NKG2D Engagement Alone Is Sufficient to Activate Cytokine-Induced Killer Cells While 2B4 Only Provides Limited Coactivation.

Cytokine-induced killer (CIK) cells are an ex vivo expanded heterogeneous cell population with an enriched NK-T phenotype (CD3+CD56+). Due to the convenient and relatively inexpensive expansion capability, together with low incidence of graft versus host disease (GVHD) in allogeneic cancer patients, CIK cells are a promising candidate for immunotherapy. It is well known that natural killer group 2D (NKG2D) plays an important role in CIK cell-mediated antitumor activity; however, it remains unclear whether its engagement alone is sufficient or if it requires additional co-stimulatory signals to activate the CIK cells. Likewise, the role of 2B4 has not yet been identified in CIK cells. Herein, we investigated the individual and cumulative contribution of NKG2D and 2B4 in the activation of CIK cells. Our analysis suggests that (a) NKG2D (not 2B4) is implicated in CIK cell (especially CD3+CD56+ subset)-mediated cytotoxicity, IFN-γ secretion, E/T conjugate formation, and degranulation; (b) NKG2D alone is adequate enough to induce degranulation, IFN-γ secretion, and LFA-1 activation in CIK cells, while 2B4 only provides limited synergy with NKG2D (e.g., in LFA-1 activation); and (c) NKG2D was unable to costimulate CD3. Collectively, we conclude that NKG2D engagement alone suffices to activate CIK cells, thereby strengthening the idea that targeting the NKG2D axis is a promising approach to improve CIK cell therapy for cancer patients. Furthermore, CIK cells exhibit similarities to classical invariant natural killer (iNKT) cells with deficiencies in 2B4 stimulation and in the costimulation of CD3 with NKG2D. In addition, based on the current data, the divergence in receptor function between CIK cells and NK (or T) cells can be assumed, pointing to the possibility that molecular modifications (e.g., using chimeric antigen receptor technology) on CIK cells may need to be customized and optimized to maximize their functional potential.

A CTLA-4 blocking strategy based on Nanoboby in dendritic cell-stimulated cytokine-induced killer cells enhances their anti-tumor effects.

BACKGROUND: Cytokine-induced killer cells induced with tumor antigen-pulsed dendritic cells (DC-CIK) immunotherapy is a promising strategy for the treatment of malignant tumors. However, itsefficacy isrestricted by the immunosuppression, which is mediated by the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) pathway. In order to overcome the negative co-stimulation from these T cells,we screened a nanobody targeted for CTLA-4 (Nb36) and blocked the CTLA-4 signaling with Nb36. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors to beused to induce CIK cells in vitro, after which they were co-cultured with DC cells that had received tumor antigens. In addition, wetested whether blocking CTLA-4 signaling with Nb36 could promote in vitro DC-CIK cells proliferation, pro-inflammatory cytokine production and cytotoxicity,or not. For the in vivo experiments, we constructed a subcutaneously transplanted tumor model and placed it in NOD/SCID mice to verify the anti-tumor effect of this therapy. RESULTS: After stimulation with Nb36, the DC-CIK cells presented enhanced proliferation and production of IFN-γ in vitro, which strengthened the killing effect on the tumor cells. For the in vivo experiments, it was found that Nb36-treated DC-CIK cells significantly inhibited the growth of subcutaneously transplanted livercancer tumors, as well as reduced the tumor weight and prolonged the survival of tumor-bearing NOD/SCID mice. CONCLUSIONS: Ourfindings demonstrated that in response to CTLA-4 specific nanobody stimulation, DC-CIK cells exhibited a better anti-tumor effect. In fact, this Nb-based CTLA-4 blocking strategy achieved an anti-tumor efficacy close to that of monoclonal antibodies. Our findings suggest that DC-CIK cells + Nb36 have the potential totreatmalignant tumors through in vivo adoptive therapy.

Novel Immune Cell-Based Therapies to Eradicate High-Risk Acute Myeloid Leukemia.

