Tolga Sutlu

Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study

BACKGROUND Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 μg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.

Natural killer cell‐based immunotherapy in cancer: current insights and future prospects

As our understanding of the molecular mechanisms governing natural killer (NK) cell activity increases, their potential in cancer immunotherapy is growing increasingly prominent. This review analyses the currently available preclinical and clinical data regarding NK cell‐based immunotherapeutic approaches in cancer starting from a historical background and an overview of molecular mechanisms taking part in NK cell responses. The status of NK cells in cancer patients, currently investigated clinical applications such as in vivo modulation of NK cell activity, ex vivo purification/expansion and adoptive transfer as well as future possibilities such as genetic modifications are discussed in detail.

Autologous antitumor activity by NK cells expanded from myeloma patients using GMP-compliant components

Multiple myeloma (MM) is an incurable plasma cell malignancy with poor outcome. The most promising therapeutic options currently available are combinations of transplantation, targeted pharmacotherapy, and immunotherapy. Cell-based immunotherapy after hematopoietic stem-cell transplantation has been attempted, but with limited efficacy. Natural killer (NK) cells are interesting candidates for new means of immunotherapy; however, their potential clinical use in MM has not been extensively studied. Here, we explored the possibility of expanding NK cells from the peripheral blood of 7 newly diagnosed, untreated MM patients, using good manufacturing practice (GMP)-compliant components. After 20 days of culture, the number of NK cells from these patients had expanded on average 1600-fold. Moreover, expanded NK cells showed significant cytotoxicity against primary autologous MM cells, yet retained their tolerance against nonmalignant cells. Based on these findings, we propose that autologous NK cells expanded ex vivo deserve further attention as a possible new treatment modality for MM.

Clinical-grade, large-scale, feeder-free expansion of highly active human natural killer cells for adoptive immunotherapy using an automated bioreactor

BACKGROUND AIMS Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. Ex vivo expansion and activation of NK cells under good manufacturing practice (GMP) conditions are crucial for facilitating large clinical trials. The goal of this study was to optimize a large-scale, feeder-free, closed system for efficient NK cell expansion. METHODS Peripheral blood mononuclear cells (PBMCs) from healthy donors and myeloma patients were cultured for 21 days using flasks, cell culture bags and bioreactors. Final products from different expansions were evaluated comparatively for phenotype and functionality. RESULTS Significant NK cell expansions were obtained in all systems. The bioreactor yielded a final product rich in NK cells (mean 38%) ensuring that a clinically relevant cell dose was reached (mean 9.8 x 10⁹ NK cells). Moreover, we observed that NK cells expanded in the bioreactor displayed significantly higher cytotoxic capacity. It was possible to attribute this partially to a higher expression level of NKp44 compared with NK cells expanded in flasks. CONCLUSIONS These results demonstrate that large amounts of highly active NK cells for adoptive immunotherapy can be produced in a closed, automated, large-scale bioreactor under feeder-free current GMP conditions, facilitating clinical trials for the use of these cells.

Safety analysis of ex vivo-expanded NK and NK-like T cells administered to cancer patients: a Phase I clinical study

The chimeric state after allogeneic hematopoietic stem cell transplantation provides a platform for adoptive immunotherapy using donor-derived immune cells. The major risk with donor lymphocyte infusions (DLIs) is the development of graft-versus-host disease (GvHD). Development of new DLI products with antitumor reactivity and reduced GvHD risk represents a challenging task in cancer immunotherapy. Although natural killer (NK) and NK-like T cells are promising owing to their antitumor activity, their low concentrations in peripheral blood mononuclear cells reduces their utility in DLIs. We have recently developed a system that allows expansion of clinical-grade NK and NK-like T cells in large numbers. In this study, the safety of donor-derived long-term ex vivo-expanded human NK and NK-like T cells given as DLIs was investigated as immunotherapy for cancer in five patients following allogeneic stem cell infusion. Infusion of the cells was safe whether administered alone or with IL-2 subcutaneously. No signs of acute GvHD were observed. One patient with hepatocellular carcinoma showed markedly decreased serum alpha-fetoprotein levels following cell infusions. These findings suggest that the use of ex vivo-expanded NK and NK-like T cells is safe and appears an attractive approach for further clinical evaluation in cancer patients.

