CAR-Expressing Natural Killer Cells for Cancer Retargeting

Since the approval in 2017 and the outstanding success of Kymriah® and Yescarta®, the number of clinical trials investigating the safety and efficacy of chimeric antigen receptormodified autologous T cells has been constantly rising. Currently, more than 200 clinical trials are listed on clinicaltrial. gov. In contrast to CAR-T cells, natural killer (NK) cells can be used from allogeneic donors as an “off the shelf product” and provide alternative candidates for cancer retargeting. This review summarises preclinical results of CAR-engineered NK cells using both primary human NK cells and the cell line NK-92, and provides an overview about the first clinical CAR-NK cell studies targeting haematological malignancies and solid tumours, respectivel

Taking Lessons from CAR-T Cells and Going Beyond: Tailoring Design and Signaling for CAR-NK Cells in Cancer Therapy

Cancer immunotherapies utilize the capabilities of the immune system to efficiently target malignant cells. In recent years, chimeric antigen receptor (CAR) equipped T cells showed promising results against B cell lymphomas. Autologous CAR-T cells require patientspecific manufacturing and thus extensive production facilities, resulting in high priced therapies. Along with potentially severe side effects, these are the major drawbacks of CAR-T cells therapies. Natural Killer (NK) cells pose an alternative for CAR equipped immune cells. Since NK cells can be safely transferred from healthy donors to cancer patients, they present a suitable platform for an allogeneic “off-the-shelf” immunotherapy. However, administration of activated NK cells in cancer therapy has until now shown poor anti-cancer responses, especially in solid tumors. Genetic modifications such as CARs promise to enhance recognition of tumor cells, thereby increasing anti-tumor effects and improving clinical efficacy. Although the cell biology of T and NK cells deviates in many aspects, the development of CAR-NK cells frequently follows within the footsteps of CART cells, meaning that T cell technologies are simply adopted to NK cells. In this review, we underline the unique properties of NK cells and their potential in CAR therapies. First, we summarize the characteristics of NK cell biology with a focus on signaling, a fine-tuned interaction of activating and inhibitory receptors. We then discuss why tailored NK cellspecific CAR designs promise superior efficacy compared to designs developed for T cells. We summarize current findings and developments in the CAR-NK landscape: different CAR formats and modifications to optimize signaling, to target a broader pool of antigens or to increase in vivo persistence. Finally, we address challenges beyond NK cell engineering, including expansion and manufacturing, that need to be addressed to pave the way for CAR-NK therapies from the bench to the clinics

Production and Application of CAR T Cells: Current and Future Role of Europe

Rapid developments in the field of CAR T cells offer important new opportunities while at the same time increasing numbers of patients pose major challenges. This review is summarizing on the one hand the state of the art in CAR T cell trials with a unique perspective on the role that Europe is playing. On the other hand, an overview of reproducible processing techniques is presented, from manual or semi-automated up to fully automated manufacturing of clinical-grade CAR T cells. Besides regulatory requirements, an outlook is given in the direction of digitally controlled automated manufacturing in order to lower cost and complexity and to address CAR T cell products for a greater number of patients and a variety of malignant diseases

Bilateral Crystalline Corneal Deposits as First Clinical Manifestation of Monoclonal Gammopathy: A Case Report

Aims: To report the clinical and diagnostic findings of a patient with bilateral corneal deposits caused by an underlying monoclonal gammopathy. Methods: Slit-lamp biomicroscopy, confocal microscopy and additional serological tests were performed on a 35-year-old man presenting with bilateral crystalline corneal deposits. Results: The patient was diagnosed as having monoclonal gammopathy based on elevated levels of serum immunoglobulin G. Confocal microscopy showed highly reflective (protein) deposits throughout the entire cornea, with the highest density in the epithelium and anterior stromal keratocytes. Conclusions: Monoclonal gammopathy, a potential sign of a life-threatening disease, can lead to dense, bilateral corneal deposits. As such changes can occur long before ocular or systemic discomforts appear, an early diagnosis is crucial. Ophthalmologists should be aware of corneal deposits as potential warning signs of monoclonal gammopathy

Score-based prediction of genomic islands in prokaryotic genomes using hidden Markov models

