scholarly journals In situ multiplex RNA fluorescence imaging of SHIV1157ipd3N4 and anti-HIV CAR T cells to study CAR T cell trafficking to sites of viral reservoir in macaque lymphoid tissues

2017 ◽  
Vol 3 ◽  
pp. 50
Author(s):  
K. Eichholz ◽  
C. Peterson ◽  
T. Wagner ◽  
D. Rawlings ◽  
J. Zhu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Viral Reservoir ◽  
Lymphoid Tissues ◽  
Cell Trafficking ◽  
T Cell Trafficking ◽  
Car T Cells ◽  
Car T ◽  
Anti Hiv

scholarly journals Feasibility of real-time in vivo89Zr-DFO-labeled CAR T-cell trafficking using PET imaging

2019 ◽  
Author(s):  
Suk Hyun Lee ◽  
Hyunsu Soh ◽  
Jin Hwa Chung ◽  
Eun Hyae Cho ◽  
Sang Joo Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Real Time ◽  
Pet Imaging ◽  
Cell Trafficking ◽  
T Cell Trafficking ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

AbstractIntroductionChimeric antigen receptor (CAR) T-cells have been developed recently, producing impressive outcomes in patients with hematologic malignancies. However, there is no standardized method for cell trafficking and in vivo CAR T-cell monitoring. We assessed the feasibility of real-time in vivo89Zr-p-Isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS, DFO) labeled CAR T-cell trafficking using positron emission tomography (PET).ResultsThe 89Zr-DFO radiolabeling efficiency of Jurkat/CAR and human peripheral blood mononuclear cells (hPBMC)/CAR T-cells was 70–79%, and cell radiolabeling activity was 98.1–103.6 kBq/106 cells. Cell viability after radiolabeling was >95%. Compared with unlabeled cells, cell proliferation was not significantly different during the early period after injection; however, the proliferative capacity decreased over time (p = 0.02, day 7 after labeling). IL-2 or IFN-γ secretion was not significantly different between unlabeled and labeled CAR T-cells. PET/magnetic resonance images in the xenograft model showed that most of the 89Zr-DFO-labeled Jurkat/CAR T-cells were distributed in the lung (24.4% ± 3.4%ID) and liver (22.9% ± 5.6%ID) by 1 hour after injection. The cells gradually migrated from lung to the liver and spleen by day 1, and remained stably until day 7 (on day 7: lung 3.9% ± 0.3%ID, liver 36.4% ± 2.7%ID, spleen 1.4% ± 0.3%ID). No significant accumulation of labeled cells was identified in tumors. A similar pattern was observed in ex vivo biodistributions on day 7 (lung 3.0% ± 1.0%ID, liver 19.8% ± 2.2%ID, spleen 2.3% ± 1.7%ID). 89Zr-DFO-labeled hPBMC/CAR T-cells showed the similar distribution on serial PET images as Jurkat/CAR T-cells. The distribution of CAR T-cells was cross-confirmed by flow cytometry, Alu polymerase chain reaction, and immunohistochemistry.ConclusionUsing PET imaging of 89Zr-DFO-labeled CAR T-cells, real time in vivo cell trafficking is feasible. It can be used to investigate cellular kinetics, initial in vivo biodistribution, and the safety profile in future CAR T-cell development.

scholarly journals STING agonist promotes CAR T cell trafficking and persistence in breast cancer

2020 ◽  
Vol 218 (2) ◽  
Author(s):  
Nuo Xu ◽  
Douglas C. Palmer ◽  
Alexander C. Robeson ◽  
Peishun Shou ◽  
Hemamalini Bommiasamy ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Trafficking ◽  
T Cell Therapy ◽  
T Cell Trafficking ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

CAR T therapy targeting solid tumors is restrained by limited infiltration and persistence of those cells in the tumor microenvironment (TME). Here, we developed approaches to enhance the activity of CAR T cells using an orthotopic model of locally advanced breast cancer. CAR T cells generated from Th/Tc17 cells given with the STING agonists DMXAA or cGAMP greatly enhanced tumor control, which was associated with enhanced CAR T cell persistence in the TME. Using single-cell RNA sequencing, we demonstrate that DMXAA promoted CAR T cell trafficking and persistence, supported by the generation of a chemokine milieu that promoted CAR T cell recruitment and modulation of the immunosuppressive TME through alterations in the balance of immune-stimulatory and suppressive myeloid cells. However, sustained tumor regression was accomplished only with the addition of anti–PD-1 and anti–GR-1 mAb to Th/Tc17 CAR T cell therapy given with STING agonists. This study provides new approaches to enhance adoptive T cell therapy in solid tumors.

