Publications by authors named "Ulrike Köhl"

41 Publications

The noncoding RNA LINC00152 conveys contradicting effects in different glioblastoma cells.

Sci Rep 2021 09 16;11(1):18499. Epub 2021 Sep 16.

Institute of Clinical Immunology, University of Leipzig, Leipzig, Germany.

Glioblastoma multiforme (GBM) is an extremely aggressive brain tumor, characterized by its high genetic heterogeneity. In search of novel putative therapeutic RNA targets we investigated the role of the oncogenic long noncoding RNA LINC00152 (CYTOR, and STAiR18) in A172 glioblastoma cells. Here, we are the first to describe, that LINC00152 unexpectedly acts in a tumor suppressive manner in this cell line. SiRNA-based knockdown of LINC00152 enhanced malignant tumor behaviors including proliferation, cell cycle entry, migration, and invasion, contradicting previous studies using U87-MG and LN229 glioblastoma cells. Furthermore, LINC00152 knockdown had no influence on survival of A172 glioblastoma cells. In a genome wide transcription analysis of A172 and U87-MG glioblastoma cells, we identified 70 LINC00152 target genes involved in locomotion, cell migration, and motility in A172 cells, whereas in U87-MG cells only 40 target genes were detected. The LINC00152-regulated genes found in A172 differed from those identified in U87-MG glioblastoma cells, none of them being regulated in both cell lines. These findings underline the strong genetic heterogeneity of glioblastoma and point to a potential, yet unknown risk addressing LINC00152 lncRNA as a prospective therapeutic target in GBM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-97533-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8446032PMC
September 2021

Distinct Immune Signatures Indicative of Treatment Response and Immune-Related Adverse Events in Melanoma Patients under Immune Checkpoint Inhibitor Therapy.

Int J Mol Sci 2021 Jul 27;22(15). Epub 2021 Jul 27.

Department of Dermatology, Venereology and Allergology, University Medical Center Leipzig, Philipp-Rosenthal-Str. 23, 04103 Leipzig, Germany.

To identify potential early biomarkers of treatment response and immune-related adverse events (irAE), a pilot immune monitoring study was performed in stage IV melanoma patients by flow cytometric analysis of peripheral blood mononuclear cells (PBMC). Overall, 17 patients were treated with either nivolumab or pembrolizumab alone, or with a combination of nivolumab and ipilimumab every three weeks. Of 15 patients for which complete response assessment was available, treatment responders ( = 10) as compared to non-responders ( = 5) were characterized by enhanced PD-1 expression on CD8 T cells immediately before treatment (median ± median absolute deviation/MAD 26.7 ± 10.4% vs. 17.2 ± 5.3%). Responders showed a higher T cell responsiveness after T cell receptor ex vivo stimulation as determined by measurement of programmed cell death 1 (PD-1) expression on CD3 T cells before the second cycle of treatment. The percentage of CD8 effector memory (CD8CD45RACD45ROCCR7) T cells was higher in responders compared to non-responders before and immediately after the first cycle of treatment (median ± MAD 39.2 ± 7.3% vs. 30.5 ± 4.1% and 37.7 ± 4.6 vs. 24.0 ± 6.4). Immune-related adverse events (irAE) were accompanied by a higher percentage of activated CD4 (CD4CD38HLADR) T cells before the second treatment cycle (median ± MAD 14.9 ± 3.9% vs. 5.3 ± 0.4%). In summary, PBMC immune monitoring of immune-checkpoint inhibition (ICI) treatment in melanoma appears to be a promising approach to identify early markers of treatment response and irAEs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22158017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8348898PMC
July 2021

Detection of Immune Checkpoint Receptors - A Current Challenge in Clinical Flow Cytometry.

Front Immunol 2021 1;12:694055. Epub 2021 Jul 1.

Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany.

Immunological therapy principles are increasingly determining modern medicine. They are used to treat diseases of the immune system, for tumors, but also for infections, neurological diseases, and many others. Most of these therapies base on antibodies, but small molecules, soluble receptors or cells and modified cells are also used. The development of immune checkpoint inhibitors is amazingly fast. T-cell directed antibody therapies against PD-1 or CTLA-4 are already firmly established in the clinic. Further targets are constantly being added and it is becoming increasingly clear that their expression is not only relevant on T cells. Furthermore, we do not yet have any experience with the long-term systemic effects of the treatment. Flow cytometry can be used for diagnosis, monitoring, and detection of side effects. In this review, we focus on checkpoint molecules as target molecules and functional markers of cells of the innate and acquired immune system. However, for most of the interesting and potentially relevant parameters, there are still no test kits suitable for routine use. Here we give an overview of the detection of checkpoint molecules on immune cells in the peripheral blood and show examples of a possible design of antibody panels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2021.694055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8281132PMC
July 2021

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing.

Front Immunol 2021 4;12:684052. Epub 2021 Jun 4.

