Cytokine-Induced Killer Cells: A Unique Platform for Adoptive Cell Immunotherapy after Allogeneic Hematopoietic Stem Cell Transplantation.

Historical Background

More than 30 years ago, in 1986, Phillips and Lanier dissected and identified the contribution of peripheral blood (PB) natural killer (NK) and T cells to the cytolysis of cancer following lymphokine activation. Pioneering work in the context of cytokine-induced killer (CIK) cell generation and their clinical application in cancer patients was then conducted in 1991 and 1999 by Schmidt-Wolf and colleagues [ 1 , 2 ].

Both the conduct-depended factor (CD58/lymphocyte function-associated antigen [LFA]-3) and the soluble factor (interleukin [IL]-12) provided by interferon-γ (IFNγ)-activated monocytes were crucial for the in vitro expansion of T-NK cells from PB mononuclear cells [ 3 ]. Further mitogenic signals for T lymphocytes were then applied in vitro by low-dose anti-CD3 antibodies and repetitive interleukin 2 (IL-2) stimulation every 3 to 4 days for a total of 21 days [ 4 ].

The efficacy of CIK cells generated by this protocol, in general, was due to the enrichment of immune cells, namely, T cells, NK cells, and, in particular, T-NK cells, which showed natural non-major histocompatibility complex (MHC)-restricted cytotoxicity against various cancers, particularly through their interaction between the activating NK cell group receptor (NKG) 2D and its ligands, leading to the secretion of cytokines such as IFNγ [ 5 ]. However, there were additional, at that time unknown, mechanisms considered that CIK cells might use to fight cancer cells.

Current CIK Cell Expansion Protocols

CIK cells can be prepared from autologous (patient’s own) or allogeneic (donor-derived) fresh and cryopreserved leukapheresis material, PB, leukocytapheresis, bone marrow (BM), or cord blood (CB) mononuclear cells in the presence of IFNγ, anti-CD3 antibody, and IL-2. The general culture protocol requires 3 weeks. CIK cells generated according to this protocol are a heterogeneous composition of a majority fraction of CD3 + CD56 − T cells, a minority fraction of CD3 − CD56 + NK cells, and – CIK cell-specific – an increasing number of CD3 + CD56 + T-NK cells, which expands with the duration of the culture. At the end of the differentiation protocol, mature CIK cells typically are almost entirely CD3 + T lymphocytes expressing CD8 and CD56 and displaying an effector memory phenotype.

CIK effector cells, as a pharmacological tool for cancer immunotherapy, have shown potent killing activity via non-MHC-restricted NK-like and T-cell receptor (TCR)-dependent cytotoxicity pathways. In addition to their direct killing activity, CIK cells can also regulate the immune function of innate and adoptive immunity by secreting various cytokines. Hence, CIK cells are an alternative option over conventional T cells known to be safe and associated with low graft-versus-host disease (GvHD) and minimal alloreactivity in an allogeneic setting, as demonstrated by different groups [ 6 – 8 ]. In autologous settings, efforts have been made to improve CIK cells’ cytotoxicity, including the combination with dendritic cells (DC), checkpoint inhibitors (CIs), antibodies, chemotherapeutic agents, and chimeric antigen receptors (CARs) ( Table 1 ).

CIK Cell Expansion to Clinical-Grade Cultures

There is an urgent need to standardize protocols for CIK cell generation to achieve greater acceptance and comparability of this unique therapy. However, a major hurdle, at least in the autologous setting, is the difficulty in generating clinically relevant numbers of highly effective CIK cells from heavily pretreated patients [ 9 ]. New protocols have been reported to decrease regulatory T cells (Tregs) and simultaneously increase the proliferation ability and cytotoxicity of CIK cells. Strategies to further improve the expansion of CIK cells include adding new cytokines to the culture medium such as IL-1α, IL-6, IL-7, IL-12, and IL-21. Lu et al. reported the expansion of highly efficient cytotoxic CIK cells by culturing lymphocytes in the presence of IL-1α [ 10 ]. The addition of IL-6 significantly decreased the number of Tregs, leading to an increased proliferative and cytotoxic capability of CIK cells against hepatocellular carcinoma [ 11 ]. The addition of IL-7 resulted in the upregulation of LFA-1 (CD11a/CD18) and CD28 in CIK cells, which played an important costimulatory role in T-cell activation [ 12 ]. Application of IL-21 increased the expression of IFNγ, tumor necrosis factor (TNF)-α, perforin, and granzyme B, thereby enhancing cell cytotoxicity [ 13 ]. Furthermore, thymoglobulin was compared with αCD3 (OKT3) monoclonal antibody (mAb) in clinical-grade culture protocols [ 14 ]. Han and colleagues recently used retronectin for CIK cell activation, resulting in a higher percentage of CD3 + CD4 + cells and CD3 + CD28 + cells compared with conventional types of CIK cells [ 15 ]. Finally, studies with IL-15-activated CIK cells displayed improved proliferation and cytotoxic capacity against hematologic and solid malignancies [ 16 – 21 ].

