Clinical Studies on Cytokine-Induced Killer Cells: Lessons from Lymphoma Trials

Cancer is a complex disease where resistance to therapies and relapses often pose a serious clinical challenge. The scenario is even more complicated when the cancer type itself is heterogeneous in nature, e.g., lymphoma, a cancer of the lymphocytes which constitutes more than 70 different subtypes. Indeed, the treatment options continue to expand in lymphomas. Herein, we provide insights into lymphoma-specific clinical trials based on cytokine-induced killer (CIK) cell therapy and other pre-clinical lymphoma models where CIK cells have been used along with other synergetic tumor-targeting immune modules to improve their therapeutic potential. From a broader perspective, we will highlight that CIK cell therapy has potential, and in this rapidly evolving landscape of cancer therapies its optimization (as a personalized therapeutic approach) will be beneficial in lymphomas.

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 Nanobody 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, it sefficacy is restricted 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, we tested 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 Our findings 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 to treat malignant tumors through in vivo adoptive therapy.

Efficacy of Enhanced Cytokine-Induced Killer Cells as an Adjuvant Immunotherapy for Renal Cell Carcinoma: Preclinical and Clinical Studies

Cytokine-induced killer (CIK) cells have been proved to be an effective method of tumor immunotherapy in numerous preclinical and clinical studies. In our previous study, a new method was developed to prime and propagate CIK cells by the combination of IL-2 and IL-15, and this kind of CIK cells had enhanced antitumor effect on lung cancer. For renal cell carcinoma (RCC), immunotherapy plays an important role because of the poor efficacy of radiotherapy and chemotherapy. In this study, we further evaluated the antitumor effects of these enhanced CIK cells against RCC. Enhanced CIK cells were generated by IL-2 combined with IL-15 and identified by flow cytometry. HEK-293 and ACHN cell lines were used to verify the efficiency of CIK cells in vitro, and then the ACHN tumor xenograft model was also employed for in vivo study. In addition, the secreted cytokines including IFN-γ, granzyme B, TNF-α, and perforin, as well as the local microstructure were also studied. Subsequently, 20 patients with RCC were enrolled into our study, and 11 patients were randomly divided into the autologous CIK treatment group for clinical research. The results showed that enhanced CIK cells exert better antitumor effects in RCC in vitro ( p < 0.01 in HEK-293 and p < 0.05 in ACHN）and in vivo ( p < 0.05 ). Patients benefit overall survival from enhanced CIK therapy in our clinical study. Our present preclinical and clinical studies for the first time elucidated that these enhanced CIK cells would be used as an effective adjuvant therapy in the treatment of RCC.

Innovative therapeutic strategy for B-cell malignancies that combines obinutuzumab and cytokine-induced killer cells

BackgroundPatients affected by aggressive B-cell malignancies who are resistant to primary or salvage chemoimmunotherapy have an extremely poor prognosis and limited therapeutic options. Promising therapeutic success has been achieved with the infusion of CD19 chimeric antigen receptor-T cells, but several limits still restrain the administration to a limited proportion of patients. This unmet clinical need might be fulfilled by an adoptive immunotherapy approach that combines cytokine-induced killer (CIK) cells and monoclonal antibodies (mAb) to the CD20 antigen. Indeed, CIK cells are an effector population endowed with antitumor activity, which can be further improved and antigen-specifically redirected by clinical-grade mAb triggering antibody-dependent cell-mediated cytotoxicity.MethodsCIK cells were generated from peripheral blood of patients affected by different B-cell malignancies using a blinatumomab-based cell culture protocol. Effector cells were combined with the anti-CD20 mAb obinutuzumab and their therapeutic activity was assessed both in vitro and in vivo.ResultsCIK cells were successfully expanded in clinically relevant numbers, starting from small volumes of peripheral blood with extremely low CD3+ counts and high tumor burden. This relied on the addition of blinatumumab in culture, which leads to the simultaneous expansion of effector cells and the complete elimination of the neoplastic component. Moreover, CIK cells were highly cytotoxic in vitro against both B-cell tumor cell lines and autologous neoplastic targets, and had a significant therapeutic efficacy against a B-cell malignancy patient-derived xenograft on in vivo transfer.ConclusionsThe combination of an easily expandable CIK cell effector population with a mAb already in clinical use establishes a tumor antigen-specific redirection strategy that can be rapidly translated into clinical practice, providing an effective therapeutic alternative for B-cell malignancies without any need for genetic modifications. Additionally, the approach can be potentially applied to an extremely vast array of different tumors by simply substituting the targeting mAb.

