Academic literature on the topic 'Cytokine-induced killer (CIK) cell'

Abstract Although much has been learned about the cytotoxic mechanisms of cytokine-induced killer (CIK) and natural killer (NK) cells, little is known about how they kill cancer cells at the single-cell level. In the present study, we examined the contact dynamics of CIK and NK cells at the single-cell level by using time-lapse imaging. CIK cells killed MHC-I-negative and -positive cancer cells, but NK cells destroyed MHC-I-negative cells only. Moreover, CIK cells moved in all directions and showed longer tracks than did NK cells. CIK cells showed higher displacement and straightness scores than did NK cells, which indicates long-distance random migration of CIK cells. CIK and NK cells moved at 6.7 mm/min and 4.5 mm/min on average, respectively. These data suggest that CIK cells are likely moving more actively than NK cells. The average threshold number of CIK cells required to kill an individual cancer cell was 6.7 for MHC-I-negative cells and 6.9 for MHC-I-positive cells. That of NK cells was 2.4 for MHC-I-negative cells. Likely due to the higher threshold numbers, killing by CIK cells was delayed in comparison with NK cells: 40% of MHC-negative target cells were killed after 5 h when co-cultured with CIK cells and after 2 h with NK cells. Our data have implications for the rational design of CIK or NK cell–based immunotherapy of cancer patients

Adoptive cell therapy (ACT) using autologous cytokine-induced killer (CIK) cells is a promising treatment for metastatic carcinomas. In this study, we investigated the impact of RetroNectin on the proliferation, phenotype alternation, cytokine secretion, and cytotoxic activity of CIK cells from pancreatic cancer patients. Furthermore, we treated 13 patients with metastatic or locally advanced pancreatic cancer using autologous RetroNectin-activated CIK cells (R-CIK cells) alone or in combination with chemotherapy. Compared with only CD3 activated CIK cells (OKT-CIK cells), R-CIK cells showed stronger and faster proliferative ability, with a lower ratio of spontaneous apoptosis. Moreover, this ability continued after IL-2 was withdrawn from the culture system. R-CIK cells could also secrete higher levels of IL-2 and lower levels of IL-4 and IL-5 versus OKT-CIK cells. There was no difference between OKT-CIK and R-CIK cells in cytotoxic ability against lymphoma cell line K562. In patients who received auto-R-CIK cell infusion therapy, the overall objective response rate was 23.1%. Median survival time (mOS) after first R-CIK cell infusion was 10.57 months; the 1-year survival rate was 38.5%. No serious toxicity was associated with R-CIK cell infusion. In conclusion, RetroNectin may enhance antitumor activity of CIK cells: it is safe for use in treating pancreatic cancer.

Cytokine induced killer (CIK) cells has been increasingly used in adoptive immunotherapy against various cancers and viral infections. This review summarizes the basic overview of CIK cells as a therapeutic immunocyte. Herein, the basic concepts on CIK cells, their general characteristics, approaches in enhancing their functions, cytotoxic mechanism of CIK cells and their therapeutic benefits in tumors and viral infections are explored. J-GMC-N | Volume 11 | Issue 01 | January-June 2018, Page: 58-63

Objective: To investigate the efficacy of cytokine-induced killer cell-based immunotherapies in patients with advanced malignant solid tumors and the difference in clinical efficiency among 3 kinds of cytokine-induced killer cell-based immunotherapies. Methods: One hundred forty-six cases with advanced solid tumor, 230 cycles of cytokine-induced killer cell-based immunotherapies, were involved in this study. T-lymphocyte subsets, carcinoembryonic antigen, and adverse reactions were recorded. Results: CD3+ T lymphocyte, Th, NKT, and Th/Tc were increased after cytokine-induced killer cell-based treatment, from 55.67 ± 3.64 to 84.12 ± 5.15, 26.56 ± 4.47 to 42.76 ± 3.68, 1.82 ± 0.58 to 7.08 ± 0.92, 0.79 ± 3.64 to 1.35 ± 0.20, respectively ( P < .001). Carcinoembryonic antigen was decreased from 398.39 ± 219.16 to 127.26 ± 153.41 ( P < .001). Difference values were greater than 0 ( P < .001). Difference value of carcinoembryonic antigen was obviously less than 0 ( P < .001). There was no obvious difference in all variations between cytokine-induced killer cell and DC+CIK groups ( P > .05). The highest amount of CD3+ T lymphocyte and Th was recorded after at least 4 cycles of immunotherapy. And CD8+ T/CD4+ T also began to decrease after 4 cycles of immunotherapy. Difference value of T lymphocyte and Tc of patients with surgery is higher than that of patients without surgery. Conclusion: Cytokine-induced killer cell-based immunotherapy is capable of increasing T-lymphocyte subsets, recovering cellular immunity without severe side effects, and is suitable for different kinds of solid cancer. Clinical efficiency of cytokine-induced killer cell-based immunotherapy is influenced by many factors such as surgery, stage.

