19F Magnetic Resonance Imaging platform to quantify T cell therapy biodistribution, persistence and efficacy

Cancer is responsible for over 8 million deaths annually and the number of new cases is expected to increase by approximately 70% over the next 20 years. Surgery, chemotherapy and radiotherapy have been used for decades as primary strategies against cancer in patients; however, cancer cell drug and radiation resistance development often leads to lower remission and higher relapse incidence. Cell therapies for cancer are emerging approaches to enhance tumor-specific killing and include expansion of patients’ tumor-infiltrating lymphocytes or complex engineering of the T cell receptor or Chimeric Antigen Receptors (CAR). The latter have shown unprecedented progress towards treating incurable cancers and are currently being examined in over 200 clinical trials. Developing successful therapeutic strategies using live cells entails the ability to determine their in vivo biodistribution and persistence after systemic administration. Non-invasive imaging techniques such as Magnetic Resonance Imaging (MRI) and Positron Emission Tomography are the best candidates for real time, quantitative assessment of tumor response. This thesis describes fluorinated imaging probes and methods for labeled cell tracking by 19F MRI. Perfluorocarbon (PFC) probes are composed of numerous fluorine atoms, which are not naturally present in the body and allow for background free quantitation of the signal. Following transfer to the subject, 19F MRI allows direct visualization of instant labeled-cell fate as well as quantification of local and systemic accumulation over time. The principle bottleneck that remains for longitudinal tracking of transferred immune cells is 19F MRI sensitivity. This work depicts strategies to increase cell loading through cell penetrating peptides. In addition, one can exploit the properties of PFC nanoemulsions to indirectly detect adoptive cell therapy cytotoxicity against cancer cells. Overall, 19F MR imaging is a versatile technique that can provide insights into the survival and modes of actions of cell therapy against cancer

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Contribution à l'étude des relations entre hypothyroïdie canine et troubles du comportement

TOULOUSE3-BU Santé-Centrale (315552105) / SudocTOULOUSE-EN Vétérinaire (315552301) / SudocSudocFranceF

Fluorine-19 MRI for detection and quantification of immune cell therapy for cancer

Abstract Over the past two decades, immune cell therapy has emerged as a potent treatment for multiple cancers, first through groundbreaking leukemia therapy, and more recently, by tackling solid tumors. Developing successful therapeutic strategies using live cells could benefit from the ability to rapidly determine their in vivo biodistribution and persistence. Assaying cell biodistribution is unconventional compared to traditional small molecule drug pharmacokinetic readouts used in the pharmaceutical pipeline, yet this information is critical towards understanding putative therapeutic outcomes and modes of action. Towards this goal, efforts are underway to visualize and quantify immune cell therapy in vivo using advanced magnetic resonance imaging (MRI) techniques. Cell labeling probes based on perfluorocarbon nanoemulsions, paired with fluorine-19 MRI detection, enables background-free quantification of cell localization and survival. Here, we highlight recent preclinical and clinical uses of perfluorocarbon probes and 19F MRI for adoptive cell transfer (ACT) studies employing experimental T lymphocytes, NK, PBMC, and dendritic cell therapies. We assess the forward looking potential of this emerging imaging technology to aid discovery and preclinical phases, as well as clinical trials. The limitations and barriers towards widespread adoption of this technology, as well as alternative imaging strategies, are discussed

Fluorine-19 nuclear magnetic resonance of chimeric antigen receptor T cell biodistribution in murine cancer model.

Discovery of effective cell therapies against cancer can be accelerated by the adaptation of tools to rapidly quantitate cell biodistribution and survival after delivery. Here, we describe the use of nuclear magnetic resonance (NMR) 'cytometry' to quantify the biodistribution of immunotherapeutic T cells in intact tissue samples. In this study, chimeric antigen receptor (CAR) T cells expressing EGFRvIII targeting transgene were labeled with a perfluorocarbon (PFC) emulsion ex vivo and infused into immunocompromised mice bearing subcutaneous human U87 glioblastomas expressing EGFRvIII and luciferase. Intact organs were harvested at day 2, 7 and 14 for whole-sample fluorine-19 (19F) NMR to quantitatively measure the presence of PFC-labeled CAR T cells, followed by histological validation. NMR measurements showed greater CAR T cell homing and persistence in the tumors and spleen compared to untransduced T cells. Tumor growth was monitored with bioluminescence imaging, showing that CAR T cell treatment resulted in significant tumor regression compared to untransduced T cells. Overall, 19F NMR cytometry is a rapid and quantitative method to evaluate cell biodistribution, tumor homing, and fate in preclinical studies

