanced and improved by DC vaccination and when transfected with IL-2 gene or activated by Retro Nectin, these CIK cells demonstrate a higher proliferation rate and efficient cytotoxic activity.^{54,56,58-60,62-64,67,68,72,75,84} In general, all these published studies have demonstrated that CIK cell-based immunotherapy utilizes the body's natural ability to eliminate tumor cells by stimulating and restoring the ability of the immune system to recognize and kill tumor cells. In contrast to conventional therapeutic strategies, the application of CIK cells is associated with minimal side effects and no severe adverse events have been reported. Interestingly, a reduction of Hepatitis B virus load is observed in patients undergoing treatment with CIK cells in our laboratory and elsewhere also.⁵⁴

Owing to its easy availability and potent anti-tumor activity, CIK cell therapy is emerging as a promising immunotherapy approach in oncology and may improve the quality of life of tumor patients, when combined with standard therapeutic procedures.^98,99 However, the lack of large-scale clinical experiments and the standard period for immunotherapy are the main shortcomings. Currently, our institution is in the process of collecting huge amount of data from clinical experiments, with a hope to reveal new insights into the efficacy of the technique in future.

Conclusions

At present and in the immediate future, it appears that immunotherapy will hold a place second to standard cancer therapy, but not as an alternative.¹⁰⁰ CIK cell-based immunotherapy has demonstrated potential in improving clinical outcomes and relieving the major side effects of standard treatment options. Moreover, CIK cells in combination with other conventional and established therapeutic options represent an innovative approach and will probably provide a new insight into research in the future. In this article, we have discussed the complete SOP of CIK cell-based immunotherapy with a hope that a comprehensive criterion would be established as soon as possible. Furthermore, clinical trials suggest that the combination of conventional chemotherapy and CIK cell-based immunotherapy could represent a valuable option to overcome the first-line chemotherapy failure, which is frequently observed in cancer treatment.^74,90 Furthermore, the number of transfused CIK cells and frequency of transfusion seem to play an important role in enhancing antitumor activity.²⁸

However, there are also some limitations to our study. First, as the CIK cell-based therapy is restricted to Asian countries and doesn't involve clinical samples from different regions of the world, it fails to completely depict the effects of the treatment. Second, in most clinical trials, a limited number of patients are included, focusing only on a certain type of tumor. Third, the clinical data discussed here are selected from published papers rather than drawn directly from patient records. More large-scale clinical trials are required to confirm the role of CIK cell-based immunotherapy in cancer treatment. In conclusion, the combination of CIK cell-based immunotherapy and other constructive therapeutic procedures is likely to make a significant contribution to the treatment of patients with tumors.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgment

We thank Professor Fengping Shan for his professional suggestion and constructive comments on this manuscript.

Funding

This research work was supported by Doctoral Scientific Research Foundation of Liaoning Province (201601412 to Yiming Meng).