Method for estimation of apoptotic cell fraction of cytotherapy using in vivo fluorine-19 magnetic resonance: pilot study in a patient with head and neck carcinoma receiving tumor-infiltrating lymphocytes labeled with perfluorocarbon nanoemulsion.

BACKGROUND: Adoptive transfer of T cells is a burgeoning cancer therapeutic approach. However, the fate of the cells, once transferred, is most often unknown. We describe the first clinical experience with a non-invasive biomarker to assay the apoptotic cell fraction (ACF) after cell therapy infusion, tested in the setting of head and neck squamous cell carcinoma (HNSCC). A patient with HNSCC received autologous tumor-infiltrating lymphocytes (TILs) labeled with a perfluorocarbon (PFC) nanoemulsion cell tracer. Nanoemulsion, released from apoptotic cells, clears through the reticuloendothelial system, particularly the Kupffer cells of the liver, and fluorine-19 (19F) magnetic resonance spectroscopy (MRS) of the liver was used to non-invasively infer the ACF. METHODS: Autologous TILs were isolated from a patient in their late 50s with relapsed, refractory human papillomavirus-mediated squamous cell carcinoma of the right tonsil, metastatic to the lung. A lung metastasis was resected for T cell harvest and expansion using a rapid expansion protocol. The expanded TILs were intracellularly labeled with PFC nanoemulsion tracer by coincubation in the final 24 hours of culture, followed by a wash step. At 22 days after intravenous infusion of TILs, quantitative single-voxel liver 19F MRS was performed in vivo using a 3T MRI system. From these data, we model the apparent ACF of the initial cell inoculant. RESULTS: We show that it is feasible to PFC-label ~70×1010 TILs (F-TILs) in a single batch in a clinical cell processing facility, while maintaining >90% cell viability and standard flow cytometry-based release criteria for phenotype and function. Based on quantitative in vivo 19F MRS measurements in the liver, we estimate that ~30% cell equivalents of adoptively transferred F-TILs have become apoptotic by 22 days post-transfer. CONCLUSIONS: Survival of the primary cell therapy product is likely to vary per patient. A non-invasive assay of ACF over time could potentially provide insight into the mechanisms of response and non-response, informing future clinical studies. This information may be useful to developers of cytotherapies and clinicians as it opens an avenue to quantify cellular product survival and engraftment.

Options for imaging cellular therapeutics in vivo: a multi-stakeholder perspective.

Cell-based therapies have been making great advances toward clinical reality. Despite the increase in trial activity, few therapies have successfully navigated late-phase clinical trials and received market authorization. One possible explanation for this is that additional tools and technologies to enable their development have only recently become available. To support the safety evaluation of cell therapies, the Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee, a multisector collaborative committee, polled the attendees of the 2017 International Society for Cell & Gene Therapy conference in London, UK, to understand the gaps and needs that cell therapy developers have encountered regarding safety evaluations in vivo. The goal of the survey was to collect information to inform stakeholders of areas of interest that can help ensure the safe use of cellular therapeutics in the clinic. This review is a response to the cellular imaging interests of those respondents. The authors offer a brief overview of available technologies and then highlight the areas of interest from the survey by describing how imaging technologies can meet those needs. The areas of interest include imaging of cells over time, sensitivity of imaging modalities, ability to quantify cells, imaging cellular survival and differentiation and safety concerns around adding imaging agents to cellular therapy protocols. The Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee believes that the ability to understand therapeutic cell fate is vital for determining and understanding cell therapy efficacy and safety and offers this review to aid in those needs. An aim of this article is to share the available imaging technologies with the cell therapy community to demonstrate how these technologies can accomplish unmet needs throughout the translational process and strengthen the understanding of cellular therapeutics.

Integrating a 19F MRI Tracer Agent into the Clinical Scale Manufacturing of a T-Cell Immunotherapy.