Cell penetrating peptide functionalized perfluorocarbon nanoemulsions for targeted cell labeling and enhanced fluorine‐19 MRI detection

PurposeA bottleneck in developing cell therapies for cancer is assaying cell biodistribution, persistence, and survival in vivo. Ex vivo cell labeling using perfluorocarbon (PFC) nanoemulsions, paired with 19 F MRI detection, is a non-invasive approach for cell product detection in vivo. Lymphocytes are small and weakly phagocytic limiting PFC labeling levels and MRI sensitivity. To boost labeling, we designed PFC nanoemulsion imaging probes displaying a cell-penetrating peptide, namely the transactivating transcription sequence (TAT) of the human immunodeficiency virus. We report optimized synthesis schemes for preparing TAT co-surfactant to complement the common surfactants used in PFC nanoemulsion preparations.MethodsWe performed ex vivo labeling of primary human chimeric antigen receptor (CAR) T cells with nanoemulsion. Intracellular labeling was validated using electron microscopy and confocal imaging. To detect signal enhancement in vivo, labeled CAR T cells were intra-tumorally injected into mice bearing flank glioma tumors.ResultsBy incorporating TAT into the nanoemulsion, a labeling efficiency of ~1012 fluorine atoms per CAR T cell was achieved that is a &gt;8-fold increase compared to nanoemulsion without TAT while retaining high cell viability (~84%). Flow cytometry phenotypic assays show that CAR T cells are unaltered after labeling with TAT nanoemulsion, and in vitro tumor cell killing assays display intact cytotoxic function. The 19 F MRI signal detected from TAT-labeled CAR T cells was 8 times higher than cells labeled with PFC without TAT.ConclusionThe peptide-PFC nanoemulsion synthesis scheme presented can significantly enhance cell labeling and imaging sensitivity and is generalizable for other targeted imaging probes

Assessing Oximetry Response to Chimeric Antigen Receptor T-cell Therapy against Glioma with 19F MRI in a Murine Model

PurposeTo assess the cell-specific, intracellular partial pressure of oxygen (Po2) dynamics of both tumor and chimeric antigen receptor (CAR) T cells in a murine immunotherapy model.Materials and methodsHuman glioblastoma cells or human T cells were intracellularly labeled with perfluorocarbon nanoemulsion droplet sensors prior to in vivo injection in severe combined immunodeficient mice to measure Po2 in the two cell types in response to treatment. Two main sets of experiments were performed: (a) mice were injected in the flank with perfluorocarbon-labeled human glioblastoma cells and were then inoculated with either CAR T cells or untransduced T cells or were untreated 5 days after tumor inoculation; and (b) mice with unlabeled glioblastoma tumors were inoculated with perfluorocarbon-labeled CAR T cells or untransduced T cells 5 days after tumor inoculation. Longitudinal fluorine 19 (19F) spin-lattice relaxation time measurements of the tumor mass were used to ascertain absolute Po2 in vivo. Results were analyzed for significance using an analysis of variance, a linear mixed-effect model, and a Pearson correlation coefficient test, as appropriate.ResultsThe intracellular tumor cell Po2 temporal dynamics exhibited delayed, transient hyperoxia at 3 days after infusion of CAR T cells, commensurate with significant tumor cell killing and CAR T-cell infiltration, as observed by bioluminescence imaging and histologic findings. Conversely, no significant changes were detected in CAR or untransduced T-cell intracellular Po2 over time in tumor using these same methods. Moreover, it was observed that the total 19F tumor cell signal quenches with treatment, consistent with rapid tissue clearance of probe from apoptotic tumor cells.ConclusionCell-specific Po2 measurements using perfluorocarbon probes can provide insights into effector cell function and tumor response in cellular immunotherapeutic cancer models.Keywords: Animal Studies, MR-Imaging, MR-Spectroscopy, Molecular Imaging-Cancer, Molecular Imaging-Immunotherapy Supplemental material is available for this article. © RSNA, 2021See also commentary by Bulte in this issue