Adverse genetic risk acute myeloid leukemia (AML) includes a wide range of clinical-pathological entities with extremely poor outcomes; thus, novel therapeutic approaches are needed. Promising results achieved by engineered chimeric antigen receptor (CAR) T cells in other blood neoplasms have paved the way for the development of immune cell-based therapies for adverse genetic risk AML. Among these, adoptive cell immunotherapies with single/multiple CAR-T cells, CAR-natural killer (NK) cells, cytokine-induced killer cells (CIK), and NK cells are subjects of ongoing clinical trials. On the other hand, allogeneic hematopoietic stem cell transplantation (allo-HSCT) still represents the only curative option for adverse genetic risk AML patients. Unfortunately, high relapse rates (above 50%) and associated dismal outcomes (reported survival ~10-20%) even question the role of current allo-HSCT protocols and emphasize the urgency of adopting novel effective transplant strategies. We have recently demonstrated that haploidentical allo-HSCT combined with regulatory and conventional T cells adoptive immunotherapy (Treg-Tcon haplo-HSCT) is able to overcome disease-intrinsic chemoresistance, prevent leukemia-relapse, and improve survival of adverse genetic risk AML patients. In this Perspective, we briefly review the recent advancements with immune cell-based strategies against adverse genetic risk AML and discuss how such approaches could favorably impact on patients' outcomes.

Clinical efficacy analysis of Dendritic Cell-Cytokine Induced Killer Cell Immunotherapy Combined with Paclitaxel-Cisplatin Chemotherapy in patients with Advanced Ovarian Cancer.

PURPOSE: To explore the efficacy and safety of dendritic cell-cytokine induced killer cell (DC-CIK) immunotherapy combined with paclitaxel-cisplatin chemotherapy in the treatment of advanced ovarian cancer. METHODS: Patients with advanced ovarian cancer formed the Chemotherapy group (paclitaxel-cisplatin, n=68) and the other 68 patients formed the DC-CIK group (DC-CIK + paclitaxel-cisplatin, n=68). The short-term efficacy and changes in the levels of immune function indexes, serum CD133 and DEAD-box helicase 4 (DDX4) were analyzed. RESULTS: Both overall response rate (ORR) and disease control rate (DCR) in the DC-CIK group were significantly better than those in the Chemotherapy group. After treatment, the proportions of CD3+ T lymphocytes, CD3+ CD4+ T lymphocytes and NK cells and the CD4/CD8 ratio were evidently higher, while the proportion of CD4+ CD25+ T lymphocytes was evidently lower in the DC-CIK group than those in the Chemotherapy group. After treatment, the levels of basic fibroblast growth factor (bFGF), carbohydrate antigen 19-9 (CA19-9), CA125, human epididymis protein 4 (HE4), cytokeratin 19 fragment antigen 21-1 (CYFRA21-1), DDX4 and CD133 markedly declined in the two groups, and they were markedly lower in the DC-CIK group than in the Chemotherapy group. At 1 month after treatment, the score of emotional functioning in function module of QLQ-C30 scale was greatly higher in the DC-CIK group than that in the Chemotherapy group, while the score of nausea and vomiting in symptom module was greatly lower in the DC-CIK group than that in the Chemotherapy group. The follow-up analysis showed that the overall survival (OS) rate in the DC-CIK group was remarkably superior than in the Chemotherapy group. CONCLUSIONS: DC-CIK immunotherapy combined with paclitaxel-cisplatin chemotherapy can greatly improve the clinical efficacy, enhance the immune function, reduce the levels of serum tumor markers, raise the quality of life and prolong the survival in patients with advanced ovarian cancer.

Cytokine-induced killer cells/dendritic cells and cytokine-induced killer cells immunotherapy for the treatment of esophageal cancer: A meta-analysis.

