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Chimeric antigen receptor (CAR) T cell therapy is a potent new treatment option for relapsed or refractory hematologic malignancies. As the monitoring of CAR T cell kinetics can provide insights into the activity of the therapy, appropriate CAR T cell detection methods are essential. Here, we report on the comprehensive validation of a flow cytometric assay for peripheral blood CD19 CAR T cell detection. Further, a retrospective analysis (n = 30) of CAR T cell and B cell levels over time has been performed, and CAR T cell phenotypes have been characterized. Serial dilution experiments demonstrated precise and linear quantification down to 0.05% of T cells or 22 CAR T cell events. The calculated detection limit at 13 events was confirmed with CAR T cell negative control samples. Inter-method comparison with real-time PCR showed appreciable correlation. Stability testing revealed diminished CAR T cell values already one day after sample collection. While we found long-term CAR T cell detectability and B cell aplasia in most patients (12/17), some patients (5/17) experienced B cell recovery. In three of these patients the coexistence of CAR T cells and regenerating B cells was observed. Repeat CAR T cell infusions led to detectable but limited re-expansions. Comparison of CAR T cell subsets with their counterparts among all T cells showed a significantly higher percentage of effector memory T cells and a significantly lower percentage of naïve T cells and T EMRA cells among CAR T cells. In conclusion, flow cytometric CAR T cell detection is a reliable method to monitor CAR T cells if measurements start without delay and sufficient T cell counts are given.
Cytokine-induced killer (CIK) cells are an immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (HSCT) in acute leukemia or myelodysplastic syndrome (MDS) patients. Prompt and sequential administration of escalating cell doses improves the efficacy of CIK cell therapy without exacerbating graft vs. host disease (GVHD). This study addresses manufacturing-related issues and aimed to develop a time-, personal- and cost-saving good manufacturing process (GMP)-compliant protocol for the generation of ready-for-use therapeutic CIK cell doses starting from one unstimulated donor-derived peripheral blood (PB) or leukocytapheresis (LP) products. Culture medium with or without the addition of either AB serum, fresh frozen plasma (FFP) or platelet lysate (PL) was used for culture. Fresh and cryopreserved CIK cells were compared regarding expansion rate, viability, phenotype, and ability to inhibit leukemia growth. Cell numbers increased by a median factor of 10-fold in the presence of FFP, PL, or AB serum, whereas cultivation in FFP/PL-free or AB serum-free medium failed to promote adequate CIK cell proliferation (p < 0.01) needed to provide clinical doses of 1 × 106 T cells/kG, 5 × 106 T cells/kG, 1 × 107 T cells/kG, and 1 × 108 T cells/kG recipient body weight. CIK cells consisting of T cells, T- natural killer (T-NK) cells and a minor fraction of NK cells were not significantly modified by different medium supplements. Moreover, neither cytotoxic potential against leukemic THP-1 cells nor cell activation shown by CD25 expression were significantly influenced. Moreover, overnight and long-term cryopreservation had no significant effect on the composition of CIK cells, their phenotype or cytotoxic potential. A viability of almost 93% (range: 89–96) and 89.3% (range: 84–94) was obtained after freeze-thawing procedure and long-term storage, respectively, whereas viability was 96% (range: 90-97) in fresh CIK cells. Altogether, GMP-complaint CIK cell generation from an unstimulated donor-derived PB or LP products was feasible. Introducing FFP, which is easily accessible, into CIK cell cultures was time- and cost-saving without loss of viability and potency in a 10-12 day batch culture. The feasibility of cryopreservation enabled storage and delivery of sequential highly effective ready-for-use CIK cell doses and therefore reduced the number of manufacturing cycles.
Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Despite therapeutic progress, prognosis in high-risk NB is poor and innovative therapies are urgently needed. Therefore, we addressed the potential cytotoxic capacity of interleukin (IL)-activated natural killer (NK) cells compared to cytokine-induced killer (CIK) cells for the treatment of NB. NK cells were isolated from peripheral blood mononuclear cells (PBMCs) by indirect CD56-enrichment or CD3/CD19-depletion and expanded with different cytokine combinations, such as IL-2, IL-15, and/or IL-21 under feeder-cell free conditions. CIK cells were generated from PBMCs by ex vivo stimulation with interferon-γ, IL-2, OKT-3, and IL-15. Comparative analysis of expansion rate, purity, phenotype and cytotoxicity was performed. CD56-enriched NK cells showed a median expansion rate of 4.3-fold with up to 99% NK cell content. The cell product after CD3/CD19-depletion consisted of a median 43.5% NK cells that expanded significantly faster reaching also 99% of NK cell purity. After 10–12 days of expansion, both NK cell preparations showed a significantly higher median cytotoxic capacity against NB cells relative to CIK cells. Remarkably, these NK cells were also capable of efficiently killing NB spheroidal 3D culture in long-term cytotoxicity assays. Further optimization using a novel NK cell culture medium and a prolonged culturing procedure after CD3/CD19-depletion for up to 15 days enhanced the expansion rate up to 24.4-fold by maintaining the cytotoxic potential. Addition of an IL-21 boost prior to harvesting significantly increased the cytotoxicity. The final cell product consisted for the major part of CD16−, NCR-expressing, poly-functional NK cells with regard to cytokine production, CD107a degranulation and antitumor capacity. In summary, our study revealed that NK cells have a significantly higher cytotoxic potential to combat NB than CIK cell products, especially following the synergistic use of IL-15 and IL-21 for NK cell activation. Therefore, the use of IL-15+IL-21 expanded NK cells generated from CD3/CD19-depleted apheresis products seems to be highly promising as an immunotherapy in combination with haploidentical stem cell transplantation (SCT) for high-risk NB patients.
Background: Prolonged 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.
Methods: CIK 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.
Results: Pre-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.
Discussion: The 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.
Despite the availability of new antifungal compounds, invasive fungal infection remains a significant cause of morbidity and mortality in children and adults undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Allogeneic HSCT recipients suffer from a long lasting defect of different arms of the immune system, which increases the risk for and deteriorates the prognosis of invasive fungal infections. In turn, advances in understanding these immune deficits have resulted in promising strategies to enhance or restore critical immune functions in allogeneic HSCT recipients. Potential approaches include the administration of granulocytes, since neutropenia is the single most important risk factor for invasive fungal infection, and preliminary clinical results suggest a benefit of adoptively transferred donor-derived antifungal T cells. In vitro data and animal studies demonstrate an antifungal effect of natural killer cells, but clinical data are lacking to date. This review summarizes and critically discusses the available data of immunotherapeutic strategies in allogeneic HSCT recipients suffering from invasive fungal infection.
Allogeneic stem cell transplantation (allo-SCT) has become an important treatment modality for patients with high-risk acute myeloid leukemia (AML) and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD) status ultimately leading to patients’ relapse. Adoptive donor lymphocyte infusions based on MRD status using IL-15-expanded cytokine-induced killer (CIK) cells may prevent relapse without causing graft-versus-host-disease (GvHD). To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL-2Rγc−, NSG) were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS) cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow followed by liver, lung, spleen, peripheral blood (PB), and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at equal amounts were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells 250 times more CIK than THP-1 cells were needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliable 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells have potent cytotoxic capacity against AML and RMS cells without causing GvHD.