Considering the urgent need for treatment options for defined patients and to enable sequential and dose-increased CIK cell therapy, we established good manufacturing practice-compliant procedures for the robust generation of fresh and cryopreserved allogeneic ready-for-use therapeutic CIK cell doses within just 10–12 days [ 22 ]. By adding IL-15 instead of IL-2 from day four of culture, cells with a CIK cell phenotype were expanded within 10–12 days. Compared to the 3-week culture system, numbers of T- NK cells were lower, while numbers of CD3 + CD8 + TCR α/β + T cells were higher, potentially associated with increased alloreactive potential. These short-term cell preparations contained a predominant proportion of T cells which showed homing to leukemia sites and complete leukemia control when repeatedly administered in several established mouse models of human leukemia xenografts [ 16 , 23 ]. Our data demonstrated that IL-15 was as effective as IL-2 in promoting cell proliferation and viability. There was no significant alteration in the CIK cell composition. CIK cells generated from cryopreserved unstimulated leukocytapheresis instead of from fresh PB or leukocytapheresis material were associated with limited expansion of NK and T-NK cells in the final product.

As no systematic information on the composition of CIK cells after expansion with different clinical culture protocols can be found in the literature, this information is not included in this review. However, the three aforementioned subgroups of the bulk CIK population could differ depending on donor source material and CIK cell generation protocols, making it difficult to compare this unique therapy.

CIK Cell Mechanisms to Fight Cancer Cells

The dual-functional capability of CIK cells was reported by Pievani et al. [ 24 ], involving both T cell and NK cell-like cytotoxicity mechanisms mediated through the TCR and LFA-1, as well as through NK cell-activating receptors, such as DNAX accessory molecule-1, NKp30, NKp46, and the NKG2D receptor. This results in granule exocytosis, cytokine secretion, and cytotoxicity via apoptosis induction. As T-cell-mediated alloreactivity and NK-like antitumor activity segregate within defined CIK cell subsets, alloreactive subpopulations may consequently be separated before being applied across human leukocyte antigen (HLA)-mismatched barriers due to safety concerns [ 25 ].

However, we found that after CD56+ selection or TCRα/β-depletion, antitumor activity was substantially reduced compared with the tested bulk CIK cell population, whereas alloreactivity of CD56+, TCRα/β-, and bulk CIK cells against mismatched PB mononuclear cells or fibroblasts remained low [ 23 ]. Recently, the CD4+ T-cell subset was identified as responsible for killing tumor cells through the phosphorylation of the AKT signaling pathway, secretion of IL-17A, and upregulation of T-box transcription factors T-box expressed in T cells and eomesodermin (T-BET and EOMES). Consequently, the exhausted phenotype (programmed cell death protein [PD]-1+ and T-cell immunoglobulin and mucin domain [TIM]-3+) of CD8+/CD3+CD56+ T-NK cells within the CIK cell population was reduced in the presence of CD4+ T cells, at least in non-small cell lung cancer [ 26 ].

Overall, both autologous and particularly allogeneic bulk CIK cells are capable of a direct TCR-dependent and broad MHC-unrestricted antitumor activity, depending on the raw material as well as the donor- and manufacturing-specific composition of CIK cells. Additionally, CIK cells can indirectly regulate immune responses by secreting various cytokines.

Combinational Treatment Strategies for Enhanced Toxicity

Retargeting CIK cells with mAbs, Fc-engineered mAbs, and bispecific antibodies (bsAb) such as cetuximab [ 27 ], trastuzumab, and human epidermal growth factor receptor (Her)-2xCD3 bsAb enhances the cytotoxicity of CIK cells against various Her2+ cancers, including glioblastoma [ 28 ]. BsAbs against cancer antigen-125 and Her2 significantly enhance the cytotoxicity of CIK cells in primary ovarian cancer [ 29 ]. BsAb epidermal growth factor receptor/CD3 improved cytotoxic activity toward gastric cancer cells [ 30 ] and anti-CD3/anti-CD133 BbsAb against CD133 high pancreatic and hepatic cancer cells [ 31 ].

In terms of hematologic malignancies, the use of anti-CD20 mAbs [ 32 ] increased the expression of components of the mitogen-activated protein kinases/extracellular signal-regulated kinases and signal transducers and activators of transcription signaling pathways [ 33 ]. Blinatumomab (Blina, CD19xCD3) [ 34 ] or bsAb CD19xCD5 (HD37xT5.16) have been reported to increase the cytolytic activity of CIK cells against B-leukemia/lymphoma cells [ 35 ]. Combinational treatment with cell components, such as DC with or without delivering tumor-associated antigen as vaccines, and CIK cells, proved to be safe and effective. This promotes NK cell antitumor effects through direct cell-to-cell contact [ 36 , 37 ], increases the secretion of IL-12 [ 38 ], and decreases the number of Tregs. Adding myeloid-derived suppressor cell (MDSC)-reducing chemotherapy to CIK cell therapy improved survival in patients with metastatic renal and pancreatic cancer [ 39 ].