OBJECTIVES: This meta-analysis was designed to systematically evaluate whether autologous cytokine-induced killer cells (CIK) or dendritic cells and cytokine-induced killer cells (DC-CIK) immunotherapy combined with chemotherapy can improve the therapeutic effect and safety of chemotherapy in esophageal cancer (EC). MATERIALS AND METHODS: Randomized controlled trials (RCTs) were electronically searched databases including CNKI, WanFang, WeiPu, CBMDisc, PubMed, Web of Science, EMbase, the Cochrane Library, and Clinical Trials. The databases were searched for articles published until June 2019. Two researchers independently screened the literature, extracted data, and evaluated the quality of the included literature. Meta-analysis was performed using RevMan5.3. RESULTS: Seventeen studies (1416 participants) were included. The differences between CIK/DC-CIK combination chemotherapy and chemotherapy alone were significant. The results displayed that the number of CD3+, CD4+, CD4+/CD8+, and NK cells was significantly increased after 1 to 2 weeks of treatment with CIK/DC-CIK cells in the treatment group (all P < .05). In addition, the results shown that 1-year overall survival was significantly prolonged (P < .0001) and quality of life was improved (P = .001) in EC chemotherapy combined with immunotherapy groups compared with conventional treatment. Furthermore, cytokine expression levels of interleukin 2 (IL-2), tumor necrosis factor α (TNF-α), and interleukin 12 (IL-12) were significantly increased (P = .0003) as well as the levels of immunoglobulins were elevated (P < .00001). Serum levels of tumor marker molecules, carcinoembryonic antigen (CEA), carbohydrate antigen (CA)-199, and CA-125 were lower in treatment groups than that of control groups (P < .00001). No fatal adverse reactions were noted (P = .04). CONCLUSIONS: It is safe and effective for patients to use chemotherapy combined with CIK/DC-CIK immunotherapy. Immunotherapy can simultaneously improve the antitumor immune response. Specifically, DC-CIK cells can increase T lymphocyte subsets, CIK cells, NK cells, and immunoglobulins in peripheral blood to enhance antitumor immunity. Therefore, combination therapy enhances the immune function and improves the therapeutic efficacy of patients with EC.

High Complete Response Rate in Patients With Metastatic Renal Cell Carcinoma Receiving Autologous Cytokine-Induced Killer Cell Therapy Plus Anti-Programmed Death-1 Agent: A Single-Center Study.

Background and Objective: The results of the CheckMate 025 trial established the status of nivolumab in the second-line treatment of metastatic renal cell carcinoma (mRCC), with an objective response rate (ORR) of 25% and a complete response (CR) rate of 1%. Thus, the efficacy of anti-programmed death (PD)-1 antibodies in the second-line treatment of mRCC requires improvement. The purpose of this study was to explore the clinical efficacy and safety of anti-PD-1 agents combined with cytokine-induced killer (CIK) cell therapy for refractory mRCC. Patients and Methods: Patients with mRCC refractory to previous targeted therapy were included in this study. All patients received anti-PD-1 plus CIK cell therapy. The ORR and CR rate, progression-free survival (PFS), overall survival (OS), and safety were assessed. Results: CR was observed in seven of the 29 patients, and partial response was observed in five patients. The ORR was 41.4% and the median PFS was 15.0 months. Up to the last follow-up, 15 patients died with an average survival time of 37 months. Among the patients who achieved a CR, one experienced cerebellar metastasis 18.8 months after discontinuation, but achieved CR again after localized gamma knife and 1-month axitinib treatment. This regimen was tolerated well and there was no treatment-related death. Conclusions: Combination therapy with anti-PD-1 and CIK cell therapy is safe and effective in patients with mRCC refractory to previous targeted therapy. The high CR rate and long disease-free survival even after long-term discontinued therapy suggest that this combination treatment may represent a potential curative regimen for this type of malignancy.

Use of an anti-CD200-blocking antibody improves immune responses to AML in vitro and in vivo.

Treatment of relapsed/resistant acute myeloid leukaemia (AML) remains a significant area of unmet patient need, the outlook for most patients remaining extremely poor. A promising approach is to augment the anti-tumour immune response in these patients; most cancers do not activate immune effector cells because they express immunosuppressive ligands. We have previously shown that CD200 (an immunosuppressive ligand) is overexpressed in AML and confers an inferior overall survival compared to CD200low/neg patients. Here we show that a fully human anti-CD200 antibody (TTI-CD200) can block the interaction of CD200 with its receptor and restore AML immune responses in vitro and in vivo.

Analysis of the Clinical Efficacy of Dendritic Cell -cytokine Induced Killer Cell-based Adoptive Immunotherapy for Colorectal Cancer.