BACKGROUND: Horizontal gene transfer (HGT) is considered a strong evolutionary force shaping the content of microbial genomes in a substantial manner. It is the difference in speed enabling the rapid adaptation to changing environmental demands that distinguishes HGT from gene genesis, duplications or mutations. For a precise characterization, algorithms are needed that identify transfer events with high reliability. Frequently, the transferred pieces of DNA have a considerable length, comprise several genes and are called genomic islands (GIs) or more specifically pathogenicity or symbiotic islands. RESULTS: We have implemented the program SIGI-HMM that predicts GIs and the putative donor of each individual alien gene. It is based on the analysis of codon usage (CU) of each individual gene of a genome under study. CU of each gene is compared against a carefully selected set of CU tables representing microbial donors or highly expressed genes. Multiple tests are used to identify putatively alien genes, to predict putative donors and to mask putatively highly expressed genes. Thus, we determine the states and emission probabilities of an inhomogeneous hidden Markov model working on gene level. For the transition probabilities, we draw upon classical test theory with the intention of integrating a sensitivity controller in a consistent manner. SIGI-HMM was written in JAVA and is publicly available. It accepts as input any file created according to the EMBL-format. It generates output in the common GFF format readable for genome browsers. Benchmark tests showed that the output of SIGI-HMM is in agreement with known findings. Its predictions were both consistent with annotated GIs and with predictions generated by different methods. CONCLUSION: SIGI-HMM is a sensitive tool for the identification of GIs in microbial genomes. It allows to interactively analyze genomes in detail and to generate or to test hypotheses about the origin of acquired genes

The Epitope-Specific Anti-human CD4 Antibody MAX.16H5 and Its Role in Immune Tolerance

T cell modulation in the clinical background of autoimmune diseases or allogeneic cell and organ transplantations with concurrent preservation of their natural immunological functions (e.g., pathogen defense) is the major obstacle in immunology. An anti-human CD4 antibody (MAX.16H5) was applied intravenously in clinical trials for the treatment of autoimmune diseases (e.g., rheumatoid arthritis) and acute late-onset rejection after transplantation of a renal allograft. The response rates were remarkable and no critical allergic problems or side effects were obtained. During the treatment of autoimmune diseases with the murine MAX.16H5 IgG1 antibody its effector mechanisms with effects on lymphocytes, cytokines, laboratory and clinical parameters, adverse effects as well as pharmacodynamics and kinetics were studied in detail. However, as the possibility of developing immune reactions against the murine IgG1 Fc-part remains, the murine antibody was chimerized, inheriting CD4-directed variable domains of the MAX.16H5 IgG1 connected to a human IgG4 backbone. Both antibodies were studied in vitro and in specific humanized mouse transplantation models in vivo with a new scope. By ex vivo incubation of an allogeneic immune cell transplant with MAX.16H5 a new therapy strategy has emerged for the first time enabling both the preservation of the graft-vs.-leukemia (GVL) effect and the permanent suppression of the acute graft-vs.-host disease (aGVHD) without conventional immunosuppression. In this review, we especially focus on experimental data and clinical trials obtained from the treatment of autoimmune diseases with the murine MAX.16H5 IgG1 antibody. Insights gained from these trials have paved the way to better understand the effects with the chimerized MAX.16H5 IgG4 as novel therapeutic approach in the context of GVHD prevention

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

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

One-Step Liquid Phase Polymerization of HEMA by Atmospheric-Pressure Plasma Discharges for Ti Dental Implants

Dental implants can fail due to various factors, in which bad tissue integration is believed to have a significant role. Specific properties of the implant surface, such as its chemistry and roughness, are of paramount importance to address specific cell responses, such as the adsorption of proteins, as well as the adhesion and differentiation of cells, which are suitable for biomaterial and tissue engineering. In this study, an acrylate-containing coating was produced on titanium surfaces through the atmospheric pressure plasma treatment of a liquid precursor, 2-hydroxyethyl methacrylate. A hydrophilic coating was obtained, showing retention of the monomer chemistry as assessed by FTIR analysis and XPS. Enhanced fibroblast adhesion and decreased Staphylococcus aureus and Escherichia coli adhesion were recorded, showing that this is a suitable method to produce biocompatible coatings with a reduced bacterial adhesion

Allogeneic Non-Adherent Bone Marrow Cells Facilitate Hematopoietic Recovery but Do Not Lead to Allogeneic Engraftment

Background Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4+, murine CD4?/?, HLA-DR3+ mice. Methodology/Principal Findings Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45+ showed depletion of CD4+, CD8+, CD19+, and CD117+ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4+ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death. Conclusions/Significance NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies