Cytokine-induced Killer Cell Infusion Research Articles

Impact of natural killer cells on outcomes after allogeneic hematopoietic stem cell transplantation: A systematic review and meta-analysis.

BackgroundNatural killer (NK) cells play a vital role in early immune reconstitution following allogeneic hematopoietic stem cell transplantation (HSCT).MethodsA literature search was performed on PubMed, Cochrane, and Clinical trials.gov through April 20, 2022. We included 21 studies reporting data on the impact of NK cells on outcomes after HSCT. Data was extracted following the PRISMA guidelines. Pooled analysis was done using the meta-package (Schwarzer et al.). Proportions with 95% confidence intervals (CI) were computed.ResultsWe included 1785 patients from 21 studies investigating the impact of NK cell reconstitution post-HSCT (8 studies/1455 patients), stem cell graft NK cell content (4 studies/185 patients), therapeutic NK cell infusions post-HSCT (5 studies/74 patients), and pre-emptive/prophylactic NK cell infusions post-HSCT (4 studies/77 patients). Higher NK cell reconstitution was associated with a better 2-year overall survival (OS) (high: 77%, 95%CI 0.73-0.82 vs low: 55%, 95%CI 0.37-0.72; n=899), however, pooled analysis for relapse rate (RR) or graft versus host disease (GVHD) could not be performed due to insufficient data. Higher graft NK cell content demonstrated a trend towards a better pooled OS (high: 65.2%, 95%CI 0.47-0.81 vs low: 46.5%, 95%CI 0.24-0.70; n=157), lower RR (high: 16.9%, 95%CI 0.10-0.25 vs low: 33%, 95%CI 0.04-0.72; n=157), and lower acute GVHD incidence (high: 27.6%, 95%CI 0.20-0.36 vs low: 49.7%, 95%CI 0.26-0.74; n=157). Therapeutic NK or cytokine-induced killer (CIK) cell infusions for hematologic relapse post-HSCT reported an overall response rate (ORR) and complete response (CR) of 48.9% and 11% with CIK cell infusions and 82.8% and 44.8% with NK cell infusions, respectively. RR, acute GVHD, and chronic GVHD were observed in 55.6% and 51.7%, 34.5% and 20%, and 20.7% and 11.1% of patients with CIK and NK cell infusions, respectively. Pre-emptive donor-derived NK cell infusions to prevent relapse post-HSCT had promising outcomes with 1-year OS of 69%, CR rate of 42%, ORR of 77%, RR of 28%, and acute and chronic GVHD rates of 24.9% and 3.7%, respectively.ConclusionNK cells have a favorable impact on outcomes after HSCT. The optimal use of NK cell infusions post-HSCT may be in a pre-emptive fashion to prevent disease relapse.

Autologous cytokine-induced killer (CIK) cells enhance the clinical response to PD-1 blocking antibodies in patients with advanced non-small cell lung cancer: A preliminary study.

BackgroundProgrammed death‐1 (PD‐1) blocking antibodies have been shown to improve progression‐free survival (PFS) and overall survival in a subset of patients with non–small cell lung cancer (NSCLC). However, the objective response rate with these agents remains low, and the vast majority of NSCLC patients require alternative combination treatment regimens to prolong their survival. The purpose of this study was to evaluate the clinical efficacy of autologous cytokine‐induced killer (CIK) cell infusions combined with PD‐1 blocking antibodies in patients with NSCLC.MethodsIn this preliminary study, we investigated the safety and immune function effectiveness of PD‐1 blockade antibodies pembrolizumab or nivolumab administered in combination with or without autologous CIK cell infusions in 18 patients with advanced NSCLC. The peripheral blood mononuclear cells were isolated from these patients and the expression level of some cell surface molecules like PD‐1 were detected using flow cytometry to reflect the effectiveness of this combination regimen.ResultsNo treatment‐related deaths occurred in either cohort. In comparison with the pretreatment level, CD3+CD56+CD16+ T cells were significantly increased with the combination therapy, while myeloid‐derived suppressor cells were significantly increased with PD‐1 blocking antibody therapy alone but not with combination therapy. Although the serum interleukin‐4 level was downregulated following treatment with the combination regimen, interferon‐γ levels were unchanged.ConclusionsThe purpose of this clinical study was to report the clinical efficacy and lack of exacerbated autoimmune adverse events with a combination of PD‐1 blockade and CIK cell infusions in patients with advanced NSCLC, further supporting assessments of this combination in future clinical trials.

418P Analysis of the efficacy of cytokine-induced killer cell therapy in adjuvant treatment of colorectal cancer