Background: To analyze the efficacy and safety of dendritic cell - cytokine - induced killer (DC-CIK) immunotherapy combined with chemotherapy for colorectal cancer. Method: A retrospective analysis was conducted in 116 patients from February 2012 to December 2017, who were divided into postoperative adjuvant chemotherapy group alone, combined DC-CIK immunotherapy group, advanced cancer palliative care group, and palliative care + DC-CIK immunotherapy group, to evaluate cellular immune function, disease-free survival(DFS) and overall survival(OS). Results: In the adjuvant therapy and palliative care group, the percentages of CD3+, CD8+ and NK cells after treatment were significantly lower than before, whereas in the other two groups given DC-CIK immunotherapy, the percentages of CD3+, CD8+, NK and NKT cells after treatment were all higher than before, with a significant increase compared with the chemotherapy group (P < .05). DFS (42.4 ± 5.26 m) in the group receiving postoperative adjuvant chemotherapy + DC-CIK immunotherapy was significantly longer than that (23.5 ± 2.79 m) in the group only given postoperative adjuvant chemotherapy (P < .05). OS in the group receiving palliative care + DC-CIK immunotherapy was slightly longer than that in the group only given palliative care for advanced cancer (29 m vs 26 m, P > .05).Conclusion: Combination with DC-CIK immunotherapy could effectively improve cellular immune function. Postoperative adjuvant chemotherapy in combination with DC-CIK immunotherapy could significantly prolong DFS, but palliative care in combination with DC-CIK immunotherapy did not significantly prolong OS in patients with advanced cancer.

MiR-374b increases the CIK expression and mediates biological function changes in cervical cancer cells by targeting the PD-1/PD-L1 signaling pathway.

OBJECTIVE: To investigate the role of miR-374b in medicating biological function changes in cervical cancer cells by increasing the cytokine-induced killer (CIK) expression. METHODS: Venous blood of 62 cervical cancer patients and 58 healthy individuals including Human cervical cancer cell line (HeLa) and normal human uterine smooth muscle cells (HUSMC) were tested for expression of miR-374b, PD-1, and PD-L1. sh-PD-1, si-PD-1, NC, miR-374b-inhibitor, and miR-374b-mimics were transfected into HeLa cells, and expression of miR-374b, PD-1, and PD-L1 was determined by a qRT-PCR assay, and the proliferation and apoptosis of the cells were detected using a MTT assay and flow cytometry, respectively. RESULTS: PD-1 was highly expressed in cervical cancer, while miR-374b is lowly expressed in it, and the area-under-the-curve (AUC) of both PD-1 and miR-374b was larger than 0.8. The dual luciferase reporter assay confirmed relationship between PD-1 and miR-374b. Expression of PD-1 in HeLa cells was significantly down-regulated after transfection of miR-374b-mimics. Compared with the CIK + NC group, the CIK + miR-374b-mimics group and the CIK + siRNA-PD-1 group showed a significant decrease in the relative mRNA expression of PD-1, compared with other group showed significantly lowered activity of HeLa cells, and the two groups showed significantly reduced tumor volume. CONCLUSION: MiR-374b increases the CIK expression and mediates biological function changes in cervical cancer cells by targeting the PD-1/PD-L1 signaling pathway, so it is expected to be a potential therapeutic target for cervical cancer.

Efficacy of dendritic cell-cytokine induced killer cells combined with concurrent chemoradiotherapy on locally advanced non-small cell lung cancer.