Department for GMP Process Development/ATMP Design, Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany.

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these "off-the-shelf" therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function.

Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells flow cytometry, cytotoxicity assays, and comet assays, amongst others.

Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30.

Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2021.684052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8212864PMC
June 2021

First-in-human intracochlear application of human stromal cell-derived extracellular vesicles.

J Extracell Vesicles 2021 06 4;10(8):e12094. Epub 2021 Jun 4.

Department of Transfusion Medicine University Hospital Salzburger Landeskliniken GesmbH (SALK) and Paracelsus Medical University (PMU) Salzburg Austria.

Extracellular vesicles (EVs) derived from the secretome of human mesenchymal stromal cells (MSC) contain numerous factors that are known to exert anti-inflammatory effects. MSC-EVs may serve as promising cell-based therapeutics for the inner ear to attenuate inflammation-based side effects from cochlear implantation which represents an unmet clinical need. In an individual treatment performed on a 'named patient basis', we intraoperatively applied allogeneic umbilical cord-derived MSC-EVs (UC-MSC-EVs) produced according to good manufacturing practice. A 55-year-old patient suffering from Menière's disease was treated with intracochlear delivery of EVs prior to the insertion of a cochlear implant. This first-in-human use of UC-MSC-EVs demonstrates the feasibility of this novel adjuvant therapeutic approach. The safety and efficacy of intracochlear EV-application to attenuate side effects of cochlea implants have to be determined in controlled clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jev2.12094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178433PMC
June 2021

Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL.

Mol Ther Methods Clin Dev 2021 Jun 9;21:621-641. Epub 2021 Apr 9.

Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, 30625 Hannover, Germany.

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent "induced DCs" (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14 monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34 cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtm.2021.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142053PMC
June 2021

[CAR T cells as drugs for novel therapies (advanced therapy medicinal products)].

Internist (Berl) 2021 Apr 15;62(4):449-457. Epub 2021 Feb 15.

Regensburger Centrum für Interventionelle Immunologie (RCI), Abteilung für Gen-Immuntherapie, Universitätsklinikum Regensburg, Franz-Josef-Strauß-Allee 11, 93053, Regensburg, Deutschland.

Background: Two commercial chimeric antigen receptor (CAR) T cell products, axicabtagene-ciloleucel (Yescarta®) and tisagenlecleucel (Kymriah®), are registered for the treatment of B cell neoplasia, for which an increased supply of CAR T cell products is required.

Problem: The production of patient-specific CAR T cells as advanced therapy medicinal products (ATMPs) poses considerable challenges with respect to logistics, regulation, and manufacturing.

Method: Review of the CAR T cell manufacturing process and the regulatory network, the current challenges, and future development capabilities of CAR T cells for adoptive immunotherapy.

Results: CAR T cells are manufactured under individualized, laborious, good manufacturing practice-conforming processes in decentralized or in specialized centers. Starting from the patient's leukapheresis product, T cells are genetically engineered ex vivo with a CAR, amplified, and after extensive quality control re-applied to the patient. Most CAR T cell products are manufactured in a manual or semi-automated process; fully automated, supervised, and closed systems are increasingly applied to meet the need for a growing number of CAR T cell products. In this setting, research aims at providing allogeneic CAR T cell products or non-T cells such as natural killer cells for broad applications.

Conclusion: The significance of CAR T cells in adoptive immunotherapy is continuously growing. As individualized cell products, manufacturing requires highly efficient processes under the control of harmonized protocols and regulations so as to ensure the quality of the ATMP in view of increasing demand and to develop new fields in therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00108-021-00953-xDOI Listing
April 2021

Extracellular vesicles from human multipotent stromal cells protect against hearing loss after noise trauma in vivo.

Clin Transl Med 2020 Dec;10(8):e262

GMP Unit, Spinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria.

The lack of approved anti-inflammatory and neuroprotective therapies in otology has been acknowledged in the last decades and recent approaches are heralding a new era in the field. Extracellular vesicles (EVs) derived from human multipotent (mesenchymal) stromal cells (MSC) can be enriched in vesicular secretome fractions, which have been shown to exert effects (eg, neuroprotection and immunomodulation) of their parental cells. Hence, MSC-derived EVs may serve as novel drug candidates for several inner ear diseases. Here, we provide first evidence of a strong neuroprotective potential of human stromal cell-derived EVs on inner ear physiology. In vitro, MSC-EV preparations exerted immunomodulatory activity on T cells and microglial cells. Moreover, local application of MSC-EVs to the inner ear significantly attenuated hearing loss and protected auditory hair cells from noise-induced trauma in vivo. Thus, EVs derived from the vesicular secretome of human MSC may represent a next-generation biological drug that can exert protective therapeutic effects in a complex and nonregenerating organ like the inner ear.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ctm2.262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752163PMC
December 2020

Editorial: Modulation of Human Immune Parameters by Anticancer Therapies.