In the past decade, cell-based immunotherapy has been reported to improve clinical outcomes in cancer patients by altering tumour immune responses and improving prognosis and overall survival rates. This retrospective study aimed to evaluate the efficacy of cytokine-induced killer (CIK) cell infusion as an adjuvant therapy in patients with colorectal cancer. A total of 764 patients with colorectal cancer admitted to the Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute from June 2006 to September 2017 were retrospectively analysed. All patients were treated with surgery for primary lesions, and then adjuvant chemotherapy was administered according to the NCCN guidelines. Among these patients, 394 patients received CIK therapy (CIK group), while the other 370 patients who had similar demographic and clinical characteristics did not receive CIK cell infusion therapy (non-CIK group). Patients were followed up and data were analysed by χ2 test, Kaplan-Meier and Cox regression. Our results showed that the 3- and 5-year OS rates were 88.71% and 83.68%, respectively, in the CIK group, and 79.24% and75.3%, respectively, in the non-CIK group. The OS in the CIK group was significantly higher than in the non-CIK group (P<0.01). At different stages according to AJCC 8 Cancer Staging Manual, the OS for patients with stage I CRC was not statistically significant between the two groups, P=0.195. The OS of patients with stage II∼IV CRC in the CIK group was significantly higher than in the non-CIK group, P values were 0.001, 0.033 and 0.008, respectively. Differentiation of primary tumour and CIK cell treatment were significantly associated with improved OS in the univariate analysis. Further multivariate survival analysis indicated that CIK cell treatment remained associated with improved OS. In addition, the OS in the chemotherapy plus CIK group was significantly higher than in the chemotherapy alone group (P < 0.001). The side effects in the chemotherapy plus CIK group were lower than in the chemotherapy alone group. The 5-year OS increased with the increase in cycles of treatment, which was statistically significant. CIK cell therapy can improve the efficacy of adjuvant chemotherapy in patients with colorectal cancer. Multicentre, large-sample randomized controlled trials are still needed to confirm these results.

Analysis of the efficacy of CIK therapy in adjuvant treatment of colorectal cancer.

e15022 Background: In the past decade, cell-based immunotherapy has been reported to improve the clinical outcomes by altering tumor immune responses, improving prognosis and overall survival rates in cancer patients.This retrospective study aimed to evaluate the efficacy of cytokine-induced killer (CIK) cell infusion as an adjuvant therapy in patients with colorectal cancer. Methods: A total of 370 patients with colorectal cancer admitted to Cancer Hospital of China Medical University, Liaoning Cancer Hospital &amp; Insititute from March 2013 to April 2015 were retrospectively analyzed.All patients were treated with surgery for primary lesions, and then adjuvant chemotherapy was determined according to the guidelines. Among these patients, 201 patients received CIK therapy (CIK group),while the other 169 patients who had similar demographic and clinical characteristics did not receive a CIK cell infusion therapy (non-CIK group). Then we followed up these patients. Data were analyzed by Kaplan-Meier. Results: Our results showed that the 1,3,5-year overall survival (OS) rate for the CIK group versus the non-CIK group was 98.46 versus 93.61%, 85.4 versus 71.61%, 79.39 versus 67.9%, respectively. The OS was significantly longer in CIK group than in non-CIK group (P = 0.009). Meanwell, the OS of stage II and III colorectal cancer was significant in CIK group than in non-CIK group (P = 0.02 and 0.02). But the OS was not significant in Stage I and IV colorectal cancer (P = 0.347 and 0.285). We analyzed the patients between chemotherapy plus CIK therapy group and chemotherapy only group. We find that the OS has significant difference in two groups (P &lt; 0.001). Conclusions: CIK therapy can improve the efficacy of adjuvant therapy in patients with colorectal cancer. But multicenter, large-sample randomized controlled trials are still needed to confirm it.

Restoration of CD3+CD56+ cell level improves skin lesions in severe psoriasis: A pilot clinical study of adoptive immunotherapy for patients with psoriasis using autologous cytokine-induced killer cells

Psoriasis is a chronic inflammatory skin disorder mediated by the cells and molecules of both the innate and adaptive immune systems. Autologous cytokine-induced killer (CIK) cell infusion is considered an effective and safe cancer treatment and is licensed for this use in China. Accumulated evidence indicating that CD3+CD56+ cells are significantly decreased in psoriatic patients prompted us to investigate if the restoration of CD3+CD56+ cells may be beneficial for psoriatic patients. We designed a clinical trial for psoriasis treatment that involved CIK cell infusion because CIK cells include a large amount of CD3+CD56+ T cells (NCT01894373 at www.clinicaltrials.gov). Six patients with severe psoriasis were initially enrolled, and four of them exhibited markedly lower levels of CD3+CD56+ cells in their peripheral blood (PB) relative to healthy donors. CIK cell infusion-associated toxicity was not observed in any infusion. The percentage of CD3+CD56+ cells in the PB markedly increased and the psoriasis area and severity index (PASI) synchronously decreased in four patients with lower CD3+CD56+ cell contents, and two of them obtained a more than 4-month PASI75 after completing a four-cycle treatment. However, a decrease in the CD3+CD56+ cells was observed concomitantly with disease recurrence after short-term amelioration. In contrast, no obvious improvement was observed in the two patients with nearly normal CD3+CD56+ cells in the PB before treatment. These observations suggest that the normalization of the CD3+CD56+ cell level may improve the skin lesions of severe psoriasis and warrant further clinical trials for severe psoriasis using repeated CIK adoptive immunotherapy.