scholarly journals Novel Immunotherapeutic Approaches for Hodgkin Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-9-SCI-9
Author(s):  
Barbara Savoldo ◽  
Natalie S Grover ◽  
Carlos A. Ramos ◽  
Gianpietro Dotti
Keyword(s):  
T Cells ◽  
T Cell ◽  
Checkpoint Inhibitors ◽  
Cell Transplant ◽  
Cell Trafficking ◽  
Antigen Specificity ◽  
Advisory Committees ◽  
T Cell Trafficking ◽  
Car T Cells ◽  
Car T

The outcome of patients with relapsed/refractory Hodgkin Lymphoma (HL) for whom salvage therapy or stem cell transplant have failed is poor. HL tumor cells (HRS) cells universally express CD30, which is an excellent target for directed therapy such as the antibody drug conjugate brentuximab vedotin (BV). Patients with relapsed HL can also respond to checkpoint inhibitors, emphasizing the susceptibility of this tumor to T-cell-mediated immune control. The adoptive transfer of chimeric antigen receptor T (CAR-T) cells, which combine antibody-mediated antigen specificity with the effector function and replication potential of T lymphocytes, offers the opportunity to infuse large numbers of T cells with defined antigen-specificity and MHC-independent tumor targeting. In two parallel and simultaneously conducted phase 1/2 dose-escalation studies, we administered CD30-specific CAR-T (CD30.CAR-T) cells after lymphodepletion to patients with HL that had relapsed after multiple lines of therapy, including BV, checkpoint inhibitors and stem cell transplant. We observed an excellent safety profile, with modest and self-limited cytokine release syndrome and no neurologic toxicity. The overall response rate was 78%, including 18 patients (67%) with complete responses, and the 1 year progression free survival was 47% (95% CI: 25% - 67%). Despite these promising results, unsolved obstacles include CAR-T cell trafficking and persistence in the tumor site. HL is characterized by a unique tumor microenvironment that creates a physical barrier and a hostile niche for CD30.CAR-T cells. Expression of PD-1 on CD30.CAR-T cells indicates that these cells likely remain susceptible to inhibition by PD-L1 in the tumor, and suggests that the combination of CD30.CAR-T cells with checkpoint blockade should be investigated. Enhancing CAR-T cell trafficking to tumor sites should also tip the delicate balance between effector and inhibitory mechanisms towards the former. Since the tumor microenvironment of HL is rich in TARC (thymus and activation regulated chemokine/CC chemokine ligand 17), we have initiated a clinical study in which CD30.CAR-T cells are further modified to co-express the cognate receptor (CCR4) for TARC to improve their tumor homing (NCT03062157). Disclosures Savoldo: Baylor College of Medicine: Patents & Royalties: CAR.CD30 patent; Bluebirdbio: Other: research agreement; Cell Medica: Other: Research Agreement; Bellicum: Other: Research Agreement. Grover:Seattle Genetics: Consultancy. Ramos:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding.

scholarly journals A CAR RNA FISH assay reveals functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

2020 ◽  
Author(s):  
Karsten Eichholz ◽  
Alvason Zhenhua Li ◽  
Kurt Diem ◽  
Semih U. Tareen ◽  
Michael C. Jensen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Rna Fish ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer and HIV sanctuary sites. Here, we describe a CAR RNA FISH histocytometry platform combined with a dnnRRS image analysis algorithm to quantitate spatial distribution and in vivo functional ability of a CAR T cell population at a single cell resolution. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions engineered cells in situ with their target cells in tissues with high statistical power and thus, as an important tool for preclinical and potentially clinical assessment of CAR T cell effectiveness.One Sentence SummaryWe developed a CAR RNA FISH assay to study chimeric antigen receptor T cell trafficking and function in human and mouse tissue.Impact statementWe developed an imaging platform and analysis pipeline to study large populations of engineered cells on a single cell level in situ.

scholarly journals A CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karsten Eichholz ◽  
Alvason Zhenhua Li ◽  
Kurt Diem ◽  
Michael Claus Jensen ◽  
Jia Zhu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Rna Fish ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness.

scholarly journals Editor's Pick: Tumour-Associated Hypoxia: Can We Give Chimeric Antigen Receptor T Cells More Breathing Space?