Metastatic renal cell carcinoma (RCC) seems to be resistant to conventional chemo- and radiotherapy and the general treatment regimen of cytokine therapy produces only modest responses while inducing severe side effects. Nowadays standard of care is the treatment with VEGF-inhibiting agents or mTOR inhibition; nevertheless, immunotherapy can induce complete remissions and long-term survival in selected patients. Among different adoptive lymphocyte therapies, cytokine-induced killer (CIK) cells have a particularly advantageous profile as these cells are easily available, have a high proliferative rate, and exhibit a high antitumor activity. Here, we reviewed clinical studies applying CIK cells, either alone or with standard therapies, for the treatment of RCC. The adverse events in all studies were mild, transient, and easily controllable.In vitrostudies revealed an increased antitumor activity of peripheral lymphocytes of participants after CIK cell treatment and CIK cell therapy was able to induce complete clinical responses in RCC patients. The combination of CIK cell therapy and standard therapy was superior to standard therapy alone. These studies suggest that CIK cell immunotherapy is a safe and competent treatment strategy for RCC patients and further studies should investigate different treatment combinations and schedules for optimal application of CIK cells.

There is growing interest in cytokine-induced killer (CIK) cells on the integrated therapy of patients with RCC, especially those in the late stage or refractory to conventional chemotherapy and radiotherapy. In this review, a total of 15 clinical studies including 681 patients enrolled in CIK cell immunotherapy were outlined. Three-hundred-and-eighty-two patients with RCC were treated with CIK cells alone or in combination with DC vaccination, targeted agents sunitinib or sorafenib, and the PD-1 inhibitor pembrolizumab. Significantly improved 3-year overall survival rate was reported in four trials, whereas remarkably longer median progression-free survival was observed in three studies. Adverse reactions were mild and usually controllable fever and fatigue. Besides, preclinical research progresses were reviewed to increase our understanding about the underlying mechanisms of CIK cell cytotoxicity and identify potential targets to enhance their anti-tumor activity. These studies suggest that CIK cell-based immunotherapy has potential clinical benefits with a good safety profile and could become a promising approach in the combined therapies of RCC patients. However, further large-scale studies are required to evaluate the clinical efficacy of CIK cells and more efforts should be performed to identify the optimal CIK cell-based therapeutic regimen for RCC patients.

Abstract Abstract 5119 Cytokine-induced killer (CIK) cells are T lymphocytes enriched in CD3+CD56+ cells, which can be easily and rapidly expanded in vitro from human peripheral blood, bone marrow or cord blood mononuclear cells with the sequential addition of interferon (IFN)-{gamma}, OKT-3 and high doses of interleukin (IL)-2. The cytokine-induced killer (CIK) cells have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanism of CIK cell recognizing MM cells remains unknow. Recent studies indicated that ligation of NKG2D on immunological cells directiy induce cytotoxicity. We suspect whether NKG2D receptor induction on CIK cells by cytokines is responsible for the killing of MM cells by CIK. We expended CIK cells from healthy controlswith interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and IL-2, and checked expression of NK cell receptors on CIK cells by flow cytometry. We found higher expression of NKG2D receptor and lower other NK receptors, such as CD158a,CD158b and NCRs on expanded CIK. These CIK cells showed higer cytotoxicity to multiple myleoma cell line U266 expressing NKG2D ligands. Interestingly, when cocultured with U266 cells, only NKG2D expressing CIK cells released IFN-γ detected by flow cytometry. We next analyzed NKG2D ligands expression on primary plasma cells in 22 MM patients by flow cytometry, the primary plasma cells in 16/22 (72.7%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. CIK cells showed higher cytotoxicity (12.5%) to NKG2D ligands expressing primary plasma cells compared to those did not express NKG2D ligands. The killing of CIK against MM cells were partially blocked by treatment of CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligangd interaction may be one of the mechanisms by which CIK cells kill MM cells. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3719 Cytokine-induced killer (CIK) cells are ex vivo–expanded T lymphocytes expressing both natural killer (NK)– and T-cell markers. We have reported that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft versus host disease (GVHD) with retention of antitumor activity mediated by NKG2D, which is an activating receptor expressed on NK cells. With the purpose of potential application of CIK cells in a clinical hematopoietic stem cell transplantation, the problem we have to consider next is whether CIK cells could promote an engraftment and facilitate an immune reconstitution. To this end, lethally irradiated BALB/c mice were injected with minimal number of MHC incompatible C57BL/6 bone marrow (BM) cells alone, in which almost half of mice died because of graft failure, or with CIK concurrently. The mice receiving BM plus CIK cells survived with 93% without GVHD, demonstrating that CIK cells significantly promote an engraftment (P<0.05). In particular, recovery of CD3+ T-cells was significantly faster (p<0.05) in the mice receiving BM plus CIK cells than those receiving BM cells alone. Next we further evaluated whether CIK cells also promote an engraftment in the mice receiving non-myeloablative conditioning using low dose total body irradiation. As expected, CIK cells promoted an engraftment and favored immunoreconstitution, especially from the early time point after BMT. In conclusion, our study clearly demonstrated that CIK cells promote an engraftment and facilitate an immure reconstitution without GVHD. As recent studies demonstrated that sufficient number of CIK cells could be expanded even from washouts of cord blood units bags, infusion of CIK cells would be a potent strategy for preventing a graft-failure in clinical settings, especially after cord blood transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract Disease relapse remains a major cause of treatment failure following autologous hematopoietic cell transplantation (HCT) for patients with high-risk malignancies. Cellular immunotherapy using activated autologous effector cells to recognize and kill tumor targets in a minimal disease state after transplant is a novel strategy to reduce relapse and improve survival. In this phase I/II study, we investigated the safety and efficacy of administering ex-vivo expanded autologous cytokine-induced killer (CIK) cells as post-transplant cellular immunotherapy. Patients with high risk recurrent or refractory Hodgkin’s disease (HD)- defined as B symptoms at relapse, pulmonary or bone marrow disease at relapse and more than minimal disease at the time of transplantation- and patients with multiple myeloma (MM) and acute myelogenous leukemia undergoing single autologous HCT were eligible. The autologous CIK cells were ex-vivo expanded from an aliquot of the patient’s autologous apheresis product for 21–28 days in the presence of IFN-γ, IL-2, and OKT-3 to a maximal cell dose of 2×108 CIK cells/kg. The CIK cell population is uniquely characterized by having the functional and phenotypic properties of both T cells and NK cells, (i.e. expressing CD3 and CD56 molecules) and kills tumor targets through an NKG2D-mediated mechanism. A single infusion of CIK cells was given on day 42–63 following autologous transplantation. Monitoring for infusion-related toxicities was performed for the first 28 days. Disease response assessments were done at day 90, 180, one year and annually. Correlative studies on cytokine profile, primary tumor cytotoxicity and NKG2D ligand expression are being performed. Thirteen patients (ages 19–64) have received autologous CIK cell infusions to date. Nine patients have high risk HD; four patients have MM with a single autologous HCT. The median CIK cell dose for infusion was 0.9×108 cells/kg (range 0.2–2×108/kg). The only infusional toxicities observed have been one grade 1 headache and one grade 1 nausea- both were transient and resolved. Hematologic parameters have not been affected by the infusion. In vitro cytotoxicity of cultured CIK cells was assessed against a panel of leukemia and lymphoma cell lines including Jurkat, DB, OCI-Ly8 and SUDHL4. Median 51-Cr release at 40:1 (E:T) ranged from 71% killing against Jurkat cells to 21% against DB. The first patient on the trial with high risk HD involving the lung and bone marrow is one year from HCT without evidence of disease relapse. Four other patients (all high risk HD) have completed day 180 disease response assessments and have no evidence of disease relapse. One HD patient had progressive disease at day 100. The three evaluable MM patients have no disease progression at day 90–180. In this first phase of study, we have confirmed the safety of administering autologous CIK cells at a single maximal dose at day 42–63 post-transplant. Maintenance of disease response in these high risk patients is encouraging in the first year following transplantation.