Epstein-Barr virus–specific cytokine-induced killer cells for treatment of Epstein-Barr virus–related malignant lymphoma

BackgroundProlonged immunosuppression or delayed T-cell recovery may favor Epstein-Barr virus (EBV) infection or reactivation after allogeneic hematopoietic stem cell transplantation (HSCT), which can lead to post-transplant lymphoproliferative disease (PTLD) and high-grade malignant B-cell lymphoma. Cytokine-induced killer (CIK) cells with dual specific anti-tumor and virus-specific cellular immunity may be applied in this context. MethodsCIK cells with EBV-specificity were generated from peripheral blood mononuclear cells (PBMCs), expanded in the presence of interferon-γ, anti-CD3, interleukin (IL)-2 and IL-15 and were pulsed twice with EBV consensus peptide pool. CIK cells with EBV-specificity and conventional CIK cells were phenotypically and functionally analyzed. Additionally, CIK cells with EBV-specificity were applied to a patient with EBV-related PTLD rapidly progressing to highly aggressive B-cell lymphoma on a compassionate use basis after approval and agreement by the regulatory authorities. ResultsPre-clinical analysis showed that generation of CIK cells with EBV-specificity was feasible. In vitro cytotoxicity analyses showed increased lysis of EBV-positive target cells, enhanced proliferative capacity and increased secretion of cytolytic and proinflammatory cytokines in the presence of EBV peptide-displaying target cells. In addition, 1 week after infusion of CIK cells with EBV-specificity, the patient's highly aggressive B-cell lymphoma persistently disappeared. CIK cells with EBV-specificity remained detectable for up to 32 days after infusion and infusion did not result in acute toxicity. DiscussionThe transfer of both anti-cancer potential and T-cell memory against EBV infection provided by EBV peptide-induced CIK cells might be considered a therapy for EBV-related PTLD.

Enhanced antitumor effects and improved immune status of dendritic cell and cytokine-induced killer cell infusion in advanced cancer patients.

Little progress has been made in the treatment of advanced cancer. Dendritic cells (DCs) plus cytokine-induced killer (CIK) cells have exhibited antitumor effects. Thus, the aim of the present study was to evaluate the clinical efficacy of DC-CIK cell treatment in patients with advanced cancer. A paired study including 57 patients treated with DC-CIK cells (DC-CIK group) and 33 patients treated with best supportive care alone (BSC group) was performed. The patients in the DC-CIK group were matched to those in the control group in terms of sex, age, tumor type and clinical stage. T-cell subsets were detected and overall survival (OS) was compared between the two groups. The results demonstrated that CD4+/CD25+ and CD8+/CD28- subsets significantly decreased following DC-CIK immunotherapy (P<0.05). The CD3+, CD3+/CD8+, CD8+/CD28+ and CD3+/CD56+ T-cell subsets were significantly increased in the DC-CIK group compared with the BSC group, while the CD8+/CD28- subset was significantly decreased. Univariate analysis demonstrated that a lower CD8+/CD28- and a higher CD8+/CD28+ ratio were associated with prolonged OS in advanced cancer patients. In addition, DC-CIK treatment administration, age (>60 vs. <60 years), clinical stage and the frequency of CIK treatment significantly affected the OS of patients in the DC-CIK group. A CD8+/CD28- ratio of <21.12 was found to decrease the hazard ratio (HR) of OS to 0.50 [95% confidence interval (CI): 0.29-0.87] and a CD8+/CD28+ ratio >9.04 was found to decrease the HR of OS to 0.45 (95% CI: 0.21-0.98). No serious side effects were observed in the DC-CIK group. Taken together, these data indicate that DC-CIK infusions were able to change the ratios of the T-cell subsets, which increased the T helper cell and cytotoxic T lymphocyte subsets, while it decreased regulatory T lymphocyte subsets. Thus, this method of immunotherapy was found to improve the imbalance in the immune system and prolong the OS in patients with advanced cancer.

Effects of Autologous Cytokine-Induced Killer Cells Infusion in Colorectal Cancer Patients: A Prospective Study.

To evaluate the efficacy and safety of postoperative adjuvant immunotherapy with cytokine-induced killer (CIK) cells in combination with chemotherapy (CT) in colorectal cancer (CRC) patients. A total of 46 patients were randomly assigned to either group 1 (control group) or group 2 (CIK group) using blocked randomization. Both groups received the FOLFOX4 (5-fluorouridine, leucovorin, and oxaliplatin) CT. In the CIK group, patients were given CIK cell infusion after FOLFOX4 CT. Treatment efficacy, adverse effects, and quality of life (QOL) were assessed. During the first 2 years of follow-up, the recurrence rate in the CIK group (26.1%, 6 in 23 cases) was significantly lower than the control group (43.5%, 10 in 23). The survival time was significantly longer in the CIK group (41.9 months, 95% confidence interval [CI]: 38.2-45.7) than in the control group (33.8 months, 95% CI: 28.4-39.2). Although QOL was reduced in both treatment groups, adjuvant CIK cell transfusion significantly improved the QOL in patients with CRC. Toxicity was mild in patients with CIK treatment. Immunotherapy with CIK cells may serve as an adjuvant treatment in patients with CRC after CT with prolonged survival of patients, limited side-effects, and improved QOL.

Phase II Study of Sequential Infusion of Donor Lymphocyte Infusion and Cytokine-Induced Killer Cells for Patients Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.

Cytokine-induced killer cell therapy for modulating regulatory T cells in patients with non-small cell lung cancer.