Triticum durum Metallothionein ISOLATION OF THE GENE AND STRUCTURAL CHARACTERIZATION OF THE PROTEIN USING SOLUTION SCATTERING AND MOLECULAR MODELING

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 ± 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.

Inhibition of Intracellular Antiviral Defense Mechanisms Augments Lentiviral Transduction of Human Natural Killer Cells: Implications for Gene Therapy

Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approach for cancer treatment. Yet, optimization of highly efficient and clinically applicable gene transfer protocols for NK cells still presents a challenge. In this study, we aimed at identifying conditions under which optimum lentiviral gene transfer to NK cells can be achieved. Our results demonstrate that stimulation of NK cells with interleukin (IL)-2 and IL-21 supports efficient transduction using a VSV-G pseudotyped lentiviral vector. Moreover, we have identified that inhibition of innate immune receptor signaling greatly enhances transduction efficiency. We were able to boost the efficiency of lentiviral genetic modification on average 3.8-fold using BX795, an inhibitor of the TBK1/IKKɛ complex acting downstream of RIG-I, MDA-5, and TLR3. We have also observed that the use of BX795 enhances lentiviral transduction efficiency in a number of human and mouse cell lines, indicating a broadly applicable, practical, and safe approach that has the potential of being applicable to various gene therapy protocols.

Clinical impact of chromosomal aberrations in multiple myeloma.

Abstract.  Nahi H., Sutlu T., Jansson M., Alici E., Gahrton G. (Karolinska Institutet, Karolinska University Hospital Huddinge; Hematology Centre, Karolinska University Hospital, Huddinge, Stockholm, Sweden) Clinical impact of chromosomal aberrations in multiple myeloma (Review). J Intern Med 2011; 269: 137–147.

The prognostic significance of 8p21 deletion in multiple myeloma.