Front Immunol 2020 2;11:621556. Epub 2020 Dec 2.

Department of Hematology and Oncology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.621556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738630PMC
June 2021

Flow cytometric measurement of STAT5 phosphorylation in cytomegalovirus-stimulated T cells.

Cytometry A 2021 Aug 20;99(8):774-783. Epub 2020 Dec 20.

Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany.

Cytomegalovirus (CMV)-specific T cells expand with CMV reactivation and are probably prerequisite for control and protection. Given the critical role STAT5A phosphorylation (pSTAT5A) in T cell proliferation, this study presents a simple and sensitive flow cytometric-based pSTAT5A assay to quickly identify CMV-specific T cell proliferation. We determined pSTAT5A in T cells treated with CMV-specific peptide mix (pp65 + IE1 peptides) from 20 healthy adult subjects and three immunodeficient patients with CARMIL-2 mutation. After stimulation, the percentage of pSTAT5A T cells in CMV-seropositive (CMV ) subjects significantly increased from 3.0% ± 1.9% (unstimulated) to 11.4% ± 5.9% (stimulated) for 24 h. After 7 days of stimulation, the percentage of expanded T cells amounted to 26% ± 17.2%. Conversely, the percentage of pSTAT5A T cells and T cell proliferation from CMV-seronegative (CMV ) subjects hardly changed (from 3.0% ± 1.3% to 3.7% ± 1.8% and from 4.3% ± 2.1% to 5.7% ± 1.7%, respectively). We analyzed the correlation between the percentage of pSTAT5A T cells versus (1) CMV-IgG concentrations versus (2) the percentage of expanded T cells and versus (3) the percentage of initial CMV-specific T cells. In immunodeficient patients with CARMIL-2 mutation, CMV-specific pSTAT5A and T cell proliferation were completely deficient. In conclusion, flow cytometric-based pSTAT5A assay represents an appropriate tool to quickly identify CMV-specific T cell proliferation and helps to understand dysfunctions in controlling other pathogens. Flow cytometric-based pSTAT5A assay may be a useful test in clinical practice and merits further validation in large studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.24286DOI Listing
August 2021

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

Cytotherapy 2020 11 24;22(11):653-668. Epub 2020 Aug 24.

Department of Orthopaedic Surgery, Graded Implants and Regenerative Strategies, Hannover Medical School, Hannover, Germany; Lower Saxony Centre for Biomedical Engineering, Implant Research and Development (NIFE), Hannover, Germany. Electronic address:

Background Aims: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues.

Methods: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium.

Results: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected.

Conclusions: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcyt.2020.07.005DOI Listing
November 2020

Nicotinamide Inhibits Self-renewal and Induces Granulocyte Differentiation of Multipotent Progenitor Cells.

Stem Cell Rev Rep 2020 12;16(6):1335-1342

Medical Faculty, Institute of Clinical Immunology, Leipzig University, Johannisallee 30, 04103, Leipzig, Germany.

Nicotinamide (NAM) a form of vitamin B3, is an essential precursor of NAD. This dinucleotide (pyridine nucleotide) participates in the regulation of fundamental processes including transcription, cell cycle progression and DNA repair. Here we assessed the effect of NAM on myeloid differentiation of the IL-3 dependent, multipotent hematopoietic progenitor cell line FDCP-Mix. We found that NAM reduces the pSTAT5 signaling response, cell cycling and self-renewal potential. It initiates an atypical program of myeloid differentiation that results in the emergence of granulocytic cells in the absence of added myeloid differentiation factors. NAM did not affect the expression the of cell surface granulocyte marker GR1 but led to a strong downregulation of MHC-II molecules. Taken together our data show that NAM induces a differentiation program in hematopoietic progenitors prompting them to undergo differentiation along the granulocyte path without reaching the status of fully developed granulocytes. Graphical abstract.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12015-020-10019-4DOI Listing
December 2020

From Cancer to Immune-Mediated Diseases and Tolerance Induction: Lessons Learned From Immune Oncology and Classical Anti-cancer Treatment.

Front Immunol 2020 8;11:1423. Epub 2020 Jul 8.

Institute of Clinical Pharmacology, University Hospital Frankfurt, Frankfurt, Germany.