Previous studies have reported that regulatory T cells (Tregs), which are physiologically engaged in the maintenance of immunological self-tolerance, have a critical role in the regulation of the antitumor immune response. Targeting Tregs has the potential to augment cancer vaccine approaches. The current study therefore aimed to evaluate the role of cytokine-induced killer (CIK) cell infusion in modulating Tregs in patients with non-small cell lung cancer (NSCLC). A total of 15 patients with advanced NSCLC were treated by an infusion of CIK cells derived from autologous peripheral blood mononuclear cells (PBMCs). By using flow cytometry and liquid chip analysis, subsets of T cells and natural killer (NK) cells in peripheral blood, and plasma cytokine profiles in the treated patients, were analyzed at 2 and 4 weeks after CIK cell infusion. Cytotoxicity of PBMCs (n=15) and NK cells (n=6) isolated from NSCLC patients was evaluated before and after CIK cell therapy. Progression-free survival (PFS) and overall survival (OS) were also assessed. Analysis of the immune cell populations before and after treatment showed a significant increase in NK cells (P<0.05) concomitant with a significant decrease in Tregs (P<0.01) at 2 weeks post-infusion of CIK cells compared with the baseline. NK group 2D receptor (NKG2D) expression on NK cells was also significantly increased at 2 weeks post-infusion compared with the baseline (P<0.05). There was a positive correlation between NKG2D expression and the infusion number of CIK cells (P<0.05). When evaluated at 2 weeks after CIK cell therapy, the cytotoxicity of PBMCs and isolated NK cells was significantly increased compared with the baseline (P<0.01 and P<0.05). Correspondingly, plasma cytokine profiles showed significant enhancement of the following antitumor cytokines: Interferon (IFN)-γ (P<0.05), IFN-γ-inducible protein 10 (P<0.01), tumor necrosis factor-α (P<0.001), granulocyte-macrophage colony-stimulating factor (P<0.01), monocyte chemotactic protein-3 (P<0.01) and interleukin-21 (P<0.05) at 2 weeks post-infusion, compared with the baseline. At the same time, the expression of transforming growth factor-β1, which is primarily produced by Tregs, was significantly decreased compared with the baseline (P<0.05). Median PFS and OS in the CIK cell treatment group were significantly increased compared with the control group (PFS, 9.98 vs. 5.44 months, P=0.038; OS, 24.17 vs. 20.19 months, P=0.048). No severe side-effects were observed during the treatment period. In conclusion, CIK cell therapy was able to suppress Tregs and enhance the antitumor immunity of NK cells in advanced NSCLC patients. Therefore, CIK cell treatment may improve PFS and OS in patients with advanced NSCLC. CIK cell infusion may have therapeutic value for patients with advanced NSCLC, as a treatment that can be combined with chemotherapy and radiotherapy.

Cytokine-Induced Killer Cells Kill Chemo-surviving Melanoma Cancer Stem Cells.

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses.Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4 Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models.Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP- counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT-immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01).Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277-88. ©2016 AACR.

Efficacy of cytokine-induced killer cell infusion as an adjuvant immunotherapy for hepatocellular carcinoma: a systematic review and meta-analysis.

This study was designed to evaluate the efficacy and safety of cytokine-induced killer (CIK) cell-based immunotherapy as an adjuvant therapy for hepatocellular carcinoma (HCC). Published studies were identified by searching Medline, Cochrane, EMBASE, and Google Scholar databases with the keywords: cytokine-induced killer cell, hepatocellular carcinoma, and immunotherapy. The outcomes of interest were overall survival, progression-free survival, and disease-free survival. Eight randomized controlled trials (RCTs), six prospective studies, and three retrospective studies were included. The overall analysis revealed that patients in the CIK cell-treatment group had a higher survival rate (pooled hazard ratio (HR) =0.594, 95% confidence interval [CI] =0.501–0.703, P<0.001). Patients treated with CIK cells in non-RCTs had a higher progression-free survival rate (pooled HR =0.613, 95% CI =0.510–0.738, P<0.001). However, CIK cell-treated patients in RCTs had progression-free survival rates similar to those of the control group (pooled HR =0.700, 95% CI =0.452–1.084, P=0.110). The comparison between pooled results of RCTs and non-RCTs regarding the progression-free survival rate did not reach statistical significance. Patients in the CIK cell-treatment group had lower rates of relapse in RCTs (pooled HR =0.635, 95% CI =0.514–0.784, P<0.001). Similar results were found when non-RCT and RCTs were pooled (pooled HR =0.623, 95% CI =0.516–0.752, P<0.001). Adjuvant CIK cell-based immunotherapy is a promising therapeutic approach that can improve overall survival and reduce recurrence in patients with HCC.

Final Analysis of a Multicenter Pilot Phase 2 Study of Cytokine Induced Killer (CIK) Cells for Patients with Relapse after Allogeneic Transplantation

