A TCR-based Chimeric Antigen Receptor Walseng, Even; Köksal, Hakan; Sektioglu, Ibrahim M ...
Scientific reports,
09/2017, Letnik:
7, Številka:
1
Journal Article
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Effector T cells equipped with engineered antigen receptors specific for cancer targets have proven to be very efficient. Two methods have emerged: the Chimeric Antigen Receptors (CARs) and T-cell ...Receptor (TCR) redirection. Although very potent, CAR recognition is limited to membrane antigens which represent around 1% of the total proteins expressed, whereas TCRs have the advantage of targeting any peptide resulting from cellular protein degradation. However, TCRs depend on heavy signalling machinery only present in T cells which restricts the type of eligible therapeutic cells. Hence, an introduced therapeutic TCR will compete with the endogenous TCR for the signalling proteins and carries the potential risk of mixed dimer formation giving rise to a new TCR with unpredictable specificity. We have fused a soluble TCR construct to a CAR-signalling tail and named the final product TCR-CAR. We here show that, if expressed, the TCR-CAR conserved the specificity and the functionality of the original TCR. In addition, we demonstrate that TCR-CAR redirection was not restricted to T cells. Indeed, after transduction, the NK cell line NK-92 became TCR positive and reacted against pMHC target. This opens therapeutic avenues combing the killing efficiency of NK cells with the diversified target recognition of TCRs.
Treating osteosarcoma with CAR T cells Köksal, Hakan; Müller, Elisabeth; Inderberg, Else Marit ...
Scandinavian journal of immunology,
March 2019, Letnik:
89, Številka:
3
Journal Article
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Novel therapies to treat patients with solid cancers that have developed resistance to chemotherapy represent unmet needs of considerable dimensions. In the present review, we will address the ...attempts to develop chimeric antigen receptor (CAR) targeted immunotherapy against osteosarcoma (OS). This aggressive cancer displays its peak incidence in children and young adults. The main cause of patient death is lung metastases with a 5‐year survival as low as 5%‐10% in the primary metastatic setting and 30% in the relapse situation, respectively. Effective adjuvant combination chemotherapy introduced more than 40 years ago improved the survival rates from below 20% to around 60% in patients; however, since then, no major breakthroughs have been made. The use of immune checkpoint inhibitors has been disappointing in OS, while other types of immunotherapies such as CAR T cells remain largely unexplored. Indeed, for CAR T‐cell therapy to be efficacious, two main criteria need to be fulfilled: (a) CAR T cells should target an epitope selectively expressed on the cell surface of OS in order to prevent toxicities in normal tissues and (b) the target should also be widely expressed on OS metastases. These challenges have already been undertaken in OS and illustrate the difficulties in developing tomorrow's CAR‐T treatment in a solid tumour. We will discuss the experiences with CAR‐T therapy development and efficacy to combat the clinical challenges in OS.
Success of adoptive cell therapy mainly depends on the ability of immune cells to persist and function optimally in the immunosuppressive tumor microenvironment. Although present at the cancer site, ...immune cells become exhausted and/or inhibited, due to the presence of inhibitory receptors such as PD‐L1 on malignant cells. Novel genetic strategies to manipulate the PD1/PD‐L1 axis comprise (i) PD‐1 reversion where the receptor intracellular domain is replaced with an activating unit, (ii) the use of anti‐PD‐L1 CAR or (iii) the disruption of the PD‐1 gene. We here present an alternative strategy to equip therapeutic cells with a truncated PD‐1 (tPD‐1) to abrogate PD‐1/PD‐L1 inhibition. We show that engagement of tPD‐1 with PD‐L1‐positive tumor unleashes NK‐92 activity in vitro. Furthermore, this binding was sufficiently strong to induce killing of targets otherwise not recognized by NK‐92, thus increasing the range of targets. In vivo treatment with NK‐92 tPD‐1 cells led to reduced tumor growth and improved survival. Importantly, tPD‐1 did not interfere with tumor recognition in PD‐L1 negative conditions. Thus, tPD‐1 represents a straightforward method for improving antitumor immunity and revealing new targets through PD‐L1 positivity.
CAR T cells targeting the B lymphocyte antigen CD19 have led to remarkable clinical results in B cell leukemia and lymphoma but eliminate all B lineage cells, leading to increased susceptibility to ...severe infections. As malignant B cells will express either immunoglobulin (Ig) light chain κ or λ, we designed a second-generation CAR targeting Igκ, IGK CAR. This construct demonstrated high target specificity but displayed reduced efficacy in the presence of serum IgG. Since CD19 CAR is insensitive to serum IgG, we designed various combinatorial CAR constructs in order to maintain the CD19 CAR T cell efficacy, but with IGK CAR target selectivity. The Kz-19BB design, combining CD19 CAR containing a 4-1BB costimulatory domain with an IGK CAR containing a CD3zeta stimulatory domain, maintained the target specificity of IgK CAR and was resistant to the presence of soluble IgG. Our results demonstrate that a combinatorial CAR approach can improve target selectivity and efficacy.
A Spheroid Killing Assay by CAR T Cells Dillard, Pierre; Köksal, Hakan; Inderberg, Else-Marit ...
Journal of visualized experiments,
2018-Dec-12
142
Journal Article
Recenzirano
Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells ...obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.