Tumors of the gastrointestinal system represent a significant share of solid tumors worldwide. Despite the advances in diagnosis and treatment, the prognosis of gastrointestinal tumors is still very poor and improved therapies are indispensable. Cytokine-induced killer (CIK) cells are feasible for an immunotherapeutic approach as they are easily available and have an advantageous biologic profile; they are rapidly proliferating and their high cytotoxicity is non-MHC-restricted. We summarize and discuss twenty recent clinical studies applying CIK cells for the treatment of gastric, pancreatic, hepatocellular, and colorectal cancer. Autologous CIK cells were transfused intravenously, intraperitoneally, or via the common hepatic artery. In all studies side effects and toxicity of CIK cell therapy were mild and easily controllable. The combination of CIK cell therapy with conventional adjuvant or palliative therapies was superior to the standard therapy alone, indicating the benefit of CIK cell therapy for cancer patients. Thus, CIK cells represent a promising immunotherapy for the treatment of gastrointestinal tumors. The optimal treatment schedule and ideal combination with conventional therapies should be evaluated in further clinical studies.

Cytokine-induced killer (CIK) cells are a heterogeneous population of lymphocytes obtained in vitro within 21 days from mononuclear cells under the influence of cytokines. CIK cells show potent MHC-unrestricted cytotoxicity against a variety of tumor cells, in particular hematological malignancies, and minimal tendency to induce graft-versus-host disease. The expanded bulk CIK culture consists of over 90% CD3+ cells, of which the majority coexpress CD56 and the remaining cells are CD56-. CD3+CD56+ “true” CIK cells are terminally differentiated non dividing lymphocytes which could deliver potent MHC-unrestricted cytotoxicity for the immediate destruction of tumor cells. The other less cytotoxic CD3+CD56- cell subset represents a progenitor reservoir that could proliferate and differentiate into CD3+CD56+ CIK cells. CD3+CD56+ CIK cells express activating NK receptors including NKG2D, DNAM-1 and low levels of NKp30. Cell signalling not only through TCR/CD3, but also through NKG2D, DNAM-1 and NKp30, leads to CIK cell activation resulting in granule exocytosis and cytotoxicity. Antibody blocking experiments revealed that NKG2D, DNAM-1 and NKp30 are actually involved in tumor cell recognition and killing. Anti-CMV specific CIK cells could be expanded in standard CIK conditions and mediate both specific, MHC-restricted recognition of a CMV-pulsed autologous target and NK-like non specific cytolytic activity against leukemic cell targets. Antibody blocking of NKG2D and NKp30 only inhibited NK-like cytotoxicity. Their dual effector function suggests that CIK cells, when used in a clinical setting, may control both neoplastic relapses and viral infections, two frequently associated complications in transplanted patients. B-cell non-Hodgkin lymphoma is only partially susceptible to CIK-mediated lysis. The addition of anti-CD20 monoclonal antibodies GA101 or rituximab increased cytotoxicity mediated by CIK cell cultures by 35% and 15%, respectively. This enhancement was mainly due to antibody-dependent cytotoxicity mediated by the 1%-10% NK cells contaminating CIK cultures. The addition of human serum inhibited NK-cell activation induced by rituximab, but not activation induced by GA101. Overall lysis in presence of serum, even of a resistant B-NHL cell line, was significantly increased by 100 mcg/mL of rituximab, but even more so by GA101, with respect to CIK cultures alone. The combined use of CIK cells with anti-CD20 mAbs could represent a novel immunotherapy protocol for the treatment of B lymphoma patients with resistant disease.