Introduction: Patients with disease relapse after allogeneic transplantation (alloHSCT) have a poor prognosis. Donor lymphocyte infusion (DLI) is one of the main clinical options to salvage patients in relapse after transplant. Cytokine Induced Killer (CIK) cells are in vitro activated and expanded T lymphocytes which have acquired NK like cytotoxicity as well as CD56 expression. CIK cells have shown Graft versus Leukemia (GvL) activity with little GvHD and therefore may represent an ideal candidate to treat post-transplant relapse. We report the final results of a phase II multicenter pilot study, whose objective was to evaluate the safety and efficacy of sequential administration of donor derived unmanipulated DLI and CIK cells in patients with recurrent hematologic cancers after alloHSCT.MethodsSeventy-four patients relapsed after alloHSCT, performed using either a matched related (N=42) or unrelated donor (n= 32), were enrolled in the study. This phase II multicenter study was authorized by Istituto Superiore di Sanità, as for Advanced Therapeutic Medicinal Product (ATMP) regulations, and approved by the Agenzia Italiana del Farmaco (AIFA). The trial was registered as (EUDRACT number 2008-003185-26, ClinicalTrial.gov : NCT01186809).ResultsAmong the 74 patients (including 16 children and 58 adults) enrolled into this study (median age 45, range 1-67), 20 had a diagnosis of ALL (27%), 41 of AML (55%), 4 of MM (5%), 3 of HD (4%), 2 of NHL (3%) and 4 of MPN (5%). All patients relapsed after matched allogeneic transplants (32 unrelated and 42 sibling), of whom 44 (59%) suffered from a hematological, 4 (5%) from a cytogenetic and 26 (35%) from a molecular relapse. The therapeutic strategy consisted of two infusions of unmanipulated DLI (each of 1 x 106/kg cells) at 3 weeks interval, followed by three infusions of donor derived CIK cells given at 3 weeks interval. The first 12 patients were treated with increasing numbers of CIK cells, in groups of three patients per dose level. Since dose limiting toxicity (DLT) was never observed (acute GVHD of grade III or more), the highest dose planned (5 x 106/kg, 5 x 106/kg and 10 x 106/kg) was then administered to all patients.Ten patients died for disease progression, 1 patient developed aGVHD (grade I, skin only) and 1 withdrawn for medical decision before or during the DLI treatment and could not proceed to the planned subsequent CIK administration. Sixty-two patients received at least one infusion of CIK cells, of whom 43 patients (61%) completed the cell therapy program, while 3 patients are still under treatment. The study flow is outlined in Figure 1.As per protocol, clinical response was determined 100 days after the last CIK administration and the study was analyzed on an intent to treat basis. An early death occurred in 24 (32%) patients (4 during the DLI), no response was observed in 18 (24%) patients, a stable disease in 1 patient (1%), a complete remission in 21 (28%) and a partial remission in 6 (8%), for an overall response rate of 36%. In 4 patients clinical response could not be evaluated (3 patients still in treatment and 1 withdrawn from the protocol). Acute GVHD was observed in a total of 11 patients (15%): grade 1 (n=4), 2 (n=2) and 3-4 (n=5). During follow up, chronic GVHD was observed in 8 patients (11 %) (3 mild, 4 moderate and 1 severe).By univariate analysis, progression free survival (PFS) and overall survival (OS) were significantly associated (p<0.0001) with the type of relapse since at 3 years it was 11% and 23% vs. 54% and 77 % for patients enrolled due to a hematologic vs. a molecular/cytogenetic relapses, respectively (Figure 2A-B). By multivariate analysis, the type of relapse remained the only significant predictor of survival (p=0.0019).ConclusionOur study shows that administration of CIK cells is feasible in patients with recurrent hematologic cancer after alloHSCT with a relatively low toxicity in terms of GvHD. Particularly in the setting of the molecularly relapsed patients, long-term survival can be achieved. In future studies, we are planning to test CIK cells in preventing post-transplantation relapse in high risk AML. Finally, CIK cells may represent an innovative platform to transduce chimeric antigen receptors in allogeneic T cells with a reduced risk to induce GvHD. [Display omitted] [Display omitted] DisclosuresBiondi:Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board.

Third Party Cytokine-Induced Killer Cells Eliminate Highly Activated T-Cells, Leading to Protect from Murine Lethal Graft-Versus-Host Disease

Protecting from lethal graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects by adoptive immunotherapy would be an ideal goal in allogeneic hematopoietic stem cell transplantation (HSCT), especially using third party derived cells, because the use of third party cells would open a huge source of availability and feasibility across major histocompatibility barriers. We have demonstrated that third party cytokine-induced killer (CIK) cells protected from murine lethal GVHD with faster hematological recovery, which are ex vivo-expanded T lymphocytes expressing both natural killer and T-cell markers with strong cytotoxic activity both against autologous and allogeneic tumor cells. However the mechanism responsible for ameliorating acute GVHD by third party CIK cells are still unknown.First, lethally irradiated Balb/c (H-2d) mice were given C3H (H-2k) bone marrow cells with C3H whole splenocytes to induce lethal GVHD with/without third party (C57/BL6 (H-2b)) CIK cells or third party whole splenocytes on day 0 or day4. Mice receiving third party CIK cells showed much less GVHD and significant survival benefit compared those with third party splenocytes as shown below (p<0.05). On day7 after BMT, the percentage of CD69 on CD8 T-cells in peripheral blood (PB) in the mice receiving third party CIK cells was significantly lower than those in GVHD control mice as shown below (p<0.05), meanwhile donor derived not-activated T-cells were preserved in both CD4 and CD8 T-cells. From these results, we hypothesized that third party CIK cells prevent acute GVHD by selectively depleting highly proliferative alloreactive T-cells stimulated early after transplant, while sparing naïve T-cells. To test this, we performed mixed lymphocyte reaction (MLR) by adding third party CIK cells or third party splenocytes (Stimulator: Balb/c splenocytes, Responder: C3H splenocytes, third party cells: C57/BL6). The percentage of CD69 on responder derived CD8 T-cells in the culture mixture with third party CIK cells was much less compared to that with third party splenocytes as shown below (p<0.05). We have previously shown that allogeneic CIK cells had cytotoxicity against cultured dendritic cells (DCs) by 51Cr release assay. Therefore reduced alloreaction by third party CIK cells might be due in part to the elimination of antigen presenting cells such as DCs. Next, to demonstrate that third party CIK cells could eliminate responder cells directly and then reduce the alloreaction, we performed MLR like experiments using PHA to activate T-cells strongly instead of antigen presenting cells as a stimulator. As shown below, we have demonstrated that the percentage of highly activated T-cells (CD8+CD69+) in third party CIK added plates was significantly lower compared to that in third party splenocytes added plates, indicating that third party CIK cells could have direct effects against highly activated T-cells.To further clarify the mechanisms of how third party CIK cells reduce the highly activated T-cells, we performed MLR as mentioned above with/without the neutralizing antibody against NKG2D, which is an activator receptor expressed on NK cells. Although CIK cells have cytotoxicity against tumor cells via NKG2D signaling pathway in part, adding NKG2D antibody did not show a statistical significant difference in MLR assays. It suggested that third party CIK cells could suppress alloreactive T-cells by several pathways other than NKG2D signaling pathway.Finally, we identified the in vivo kinetics of third party CIK cells after BMT. Third party derived CIK cells could be identified as a rare subpopulation in recipient peripheral blood for some days after CIK infusion, but all of them could not be detected anymore by day 7 after infusion. Whereas third party whole splenocytes expanded in recipient peripheral blood and mice received third party splenocytes with donor BM and splenocytes died until day7 because of GVHD induced by third party splenocytes.In conclusion, infusion of third party CIK cells has strong potential to prevent murine lethal GVHD probably due to selectively depleting highly proliferative alloreactive T-cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Selective effect of cytokine-induced killer cells on survival of patients with early-stage melanoma.

