Abstract Chimeric antigen receptors (CAR) can transmit signals akin to those from activated T-cell receptors when bound to a cell surface target. CAR-expressing T cells against CD19 can cause curative effects in leukemia and lymphoma and is approved for clinical use. However, no CAR-T therapy is currently approved for use in solid tumors. We hypothesize that the resistance of solid tumors to CAR-T can be overcome by similar means as those used to reactivate tumor-infiltrating T lymphocytes (TIL), for example, by cytokines or immune checkpoint blockade. Here we demonstrate that CAR-T cells directed against HER2 can kill uveal and cutaneous melanoma cells in vitro and in vivo. Curative effects in vivo were only observed in xenografts grown in a NOD/SCID IL2 receptor gamma (NOG) knockout mouse strain transgenic for human IL2. The effect was target-specific, as CRISPR/Cas9-mediated disruption of HER2 in the melanoma cells abrogated the killing effect of the CAR-T cells. The CAR–T cells were also able to kill melanoma cells from patients resistant to adoptive T-cell transfer (ACT) of autologous TILs. Thus, CAR-T therapy represents an option for patients that do not respond to immunotherapy with ACT of TIL or immune checkpoint blockade. In addition, our data highlight the use of IL2 transgenic NOG mice as models to prove efficacy of CAR-T-cell products, possibly even in a personalized manner. Significance: These findings demonstrate that a novel humanized mouse model can help clinical translation of CAR-T cells against uveal and cutaneous melanoma that do not respond to TIL therapy or immune checkpoint blockade.
Immune cells in primary uveal melanoma (PUM) have mostly been studied in enucleated tissue, thereby precluding material from thinner, low-risk PUM tumors for research purposes. Here, we investigated the feasibility of using tumor tissue acquired by transvitreal retinochoroidal (TVRC) tumor biopsies to study the tumor immune microenvironment and relation to genetic risk class. Collected tumor biopsies of 41 patients were tested for genetic aberrations to determine high-risk (n = 19), medium-risk (n = 12), and low-risk (n = 9) tumors and were digested for flow cytometry analysis of immune cell and tumor markers. In addition, 13 patient-matched enucleated tumors were stained using multiplex immunohistochemistry. Tumor biopsies showed a high variability in the degree of immune infiltration. The tumor-specific lymphocyte infiltration pattern correlated well with the infiltration pattern in patient-matched enucleations. High-risk tumors tended to have a higher abundance of CD8 T cells, which expressed activation markers CD39, CD69, and PD-1, with reduced CD127 expression. In general, low-risk tumors exhibited decreased human leukocyte antigen (HLA) class I expression, coinciding with a higher abundance of natural killer (NK) cells (P = 0.0049), indicating a different lymphocyte infiltration pattern between low-, medium- and high-risk tumors. TVRC biopsies are a suitable and valuable source of PUM tissue for research purposes. An abundance of CD8 T cells was found in high-risk PUM tumors. Further, medium- and low-risk PUM tumors are characterized by decreased HLA class I expression with a concomitant increase in NK cell infiltration as compared to high-risk PUM tumors, correlating with decreased risk of disease recurrence.
Despite the improvements in HCV-therapy achieved in the last 20 years, the occurrence of high frequency of non-responders and of therapy-related side effects has lead to an ongoing interest in optimizing duration and dosage of current antiviral regimens as well as to the research and development of new antiviral treatment. Recently, the discovery of a system for in vitro HCV replication provided a useful tool for a better understanding of the viral life cycle followed by the discovery of new compounds that unlike classical drugs specifically target fundamental steps of this process. The aim of this review is to provide an update on the preclinical and clinical development of novel anti-HCV treatments targeting the first steps of the viral life cycle. The recent patents in this review article discuss the new perspectives in HCV therapy. Keywords: HCV, entry inhibitors, preclinical development, clinical development, HCV-therapy, antiviral treatment, replication, anti-HCV treatments, flavivirus, Chronic, acute, cirrhosis, hepatocellular, glomerulonephritis, cryoglobulinemia, porphyria cutanea tarda, lymphoproliferative, liver transplantation, HCC, prognosis, fibrosis, IFN-alpha, ribavirin, peginterferon, PEG-IFN, viral RNA, anaemia, thrombocytopenia, Hepacivirus, Flaviviridae, heterodimer, glycosaminoglycans, glycoprotein, drug design, albumin, Boceprevir, Telaprevir, Albinterferon alpha-2b, luciferase assay, liver-uPA-SCID, Viral Envelope, epitopes, monoclonal antibodies, plasma donor antibodies, polyclonal antibodies, HCV-genotypes, Glycosylation, glycans, cyanovirin-N, high-mannose oligosaccharides, iminosugars, BVDV, adenovirus, vaccinia virus, canary pox virus, alphavirus, CD81, tetraspanin, endocytosis, Claudin-1, Occludin, CLDN1, chlorpromazine, chloroquine, concanamycin A, bafilomycin, Heparin, Heparinase, Lamiridosins, Phosphorothioate, Oligonucleotides (PS-ONs), Arbidol, C5A, amphipathic-helical peptide, paramyxoviruses, SP-30, PRO 206, Civacir, ITX5061, REP 9AC, JTK-652, immune serum globulin, placebo, monotherapy, clinical trials, Lamiaceae
Abstract Rapamycin is a macrocyclic lactone currently used for the treatment of cancer and for the prevention of transplant rejection. The primary pharmacological mode of action of rapamycin occurs through the inhibition (blocking) of the mammalian target of rapamycin ( mTOR ). By doing so, rapamycin interferes with the phosphoinositide 3‐kinase ( PI 3 K )‐ A kt‐ mTOR axis that controls several cellular functions involving cell growth, proliferation and angiogenesis. The frequent activation of the phosphoinositide 3‐kinase ( PI 3 K )/ AKT pathway in advanced prostate cancer has provided a rationale for the use of mTOR inhibitors in this setting. We carried out a comparative study on the effects of rapamycin and temsirolimus on the in vitro and in vivo growth of the prostate cancer cell lines, L n C ap and PC 3. Our results demonstrate that rapamycin and temsirolimus exert similar in vitro and in vivo anti‐proliferative effects against prostate cancer cells.
Summary Analysis of antigen‐specific T‐cell populations by flow cytometry with peptide– MHC ( pMHC ) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of ‘tricks’ that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of ‘boosting’ staining by inclusion of an antibody against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T‐cell receptor is over 20‐fold weaker ( K D > 1 m m ) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anti‐cancer or autoimmune T‐cell populations, which tend to bear lower affinity T‐cell receptors. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log‐fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them.
Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II-restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR-pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.