Success in cancer treatment over the last four decades has ranged from improvements in classical drug therapy to immune oncology. Anti-cancer drugs have also often proven beneficial for the treatment of inflammatory and autoimmune diseases. In this review, we report on challenging examples that bridge between treatment of cancer and immune-mediated diseases, addressing mechanisms and experimental models as well as clinical investigations. Patient-derived tumor xenograft (PDX) (humanized) mouse models represent useful tools for preclinical evaluation of new therapies and biomarker identification. However, new developments using human approaches modeling cancer, for example in microfluidic human organs-on-chips, promise to identify key molecular, cellular and immunological features of human cancer progression in a fully human setting. Classical drugs which bridge the gap, for instance, include cytotoxic drugs, proteasome inhibitors, PI3K/mTOR inhibitors and metabolic inhibitors. Biologicals developed for cancer therapy have also shown efficacy in the treatment of autoimmune diseases. In immune oncology, redirected chimeric antigen receptor (CAR) T cells have achieved spectacular remissions in refractory B cell leukemia and lymphoma and are currently under development for tolerance induction using cell-based therapies such as CAR Tregs or NK cells. Finally, a brief outline will be given of the lessons learned from bridging cancer and autoimmune diseases as well as tolerance induction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.01423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360838PMC
April 2021

The Role of lncRNAs TAPIR-1 and -2 as Diagnostic Markers and Potential Therapeutic Targets in Prostate Cancer.

Cancers (Basel) 2020 Apr 30;12(5). Epub 2020 Apr 30.

Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Johannisallee 30, D-04103 Leipzig, Germany.

In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA ( and . To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and -damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting significantly reduced tumor growth. These findings point to a crucial role of and in PCa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12051122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280983PMC
April 2020

Management of adults and children undergoing chimeric antigen receptor T-cell therapy: best practice recommendations of the European Society for Blood and Marrow Transplantation (EBMT) and the Joint Accreditation Committee of ISCT and EBMT (JACIE).

Haematologica 2020 31;105(2):297-316. Epub 2020 Jan 31.

Department of Stem Cell Transplantation, University Medical Center Hamburg, Hamburg, Germany.

Chimeric antigen receptor (CAR) T cells are a novel class of anti-cancer therapy in which autologous or allogeneic T cells are engineered to express a CAR targeting a membrane antigen. In Europe, tisagenlecleucel (Kymriah™) is approved for the treatment of refractory/relapsed acute lymphoblastic leukemia in children and young adults as well as relapsed/refractory diffuse large B-cell lymphoma, while axicabtagene ciloleucel (Yescarta™) is approved for the treatment of relapsed/refractory high-grade B-cell lymphoma and primary mediastinal B-cell lymphoma. Both agents are genetically engineered autologous T cells targeting CD19. These practical recommendations, prepared under the auspices of the European Society of Blood and Marrow Transplantation, relate to patient care and supply chain management under the following headings: patient eligibility, screening laboratory tests and imaging and work-up prior to leukapheresis, how to perform leukapheresis, bridging therapy, lymphodepleting conditioning, product receipt and thawing, infusion of CAR T cells, short-term complications including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, antibiotic prophylaxis, medium-term complications including cytopenias and B-cell aplasia, nursing and psychological support for patients, long-term follow-up, post-authorization safety surveillance, and regulatory issues. These recommendations are not prescriptive and are intended as guidance in the use of this novel therapeutic class.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2019.229781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012497PMC
April 2021

Functionality and Cell Senescence of CD4/ CD8-Selected CD20 CAR T Cells Manufactured Using the Automated CliniMACS Prodigy® Platform.

Transfus Med Hemother 2019 Feb 4;46(1):47-54. Epub 2019 Feb 4.

Cellular Therapy Centre, Institute of Cellular Therapeutics, Hannover Medical School (MHH), Hanover, Germany.

Clinical studies using autologous CAR T cells have achieved spectacular remissions in refractory CD19+ B cell leukaemia, however some of the patient treatments with CAR T cells failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes for CD20 CAR T cells from healthy donor leukapheresis using the automated CliniMACS Prodigy® platform. Starting with a CD4/CD8-positive selection, a high purity of a median of 97% T cells with a median 65-fold cell expansion was achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached in a median of 23%. CD20 CAR T cells showed a good specific IFN-γ secretion after cocultivation with CD20+ target cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase in telomere length during the manufacturing process, while telomere length remained consistent in one and decreased in another process. In conclusion, this shows for the first time that beside heterogeneity among healthy donors, CAR T cell products also differ regarding cell senescence, even for cells manufactured in a standardised automated process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000495772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558326PMC
February 2019

Cellular Therapeutics - Living Drugs: A Rising Star at the Horizon of Immunotherapy in Hematology and Oncology.

Transfus Med Hemother 2019 Feb 4;46(1). Epub 2019 Feb 4.

Institute of Cellular Therapeutics, Hannover Medical School (MHH), Hannover, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000497051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558335PMC
February 2019

Evaluating STAT5 Phosphorylation as a Mean to Assess T Cell Proliferation.

Front Immunol 2019 5;10:722. Epub 2019 Apr 5.

Medical Faculty, Institute of Clinical Immunology, University of Leipzig, Leipzig, Germany.