Adoptive immunotherapy using cytokine-induced killer (CIK) cells has shown potential antitumor ability against several kinds of cancers, including melanoma. However, little is known about the achievable outcome of CIK cells in melanoma patients at different pathological stages. Here we recruited 55 patients treated with conventional therapy plus CIK cells as the CIK group, and 49 patients treated with conventional therapy alone as the control group. The pathological characteristics were comparable between two groups, with a follow-up period up to 40months. Survival data and immune responses were evaluated after CIK cell treatment. In this study, CIK cells were successfully generated from peripheral blood of melanoma patients after in vitro culture for 14days. The cultured CIK cells not only produced high levels of pro-inflammatory cytokines upon in vitro stimulation but also efficiently killed human melanoma cell lines. No serious side events were observed in all patients treated with CIK cells. Furthermore, infusions of CIK cells improved the quality of life in some patients, including advanced cases. More importantly, the CIK group exhibited better survival rates compared to the control group among early-stage melanoma patients, in consistent with the increased frequency of peripheral CD4+ T cells. However, the patients with advanced-stage melanoma did not benefit from the CIK cell therapy in terms of survival rate. In conclusion, CIK cells combined with conventional treatments may prolong the survival of early-stage melanoma patients and improve the quality of life for some advanced cases in a safe way.

Cytokine-induced killer cell infusion combined with conventional treatments produced better prognosis for hepatocellular carcinoma patients with barcelona clinic liver cancer B or earlier stage: A systematic review and meta-analysis

Background aimsTo investigate the clinical benefits of cytokine-induced killer (CIK) cell infusions on hepatocellular carcinoma (HCC) patients, combined with other conventional treatments. MethodsThis was a systematic review and meta-analysis conducted among phase II and III randomized control trials worldwide. Review manager 5.2 version was used to pool the effect size across studies. Sensitivity analyses and risk of bias were estimated among included studies. Egger's test was used to characterize the publication bias. ResultsEight randomized controlled trials and 945 patients with HCC were included in the study. CIK infusion reduced cancer recurrence risk to 0.74 (95% confidence interval [CI] 0.5–0.92), I2 75% (P < 0.001), and reduced cancer death risk to 0.76 (95% CI 0.65–0.88), I2 50% (P = 0.09). Among studies blinded for outcome assessment and Barcelona Clinic Liver Cancer stages of 0, A and B, CIK infusion reduced recurrence risk by 18% (relative risk [RR] = 0.82, 95% CI 0.70–0.96) and death risk by 37% (RR = 0.63, 95% CI 0.47–0.85); heterogeneity was 0% and 39%, respectively (P > 0.05). The intercepts of linear regressions for recurrence and death were −2.17 and −2.07, respectively, but the P value was 0.17 and 0.38; no significant publication bias was observed with Egger's test. DiscussionAmong hepatocellular carcinoma patients with Barcelona Clinic Liver Cancer score of B or less, CIK cell infusions combined with conventional treatments significantly prolonged recurrence-free and overall survival. This adoptive immunotherapy could be recommended to HCC patients.