2020 ◽  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Solid Tumours ◽  
Cell Trafficking ◽  
T Cell Trafficking ◽  
Car T Cells ◽  
Breathing Space ◽  
Car T ◽  
Engineered T Cells

Immunotherapy using chimeric antigen receptor (CAR)-engineered T cells has encountered important limitations in the transition of their use from liquid to solid tumours. Success is dependent upon T-cell trafficking to, and functional persistence within, tumours that often present a metabolically and immunologically hostile microenvironment. Moreover, CAR targets that are tumour specific are extremely scarce. To address these issues, several strategies have been proposed to improve both tumour selectivity and safety. One approach involves the engineering of CAR-T cells that only deploy their effector function at tumour sites. Conceptually, a solution for this exploits the oxygen-limited nature of advanced tumour deposits through the engineering of CAR that are exclusively expressed or activated under conditions of profound hypoxia. T cells have a complex inter-relationship with oxygen, which also needs to be factored into the refinement of these technologies. Ideally, oxygen-sensing CAR should only function when oxygen tension is below 2%, as is commonly the case in solid tumours but rare in healthy tissue. Successful advancement of such technologies presents opportunities for solid tumour immunotherapy because it should broaden the target repertoire that may safely be exploited in this context.

Preclinical CAR-T cell target safety, biodistribution, and tumor infiltration analysis using in situ hybridization technology.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 112-112 ◽  
Author(s):  
Helly Pimentel ◽  
Helen Jarnagin ◽  
Hailing Zong ◽  
Courtney Todorov ◽  
Courtney M. Anderson ◽  
...  
Keyword(s):  
T Cells ◽  
In Situ Hybridization ◽  
T Cell ◽  
Mouse Lung ◽  
Xenograft Tumor ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Low Levels

112 Background: Chimeric antigen receptor (CAR) T cell therapy is highly effective in treating hematologic malignancies, and major efforts are being made to achieve similar efficacy in solid tumors. The greater potency of CAR-T cells compared to antibody therapeutics demands a more stringent CAR-T target safety assessment to avoid adverse events resulting from “on-target/off-tumor” activity. Furthermore, it is critical to track and monitor CAR+ T cells within intact tissue and tumor to understand the mechanisms underlying off-tumor toxicity and efficacy in tumor killing. Methods: We employed the RNAscope in situ hybridization (ISH) technology to assess target expression specificity and to track CAR-T cell distribution and activation in xenograft and host tissues using the RPMI-8226 xenograft mouse model. Results: For the CAR-T target candidates, Target X and Target Y, RNA ISH revealed that Target X was only expressed in the xenograft tumor and in no mouse organs, while Target Y was found to be expressed at low levels in mouse lung and liver, as well as in the xenograft tumor. Duplex RNA ISH assays with probes targeting the CAR 3’ UTR and either IFNG or GZMB allowed for highly sensitive and specific detection of CAR-T cells and their activation state in both tumor and normal tissues from vehicle, Target X CAR-T cell, or Target Y CAR-T cell treated mice. Activated Target X CAR-T cells expressing GZMB and IFNG were found only in the xenograft tumor, where Target X was expressed. In contrast, activated Target Y CAR-T cells were found almost exclusively in mouse lung and liver, with very few Target Y CAR-T cells being found in the xenograft tumor. Lastly, a multiplex ISH-IHC approach confirmed the presence of activated Target X CAR-T cells in the xenograft tumor through simultaneous detection of the Target X CAR 3’ UTR, IFNG, GZMB, and CD3. Conclusions: These data demonstrate how the RNAscope assay can be utilized for CAR-T cell efficacy and safety/toxicity assessment in preclinical models by detecting very low levels of target antigen expression in off-tumor tissues and monitoring CAR-T cell pharmacodynamics and activation in tumor models and can also be applied for assessing TCR-T cell activity in tumors.