Here we present a simple and sensitive flow cytometric-based assay to assess T cell proliferation. Given the critical role STAT5A phosphorylation in T cell proliferation, we decided to evaluate phosphorylation of STAT5A as an indicator of T cell proliferation. We determined pSTAT5A in T cell treated with either CD3/CD28 or PHA. After stimulation, T cells from adult healthy donors displayed a strong long-lasting phosphorylation of STAT5A, reaching a peak value after 24 h. The median fluorescence intensity (MFI) of pSTAT5A increased from 112 ± 17 to 512 ± 278 (CD3/CD28) (24 h) and to 413 ± 123 (PHA) (24 h), the IL-2 receptor-α (CD25) expression was greatly enhanced and after 72 h T cell proliferation amounted to 52.3 ± 10.3% (CD3/CD28) and to 48.4 ± 9.7% (PHA). Treatment with specific JAK3 and STAT5 inhibitors resulted in a complete blockage of phosphorylation of STAT5A, CD25 expression, and suppression of T cell proliferation. Compared with currently available methods, STAT5A phosphorylation is well-suited to predict T cell proliferation. Moreover, the method presented here is not very time consuming (several hours) and delivers functional information from which conclusions about T cell proliferation can be drawn.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.00722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460883PMC
September 2020

Analysis of the therapeutic potential of different administration routes and frequencies of human mesenchymal stromal cells in the SOD1 mouse model of amyotrophic lateral sclerosis.

J Tissue Eng Regen Med 2019 04 20;13(4):649-663. Epub 2019 Mar 20.

Department of Neurology, Hannover Medical School, Hannover, Germany.

Cellular therapy represents a novel option for the treatment of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Its major aim is the generation of a protective environment for degenerating motor neurons. Mesenchymal stromal cells secrete different growth factors and have antiapoptotic and immunomodulatory properties. They can easily and safely be isolated from human bone marrow and are therefore considered promising therapeutic candidates. In the present study, we compared intraventricular application of human mesenchymal stromal cells (hMSCs) versus single and repeated intraspinal injections in the mutant SOD1 transgenic ALS mouse model. We observed significant reduction of lifespan of animals treated by intraventricular hMSC injection compared with the vehicle treated control group, accompanied by changes in weight, general condition, and behavioural assessments. A potential explanation for these rather surprising deleterious effects lies in increased microgliosis detected in the hMSC treated animals. Repeated intraspinal injection at two time points resulted in a slight but not significant increase in survival and significant improvement of motor performance although no hMSC-induced changes of motor neuron numbers, astrogliosis, and microgliosis were detected. Quantitative real time polymerase chain reaction showed reduced expression of endothelial growth factor in animals having received hMSCs twice compared with the vehicle treated control group. hMSCs were detectable at the injection site at Day 20 after injection into the spinal cord but no longer at Day 70. Intraspinal injection of hMSCs may therefore be a more promising option for the treatment of ALS than intraventricular injection and repeated injections might be necessary to obtain substantial therapeutic benefit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/term.2846DOI Listing
April 2019

Manufacturing Mesenchymal Stromal Cells for the Treatment of Graft-versus-Host Disease: A Survey among Centers Affiliated with the European Society for Blood and Marrow Transplantation.

Biol Blood Marrow Transplant 2018 11 20;24(11):2365-2370. Epub 2018 Jul 20.

School of Cancer & Pharmaceutical Sciences, King's College London, London, United Kingdom. Electronic address:

The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients' stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbmt.2018.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299357PMC
November 2018

CAR T Cells in Trials: Recent Achievements and Challenges that Remain in the Production of Modified T Cells for Clinical Applications.

Hum Gene Ther 2018 05 5;29(5):559-568. Epub 2018 Apr 5.

4 Center for Molecular Medicine Cologne, University of Cologne , Cologne, Germany.

The adoptive transfer of chimeric antigen receptor (CAR)-modified T cells is attracting growing interest for the treatment of malignant diseases. Early trials with anti-CD19 CAR T cells have achieved spectacular remissions in B-cell leukemia and lymphoma, so far refractory, very recently resulting in the Food and Drug Administration approval of CD19 CAR T cells for therapy. With further applications and increasing numbers of patients, the reproducible manufacture of high-quality clinical-grade CAR T cells is becoming an ever greater challenge. New processing techniques, quality-control mechanisms, and logistic developments are required to meet both medical needs and regulatory restrictions. This paper summarizes the state-of-the-art in manufacturing CAR T cells and the current challenges that need to be overcome to implement this type of cell therapy in the treatment of a variety of malignant diseases and in a greater number of patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2017.254DOI Listing
May 2018

Triplebody Mediates Increased Anti-Leukemic Reactivity of IL-2 Activated Donor Natural Killer (NK) Cells and Impairs Viability of Their CD33-Expressing NK Subset.

Front Immunol 2017 8;8:1100. Epub 2017 Sep 8.

Institute for Cellular Therapeutics, IFB-Tx, Hannover Medical School (MHH), Hannover, Germany.