Leukocyte immunotherapies have made great progress in the treatment of cancer. Recent reports on the treatment of B-cell malignancies using Chimeric Antigen Receptor and affinity enhanced T-Cell Receptor therapies have demonstrated encouraging clinical results. As investigators begin to explore the treatment of solid tumors with these cells, the hurdle of evaluating T-cell homing to and persistence at the site of disease remain. Significant challenges regarding the GMP manufacture and administration of a therapeutic dose of millions to billions of transduced T-cells remain. Here we report on the application of a clinically authorized 19F MRI tracer agent to human T-cells, employing state-of-the-art methods and equipment in the manufacture of a cellular therapy. Using a general T-cell expansion protocol and clinical scale industrial bioreactors, we show 19F labeling without detriment to the product +/- cryopreservation. While the incorporation of the 19F tracer is not trivial, it is just one of the many steps that can aid in progression of a therapeutic to and though the clinic. Combining the MRI tracking capabilities, safety profiles, and clinical sensitivity of this method, this application demonstrates the ability of 19F MRI to be used in industrial scale applications to visualize the spatial fate of cellular therapeutics.

Fluorine-19 Labeling of Stromal Vascular Fraction Cells for Clinical Imaging Applications.

UNLABELLED: Stromal vascular fraction (SVF) cells are used clinically for various therapeutic targets. The location and persistence of engrafted SVF cells are important parameters for determining treatment failure versus success. We used the GID SVF-1 platform and a clinical protocol to harvest and label SVF cells with the fluorinated ((19)F) agent CS-1000 as part of a first-in-human phase I trial (clinicaltrials.gov identifier NCT02035085) to track SVF cells with magnetic resonance imaging during treatment of radiation-induced fibrosis in breast cancer patients. Flow cytometry revealed that SVF cells consisted of 25.0% ± 15.8% CD45+, 24.6% ± 12.5% CD34+, and 7.5% ± 3.3% CD31+ cells, with 2.1 ± 0.7 × 105 cells per cubic centimeter of adipose tissue obtained. Fluorescent CS-1000 (CS-ATM DM Green) labeled 87.0% ± 13.5% of CD34+ progenitor cells compared with 47.8% ± 18.5% of hematopoietic CD45+ cells, with an average of 2.8 ± 2.0 × 10¹² ¹9F atoms per cell, determined using nuclear magnetic resonance spectroscopy. The vast majority (92.7% ± 5.0%) of CD31+ cells were also labeled, although most coexpressed CD34. Only 16% ± 22.3% of CD45-/CD31-/CD34- (triple-negative) cells were labeled with CS-ATM DM Green. After induction of cell death by either apoptosis or necrosis, >95% of ¹9F was released from the cells, indicating that fluorine retention can be used as a surrogate marker for cell survival. Labeled-SVF cells engrafted in a silicone breast phantom could be visualized with a clinical 3-Tesla magnetic resonance imaging scanner at a sensitivity of approximately 2 × 106 cells at a depth of 5 mm. The current protocol can be used to image transplanted SVF cells at clinically relevant cell concentrations in patients. SIGNIFICANCE: Stromal vascular fraction (SVF) cells harvested from adipose tissue offer great promise in regenerative medicine, but methods to track such cell therapies are needed to ensure correct administration and monitor survival. A clinical protocol was developed to harvest and label SVF cells with the fluorinated (¹9F) agent CS-1000, allowing cells to be tracked with (19)F magnetic resonance imaging (MRI). Flow cytometry evaluation revealed heterogeneous ¹9F uptake in SVF cells, confirming the need for careful characterization. The proposed protocol resulted in sufficient ¹9F uptake to allow imaging using a clinical MRI scanner with point-of-care processing.

Clinical cell therapy imaging using a perfluorocarbon tracer and fluorine-19 MRI.