The Crosstalk Between Dendritic Cells, Cytokine-Induced Killer Cells And Cancer Cells From The Perspective Of Combination Therapy

Dendritic cells (DCs) are considered the most potent professional antigen-presenting cells (APCs) that elicit adaptive antitumour immunity. DCs integrate multiple environmental signals by efficiently processing tumour-associated antigens (TAAs) and migrating to draining lymph nodes (dLNs), where they present foreign antigens to T cells for priming. DCs thus serve as a major link between innate and adaptive immunity. Although DCs (mostly monocyte-derived DCs [mo-DCs]) have already been used in cancer therapies, such approaches have shown limited efficacy. Mo-DCs have the unique ability to present antigens to T cells in peripheral tissues. CD3+CD56+ cytokine-induced killer (CIK) cells are characterized by both MHC-restricted and MHC-unrestricted antitumour cytotoxicity against a broad range of cancer cells. This review presents an overview of the mechanisms by which mo-DCs and CIK cells’ interact with each other and with tumours. The maturation of DCs was identified as a crucial step in the development of effective DC-based vaccines against cancer. A further improved adoptive immunotherapy strategy involves a combination of mature mo-DCs and CIK cells. Combination therapy presents many opportunities for cancer treatment, as reported by a number of clinical trials. However, there is a lack of fundamental studies on the interaction of in vitro-generated mo-DCs with CIK cells. We discuss several methods of boosting DC-based vaccines and review the current knowledge of contact-dependent and cytokine-induced interactions of mo-DCs with CIK cells. We highlight that the combination of mo-DCs with CIK cells activates MHC-restricted and MHC-unrestricted immune responses.

Advanced Renal-Cell Carcinoma Pseudoprogression After Combined Immunotherapy: Case Report and Literature Review

Treatment with a combination of programmed cell death-1 (PD-1) blocker and cytokine-induced killer (CIK) cells has improved outcome in cancer patients but is also associated with various patterns of responses. Pseudoprogression is a unique and uncommon phenomenon with no clear criteria for rapid diagnosis. Although some reports of pseudoprogression during immunotherapy exist, there are few reports of pseudoprogression occurring twice in the same patient. Here, we report the case of 51-year-old female patient with advanced renal cell carcinoma, who received a combination treatment of PD-1 blocker and CIK cells, and where pseudoprogression of lung and brain tumors occurred successively during treatment.

A single-arm phase Ib study of autologous cytokine-induced killer (CIK) cell immunotherapy in combination with sintilimab plus chemotherapy in patients with advanced non-small cell lung cancer (NSCLC)-CCICC-002.