PURPOSE: To explore the efficacy and safety of docetaxel/cisplatin concurrent chemoradiotherapy (CCRT) combined with dendritic cell-cytokine induced killer cell (DC-CIK) immunotherapy in the treatment of locally advanced non-small cell lung cancer (LANSCLC). METHODS: The clinical data of 142 LANSCLC patients treated in our hospital from March 2014 to March 2016 were retrospectively analyzed. 71 patients were treated with docetaxel/cisplatin CCRT (CCRT group), while the remaining 71 patients underwent CCRT combined with DC-CIK immunotherapy (DC-CIK group). The clinical data of all patients were collected, the short-term efficacy, the changes in serum immunological indexes and quality of life before and after treatment, and the incidence of adverse reactions were compared between the two groups, and the overall survival (OS) and progression-free survival (PFS) were recorded during the follow-up of patients. RESULTS: After treatment, the level of cluster of differentiation 3+ (CD3+) CD4+ T lymphocytes, CD4/CD8 ratio and CD56+ natural killer (NK) cell ratio significantly rose, while the level of CD3+ CD8+ T lymphocytes significantly declined in both groups compared with those before treatment. After treatment, the level of CD3+ CD4+ T lymphocytes, CD4/CD8 ratio and CD56+ NK cell ratio were obviously higher, while the level of CD3+ CD8+ T lymphocytes was obviously lower in DC-CIK group than those in CCRT group. At 12 months after treatment, both Karnofsky performance scale (KPS) score and quality of life (QOL) score in DC-CIK group were evidently higher than those in CCRT group. In CCRT group and DC-CIK group, 1-year OS was 74.6% and 83.1%, and 1-year PFS was 70.4% and 73.2%, respectively. 2-year OS was 45.1% and 57.7%, and 2-year PFS was 38.0% and 46.5%), respectively. 3-year OS was 26.8% and 40.8%, and 3-year PFS was 15.5% and 22.5%, respectively. It can be seen that both OS and PFS in DC-CIK group were remarkably superior to those in CCRT group. CONCLUSION: Docetaxel/cisplatin CCRT combined with DC-CIK can significantly enhance the cellular immunity, improve the long-term survival rate and raise the quality of life of LANSCLC patients, with tolerable adverse reactions.

ERBB2-CAR-Engineered Cytokine-Induced Killer Cells Exhibit Both CAR-Mediated and Innate Immunity Against High-Risk Rhabdomyosarcoma.

High-risk rhabdomyosarcoma (RMS) occurring in childhood to young adulthood is associated with a poor prognosis; especially children above the age of 10 with advanced stage alveolar RMS still succumb to the disease within a median of 2 years. The advent of chimeric antigen receptor (CAR)-engineered T cells marked significant progress in the treatment of refractory B cell malignancies, but experience for solid tumors has proven challenging. We speculate that this is at least in part due to the poor quality of the patient's own T cells and therefore propose using CAR-modified cytokine-induced killer (CIK) cells as effector cells. CIK cells are a heterogeneous population of polyclonal T cells that acquire phenotypic and cytotoxic properties of natural killer (NK) cells through the cultivation process, becoming so-called T-NK cells. CIK cells can be genetically modified to express CARs. They are minimally alloreactive and can therefore be acquired from haploidentical first-degree relatives. Here, we explored the potential of ERBB2-CAR-modified random-donor CIK cells as a treatment for RMS in xenotolerant mice bearing disseminated high-risk RMS tumors. In otherwise untreated mice, RMS tumors engrafted 13-35 days after intravenous tumor cell injection, as shown by in vivo bioluminescence imaging, immunohistochemistry, and polymerase chain reaction for human gDNA, and mice died shortly thereafter (median/range: 62/56-66 days, n = 5). Wild-type (WT) CIK cells given at an early stage delayed and eliminated RMS engraftment in 4 of 6 (67%) mice, while ERBB2-CAR CIK cells inhibited initial tumor load in 8 of 8 (100%) mice. WT CIK cells were detectable but not as active as CAR CIK cells at distant tumor sites. CIK cell therapies during advanced RMS delayed but did not inhibit tumor progression compared to untreated controls. ERBB2-CAR CIK cell therapy also supported innate immunity as evidenced by selective accumulation of NK and T-NK cell subpopulations in disseminated RMS tumors, which was not observed for WT CIK cells. Our data underscore the power of heterogenous immune cell populations (T, NK, and T-NK cells) to control solid tumors, which can be further enhanced with CARs, suggesting ERBB2-CAR CIK cells as a potential treatment for high-risk RMS.

Presence of the Transmembrane Protein Neuropilin in Cytokine-induced Killer Cells.

