99 research outputs found
Underlying mechanisms of evasion from NK cells as rationale for improvement of NK cell-based immunotherapies
Natural killer (NK) cells belong to the family of innate immune cells with the
capacity to recognize and kill tumor cells. Different phenotypes and functional
properties of NK cells have been described in tumor patients, which could be
shaped by the tumor microenvironment. The discovery of HLA class I-specific
inhibitory receptors controlling NK cell activity paved the way to the fundamental
concept of modulating immune responses that are regulated by an array of
inhibitory receptors, and emphasized the importance to explore the potential of
NK cells in cancer therapy. Although a whole range of NK cell-based approaches
are currently being developed, there are still major challenges that need to be
overcome for improved efficacy of these therapies. These include escape of
tumor cells from NK cell recognition due to their expression of inhibitory
molecules, immune suppressive signals of NK cells, reduced NK cell infiltration
of tumors, an immune suppressive micromilieu and limited in vivo persistence of
NK cells. Therefore, this review provides an overview about the NK cell biology,
alterations of NK cell activities, changes in tumor cells and the tumor
microenvironment contributing to immune escape or immune surveillance by
NK cells and their underlyingmolecular mechanisms as well as the current status
and novel aspects of NK cell-based therapeutic strategies including their genetic
engineering and their combination with conventional treatment options to
overcome tumor-mediated evasion strategies and improve therapy efficacy
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
The Impact of Rubella Virus Infection on a Secondary Inflammatory Response in Polarized Human Macrophages
Macrophages (MF) are known to exhibit distinct responses to viral and bacterial infection,
but how they react when exposed to the pathogens in succession is less well understood.
Accordingly, we determined the effect of a rubella virus (RV)-induced infection followed by
an LPS-induced challenge on cytokine production, signal transduction and metabolic
pathways in human GM (M1-like)- and M (M2-like)-MF. We found that infection of both
subsets with RV resulted in a low TNF-a and a high interferon (IFN, type I and type III)
release whereby M-MF produced far more IFNs than GM-MF. Thus, TNF-a production in
contrast to IFN production is not a dominant feature of RV infection in these cells. Upon
addition of LPS to RV-infected MF compared to the addition of LPS to the uninfected cells
the TNF-a response only slightly increased, whereas the IFN-response of both subtypes
was greatly enhanced. The subset specific cytokine expression pattern remained
unchanged under these assay conditions. The priming effect of RV was also observed
when replacing RV by IFN-b one putative priming stimulus induced by RV. Small amounts
of IFN-b were sufficient for phosphorylation of Stat1 and to induce IFN-production in
response to LPS. Analysis of signal transduction pathways activated by successive
exposure of MF to RV and LPS revealed an increased phosphorylation of NFkB (MMF),
but different to uninfected MF a reduced phosphorylation of ERK1/2 (both
subtypes). Furthermore, metabolic pathways were affected; the LPS-induced increase
in glycolysis was dampened in both subtypes after RV infection. In conclusion, we show
that RV infection and exogenously added IFN-b can prime MF to produce high amounts
of IFNs in response to LPS and that changes in glycolysis and signal transduction are
associated with the priming effect. These findings will help to understand to what extent
MF defense to viral infection is modulated by a following exposure to a bacterial infection
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
Joint Modeling of Immune Reconstitution Post Haploidentical Stem Cell Transplantation in Pediatric Patients With Acute Leukemia Comparing CD34+-Selected to CD3/CD19-Depleted Grafts in a Retrospective Multicenter Study
Rapid immune reconstitution (IR) following stem cell transplantation (SCT) is essential for a favorable outcome. The optimization of graft composition should not only enable a sufficient IR but also improve graft vs. leukemia/tumor effects, overcome infectious complications and, finally, improve patient survival. Especially in haploidentical SCT, the optimization of graft composition is controversial. Therefore, we analyzed the influence of graft manipulation on IR in 40 patients with acute leukemia in remission. We examined the cell recovery post haploidentical SCT in patients receiving a CD34+-selected or CD3/CD19-depleted graft, considering the applied conditioning regimen. We used joint model analysis for overall survival (OS) and analyzed the dynamics of age-adjusted leukocytes; lymphocytes; monocytes; CD3+, CD3+CD4+, and CD3+CD8+ T cells; natural killer (NK) cells; and B cells over the course of time after SCT. Lymphocytes, NK cells, and B cells expanded more rapidly after SCT with CD34+-selected grafts (P = 0.036, P = 0.002, and P < 0.001, respectively). Contrarily, CD3+CD4+ helper T cells recovered delayer in the CD34 selected group (P = 0.026). Furthermore, reduced intensity conditioning facilitated faster immune recovery of lymphocytes and T cells and their subsets (P < 0.001). However, the immune recovery for NK cells and B cells was comparable for patients who received reduced-intensity or full preparative regimens. Dynamics of all cell types had a significant influence on OS, which did not differ between patients receiving CD34+-selected and those receiving CD3/CD19-depleted grafts. In conclusion, cell reconstitution dynamics showed complex diversity with regard to the graft manufacturing procedure and conditioning regimen
Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells
T cells are an essential part of the immune system. They determine the specificity of the immune response to foreign substances and, thus, help to protect the body from infections and cancer. Recently, T cells have gained much attention as promising tools in adoptive T cell transfer for cancer treatment. However, it is crucial not only for medical purposes but also for research to obtain T cells in large quantities, of high purity and functionality. To fulfill these criteria, efficient and robust isolation methods are needed. We used three different isolation methods to separate CD3-specific T cells from leukocyte concentrates (buffy coats) and Ficoll purified PBMCs. To catch the target cells, the Traceless Affinity Cell Selection (TACS®) method, based on immune affinity chromatography, uses CD-specific low affinity Fab-fragments; while the classical Magnetic Activated Cell Sorting (MACS®) method relies on magnetic beads coated with specific high affinity monoclonal antibodies. The REAlease® system also works with magnetic beads but, in contrast to MACS®, low-affinity antibody fragments are used. The target cells separated by TACS® and REAlease® are “label-free”, while cells isolated by MACS® still carry the cell specific label. The time required to isolate T cells from buffy coat by TACS® and MACS® amounted to 90 min and 50 min, respectively, while it took 150 min to isolate T cells from PBMCs by TACS® and 110 min by REAlease®. All methods used are well suited to obtain T cells in large quantities of high viability (>92%) and purity (>98%). Only the median CD4:CD8 ratio of approximately 6.8 after REAlease® separation differed greatly from the physiological conditions. MACS® separation was found to induce proliferation and cytokine secretion. However, independent of the isolation methods used, stimulation of T cells by anti CD3/CD28 resulted in similar rates of proliferation and cytokine production, verifying the functional activity of the isolated cells
CD44v6 specific CAR-NK cells for targeted immunotherapy of head and neck squamous cell carcinoma
Head and neck squamous cell carcinoma (HNSCC) is a major challenge for current therapies. CAR-T cells have shown promising results in blood cancers, however, their effectiveness against solid tumors remains a hurdle. Recently, CD44v6-directed CAR-T cells demonstrated efficacy in controlling tumor growth in multiple myeloma and solid tumors such as HNSCC, lung and ovarian adenocarcinomas. Apart from CAR-T cells, CAR-NK cells offer a safe and allogenic alternative to autologous CAR-T cell therapy. In this paper, we investigated the capacity of CAR-NK cells redirected against CD44v6 to execute cytotoxicity against HNSCC. Anti-CD44v6 CAR-NK cells were generated from healthy donor peripheral blood-derived NK cells using gamma retroviral vectors (gRVs). The NK cell transduction was optimized by exploring virus envelope proteins derived from the baboon endogenous virus envelope (BaEV), feline leukemia virus (FeLV, termed RD114-TR) and gibbon ape leukemia virus (GaLV), respectively. BaEV pseudotyped gRVs induced the highest transduction rate compared to RD114-TR and GaLV envelopes as measured by EGFP and surface CAR expression of transduced NK cells. CAR-NK cells showed a two- to threefold increase in killing efficacy against various HNSCC cell lines compared to unmodified, cytokine-expanded primary NK cells. Anti-CD44v6 CAR-NK cells were effective in eliminating tumor cell lines with high and low CD44v6 expression levels. Overall, the improved cytotoxicity of CAR-NK cells holds promise for a therapeutic option for the treatment of HNSCC. However, further preclinical trials are necessary to test in vivo efficacy and safety, as well to optimize the treatment regimen of anti-CD44v6 CAR-NK cells against solid tumors
Accelerating development of engineered T cell therapies in the EU: current regulatory framework for studying multiple product versions and T2EVOLVE recommendations
To accelerate the development of Advanced Therapy Medicinal Products (ATMPs) for patients suffering from life-threatening cancer with limited therapeutic options, regulatory approaches need to be constantly reviewed, evaluated and adjusted, as necessary. This includes utilizing science and risk-based approaches to mitigate and balance potential risks associated with early clinical research and a more flexible manufacturing paradigm. In this paper, T2EVOLVE an Innovative Medicine Initiative (IMI) consortium explores opportunities to expedite the development of CAR and TCR engineered T cell therapies in the EU by leveraging tools within the existing EU regulatory framework to facilitate an iterative and adaptive learning approach across different product versions with similar design elements or based on the same platform technology. As understanding of the linkage between product quality attributes, manufacturing processes, clinical efficacy and safety evolves through development and post licensure, opportunities are emerging to streamline regulatory submissions, optimize clinical studies and extrapolate data across product versions reducing the need to perform duplicative studies. It is worth noting that this paper is focusing on CAR- and TCR-engineered T cell therapies but the concepts may be applied more broadly to engineered cell therapy products (e.g., CAR NK cell therapy products)
Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications
Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved
successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to
estimate both success and severe side effects of CAR-T cell therapies, longitudinal
monitoring of the patient’s immune system including CAR-T cells is desirable to
accompany clinical staging. To conduct research on the fate and immunological impact
of infused CAR-T cells, we established standardized 13-colour/15-parameter flow
cytometry assays that are suitable to characterize immune cell subpopulations in the
peripheral blood during CAR-T cell treatment. The respective staining technology is based
on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies
of choice can be added to address specific clinical or research questions. We designed
panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a
healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells.
We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the
activation status of the T-cell compartment, the expression of co-stimulatory signaling
molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work
introduces standardized and modular flow cytometry assays for CAR-T cell clinical
research, which could also be adapted in the future as quality controls during the CART
cell manufacturing process
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