Cancer adoptive cell therapy (ACT) relies on the infusion of immune cell populations mediating direct antitumor effects, such as cytotoxic CD8+ T lymphocytes (CTL), natural killer (NK) cells and Cytokine-Induced Killer (CIK) cells. In this study, we aimed at improving CIK cell potential for adoptive immunotherapy strategies. CIK cells are a heterogeneous population of ex vivo expanded lymphocytes, which share phenotypic and functional features with both NK and T cells. They exert a potent MHC-independent antitumor activity against both hematological and solid malignancies, but not normal tissues and hematopoietic precursors. Several clinical trials have demonstrated the feasibility and the therapeutic efficacy together with low toxicity of CIK cells infusion, supporting CIK cells as a very promising cell population for adoptive immunotherapy. In this work, CIK cells were obtained from PBMCs of healthy donors by the timed addition of IFN-γ, anti-CD3 antibody and IL-2. Analyzing their phenotype, we demonstrated for the first time a relevant expression of CD16 in a donor-dependent manner and, based on this observation, we proved the ability of CIK cells to kill tumors by an Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) mechanism. Indeed, the concurrent administration of clinically therapeutic mAbs, such as trastuzumab or cetuximab, led to a significant improvement of their antitumor activity in vitro against both ovarian and breast cancer cell lines. To formally prove that the CD16 receptor is functional and directly involved in the ADCC, an anti-CD16 blocking antibody was added to the assays. NK cell depletion from bulk cultures confirmed that the ADCC activity is accountable to the CIK CD16+ subpopulation. This novel function of CIK cells, never exploited before, was assessed for therapeutic efficacy in mouse models of human ovarian carcinoma xenografted in NOD/SCID common γ chain knockout (NSG) mice. Co-administration of CIK cells and mAbs significantly increased the survival of tumor-bearing mice, as compared to animals receiving CIK cells alone. CIK cell antitumor activity in vitro was also enhanced by the combination with bispecific antibodies and immunoligands, which are able to target both a tumor-associated antigen and activating receptors expressed by effector cells. Taken together, these data envisage new perspectives for adoptive immunotherapy where antigen-specific retargeting of T cells can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. In conclusion, this new therapeutic strategy for the ACT treatment of different types of tumors could find wide implementation and application, and be expanded to the use of additional therapeutic antibodies.
La terapia cellulare adottiva (Adoptive Cell Therapy, ACT) si basa sulla somministrazione di popolazioni di cellule immunitarie in grado di mediare un effetto antitumorale in modo diretto, ad esempio linfociti T CD8+ citotossici (CTL), cellule natural killer (NK) e cellule killer indotte da citochine (Cytokine-Induced Killer cells, CIK). Lo scopo di questo lavoro è stato quello di incrementare il potenziale delle cellule CIK nelle strategie di immunoterapia adottiva. Le cellule CIK sono una popolazione eterogenea di linfociti espansi ex vivo che condividono caratteristiche fenotipiche e funzionali sia con le cellule NK sia con le cellule T. Queste cellule esercitano una potente citotossicità MHC-indipendente nei confronti di tumori sia ematologici sia solidi, ma non di tessuti normali e precursori ematopoietici. Diversi trial clinici hanno dimostrato l’attuabilità, l’efficacia terapeutica e la bassa tossicità delle infusioni di cellule CIK, supportandole come popolazione cellulare molto promettente per l’immunoterapia adottiva. In questo lavoro, le cellule CIK sono state ottenute da cellule mononucleate del sangue periferico (Pheripheral Blood Mononuclear Cells, PBMCs) di donatori sani mediante l’aggiunta di interferone gamma (Interferon-γ, IFN-γ), anticorpi anti-CD3 e interleuchina 2 (Interleukin-2, IL-2). Analizzando il fenotipo, abbiamo dimostrato per la prima volta una rilevante espressione donatore-dipendente del recettore CD16 e, basandoci su questa osservazione, abbiamo analizzato la capacità delle cellule CIK di uccidere cellule tumorali mediante citotossicità cellulo-mediata anticorpo-dipendente (Antibody-Dependent Cell-mediated Cytotoxicity, ADCC). Infatti, abbiamo osservato che la simultanea somministrazione di anticorpi monoclonali terapeutici, come il trastuzumab e il cetuximab, portano ad un significativo incremento dell’attività antitumorale in vitro delle CIK nei confronti di linee cellulari di tumore ovarico e mammario. Per dimostrare che il CD16 è funzionale ed è direttamente coinvolto nell’ADCC, è stato aggiunto al saggio un anticorpo bloccante anti-CD16. La deplezione delle cellule NK ha confermato che l’ADCC è attribuibile alla sottopopolazione CD16+ delle cellule CIK. Questa nuova funzione delle cellule CIK, descritta qui per la prima volta, è stata valutata per la sua efficacia terapeutica in un modello murino di carcinoma ovarico umano trapiantato in topi NOD/SCID knockout per la catena comune γ (topi NSG). La co-somministrazione di cellule CIK e anticorpi monoclonali ha aumentato significativamente la sopravvivenza dei topi con tumore, in confronto ai topi trattati soltanto con le CIK. Inoltre, l’attività antitumorale in vitro delle cellule CIK è stata incrementata mediante la combinazione con anticorpi bispecifici e immunoligandi, in grado di legare contemporaneamente un antigene associato al tumore e un recettore attivatore espresso dalle cellule effettrici. Complessivamente, questi dati prospettano nuove possibilità per l’immunoterapia adottiva, in cui il reindirizzamento antigene-specifico dei linfociti T può essere ottenuto mediante la combinazione di anticorpi monoclonali di utilizzo clinico, già ampiamente utilizzati per la terapia antitumorale, con popolazioni di cellule CIK, che sono facilmente espandibili, economiche, sicure e non richiedono manipolazioni genetiche. In conclusione, questa nuova strategia terapeutica per trattamento di diversi tipi di tumori mediante terapia cellulare adottiva potrà trovare ampie possibilità di implementazione e applicazione, e potrà essere estesa all’utilizzo di ulteriori anticorpi terapeutici.

Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.

Il trapianto allogenico di cellule staminali ematopoietiche (HSCT) resta ad oggi l’unico trattamento curativo delle malattie onco-ematologiche, nonostante i notevoli miglioramenti ottenuti usando la chemioterapia e più recentemente le terapie verso un bersaglio molecolare specifico. In un contesto trapiantologico allogenico è fondamentale riuscire a trovare un bilanciamento tra la potenziale induzione della graft-versus-host disease (GVHD) e gli aspetti benefici della disparità tissutale tra donatore e ricevente, la cosiddetta graft-versus-leukemia (GVL). Sfortunatamente il trapianto risulta efficace in misura molto variabile (40-80% dei casi), per refrattarietà ai farmaci o ricaduta. Quest’ultima rimane la maggiore causa di fallimento dopo HSCT per i pazienti ad alto rischio di malattie ematologiche. Inoltre, sono frequenti le complicazioni infettive, dovuta alla pesante immunosoppressione, assolutamente necessaria per la prevenzione di GVHD severa. Gli approcci di immunoterapia cellulare sono uno strumento promettente per i pazienti che ricevono un HSCT, mantenendo l’effetto benefico della GVL, componente che maggiormente influenza l’efficacia del trapianto allogenico per i tumori del sangue, dovuta principalmente al riconoscimento immunologico ed all’eliminazione delle cellule leucemiche residue da parte dei linfociti T del donatore. Le azioni per aumentare l’effetto GVL nel prevenire la ricaduta in soggetti con leucemie ad alto rischio, pur minimizzando la tossicità, si sono focalizzati sull’uso di immunoterapia antileucemica mirata. Queste strategie includono metodi per incrementare l’effetto GVL con infusione di linfociti da donatore (DLI), che tuttavia presenta limitazioni dovute al fatto che la strategia si dimostra realmente efficace solo in pazienti con CML e all’alto rischio di GVHD. Recenti studi clinici con cellule cytokine-induced killer (CIK) esplorano il duplice potenziale antitumorale ed alloreattivo di questa eterogenea popolazione di linfociti T policlonali, che potrebbe influenzare favorevolmente il bilancio tra GVL e GVHD. Oltre agli aspetti descritti, legati al rigetto ed alla ricaduta, il contesto trapiantologico allogenico porta spesso a complicazioni infettive, per la prolungata e severa immunodeficienza a cui è sottoposto il paziente: nonostante il trattamento antivirale profilattico e preventivo, le riattivazioni virali rappresentano circa il 30% delle cause di morte da infezione dopo HSCT. L’infezione da citomegalovirus umano (CMV) è una di queste complicazioni fatali. Attualmente sono in fase di studio in un setting clinico, strategie di selezione rapida per isolare linfociti T CMV-specifici (CMV-T) derivanti da donatore e i recenti miglioramenti nella produzione di terapia cellulare-T per le infezioni virali, hanno notevolmente semplificato il loro ottenimento. Diversi approcci di terapia cellulare adottiva sono attualmente oggetto di studi clinici nella loro fase conclusiva, prospettando la possibilità che il trasferimento adottivo di CMV-T possa diventare uno standard di cura dopo HSCT. Nonostante queste ottimizzazioni, devono essere sviluppate nuove strategie per superare l’impossibilità di produrre linfociti T contro CMV da donatori CMV-sieronegativi. Questi approcci si basano su: vaccinazione del ricevente con cellule dendritiche (DCs), trasferimento adottivo di linfociti T da donatore da terza parte ed espansione di cellule Vδ2neg γδT reattive verso CMV. Tra queste, la vaccinazione con DC presentanti l’antigene di interesse rappresenta un approccio immunostimolatorio potente e versatile ed alcuni risultati clinici sono già disponibili. Nel presente studio sono stati sviluppati protocolli di immunoterapia cellulare adottiva per la produzione di prodotti medicinali di terapia avanzata (ATMPs) e trattamenti cellulari di manipolazione minima per migliorare l’esito in pazienti sottoposti a trapianto allogenico. I risultati ottenuti potranno essere utilizzati per la compilazione del Investigational Medicinal Product Dossier (IMPD), richiesto da parte delle Autorità Competenti per l’approvazione di trial clinici con farmaci sperimentali in Unione Europea. In particolare, abbiamo prodotto procedure operative standard (SOPs) per la caratterizzazione e la produzione di cellule CIK, per il possibile controllo di GVHD e GVL nei pazienti a rischio dopo HSCT, in condizioni GMP-like, predisponendo la rapida traslazione clinica di questi protocolli. Vengono riportati i dati preliminari relativi all’isolamento, produzione e caratterizzazione di linfociti T specifici per CMV ed alla generazione clinical-grade di cellule dendritiche. Abbiamo messo a punto tecniche per l’isolamento e la caratterizzazione delle cellule immunitarie prima dell’infusione. Gli scopi principali di questi studi pre-clinici sono la determinazione della sicurezza ed efficacia del prodotto finito, per la traslazione clinica nel controllo della riattivazione di CMV. Questa parte è stata ampiamente sviluppata in collaborazione con il Dipartimento di Medicina Interna II dell’Ospedale Universitario di Würzburg (Germania), il primo in Europa ad usare approcci di terapia cellulare per il trattamento delle infezioni dopo HSCT. Il progetto è stato svolto presso il Laboratorio di Terapie Cellulari Avanzate (LTCA), parte del Dipartimento di Ematologia e Terapie Cellulari dell’Ospedale S. Bortolo di Vicenza, con il supporto della Fondazione Progetto Ematologia. LTCA conduce la sua attività in collaborazione con il Centro Trapianti di Midollo Osseo, accreditato dal JACIE (Joint Accreditation Committee ISCT-EBMT) per il trapianto allogenico di midollo osseo, che rappresenta uno dei principali centri di Ematologia del Nord Italia.
Although important advances have been made in the treatment of hematological malignancies using chemotherapy, and more recently with targeted therapies, curative treatments often require allogeneic hematopoietic stem cell transplantation (HSCT). One of the major challenges in HSCT is to find a balance between the harmful induction of graft-versus-host disease (GVHD) and the beneficial aspects of tissue disparity, namely graft-versus-leukemia (GVL). Unfortunately, even this intensive treatment fails to prevent relapse in 10%-60% of cases, depending on whether the disease was successfully treated early or was relapsing or refractory at the time of transplantation. Relapse remains the major cause of treatment failure after HSCT for patients with high-risk hematological malignancies. In addition, strong immunosuppression absolutely essential for the prevention of severe GVHD, leads to infectious complications. To overcome these issues, cellular based-immunotherapeutic approaches represent a promising tool for the graft recipient patients, while maintaining the GVL effect. The so-called GVL effect is a major component of the effectiveness of HSCT for hematological malignancies and is due primarily to immunologic recognition and elimination of recipient leukemia cells by donor T cells: improvements in exploiting the GVL effect to prevent relapse in high-risk leukemias while minimizing toxicity have focused on the use of targeted antileukemic immunotherapy. These strategies include methods to boost the GVL effect with donor lymphocyte infusion (DLI) but the limited spectrum of activity and high risk of GVHD remain major limitations of this approach. Recent clinical studies with cytokine-induced killer (CIK) cells explore the antitumor and alloreactive potentials of this heterogeneous population of polyclonal T lymphocytes, that might favourably affect the balance between GVL and GVHD. After HSCT, a prolonged and severe immune deficiency often leads to infectious complications: despite prophylactic or pre-emptive antiviral treatment viral reactivations consist in about 30% of death after HSCT. Human cytomegalovirus (CMV) infection is one of such life-threatening complication. Rapid selection strategies to isolate donor-derived CMV-specific T cells (CMV-T) are currently being tested in the clinical setting and recent advances in the manufacturing of T-cell therapy for viral infections have greatly simplified the production. Several approaches of adoptive cellular therapies are now being evaluated in late-phase or licensing studies, raising the prospect that adoptive transfer of CMV-T may become a standard of care after HSCT. To overcome the impossibility to produce T cells against CMV from CMV-seronegative donors, new strategies able to induce pathogen-specific responses should be developed. These approaches relies on recipient dendritic cells (DCs) vaccination, third-party adoptive T-cell transfer and on CMV-reactive Vδ2neg γδT cells expansion. Among these, vaccination with antigen-loaded DCs has been shown to be a potent and versatile immunostimulatory approach and some clinical trials results are already available. This study is aimed at the development of adoptive cellular immunotherapeutic protocols for the production of Advanced Therapy Medicinal Products (ATMPs) for the outcome improvement of patients after HSCT. In particular, we provide standard operating procedures (SOPs) to obtain fully effective and characterized CIK cells ready to use in a clinical setting under good manufacturing practice (GMP)-like conditions. Indeed, the established protocol can be used to compile the Investigational Medicinal Product Dossier (IMPD), the basis for the approval of a clinical trial with investigational new drugs by the competent authorities in the European Union. We report preliminary data on manufacturing of CMV-T and CMV-loaded DCs: we set up experimental procedures to isolate, expand and characterize the immune cells prior to infusion. The main goals of these pre-clinical studies are to determine the safety and potency profiles of the cellular product for its translation to the clinic, in the control of CMV reactivation. This part of the project was largely developed in collaboration with the Department of Internal Medicine II of the University Hospital of Würzburg (Germany), the first in Europe to use cellular therapy for treatment of infections after HSCT. The overall project was lead by the Advanced Cellular Therapy Laboratory (LTCA), part of the Cell Therapy and Hematology Department of the S. Bortolo Hospital, in collaboration with the Bone Marrow Transplant Center and with the Hematology Project Foundation.

Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.:Inhaltsverzeichnis I Abbildungsverzeichnis III Tabellenverzeichnis IV Bibliographische Beschreibung V Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 CIK-Zellen (CIK) 3 1.1.1 Merkmale von CIK-Zellen 3 1.1.2 Wirkungsmechanismen von CIK-Zellen 3 1.1.3 Studienlage 4 1.1.4 Bisherige Ansätze zur Verbesserung der Kultivierungsbedingungen 6 1.2 Mesenchymale Stammzellen (MSC) 7 1.2.1 Allgemein 7 1.2.2 Differenzierung von MSC 7 1.2.3 Heterogenität und Einflussfaktoren der MSC - Identitätsproblematik 8 1.2.4 Charakterisierung von MSC 9 1.2.5 Therapeutische Einsatzmöglichkeiten von MSC 11 2 Zielformulierung 15 3 Material und Methoden 16 3.1 Tiere 16 3.2 Materialien 17 3.2.1 Materialien für Zellkultur 17 3.2.2 Materialien für FACS-Analyse 18 3.2.3 Materialien für Zytotoxizitätsassay 19 3.2.4 Materialien für CFU-F-Assay 20 3.3 Methoden 21 3.3.1 Statistische Auswertung 21 3.3.2 Zellkultur 22 3.3.3 FACS (Fluorescence Activated Cell Sorting) 26 3.3.4 Markierung der MSC mit DiD 28 3.3.5 Zytotoxizitätsassay (LDH-Freisetzungsassay) 29 3.3.6 CFU-F-Assay 32 4 Ergebnisse 34 4.1 Beeinflussung der Wachstumskurve 34 4.1.1 Der Wachstumskurvenverlauf von CIK-Zellen (Kontrollen) 34 4.1.2 Der Wachstumskurvenverlauf von CIK-Zellen in der Kokultur mit MSC 35 4.1.3 Der Wachstumskurvenverlauf in MSC-konditioniertem Medium 37 4.1.4 Der Wachstumskurvenverlauf bei Restimulierung an Tag 14 38 4.2 Beeinflussung des Oberflächenphänotyps 40 4.2.1 Der Oberflächenphänotyp von CIK-Zellen 40 4.2.2 Vergleich Oberflächenphänotyp Kontrollen mit kokultivierten CIK 43 4.3 Beeinflussung der Vitalität 46 4.4 Beeinflussung der Zytotoxizität 48 4.5 Identifizierung der MSC 49 4.5.1 Adhärenz an Plastikoberflächen 50 4.5.2 Fibroblastenähnliche Wachstumsmorphologie 50 4.5.3 Wachstum in Colony-Forming-Units 51 4.5.4 Der Oberflächenphänotyp von MSC 53 4.6 Schicksal der MSC in der Kokultur 54 4.6.1 Der Oberflächenphänotyp der adhärenten Zellen nach Kokultur 54 4.6.2 Kokultur mit DiD gelabelten MSC 57 5 Diskussion 59 5.1 Beeinflussung der Wachstumskurve 60 5.1.1 Mechanismen der Beeinflussung des Wachstumskurvenverlaufs 60 5.1.2 Fehlerbetrachtung 68 5.2 Identifizierung der CIK sowie Beeinflussung von Phänotyp und Vitalität 69 5.3 Beeinflussung der Zytotoxizität 70 5.3.1 Vergleich Zytotoxizität Kontrollen mit Kokulturen 70 5.3.2 Fehlerbetrachtung 71 5.4 Identifizierung der MSC 72 6 Schlussfolgerung 75 7 Ausblick 77 8 Zusammenfassung 79 Literaturverzeichnis 83 Danksagung I

La leucémie aigüe lymphoblastique de précurseurs des cellules B (pré-B LAL) est le cancer le plus fréquent chez l’enfant. La transplantation de cellules souches hématopoïétiques (TCSH) est nécessaire dans environ 20 à 30 % des enfants ayant une pré-B LAL. Les rechutes après TCSH sont habituellement réfractaires aux thérapies actuelles, et par conséquent, il est important de développer et d’optimiser de nouvelles stratégies thérapeutiques. Dans cette étude, nous nous sommes intéressés aux cellules « cytokine-induced killer » (CIK). En effet, ces cellules ont été montrées comme hautement cytotoxique contre beaucoup de types de cancers. Cependant, leur activité cytotoxique contre les pré-B LAL n’est pas vraiment efficace. Par conséquent, nous avons étudié la possibilité de combiner l’immunothérapie des cellules CIK avec l’interféron alpha (IFN-α) afin d’optimiser l’activité lytique de ces cellules contre les cellules pré-B LAL. De plus, vu qu’il a été démontré que l’activité cytotoxique des cellules CIK provient de la fraction CD56+, plus particulièrement les cellules CD3+CD56+, nous avons décidé d’utiliser la fraction CD56+ (cellules CD56+) dans l’ensemble de nos expériences. Nous avons observé in vitro que les cellules CD56+ lysent mieux les lignées cellulaires pré-B LAL comparativement aux cellules CIK non purifiées. Aussi, leur activité cytotoxique peut être augmentée par le traitement avec l’IFN-α. Par ailleurs, nous avons démontré l’efficacité des cellules CD56+ traitées par l’IFN-α contre les lignées cellulaires pré- B LAL in vivo, dans le modèle de souris NOD/SCID/gamma c- (NSG). La survie des souris est significativement prolongée lorsqu’elles reçoivent les cellules pré-B LAL avec les cellules CD56+ traitées par l’IFN-α. Nous avons par la suite étudié le mécanisme d’action des cellules CD56+ contre les lignées cellulaires pré-B LAL. Nous avons observé que les cellules CD56+ provenant de sang de cordon sont plus efficaces que les cellules CD56+ provenant de sang I périphérique pour tuer les lignées cellulaires pré-B LAL. Nous avons également montré que les cellules CD56+ utilisent seulement la voie NKG2D ou bien les voies NKG2D et TRAIL selon la lignée cellulaire pré-B LAL cible et selon la provenance de la source des cellules CD56+. Par ailleurs, nous avons remarqué que les cellules CIK sont sensibles à l’apoptose par Fas, et que cette sensibilité influence leur activité cytotoxique contre les cellules tumorales. En conclusion, les cellules CD56+ sont cytotoxiques contre les lignées cellulaires pré-B LAL, et leur effet lytique est augmenté par l’IFN-α aussi bien in vitro qu’in vivo dans le modèle de souris NSG. Ces données précliniques sont encourageantes pour tester cette nouvelle approche d’immunothérapie dans le traitement contre la pré-B LAL.
Precursor B-cell acute lymphoblastic leukemia (B-ALL) is the most common form of leukemia in children. Hematopoietic stem cell transplantation (HSCT) is required in around 20 to 30% of children with a B-ALL. The relapses occuring post-HSCT are usually insensitive to current therapy. Therefore, it is important to develop and optimize a new therapeutic strategy. In this study, we were interested to study « cytokine-induced killer » (CIK) cells. These cells have been shown to be very cytotoxic against many types of tumor. However, their cytotoxic activity against B-ALL cells is not very efficient. Consequently, we have studied the effect of combining adoptive immunotherapy of CIK cells with the interferon alpha (IFN-α) to increase their lytic activity against B-ALL cells. In addition, in the literature, the cytotoxic activity of CIK cells has been shown to come from the CD56+ fraction (CD56+ CIK), in particular CD3+CD56+ cells. Therefore, we used the CD56+ fraction in all the experiments. We have observed in vitro that CD56+ CIK cells killed more efficiently B-ALL cell lines than did non-purified CIK cells. Also, their cytotoxic activity could be enhanced with IFN-α. Moreover, we have demonstrated the efficacy of IFN-α-treated-CD56+ CIK cells against B-ALL cell lines in vivo in the model of NOD/SCID/gamma c- (NSG) mice by showing that the survival of mice injected with B-ALL cell lines was significantly increased when they were injected with IFN-α-treated-CD56+ CIK cells. Subsequently, we have studied the lytic mechanism of CD56+ CIK cells against B-ALL cell lines. We have observed that CD56+ CIK cells from cord blood were more efficient than CD56+ CIK cells from peripheral blood to kill B-ALL cell lines. CD56+ CIK cells used only the NKG2D pathway or the both NKG2D and TRAIL pathways depending on the B-ALL cell line and the source of CIK cells. In addition, we showed that CIK cells were sensitive to Fas apoptosis. This sensitivity III influenced the cytotoxic activity of CIK cells against tumor cells. In conclusion, CD56+ CIK cells are cytotoxic against B-ALL cell lines, and their effect can be increased with IFN-α in vitro and in vivo. Taken together, our pre-clinical data are very interesting for testing the potential clinical utility of purified CD56+ CIK cells as an immunotherapeutic strategy for B- ALL patients.

The standard management for epithelial ovarian cancer (EOC) is a combination of aggressive debulking surgery with residual tumor of less than 1 cm and platinum-based chemotherapy. However, a high percentage of patients experience disease recurrence. Extensive efforts to find new therapeutic options have been made, albeit cancer cells develop drug resistance and malignant progression occurs. Novel therapeutic strategies are needed to enhance progression-free survival and overall survival of patients with advanced EOC. Several preclinical and clinical studies investigated feasibility and efficacy of adoptive cell therapy (ACT) in EOC. The aim of this chapter is to present an overview of ACT in EOC, focusing on Human Leukocyte Antigen (HLA)-restricted tumor infiltrating lymphocytes and MHC-independent immune effectors such as natural killer and cytokine-induced killer. The available data suggest that ACT may provide the best outcome in patients with low tumor burden, minimal residual disease, or maintenance therapy. Further preclinical studies and clinical trials are needed.