BACKGROUND/AIM: Cytokine-induced killer (CIK) cells are a heterogenous population of immune cells showing promising applications in immunotherapeutic cancer treatment. Neuropilin (NRP) proteins have been proven to play an important role in cancer development and prognosis. In this study, CIK cells were tested for expression of NRPs, transmembrane proteins playing a role in the proliferation and survival of cancer cells. MATERIALS AND METHODS: CIK cells were analyzed at different time points via flow cytometry and quantitative real-time polymerase chain reaction for neuropilin expression. RESULTS: Phenotyping results showed CIK cells having developed properly, and low levels of NRP2 were detected. On the other hand, no NRP1 expression was found. Two cancer cell lines were tested by flow cytometry: A549 cells expressed NRP1 and NRP2; U251-MG cells expressed high amounts of NRP2. CIK cell showed low levels of NRP2 expression on day 14. CONCLUSION: The presence of NRP2, but not NRP1, was shown for CIK cells. Recognizing NRP2 in CIK cells might help to improve CIK cell cytotoxicity.

Cytokine-induced killer cells-assisted tumor-targeting delivery of Her-2 monoclonal antibody-conjugated gold nanostars with NIR photosensitizer for enhanced therapy of cancer.

Maximizing the accumulation of anticancer medicine in the tumor is the priority to achieve minimal invasive cancer therapy, which raises high demands on tumor-targeting ability of drug delivery systems. Herein, we adopted an emerging "cell-drug" strategy via the nanoplatform construction to achieve high aggregation and intratumoral distribution. We fabricated gold nanostars (GNSs) with HER-2 monoclonal antibody (trastuzumab) and near-infrared region (NIR) photosensitizer indocyanine green (ICG) to obtain GNS@ICG-Ab, which combined the photothermal therapy with photodynamic therapy (PTT/PDT) that rely on enhanced photothermal conversion efficiency of GNS and 1O2 generator ICG under the exposure of a NIR laser. Tumor-tropism CIK cells loaded with GNS@ICG-Ab were able to migrate into tumors and make a difference in efficient accumulation and uniform distribution of the GNS@ICG-Ab-CIK nanoplatform inside tumors based on fluorescence, photoacoustic (PA), and computed tomography (CT) imaging observations. Encouraged by the improvements in tumor targeting and retention presented by real-time imaging, we employed the novel nanoplatform to synergistically inhibit the progression of tumors in SK-BR-3 tumor-bearing mice via PTT/PDT and immunotherapy-implemented by CIK cells for activating the immune response, and with the specific linkage between trastuzumab and SK-BR-3 tumor cells, our platform could exert a precise strike of PDT/PTT. Taken together, the integrating tri-modal imaging with tri-modal therapy endows CIK-GNS@ICG-Ab with promising potential in cancer theranostics and lays a solid foundation for the development of immune cell application in nanomedicine delivery.

CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes.

PURPOSE: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes in vitro and in immunodeficient mice. EXPERIMENTAL DESIGN: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored in vitro, in two- and three-dimensional STS cultures, and in three in vivo STS xenograft models. RESULTS: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by in silico analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior in vitro cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in vivo in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK. CONCLUSIONS: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes in vitro and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting.

Multidirectional Strategies for Targeted Delivery of Oncolytic Viruses by Tumor Infiltrating Immune Cells.

Oncolytic virus (OV) immunotherapy has demonstrated to be a promising approach in cancer treatment due to tumor-specific oncolysis. However, their clinical use so far has been largely limited due to the lack of suitable delivery strategies with high efficacy. Direct 'intratumoral' injection is the way to cross the hurdles of systemic toxicity, while providing local effects. Progress in this field has enabled the development of alternative way using 'systemic' oncolytic virotherapy for producing better results. One major potential roadblock to systemic OV delivery is the low virus persistence in the face of hostile immune system. The delivery challenge is even greater when attempting to target the oncolytic viruses into the entire tumor mass, where not all tumor cells are equally exposed to exactly the same microenvironment. The microenvironment of many tumors is known to be massively infiltrated with various types of leucocytes in both primary and metastatic sites. Interestingly, this intratumoral immune cell heterogeneity exhibits a degree of organized distribution inside the tumor bed as evidenced, for example, by the hypoxic tumor microenviroment where predominantly recruits tumor-associated macrophages. Although in vivo OV delivery seems complicated and challenging, recent results are encouraging for decreasing the limitations of systemically administered oncolytic viruses and an improved efficiency of oncolytic viral therapy in targeting cancerous tissues in vitro. Here, we review the latest developments of carrier cell-based oncolytic virus delivery using tumor-infiltrating immune cells with a focus on the main features of each cellular vehicle.