Abstract 2290: Effective immunotherapy with cytokine-induced killer cells against autologous melanoma cancer stem cells

Abstract Purpose of the study was to explore the activity of cytokine-induced killer (CIK) cells against autologous chemo-surviving melanoma cancer stem cells (mCSCs) in vivo. Elimination of mCSCs is an ambitious goal as considered responsible for chemo-resistance and relapses. CIK cells are ex vivo expanded T-NK lymphocytes with MHC-independent antitumor activity. We provided proof of concept that CIK cells can kill mCSCs in vitro (Gammaitoni et al. Clin Cancer Res 2013) and demonstration of their activity in vivo, within an autologous model, would be relevant in clinical perspective. Experimental procedures and results. In vivo activity of CIK cells against autologous mCSCs was explored in NOD/SCID mice bearing tumors generated by subcutaneous implantation of primary melanoma cells or alternatively surgically resected tumor samples (patient-derived xenografts, PDX). mCSCs were visualized by a lentiviral CSC-detector encoding eGFP under control of stem-gene promoter OCT4 (Gammaitoni et al. Clin Cancer Res 2013). Melanoma cells were engineered with the CSC-detector before implantation or alternatively, in PDX, after removal of residual tumors at the end of experimental treatments. In vivo chemotherapy (CHT) consisted of intravenous (i.v.) Fotemustine (600μg days 1;15). Immunotherapy consisted of i.v. CIK cells (1×107) every 5 days. Antitumor activity was evaluated assessing tumor proliferative index by Ki67 expression in residual tumors. Activity against mCSCs was evaluated assessing the rate of residual GFP+-mCSCs in residual tumors after treatments. In vivo infusion of CIK cells for 2 weeks determined significant tumor response (n = 6, p &amp;lt; 0.0001) that fully involved autologous eGFP+-mCSCs. On the contrary, CHT spared mCSCs that resulted significantly increased in residual tumors compared with mice treated with CIK cells (p = 0.001) or untreated controls (p = 0.001). CIK cells infused after a previous CHT treatment (n = 6) retained significant antitumor activity (p = 0.001) and were capable to revert the CHT-induced enrichment of eGFP+-mCSCs, compared to mice treated with CHT alone (p = 0.01). Similarly, in the PDX model, the infusion (n = 4) of autologous CIK cells exerted significant antitumor activity that involved eGFP+mCSCs (p = 0.009), confirmed by the absence of their increment in residual tumors (p = 0.3). In vitro CHT treatment of melanoma from 8 different patients significantly enriched eGFP+ mCSCs (2.2 ±0.2 fold, p = 0.0018) compared to untreated controls. Melanoma treated with CHT, enriched in eGFP+mCSCs, were efficiently killed by autologous CIK cells with mean killing values ranging from 94±2% (40:1 E/T) to 21%±4 (1:3 E/T). Conclusions. Our findings provide first demonstration that immunotherapy with CIK cells is active against autologous mCSCs, surviving chemotherapy, in vitro and in vivo. We provide an experimental platform to study mCSCs and rationale to design clinical studies with CIK cells against melanoma. Citation Format: Loretta Gammaitoni, Lidia Giraudo, Marco Macagno, Giulia Cattaneo, Valeria Leuci, Francesco Sassi, Alessandro Zaccagna, Alberto Pisacane, Valentina Coha, Fabrizio Carnevale-Schianca, Massimo Aglietta, Dario Sangiolo. Effective immunotherapy with cytokine-induced killer cells against autologous melanoma cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2290.

Allogeneic hematopoietic stem cell transplantation following donor CIK cell infusion: A phase I study in patients with relapsed/refractory hematologic malignancies

Patients with relapsed/refractory hematologic malignancies after allogeneic stem cell transplantation have a poor prognosis due to the high rate of relapse. Techniques capable of decreasing post-transplantation relapse rates are urgently sought. This study aimed to explore the feasibility and safety of allogeneic hematopoietic stem cell transplantation (HSCT) following infusion of donor cytokine-induced killer (CIK) cells. CIK cells were generated in vitro from donor peripheral blood mononuclear cells, and were phenotyped using flow cytometric analysis. CIK cells were administered to 15 high-risk relapsed/refractory hematologic malignancy patients who were not in complete remission in multiple infusions. These patients also received allogeneic HSCT. The side effects and outcomes were recorded. All patients achieved engraftment and complete remission. After CIK cell infusion, two patients developed graft-versus-host disease (GvHD), which was controlled by additional immunosuppressant drugs. At the last follow-up, nine patients had died and six patients were surviving at a median follow-up of 1513days (range, 771–1655days). In conclusion, allogeneic HSCT combined with sequential infusion of donor CIK cells is well tolerated in salvage relapsed/refractory hematologic malignancy patients.

Autologous cytokine-induced killer (CIK) cell immunotherapy combined with cyclophosphamide in five patients with POEMS syndrome.

The primary objective of this study was to evaluate the safety and clinical efficacy of autologous cytokine-induced killer (CIK) cells combined with cyclophosphamide in the treatment of polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes (POEMS) syndrome patients. We evaluated five POEMS syndrome patients treated with autologous CIK cell immunotherapy combined with cyclophosphamide from 1May 2012 to 30November 2014. The Overall Neuropathy Limitation Scale (ONLS), computed tomography of the chest and abdomen, ultrasound of the abdomen, serum vascular endothelial growth factor (VEGF) level and lymphocyte count findings in the five patients were recorded. The median age of the patients was 40 years (range: 25-62), and all the patients were male. CIK cells were generated routinely from peripheral blood mononuclear cells (PBMCs) of all five patients, and the numbers of CIK cells increased by approximately 105-fold after 14 days of culture. All five patients (100%) responded to their neuropathy treatment, the ONLS scores were reduced by at least 1 and a paired-sample t-test revealed a significant difference (t=5·715, P=0·003<0·01). The extravascular volume overload responses indicated partial remission (PR=60%) or stable disease (SD=40%), and no cases of progressive disease (PD) or complete remission (CR) were observed. During clinical treatment, the serum VEGF of patient 5 decreased after one cycle of transfusion within 1 month. The lymphocyte counts of all the patients increased significantly after CIK transfusion, and a paired-sample t-test revealed a significant difference (t=5·101, P=0·004<0·01). Autologous CIK cell infusion combined with cyclophosphamide was found to be highly safe and elicited no adverse reactions. CIK cells can improve both the symptoms and quality of life, decrease serum VEGF levels and increase lymphocyte counts in patients with POEMS syndrome.