PURPOSE: Cellular therapeutics are emerging as a treatment option for a host of serious human diseases. To accelerate clinical translation, noninvasive imaging of cell grafts in clinical trials can potentially be used to assess the initial delivery and behavior of cells. METHODS: The use of a perfluorocarbon (PFC) tracer agent for clinical fluorine-19 ((19) F) MRI cell detection is described. This technology was used to detect immunotherapeutic dendritic cells (DCs) delivered to colorectal adenocarcinoma patients. Autologous DC vaccines were labeled with a PFC MRI agent ex vivo. Patients received DCs intradermally, and (19) F spin-density-weighted MRI at 3 Tesla (T) was used to observe cells. RESULTS: Spin-density-weighted (19) F images at the injection site displayed DCs as background-free "hot-spot" images. (19) F images were acquired in clinically relevant scan times (<10 min). Apparent DC numbers could be quantified in two patients from the (19) F hot-spots and were observed to decrease by ∼50% at injection site by 24 h. From 3T phantom studies, the sensitivity limit for DC detection is estimated to be on the order of ∼10(5) cells/voxel in this study. CONCLUSION: These results help to establish a clinically applicable means to track a broad range of cell types used in cell therapy.

A novel probe for the non-invasive detection of tumor-associated inflammation.

A novel dual-mode contrast agent was formulated through the addition of an optical near infrared (NIR) probe to a perfluorocarbon (PFC)-based 19F magnetic resonance imaging (MRI) agent, which labels inflammatory cells in situ. A single PFC-NIR imaging agent enables both a qualitative, rapid optical monitoring of an inflammatory state and a quantitative, detailed and tissue-depth independent magnetic resonance imaging (MRI). The feasibility of in vivo optical imaging of the inflammatory response was demonstrated in a subcutaneous murine breast carcinoma model. Ex vivo optical imaging was used to quantify the PFC-NIR signal in the tumor and organs, and results correlated well with quantitative 19F NMR analyses of intact tissues. 19F MRI was employed to construct a three-dimensional image of the cellular microenvironment at the tumor site. Flow cytometry of isolated tumor cells was used to identify the cellular localization of the PFC-NIR probe within the tumor microenvironment. Contrast is achieved through the labeling of host cells involved in the immune response, but not tumor cells. The major cellular reservoir of the imaging agent were tumor-infiltrating CD11b+ F4/80low Gr-1low cells, a cell subset sharing immunophenotypic features with myeloid-derived suppressor cells (MDSCs). These cells are recruited to sites of inflammation and are implicated in immune evasion and tumor progression. This PFC-NIR contrast agent coupled to non-invasive, quantitative imaging techniques could serve as a valuable tool for evaluating novel anticancer agents.

¹9F MRI tracer preserves in vitro and in vivo properties of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) have numerous therapeutic applications including immune reconstitution, enzyme replacement, regenerative medicine, and immunomodulation. The trafficking and persistence of these cells after administration is a fundamental question for future therapeutic applications of HSCs. Here, we describe the safe and efficacious labeling of human CD34(+) HSCs with a novel, self-delivering perfluorocarbon ¹9F magnetic resonance imaging (MRI) tracer, which has recently been authorized for use in a clinical trial to track therapeutic cells. While various imaging contrast agents have been used to track cellular therapeutics, the impact of this MRI tracer on HSC function has not previously been studied. Both human CD34(+) and murine bone marrow (BM) HSCs were effectively labeled with the MRI tracer, with only a slight reduction in viability, relative to mock-labeled cells. In a pilot study, ¹9F MRI enabled the rapid evaluation of HSC delivery/retention following administration into a rat thigh muscle, revealing the dispersal of HSCs after injection, but not after surgical implantation. To investigate effects on cell functionality, labeled and unlabeled human HSCs were tested in in vitro colony forming unit (CFU) assays, which resulted in equal numbers of total CFU as well as individual CFU types, indicating that labeling did not alter multipotency. Cobblestone assay forming cell precursor frequency was also unaffected, providing additional evidence that stem cell function was preserved after labeling. In vivo tests of multipotency and reconstitution studies in mice with murine BM containing labeled HSCs resulted in normal development of CFU in the spleen, compared to unlabeled cells, and reconstitution of both lymphoid and myeloid compartments. The lack of interference in these complex biological processes provides strong evidence that the function and therapeutic potential of the HSCs are likely maintained after labeling. These data support the safety and efficacy of the MRI tracer for clinical tracking of human stem cells.