Multiple myeloma (MM) accounts for approximately 2% of all cancer deaths and 20% of deaths caused by haematological malignancies. Allogeneic stem cell transplantation might be curative for a small group of eligible patients, but the current gold standard for the treatment of patients less than 60–65 years of age is high-dose chemotherapy (HDT) followed by autologous stem cell transplantation (ASCT). Factors that predict survival in MM including b2-microglobulin (b2M), creatinine and haemoglobin (Hb) levels have been well defined (Durie et al, 2003; Chng & Fonseca, 2005). Furthermore, the occurrence of various chromosomal abnormalities among the malignant cells, such as del(13), t(4;14), t(11;14) and del(17p) have been shown to have an impact on prognosis (Avet-Loiseau et al, 2007). Nevertheless, better assessment and stratification of prognostic factors are needed in order to comprehend the different responses to therapy. Information gained this way will enhance the understanding about treatment prediction in patients with MM. This study analyzed various previously well-known chromosomal regions and translocations by fluorescent in situ hybridization (FISH) in bone marrow (BM) aspirations from newly diagnosed patients with MM, but with added probes for the 8p21 region. We evaluated the correlations between various chromosomal abnormalities as well as their effects on survival. Statistical analysis was performed using the Statistical Package for the Social Sciences (spss) 15 software (SPSS Inc. Chicago, IL, USA) and P < 0Æ05 was considered as statistically significant. The variables were analyzed by using Log-rank tests on Kaplan–Meier plots to identify their impacts on progression-free survival (PFS) and overall survival (OS). Among the analyzed chromosomal aberrations, the frequency of occurrence was highest for del(13)(q14), at 60%, followed by amp(9)(p21) (53%), amp(15)(q22) (50%), amp(19)(q13) (41%), amp(1)(q21) (38%), del(8)(p21) (24%), del(17)(p13) (22%), t(4;14) (15%), t(11;14) (14%), and lowest for del(6)(q21) (9%). According to univariate analysis del(13)(q14), amp(1)(q21) and del(8)(p21) were significantly associated with PFS while only del(13)(q14) and del(8)(p21) had significant effect on OS. Of the 65 patients evaluated with FISH, 58 had received ASCT and 44 of these patients carried normal 8p21 whereas 14 had del(8)(p21). Survival analysis revealed that loss of 8p21 was significantly associated with poor PFS (Fig 1A) and OS (Fig 1B). Other factors, such as t(4;14) (57 months vs. 34 months, P > 0Æ05), which have been shown to affect survival did not present any significance in this series. This is possibly due to the fact that a larger number of patients is needed to investigate the effect of events with such low incidence (10– 15%) (Avet-Loiseau et al, 2007), whereas del(8)(p21) seems to have a higher incidence (24%) in this series (see Table SI for the results of FISH analysis on all the chromosomal regions as well as PFS and OS values for each group). Bivariate correlations between the occurrences of chromosomal aberrations were analyzed by a two-tailed test of significance using Kendall’s tau-b coefficient. A hierarchical clustering that fits the correlations observed by this method was obtained by using average linkage between groups and the pattern difference as a measure of the distance between factors. Hierarchical clustering analysis of FISH data resulted in three main clusters of chromosomal aberrations (Fig 1C): (i) a ‘chromosomal instability’ branch carrying the closely related aberrations del(13)(q14), t(11;14) and amp(1)(q21), (ii) a ‘hypodiploid’ branch covering losses of 6q21, 17p13, 8p21 as well as the translocation t(4;14), (iii) a ‘hyperdiploid’ branch with extra copies of 15q22, 19q13 and 9p21. Prognostic factors for survival were also subjected to multivariate analysis using the Cox proportional hazards regression model with forward stepwise method. For the multivariate analysis, only FISH results that were significantly associated with PFS or OS in the univariate analysis (13q14, 1q21 and 8p21) were used along with other data that also had a significant effect on survival in this series (percentage of plasma cells in BM at the time of diagnosis, Hb levels, b2M levels) (Table I). This method also identified del(8)(p21) as an independent prognostic factor for both PFS (P = 0Æ033) and OS (P = 0Æ029). Previous studies have linked molecular dysregulations originating from changes in the 8p21 region to various malignancies including leukaemic mantle cell lymphoma (Martinez-Climent et al, 2001), B-cell lymphoma (RubioMoscardo et al, 2005) and prostate cancer (Hornstein et al, 2008). Moreover, the loss of this region has been shown to have a negative effect on survival in head and neck cancers, but has, to our knowledge, previously not been studied in MM. This study presents del(8)(p21) as a novel independent prognostic factor in MM affecting both PFS and OS. Moreover, our data also confirmed the effects of various previously defined prognostic factors, such as del(13)(q14), amp(1)(q21), percentage of plasma cells in BM at the time of diagnosis, Hb levels, b2M levels. The 8p21 region covers a number of genes that could be taking part in the pathogenesis and progression of MM. Correspondence

Retroviral Gene Transfer into Primary Human Natural Killer Cells

Modulation of intracellular signaling pathways or receptor expression in natural killer (NK) cells by genetic manipulation is an attractive possibility in studies of NK cell specificity and function. Moreover, feasible applications of these genetic manipulations in the context of gene and NK cell therapy regimens may be considered. However, efficient gene modification of primary NK cells has been largely hampered by the absence of an efficient gene-transfer protocol.A retrovirus-based easy-to-use transduction protocol that can insert the gene of interest permanently into primary NK cells would be an important tool to advance our studies in NK cell biology and NK cell-mediated therapies. We have recently described a protocol for efficient expansion of NK cells under good manufacturing practice (GMP) conditions from the healthy donors and from patients with hematological malignancies. As the active division of cells is a prerequisite for efficient retroviral insertion, the high rate of expansion in this protocol provides more efficient transduction by retroviral vectors. We hereby present this simple and efficient retroviral vector-based gene-transfer protocol for such ex vivo cultured primary human NK cells.