A Multicenter Phase II Study of Sequential Administration of Unmanipulated DLI and Donor Derived Cytokine Induced Killer (CIK) Cells in HSCT Patients, Relapsed of Disease

▪IntroductionCytokine Induced Killer (CIK) cells are memory T lymphocytes which have acquired CD56 expression and Natural Killer (NK) like unrestricted cytotoxicity, following in vitro activation by anti CD3 OKT3 and IFNg and subsequent expansion with IL-2.CIK cells have demonstrated in vitro and in vivo anti tumor activity, direct intratumor homing following iv. administration and, more importantly, a very reduced Graft Versus Host (GVH) activity, in several experimental allogeneic models.Indeed we and others have demonstrated very limited GVH activity in preliminary phase I studies, with donor derived (matched) CIK cells in patients with different hematological neoplasms, previously treated by allogeneic Hematopoietic Stem Cells (HSC) transplantations and subsequently relapsed of diseases 1.MethodsTo better delineate the toxicity profile, as well as the potential anti tumor efficacy, of donor derived CIK cells, we prospectively studied 48 patients relapsed after allogeneic stem cell transplantation performed using either a matched related (N=28) or unrelated donor (n= 20, including 1 haplo). This phase II multicenter study was authorized by Istituto Superiore di Sanità, as for Advanced Therapeutic Medicinal Product (ATMP) regulations, approved by the Agenzia Italiana del Farmaco and (AIFA). The trial was registered as (EUDRACT n 2008-003185-26, ClinicalTrial.gov: NCT01186809)ResultsIn this interim analysis, forty-eight patients (including childrens and adults) have been so far enrolled into this study protocol. The median age was 48 (range 6-67) and a diagnosis of ALL (n=9, 20%), AML (n=29, 60%), MM (n=5, 8%), HD (n=3, 6%), MPN (n=2, 4%), NHL (n=1, 2%). Reasons for being enrolled into study was a hematologic relapse in 36 (75%) or a molecular relapse in 12 (25%).The therapeutic strategy consisted of two infusions of unmanipulated DLI (each of 1 x 106/kg cells) at 3 weeks interval, followed by three infusions of donor derived CIK cells given at 3 weeks interval. The first 12 patients were treated with growing numbers of CIK cells, in groups of three patients per dose level. Since DLT was never observed (acute GVHD of grade III or more) the highest dose planned (5 x 106/kg, 5 x 106/kg and 10 x 106/kg) was then administered to subsequent consecutive 36 patients.4 patients died for disease progression and 1 patient developed aGVHD (grade I, skin only) during the DLI treatment and could not proceed to the planned subsequent CIK administration. Of the 43 patients who eventually received at least one infusion of CIK cells, 15 patients did not complete the program, 9 for disease progression and death, 3 for insurgence of grade II aGVHD (skin only in 2 cases, skin and gut in 1 case), 1 for hemolytic anemia, 1 for insufficient cell supply and 1 for medical decision. Overall, 28 patients received the complete cell therapy planned (58%).Overall, of the 48 patients enrolled, 5 (10%) suffered from aGVHD (1 grade I, 3 grade II, 1 grade III). During follow up, chronic GVHD was observed in 7 patients (14 %) (3 mild and 4 moderate).As per protocol, clinical response was determined 100 days after the last CIK administration and the study was analyzed on an intent to treat basis. An early death occurred in 13 (27%) patients (4 during the DLI), before the clinical response could be evaluated. A CR was observed in 9 (19 %) and a PR in 7 (14%) for an overall response rate of 16 (33%). No response was observed in 19 (39%).At 2 and 4 years, the event free survival of the 48 patients is 22% and 18%, while the overall survival is of 37% and 34%, respectively. For the small group of patients who achieved a complete response, the disease free survival is of 64% at 2 years and 51% at 4 years.By univariate analysis, survival was significantly associated to the type of relapse (molecular) (p 0.0081) since at 2 and 4 years it was and 24% and 27% vs. 71% and 71 % for patients enrolled for a hematologic or a molecular relapses, respectively. By multivariate analysis, the type of relapse remained the only significant predictor of survival (0.0160 p value).ConclusionThis study shows the feasibility of CIK preparation and administration as well as the relatively low toxicity of the program (10% aGVHD grades I-III) in spite of the fact that 20 patients received cells from matched unrelated donors. Finally, the study offers the suggestion that CIK cells may be efficacious to treat post-transplant relapse.1 Introna M. et al, Haematologica, 2007, 92, 7, 948. [Display omitted] DisclosuresIntrona:roche: Research Funding. Rambaldi:Roche: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Celgene: Research Funding; Pierre Fabre: Honoraria.