T cell receptor (TCR)-engineered T cell therapy is a promising cancer treatment approach. Human telomerase reverse transcriptase (hTERT) is overexpressed in the majority of tumors and a potential ...target for adoptive cell therapy. We isolated a novel hTERT-specific TCR sequence, named Radium-4, from a clinically responding pancreatic cancer patient vaccinated with a long hTERT peptide. Radium-4 TCR-redirected primary CD4+ and CD8+ T cells demonstrated in vitro efficacy, producing inflammatory cytokines and killing hTERT+ melanoma cells in both 2D and 3D settings, as well as malignant, patient-derived ascites cells. Importantly, T cells expressing Radium-4 TCR displayed no toxicity against bone marrow stem cells or mature hematopoietic cells. Notably, Radium-4 TCR+ T cells also significantly reduced tumor growth and improved survival in a xenograft mouse model. Since hTERT is a universal cancer antigen, and the very frequently expressed HLA class II molecules presenting the hTERT peptide to this TCR provide a very high (>75%) population coverage, this TCR represents an attractive candidate for immunotherapy of solid tumors.
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CD4 T cells are major players in the antitumor response. Dillard et al. target a universal cancer antigen, telomerase, with a TCR recognizing its presentation on a frequently expressed HLA class II allele. Such TCR-redirected T cells provide both helper and direct cytotoxic functions and destroy telomerase-expressing tumors in vitro and in vivo, thus offering an alternative solution for solid tumor treatment.
Ionizing radiation sources such as Solar Energetic Particles and Galactic Cosmic Radiation may cause unexpected errors in imaging and communication systems of satellites in the Space environment, as ...reported in the previous literature. In this study, the temporal variation of the speckle values on Sentinel 1 satellite images were compared with the cosmic ray intensity/count data, to analyze the effects which may occur in the electromagnetic wave signals or electronic system. Sentinel 1 Synthetic Aperture Radar (SAR) images nearby to the cosmic ray stations and acquired between January 2015 and December 2019 were processed. The median values of the differences between speckle filtered and original image were calculated on Google Earth Engine Platform per month. The monthly median “noise” values were compared with the cosmic ray intensity/count data acquired from the stations. Eight selected stations’ data show that there are significant correlations between cosmic ray intensities and the speckle amounts. The Pearson correlation values vary between 0.62 and 0.78 for the relevant stations.
Treatment of cancers has largely benefited from the development of immunotherapy. In particular, Chimeric Antigen Receptor (CAR) redirected T cells have demonstrated impressive efficacy against ...B-cell malignancies and continuous efforts are made to adapt this new therapy to solid tumors, where the immunosuppressive tumor microenvironment is a barrier for delivery. CAR T-cell validation relies on in vitro functional assays using monolayer or suspension cells and in vivo xenograft models in immunodeficient animals. However, the efficacy of CAR therapies remains difficult to predict with these systems, in particular when challenged against 3D organized solid tumors with highly intricate microenvironment. An increasing number of reports have now included an additional step in the development process in which redirected T cells are tested against tumor spheres. Here, we report a method to produce 3D structures, or cysts, out of a colorectal cancer cell line, Caco-2, which has the ability to form polarized spheroids as a validation tool for adoptive cell therapy in general. We used CD19CAR T cells to explore this method and we show that it can be adapted to various platforms including high resolution microscopy, bioluminescence assays and high-throughput live cell imaging systems. We developed an affordable, reliable and practical method to produce cysts to validate therapeutic CAR T cells. The integration of this additional layer between in vitro and in vivo studies could be an important tool in the pre-clinical workflow of cell-based immunotherapy.
A study was conducted to evaluate the effect of dietary tannic acid and barley supplementation on growth performance, intestinal viscosity, litter quality, and footpad dermatitis (FPD) in broiler ...chickens. Five hundred forty-four 1-d-old male broiler chicks were randomly assigned to dietary treatments with 8 replicated pens per treatment and 17 broiler chickens pen as a 2 × 2 factorial arrangement of 2 diets (a corn-soybean meal diet or a diet with 30% barley) and tannic acid (0 and 2g/kg) in a completely randomized design. Growth performance, intestinal viscosity, litter quality, and FPD incidence and severity were recorded. The results showed that there was no interaction between diets and tannic acid levels. Barley-based diets reduced (P < 0.05) the body weight (BW) gain at 0–42 d, and feed intake at 21–42 d and 0–42 d. At 28 d of experiment, the viscosity of intestinal contents of anterior and posterior segments was greater (P < 0.05) in broiler chickens fed diets with barley. Similarly, the viscosity of intestinal contents of posterior segment was greater (P < 0.05) at 42 d, in broiler chickens fed barley-based diets. Litter pH, moisture, and NH3 volatilization were increased (P < 0.05) in response to barley-based diets. Barley-based diets increased (P < 0.05) the incidence and severity of FPD lesions in broiler chickens at 14, 28, and 42 d of experiment. Although dietary tannic acid had no effect on performance, intestinal viscosity, and litter quality compared to those fed diets without tannic acid (P > 0.05), it tended to reduce body weight gain (P = 0.05) and increase feed conversion ratio (FCR; P = 0.09) at 0–21 d and NH3 volatilization on 28 (P = 0.08) and 42 d (P = 0.07). Dietary tannic acid supplementation prevented the FPD lesion development and reduced (P < 0.05) the total FPD lesions at d 42. Moreover, the severe lesions decreased (P = 0.08) on d 42 in broiler chickens fed tannic acid. In conclusion, barley-based diets may worsen the growth performance and litter quality, and increase the intestinal viscosity that increases the incidence and severity of FPD in broiler chickens. Dietary tannic acid supplementation may not affect the growth performance, intestinal viscosity, and litter quality. However, it may reduce the incidence and severity of FPD in broiler chickens.
•Footpad dermatitis (FPD) is a disease of poultry arising due to many predisposing factors.•Barley-based diets declined the growth performance, and increased the incidence and severity of FPD lesions.•Dietary tannic acid had no effect on growth performance, however, reduced the incidence and severity of FPD lesions on d 42.
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