Natural killer cells (NK) are essential for the elimination of resistant acute myeloid and acute lymphoblastic leukemia (AML and ALL) cells. NK cell-based immunotherapies have already successfully entered for clinical trials, but limitations due to immune escape mechanisms were identified. Therefore, we extended our established NK cell protocol by integration of the previously investigated powerful trispecific immunoligand ULBP2-aCD19-aCD33 [the so-called triplebodies (TBs)] to improve the anti-leukemic specificity of activated NK cells. IL-2-driven expansion led to strongly elevated natural killer group 2 member D (NKG2D) expressions on donor NK cells which promote the binding to ULBP2 TBs. Similarly, CD33 expression on these NK cells could be detected. Dual-specific targeting and elimination were investigated against the B-cell precursor leukemia cell line BV-173 and patient blasts, which were positive for myeloid marker CD33 and B lymphoid marker CD19 exclusively presented on biphenotypic B/myeloid leukemia's. Cytotoxicity assays demonstrated improved killing properties of NK cells pre-coated with TBs compared to untreated controls. Specific NKG2D blocking on those NK cells in response to TBs diminished this killing activity. On the contrary, the observed upregulation of surface CD33 on about 28.0% of the NK cells decreased their viability in response to TBs during cytotoxic interaction of effector and target cells. Similar side effects were also detected against CD33 T- and CD19 B-cells. Very preliminary proof of principle results showed promising effects using NK cells and TBs against primary leukemic cells. In summary, we demonstrated a promising strategy for redirecting primary human NK cells in response to TBs against leukemia, which may lead to a future progress in NK cell-based immunotherapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.01100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596090PMC
September 2017

Shaping of Natural Killer Cell Antitumor Activity by Cultivation.

Front Immunol 2017 26;8:458. Epub 2017 Apr 26.

Division for Stem Cell Transplantation and Immunology, Department for Children and Adolescents Medicine, Hospital of the Goethe University, Frankfurt, Germany.

Natural killer (NK) cells are a promising tool for the use in adoptive immunotherapy, since they efficiently recognize and kill tumor cells. In this context, cultivation is an attractive option to increase NK cells in numbers and to improve their antitumor potential prior to clinical applications. Consequently, various strategies to generate NK cells for adoptive immunotherapy have been developed. Here, we give an overview of different NK cell cultivation approaches and their impact on shaping the NK cell antitumor activity. So far, the cytokines interleukin (IL)-2, IL-12, IL-15, IL-18, and IL-21 are used to culture and expand NK cells. The selection of the respective cytokine combination is an important factor that directly affects NK cell maturation, proliferation, survival, distribution of NK cell subpopulations, activation, and function in terms of cytokine production and cytotoxic potential. Importantly, cytokines can upregulate the expression of certain activating receptors on NK cells, thereby increasing their responsiveness against tumor cells that express the corresponding ligands. Apart from using cytokines, cocultivation with autologous accessory non-NK cells or addition of growth-inactivated feeder cells are approaches for NK cell cultivation with pronounced effects on NK cell activation and expansion. Furthermore, cultivation was reported to prime NK cells for the killing of tumor cells that were previously resistant to NK cell attack. In general, NK cells become frequently dysfunctional in cancer patients, for instance, by downregulation of NK cell activating receptors, disabling them in their antitumor response. In such scenario, cultivation can be helpful to arm NK cells with enhanced antitumor properties to overcome immunosuppression. In this review, we summarize the current knowledge on NK cell modulation by different cultivation strategies focused on increasing NK cytotoxicity for clinical application in malignant diseases. Moreover, we critically discuss the technical and regulatory aspects and challenges underlying NK cell based therapeutic approaches in the clinics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.00458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405078PMC
April 2017

Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity and against chronic lymphocytic leukemia.

Oncoimmunology 2016;5(9):e1211220. Epub 2016 Jul 15.

Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany; Experimental Tumor Research, Center for Tumor Biology and Immunology, Philipps University Marburg, Marburg, Germany.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia that affects B lymphocytes in adults. Natural killer (NK) cells in CLL patients are intrinsically potent but display poor effector functions. NKG2D is an activating receptor found on NK and CD8 T cells and plays a role in immunosurveillance of CLL. In this study, we developed mono- and dual-targeting triplebodies utilizing a natural ligand for human NKG2D receptor (ULBP2) to retarget NK cells against tumor cells. Triplebodies in both formats showed better ability to induce NK-cell-dependent killing of target cells compared to bispecific counterparts. A mono-targeting triplebody ULBP2-aCD19-aCD19 successfully triggered NK cell effector functions against CLL cell line MEC1 and primary tumor cells in allogenic and autologous settings. Additionally, a dual-targeting triplebody ULBP2-aCD19-aCD33 specific for two distinct tumor-associated antigens was developed to target antigen loss variants, such as mixed lineage leukemia (MLL). Of note, this triplebody exhibited cytotoxic activity against CD19/CD33 double positive cells and retained its binding features even in the absence of one of the tumor antigens. Further, ULBP2-aCD19-aCD19 showed significant activity in immune-deficient (NSG) mouse model transplanted with CLL cell line as target cells and human immune cells as an effector population providing a proof-of-principle for this therapeutic concept.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/2162402X.2016.1211220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5049355PMC
July 2016

Comparative Analysis of Clinical-Scale IFN-γ-Positive T-Cell Enrichment Using Partially and Fully Integrated Platforms.