Cytokine-induced Killer T Cells Enhance the Cytotoxicity Against Carboplatin-resistant Ovarian Cancer Cells.

BACKGROUND/AIM: Ovarian cancer (OC) is typically diagnosed at an advanced stage with limitations for cure. Cytokine-induced killer (CIK) T cell therapy exerts significant cytotoxic effects against cancer cells and reduces the adverse effects of chemotherapy. Herein, we performed a flow cytometry-based method to evaluate the cytotoxicity of peripheral blood mononuclear cells-derived CIK cells against OC cells. MATERIALS AND METHODS: The CIK cells were induced and expanded using an interferon-γ/IL-2-based xeno-free medium system. The cytotoxicity of CIK cells or carboplatin against OC cells was examined. RESULTS: The CIK cells showed an NK-like phenotypic characteristic and dose-dependently increased cytotoxicity against OC cells. We found that the number of advanced OC cells, which were more resistant to carboplatin, was dramatically decreased by an additional one-shot CIK treatment. CONCLUSION: CIK cells have a potent cytotoxic ability that would be explored as an alternative strategy for cancer treatment in the near future.

A serum-free protocol for the ex vivo expansion of Cytokine-Induced Killer cells using gas-permeable static culture flasks.

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-γ, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.

Targeting CD33 in Chemoresistant AML Patient-Derived Xenografts by CAR-CIK Cells Modified with an Improved SB Transposon System.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.

Ten-year update of the international registry on cytokine-induced killer cells in cancer immunotherapy.

Cytokine-induced killer (CIK) cells represent an exceptional T-cell population uniting a T cell and natural killer cell-like phenotype in their terminally differentiated CD3+ CD56+ subset, which features non-MHC-restricted tumor-killing activity. CIK cells have provided encouraging results in initial clinical studies and revealed synergistic antitumor effects when combined with standard therapeutic procedures. We established the international registry on CIK cells (IRCC) to collect and evaluate clinical trials for the treatment of cancer patients in 2010. Moreover, our registry set new standards on the reporting of results from clinical trials using CIK cells. In the present update, a total of 106 clinical trials including 10,225 patients were enrolled in IRCC, of which 4,889 patients in over 30 distinct tumor entities were treated with CIK cells alone or in combination with conventional or novel therapies. Significantly improved median progression-free survival and overall survival were shown in 27 trials, and 9 trials reported a significantly increased 5-year survival rate. Mild adverse effects and graft-versus-host diseases were also observed in the studies. Recently, more efforts have been put into the improvement of antitumoral efficacy by CIK cells including the administration of immune checkpoint inhibitors and modification with chimeric antigen receptorc. The minimal toxicity and multiple improvements on their tumor-killing activity both make CIK cells a favorable therapeutic tool in the clinical practice of cancer immunotherapy.

Clinical Trials with Cytokine-Induced Killer Cells and CAR-T Cell Transplantation for Non-small Cell Lung Cancer Treatment.

The idea of utilizing the human immune system to eradicate tumors has been successfully practiced for the past decades, as reported in multiple published studies. Among cancer types, non-small cell lung cancer (NSCLC) is considered the most lethal type, leading to the necessity of finding an effective treatment for this category of cancer. Building on the success of basic and preclinical studies, numerous clinical trials of cytokine-induced killer (CIK) cells or chimera antigen receptor (CAR) T cells for NSCLC therapy have been reported. In this review paper, we will summarize those findings in the context of clinical outcomes and adverse effects. In NSCLC, compared to CAR-T cells, CIK cells show relatively stronger antitumor efficacy and lower adverse effects. More clinical studies are needed to further confirm the clinical efficiency of both types of cellular immunotherapy.