Visualizing arthritic inflammation and therapeutic response by fluorine-19 magnetic resonance imaging (19F MRI).

BACKGROUND: Non-invasive imaging of inflammation to measure the progression of autoimmune diseases, such as rheumatoid arthritis (RA), and to monitor responses to therapy is critically needed. V-Sense, a perfluorocarbon (PFC) contrast agent that preferentially labels inflammatory cells, which are then recruited out of systemic circulation to sites of inflammation, enables detection by 19F MRI. With no 19F background in the host, detection is highly-specific and can act as a proxy biomarker of the degree of inflammation present. METHODS: Collagen-induced arthritis in rats, a model with many similarities to human RA, was used to study the ability of the PFC contrast agent to reveal the accumulation of inflammation over time using 19F MRI. Disease progression in the rat hind limbs was monitored by caliper measurements and 19F MRI on days 15, 22 and 29, including the height of clinically symptomatic disease. Naïve rats served as controls. The capacity of the PFC contrast agent and 19F MRI to assess the effectiveness of therapy was studied in a cohort of rats administered oral prednisolone on days 14 to 28. RESULTS: Quantification of 19F signal measured by MRI in affected limbs was linearly correlated with disease severity. In animals with progressive disease, increases in 19F signal reflected the ongoing recruitment of inflammatory cells to the site, while no increase in 19F signal was observed in animals receiving treatment which resulted in clinical resolution of disease. CONCLUSION: These results indicate that 19F MRI may be used to quantitatively and qualitatively evaluate longitudinal responses to a therapeutic regimen, while additionally revealing the recruitment of monocytic cells involved in the inflammatory process to the anatomical site. This study may support the use of 19F MRI to clinically quantify and monitor the severity of inflammation, and to assess the effectiveness of treatments in RA and other diseases with an inflammatory component.

Functional assessment of human dendritic cells labeled for in vivo (19)F magnetic resonance imaging cell tracking.

BACKGROUND AIMS: Dendritic cells (DC) are increasingly being used as cellular vaccines to treat cancer and infectious diseases. While there have been some promising results in early clinical trials using DC-based vaccines, the inability to visualize non-invasively the location, migration and fate of cells once adoptively transferred into patients is often cited as a limiting factor in the advancement of these therapies. A novel perflouropolyether (PFPE) tracer agent was used to label human DC ex vivo for the purpose of tracking the cells in vivo by (19)F magnetic resonance imaging (MRI). We provide an assessment of this technology and examine its impact on the health and function of the DC. METHODS: Monocyte-derived DC were labeled with PFPE and then assessed. Cell viability was determined by examining cell membrane integrity and mitochondrial lipid content. Immunostaining and flow cytometry were used to measure surface antigen expression of DC maturation markers. Functional tests included bioassays for interleukin (IL)-12p70 production, T-cell stimulatory function and chemotaxis. MRI efficacy was demonstrated by inoculation of PFPE-labeled human DC into NOD-SCID mice. RESULTS: DC were effectively labeled with PFPE without significant impact on cell viability, phenotype or function. The PFPE-labeled DC were clearly detected in vivo by (19)F MRI, with mature DC being shown to migrate selectively towards draining lymph node regions within 18 h. CONCLUSIONS: This study is the first application of PFPE cell labeling and MRI cell tracking using human immunotherapeutic cells. These techniques may have significant potential for tracking therapeutic cells in future clinical trials.