Front Immunol 2016 30;7:393. Epub 2016 Sep 30.

Institute of Cellular Therapeutics, Hannover Medical School, Niedersachsen, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Niedersachsen, Germany.

Background And Aims: The infusion of enriched CMV-specific donor T-cells appears to be a suitable alternative for the treatment of drug-resistant CMV reactivation or infection after both solid organ and hematopoietic stem cell transplantation. Antiviral lymphocytes can be selected from apheresis products using the CliniMACS Cytokine-Capture-System either with the well-established CliniMACS Plus (Plus) device or with its more versatile successor CliniMACS Prodigy (Prodigy).

Methods: Manufacturing of CMV-specific T-cells was carried out with the Prodigy and Plus in parallel starting with 0.8-1 × 10 leukocytes collected by lymphapheresis ( = 3) and using the MACS GMP PepTivator HCMVpp65 for antigenic restimulation. Target and non-target cells were quantified by a newly developed single-platform assessment and gating strategy using positive (CD3/CD4/CD8/CD45/IFN-γ), negative (CD14/CD19/CD56), and dead cell (7-AAD) discriminators.

Results: Both devices produced largely similar results for target cell viabilities: 37.2-52.2% (Prodigy) vs. 51.1-62.1% (Plus) CD45/7-AAD cells. Absolute numbers of isolated target cells were 0.1-3.8 × 10 viable IFN-γ CD3 T-cells. The corresponding proportions of IFN-γ CD3 T-cells ranged between 19.2 and 95.1% among total CD3 T-cells and represented recoveries of 41.9-87.6%. Within two parallel processes, predominantly IFN-γ CD3CD8 cytotoxic T-cells were enriched compared to one process that yielded a higher amount of IFN-γ CD3CD4 helper T lymphocytes. T-cell purity was higher for the Prodigies products that displayed a lower content of contaminating IFN-γ T-cells (3.6-20.8%) compared to the Plus products (19.9-80.0%).

Conclusion: The manufacturing process on the Prodigy saved both process and hands-on time due to its higher process integration and ability for unattended operation. Although the usage of both instruments yielded comparable results, the lower content of residual IFN-γ T-cells in the target fractions produced with the Prodigy may allow for a higher dosage of CMV-specific donor T-cells without increasing the risk for graft-versus-host disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2016.00393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5044705PMC
September 2016

Biohybrid cochlear implants in human neurosensory restoration.

Stem Cell Res Ther 2016 10 7;7(1):148. Epub 2016 Oct 7.

Department of Otorhinolaryngology, Head and Neck Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.

Background: The success of cochlear implantation may be further improved by minimizing implantation trauma. The physical trauma of implantation and subsequent immunological sequelae can affect residual hearing and the viability of the spiral ganglion. An ideal electrode should therefore decrease post-implantation trauma and provide support to the residual spiral ganglion population. Combining a flexible electrode with cells producing and releasing protective factors could present a potential means to achieve this. Mononuclear cells obtained from bone marrow (BM-MNC) consist of mesenchymal and hematopoietic progenitor cells. They possess the innate capacity to induce repair of traumatized tissue and to modulate immunological reactions.

Methods: Human bone marrow was obtained from the patients that received treatment with biohybrid electrodes. Autologous mononuclear cells were isolated from bone marrow (BM-MNC) by centrifugation using the Regenlab™ THT-centrifugation tubes. Isolated BM-MNC were characterised using flow cytometry. In addition, the release of cytokines was analysed and their biological effect tested on spiral ganglion neurons isolated from neonatal rats. Fibrin adhesive (Tisseal™) was used for the coating of silicone-based cochlear implant electrode arrays for human use in order to generate biohybrid electrodes. Toxicity of the fibrin adhesive and influence on insertion, as well on the cell coating, was investigated. Furthermore, biohybrid electrodes were implanted in three patients.

Results: Human BM-MNC release cytokines, chemokines, and growth factors that exert anti-inflammatory and neuroprotective effects. Using fibrin adhesive as a carrier for BM-MNC, a simple and effective cell coating procedure for cochlear implant electrodes was developed that can be utilised on-site in the operating room for the generation of biohybrid electrodes for intracochlear cell-based drug delivery. A safety study demonstrated the feasibility of autologous progenitor cell transplantation in humans as an adjuvant to cochlear implantation for neurosensory restoration.