2531 Background: Immune checkpoint inhibitors plus chemotherapy had showed benefits for advanced non–small-cell lung cancer (NSCLC) patients without targetable mutations. Autologous cytokine-induced killer (CIK) cells can restore the antitumor immunity to improve patient outcome. Combining CIK cells with anti-PD-1 mAb plus chemotherapy may strengthen the results in patients with advanced NSCLC. Methods: This is a single-center, open-label, phase 1b trial of combination CIK cells with sintilimab (anti-PD-1 mAb) plus chemotherapy in stage IIIB-IV NSCLC patients. Systemic therapy naïve patients received platinum-based doublet chemotherapy, sintilimab, and CIK cells every 3 weeks for 4 cycles，then sintilimab, and CIK cells for maintenance therapy until disease progression or unacceptable toxicity. Results: From May 2019 to Jan 2021, 34 patients (19 squamous， 15 non-squamous NCSLC)aged 46-73 years (median age 64 years) were enrolled. Among 32 evaluable patients, the ORR was 81.3% (73.7% in squamous and 92.3% in non-squamous NSCLC)and DCR was 100%. Among the 25 PR assessed by RECIST, CMR was demonstrated in 5 (23.1%) by PET-CT. Among 3 patients with brain metastases, 2 patients achieve intracranial CR, and 1 was PR. With a median follow-up of 7.5 months, the median DOR was not reached (range 0.5m-NA), and the median PFS and OS were not mature. Grade 3 or more TRAEs included pneumonia (n = 3); thrombocytopenia, leukopenia (n = 2 each); anemia, dysphagia, cardiomyopathy, rash (n = 1 each). Biomarkers and subgroups which correlated with efficacy and AEs are being analyzed included TMB, PDL1 expression, distribution of TILs, cytokines and so on. Conclusions: CIK cells therapy in combination with sintilimab plus chemotherapy were well tolerated and showed encouraging efficacy. Further studies are warranted to confirm these preliminary results. Research Sponsor: Tianjin Medical University Cancer Institute and Hospital. Clinical trial information: NCT03987867. [Table: see text]

Efficacy and safety of MUC1 targeted CIK cells for the treatment of advanced liver cancer.

e16278 Background: To assess the efficacy and safety of CIK treatment with MUC1/CD3 bis-Ab for patients with advanced liver cancer. Methods: A total of 11 patients were enroll from when in which hospitals, including 5 male and 6 female patients with a median age of 59 years old. Trial Design and Treatment: All participants provided written informed consent before enrollment. The study protocol was approved by the institutional review board at each participating center. All methods and procedures associated with this study were conducted in accordance with the Good Clinical Practice guidelines and accorded ethically with the principles of the Declaration of Helsinki and local laws. This phase I clinical study was a single-armed open-labeled trial. All patients received targeted activated CIK cell treatment of 6x109 cells/month until the disease progressed, the patient could not tolerate the treatment, or the study endpoint had reached. PBMC were collected from each patient on D0. After PBMC collection, patients received 10 mg/day decitabine for 5 consecutive days (D0–D4). On D14 the first batch of CIK cells were harvested and the infusion of cells was performed in two consecutive days (total cells ≥ 6x109). The bispecific antibody was infused together with CIK cells. After cell infusion, the bispecific antibody was infused alone for another 3 days. Each treatment course lasted for 4 weeks, with a total of 4 courses. Results: Efficacy: 9 patients completed 4 or fewer courses of CIK bispecific antibody treatment, and 2 patients completed 9 courses of treatment. Till the data cut-off date of Dec. 31, 2020, 9 patients died, of whom 5 patients achieved SD and 4 patients had PD. Two patients (case 1 & 2) are still alive, of whom one achieved PR and one achieved SD during CIK treatment, and both achieved CR after subsequent PD-1 inhibitor treatment when the disease progressed. The PFS ranged from 1-16.7 months with a median PFS of 4 months, and the OS ranged from 3.9-32.7 months with a median OS of 13.2 months. The DCR of CIK treatment reached 63.6%. Safety: All 11 patients developed fever after cell infusion, which subsided after a few hours by oral administration of Xinhuangpian. 2 patients developed anorexia. Other AEs included debilitation, drowsiness, nausea, hepatotoxicity and dizziness. Five patients discontinued treatment due to serious AEs. Conclusions: This study showed that adjuvant CIK cell immunotherapy prolongs the survival time of patients with advanced liver cancer, which is mainly due to the local tumor chemotaxis of CIK cells via the MUC1/CD3 bispecific antibody and the decitabine pretreatment. Clinical trial information: NCT03146637.