Two-photon fluorescent polydopamine nanodots for CAR-T cell function verification and tumor cell/tissue detection

2018 ◽  
Vol 6 (40) ◽  
pp. 6459-6467 ◽  
Author(s):  
Baojin Ma ◽  
Feng Liu ◽  
Shan Zhang ◽  
Jiazhi Duan ◽  
Ying Kong ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Function ◽  
Normal Cells ◽  
Two Photon ◽  
Car T Cells ◽  
Cell Tissue ◽  
Car T

OPDA nanodots were used to verify CAR-T cells function and efficiency through visible fluorescence. PDA nanodots can in situ convert to OPDA by the oxidation of ROS in cells to identify the tumor and normal cells/tissues.

Chimeric Antigen Receptor-Engineered T-Cells - A New Way and Era for Lymphoma Treatment

2020 ◽  
Vol 14 (4) ◽  
pp. 312-323
Author(s):  
Romeo G. Mihăilă
Keyword(s):  
T Cells ◽  
T Cell ◽  
Response Rate ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
Future Developments ◽  
Car T Cell ◽  
Large B Cell Lymphoma ◽  
Car T

Background: Patients with refractory or relapsed diffuse large B-cell lymphoma have a poor prognosis with the current standard of care. Objective: Chimeric Antigen Receptor T-cells (CAR T-cells) are functionally reprogrammed lymphocytes, which are able to recognize and kill tumor cells. The aim of this study is to make progress in this area. Method: A mini-review was achieved using the articles published in Web of Science and PubMed in the last year and the new patents were made in this field. Results: The responses to CAR T-cell products axicabtagene ciloleucel and tisagenlecleucel are promising; the objective response rate can reach up to 83%, and the complete response rate ranges between 40 and 58%. About half of the patients may have serious side effects, such as cytokine release syndrome and neurotoxicity. Current and future developments include the improvement of CAR T-cell expansion and polyfunctionality, the combined use of CAR T-cells with a fusion protein between interferon and an anti-CD20 monoclonal antibody, with checkpoint inhibitors or small molecule sensitizers that have apoptotic-regulatory effects. Furthermore, the use of IL-12-expressing CAR T-cells, an improved technology for the production of CAR T-cells based on targeted nucleases, the widespread use of allogeneic CAR T-cells or universal CAR T-cells obtained from genetically engineered healthy donor T-cells are future developments actively considered. Conclusion: CAR T-cell therapy significantly improved the outcome of patients with relapsed or refractory diffuse large B-cell lymphoma. The advances in CAR T-cells production technology will improve the results and enable the expansion of this new immunotherapy.

scholarly journals Structure of the Signal Transduction Domain in Second-Generation CAR Regulates the Input Efficiency of CAR Signals

2021 ◽  
Vol 22 (5) ◽  
pp. 2476
Author(s):  
Kento Fujiwara ◽  
Masaki Kitaura ◽  
Ayaka Tsunei ◽  
Hotaka Kusabuka ◽  
Erika Ogaki ◽  
...  
Keyword(s):  
Signal Transduction ◽  
T Cells ◽  
T Cell ◽  
Second Generation ◽  
Independent Manner ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
The Impact

T cells that are genetically engineered to express chimeric antigen receptor (CAR) have a strong potential to eliminate tumor cells, yet the CAR-T cells may also induce severe side effects due to an excessive immune response. Although optimization of the CAR structure is expected to improve the efficacy and toxicity of CAR-T cells, the relationship between CAR structure and CAR-T cell functions remains unclear. Here, we constructed second-generation CARs incorporating a signal transduction domain (STD) derived from CD3ζ and a 2nd STD derived from CD28, CD278, CD27, CD134, or CD137, and investigated the impact of the STD structure and signaling on CAR-T cell functions. Cytokine secretion of CAR-T cells was enhanced by 2nd STD signaling. T cells expressing CAR with CD278-STD or CD137-STD proliferated in an antigen-independent manner by their STD tonic signaling. CAR-T cells incorporating CD28-STD or CD278-STD between TMD and CD3ζ-STD showed higher cytotoxicity than first-generation CAR or second-generation CARs with other 2nd STDs. The potent cytotoxicity of these CAR-T cells was not affected by inhibiting the 2nd STD signals, but was eliminated by placing the STDs after the CD3ζ-STD. Our data highlighted that CAR activity was affected by STD structure as well as by 2nd STD signaling.

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