Conclusion: This is the first report of the use of autologous cell transplantation to the human inner ear. Due to the simplicity of this procedure, we hope to initiate its widespread utilization in various fields.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13287-016-0408-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055669PMC
October 2016

Cetuximab Reconstitutes Pro-Inflammatory Cytokine Secretions and Tumor-Infiltrating Capabilities of sMICA-Inhibited NK Cells in HNSCC Tumor Spheroids.

Front Immunol 2015 2;6:543. Epub 2015 Nov 2.

Institute of Cellular Therapeutics, Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School , Hannover , Germany.

Immunosuppressive factors, such as soluble major histocompatibility complex class I chain-related peptide A (sMICA) and transforming growth factor beta 1 (TGF-β1), are involved in tumor immune escape mechanisms (TIEMs) exhibited by head and neck squamous cell carcinomas (HNSCCs) and may represent opportunities for therapeutic intervention. In order to overcome TIEMs, we investigated the antibody-dependent cellular cytotoxicity (ADCC), cytokine release and retargeted tumor infiltration of sMICA-inhibited patient NK cells expressing Fcγ receptor IIIa (FcγRIIIa, CD16a) in the presence of cetuximab, an anti-epidermal growth factor receptor (HER1) monoclonal antibody (mAb). Compared to healthy controls, relapsed HNSCC patients (n = 5), not currently in treatment revealed decreased levels of circulating regulatory NK cell subsets in relation to increased cytotoxic NK cell subpopulations. Elevated sMICA and TGF-β1 plasma levels correlated with diminished TNFα and IFN-γ release and decreased NKG2D (natural killer group 2 member D)-dependent killing of HNSCC cells by NK cells. Incubation of IL-2-activated patient NK cells with patient plasma containing elevated sMICA or sMICA analogs (shed MICA and recombinant MICA) significantly impaired NKG2D-mediated killing by down-regulation of NKG2D surface expression. Of note, CD16 surface expression levels, pro-apoptotic and activation markers, and viability of patient and healthy donor NK cell subpopulations were not affected by this treatment. Accordingly, cetuximab restored killing activity of sMICA-inhibited patient NK cells against cetuximab-coated primary HNSCC cells via ADCC in a dose-dependent manner. Rapid reconstitution of anti-tumor recognition and enhanced tumor infiltration of treated NK cells was monitored by 24 h co-incubation of HNSCC tumor spheroids with cetuximab (1 μg/ml) and was characterized by increased IFN-γ and TNFα secretion. This data show that the impaired NK cell-dependent tumor surveillance in relapsed HNSCC patients could be reversed by the re-establishment of ADCC-mediated effector cell activity, thus supporting NK cell-based immunotherapy in combination with antineoplastic monoclonal mAbs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2015.00543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4629470PMC
November 2015

Increased sMICA and TGFβ levels in HNSCC patients impair NKG2D-dependent functionality of activated NK cells.

Oncoimmunology 2015 Nov 29;4(11):e1055993. Epub 2015 May 29.

Institute for Cellular therapeutics; IFB-Tx; Hannover Medical School ; Hannover, Germany.

Disseminated head-and-neck squamous cell carcinoma (HNSCC) escapes immune surveillance and thus frequently manifests as fatal disease. Here, we report on the distribution of distinct immune cell subpopulations, natural killer (NK) cell cytotoxicity and tumor immune escape mechanisms (TIEMs) in 55 HNSCC patients, either at initial diagnosis or present with tumor relapse. Compared to healthy controls, the regulatory NK cells and the ratio of pro/anti-inflammatory cytokines were decreased in HNSCC patients, while soluble major histocompatibility complex Class I chain-related peptide A (sMICA) and transforming growth factor β (TGFβ) plasma levels were markedly elevated. Increased sMICA and TGFβ concentrations correlated with tumor progression and staging characteristics in 7 follow-up HNSCC patients, with significantly elevated levels of both soluble factors from the time of initial diagnosis to that of relapse. Patient plasma containing elevated sMICA and TGFβ markedly impaired NKG2D-dependent cytotoxicity against HNSCC cells upon incubation with patient-derived and IL-2 activated NK cells those derived from healthy donors. Decreased antitumor recognition was accompanied by reduced NKG2D expression on the NK cell surface and an enhanced caspase-3 activity. blocking and neutralization experiments demonstrated a synergistic negative impact of sMICA and TGFβ on NK cell functionality. Although we previously showed the feasibility and safety of transfer of allogeneic donor NK cells in a prior clinical study encompassing various leukemia and tumor patients, our present results suggest the need for caution regarding the sole use of adoptive NK cell transfer. The presence of soluble NKG2D ligands in the plasma of HNSCC patients and the decreased NK cell cytotoxicity due to several factors, especially TGFβ, indicates timely depletion of these immunosuppressing molecules may promote NK cell-based immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/2162402X.2015.1055993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589991PMC
November 2015

Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor.

Mol Ther 2015 Feb 6;23(2):330-8. Epub 2014 Nov 6.

1] Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany [2] German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2014.219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445620PMC
February 2015
-->