PD-1, cytotoxic lymphocyte-associated antigen 4, lymphocyte activation gene 3 (LAG-3), T-cell immunoreceptor with Ig and ITIM domains, and TIM-3 are immune CIs. The blockade of PD-L1/PD-1 augmented CIK cell cytotoxicity against gastric and colorectal cancer cells [ 40 ]. Notably, CIK cells combined with PD-1/PD-1 blockade were effective even in tumors with low or undetectable PD-L1 expression [ 41 ]. This is due to the dynamic and heterogeneous PD-L1 expression within tumors and on tumor-infiltrating lymphocytes, transmitting activatory and inhibitory signals to the innate and adaptive immune system [ 42 ]. In the tumor microenvironment, this suppresses neighboring T cells even in the absence of PD-L1+ myeloid cells and induces M2-like reprogramming of tumor-associated macrophages, promoting tumor growth and intratumoral immune tolerance. Clinical evidence shows that patients with PD-L1 low or negative tumors still respond to these treatments, indicating that PD-1/PD-L1 blocking therapies may benefit patients with cancers or tumor-infiltrating immune cells with negative or low expression levels of PD-L1 and PD-1 [ 43 , 44 ].

Recently, NK cell engagers, such as bispecific killer cell engagers and trispecific killer cell engagers, retargeting NK cells to tumors cells are being developed for clinical application, which may also benefit CIK cell interventions [ 45 ]. All these suggest that combinational treatment provides novel immunotherapy strategies for cancer treatment, increasing the cytotoxicity of ineffective or less effective autologous CIK cell approaches derived from heavily pretreated patients ( Fig. 1 ; Table 1 ).

Clinical CIK Cell Trials

In the last 3 decades, numerous preclinical evaluations and 158 clinical trials have been conducted, involving more than 5,000 patients and over 30 tumor types treated with CIK cells, including 17 phase III clinical trials ( ClinicalTrials.gov ) [ 46 , 47 ] Updated data, subdivided into allogeneic and autologous applications, are shown in Table 1 . Several advantages of this approach, such as encouraging response rates, acceptable safety profiles, and cost efficiency of CIK cell therapy generated from the cancer patient’s own immune cells, have led to its approval in many countries.

In the allogeneic hematopoietic stem cell transplantation (HSCT) setting, Introna et al. described interesting applications of donor-derived CIK cells, which were infused in patients with hematological malignancies who relapsed after HSCT [ 6 , 7 , 48 ]. They reported few cases of acute aGvHD (≤ grade II) and chronic (c) GvHD after the infusion of HLA-matched, donor-derived CIK cells. The observed alloreactivity raised concerns about the potential use of CIK cell therapy across major HLA barriers (e.g., in terms of HLA-haploidentical HSCT).

Allogeneic CIK Cells

In the allogeneic setting, it became increasingly evident that starting from allogeneic PB, BM, or CB mononuclear cells, ex vivo activation and expansion in sequential cytokine cocktails reduced potential toxicity, namely, GvHD, of mismatched or matched allogeneic T cells within the heterogenous CIK cell population [ 6 , 7 , 48 – 53 ]. We have recently demonstrated the safety and efficacy of CIK cells as a modified donor lymphocyte product, which we generated by short-term expansion (10–12 days) ex vivo in sequential cytokine cocktails, including IL-15, in several preclinical and clinical studies in adult and pediatric patients transplanted for hematological malignancies [ 16 , 22 , 23 , 54 – 56 ]. Even at 100-times higher doses and in mismatched HSCT settings, these cells – compared to conventional T cells – possessed multiple endogenous antigen-specific antipathogen and presumably nonspecific non-MHC-restricted antitumor reactivity, with negligible risk for GvHD despite rapid T-cell recovery observed in patients. IL-15 activation improved the cytotoxicity of CIK cells by increasing the number of T-NK cells, decreasing the number of Tregs, upregulating Toll-like receptor 4, wingless-related integration site (Wnt) 4, platelet-derived growth factor D, and increased secretion of proinflammatory cytokines like IL-35 [ 17 , 57 ].

Preliminary results of a subsequent early-phase multicenter dose-escalation study confirmed that IL-15 activation improved/shortened CIK cell expansion for clinical applications in patients allografted for hematological malignancies. Sequential infusion of IL-15-activated CIK cells in these patients was safe, with a low incidence of GvHD, and allowed for significantly increased T-cell doses even across HLA-mismatched barriers. Furthermore, CIK cells used in the early posttransplant period improved immune regeneration, particularly T-cell recovery, compared with conventional donor lymphocyte infusions. Durable remission was achieved mostly after treatment of molecular relapse, whereas, IL-15-activated CIK cell therapy in patients with hematological relapse was well tolerated and provided palliation for up to 9 months (manuscript in preparation). Thus, IL-15-activated CIK cells, described as direct killers and modulators of immune responses, can benefit patients who are ineligible for or have failed specific/targeted therapies posttransplant, such as acute myeloid leukemia (AML) patients. This will be evaluated in an upcoming clinical trial of pediatric AML patients at risk for relapse after allogeneic HSCT.

HSCT Donor-Derived CAR-CIK Cells

Using IL-15-activated CIK cells as “CAR carriers” may further augment specific antitumor activity of the innate and adoptive immune system without increasing risks for posttransplant toxicities. Donor-derived CAR-CIK cell generation may be based on our approved CIK cell manufacturing platform (§4b Abs. 3 AMG, license number: PEI.A.11630.01.1), including the use of IL-15 cytokines as opposed to conventional IL-2, resulting in a higher central/naïve memory phenotype, more fold expansion, and activation when starting from thawed or fresh unstimulated leukapheresis bags [ 22 ]. Lentiviral transduction performed on day two allows for the elimination of the lentiviral particles on day twelve of culture. The refined production process will be tested in an upcoming phase I trial to evaluate the safety of allogeneic anti-CD19-CAR-CIK cell products in pediatric patients with relapsed/refractory B-cell acute lymphoblastic leukemia after allogeneic HSCT.

Engineering CIK cells to express CARs using the nonviral Sleeping Beauty transposon system might have potential advantages over viral systems [ 58 – 60 ], including cost efficiency and safety. In a phase I–IIa dose-finding study evaluating the safety and efficacy of donor-derived anti-CD19-CAR-CIK-products in pediatric and adult patients with B-cell acute lymphoblastic leukemia relapses after allogeneic HSCT ( NCT03389035 ), conducted in collaboration between the Pediatric and Hematology Departments of Monza and Bergamo, Italy, encouraging complete molecular response rates and an excellent safety profile was shown, especially in patients with the highest CAR-CIK cell dose [ 61 ]. Cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome were observed in some patients but were manageable. Secondary GvHD never occurred after anti-CD19-CAR-CIK treatment. Additional preclinical studies of this group include a nonviral Sleeping Beauty platform for “off-the-shelf” CAR-CIK cell therapy from CB, as well as dual and armored CARs for selective targeting of AML cells and better homing into the BM niche [ 62 , 63 ]. Ongoing studies using retargeted CIK cells with CARs are whether their therapeutic efficacy is comparable to that of CAR-T cells.

Conclusion

Despite more or less standardized protocols for CIK cell generation, CIK cells are a heterogeneous, mostly donor-dependent population of CD3+T lymphocytes, depending on the raw material as well as the donor- and manufacturing-specific composition of CIK cells. However, this should not be considered as setback but rather the biggest advantage of this approach. As a heterogeneous cell population, CIK cells are able to modulate immune responses and directly kill heterogeneous tumor cells despite immune checkpoint and antigen escape, which are major drawbacks for antibody- or CAR-based immunotherapies [ 64 ].

It is increasingly realized that curing cancer with only one drug or therapeutic strategy is not effective. Hence, the combination of different drugs or therapies has become a standard, or at least a trend, in treating heterogeneous and/or relapsed/refractory cancers. Reviewing preclinical research and clinical trials over the past 30 years in multiple types of cancer, it becomes obvious that adoptive CIK cell therapy is safe but needs an allogeneic component or combination strategy with antibodies, CARs, DCs, MDSC-reducing chemotherapy, immune CIs, NK cell engagers, or epigenetic drugs to defeat high-risk cancer. Furthermore, both autologous and allogeneic CIK cell therapies are most effective on residual cancer cells. Altogether, promising outcomes achieved by adoptive CIK cell therapy and comparison with other therapies will define and guide best-in-class cellular therapies in the future.

Acknowledgments

The author acknowledges the support provided by the Mildred-Scheel-Nachwuchszentrum (MSNZ), Frankfurt am Main, Germany (70113301), and the Parents Association “Hilfe für krebskranke Kinder e.V.,” Frankfurt am Main, Germany (2022_01_COMBAT-AML).

Conflict of Interest Statement

The author has nothing to disclose.

Funding Sources

This work was supported by grants from the Mildred-Scheel-Nachwuchszentrum (MSNZ), Frankfurt am Main, Germany (70113301), and the Parents Association “Hilfe für krebskranke Kinder e.V.,” Frankfurt am Main, Germany (2022_01_COMBAT-AML).

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