ABSTRACT
Multiple myeloma is a prevalent and incurable disease, despite the development of new and effective drugs. The recent development of chimeric antigen receptor (CAR)-T cell therapy has shown impressive results in the treatment of patients with relapsed or refractory hematological B cell malignancies. In the recent years, B-cell maturation antigen (BCMA) has appeared as a promising antigen to target using a variety of immuno-therapy treatments including CART cells, for MM patients. To this end, we generated clinical-grade murine CART cells directed against BCMA, named ARI2m cells. Having demonstrated its efficacy, and in an attempt to avoid the immune rejection of CART cells by the patient, the single chain variable fragment was humanized, creating ARI2h cells. ARI2h cells demonstrated comparable in vitro and in vivo efficacy to ARI2m cells, and superiority in cases of high tumor burden disease. In terms of inflammatory response, ARI2h cells showed a lower TNFα production and lower in vivo toxicity profile. Large-scale expansion of both ARI2m and ARI2h cells was efficiently conducted following Good Manufacturing Practice guidelines, obtaining the target CART cell dose required for treatment of multiple myeloma patients. Moreover, we demonstrate that soluble BCMA and BCMA released in vesicles impacts on CAR-BCMA activity. In summary, this study sets the bases for the implementation of a clinical trial (EudraCT code: 2019-001472-11) to study the efficacy of ARI2h cell treatment for multiple myeloma patients.
Subject(s)
Multiple Myeloma , Receptors, Chimeric Antigen , Animals , B-Cell Maturation Antigen , Humans , Immunotherapy, Adoptive , Mice , Multiple Myeloma/therapy , Receptors, Chimeric Antigen/genetics , T-LymphocytesABSTRACT
BACKGROUND AIMS: The genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: Cryptococcus gattii and Cryptococcus neoformans. To establish invasive human disease, inhaled cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infection's mortality rate ranges from 20% to 70%. Many HIV+/AIDS patients are affected by Cryptococcus infections, with 220,000 cases of cryptococcal meningitis reported worldwide in this population every year (C. neoformans infection statistics, via the Centers for Disease Control and Prevention, https://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/statistics.html). To escape from host immune cell attack, Cryptococcus covers itself in a sugar-based capsule composed primarily of glucuronoxylomannan (GXM). To evade phagocytosis, yeast cells increase to a >45-µm perimeter and become titan, or giant, cells. Cryptococci virulence is directly proportional to the percentage of titan/giant cells present during Cryptococcus infection. To combat cryptococcosis, the authors propose the redirection of CD8+ T cells to target the GXM in the capsule via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR). RESULTS: GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk in the extracellular domain, a CD28 transmembrane domain and CD28 and CD3-ς signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR+ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined with the use of brightfield microscopy. Large clusters of proliferating GXMR-CAR+ T cells were observed in GXM-treated cells, whereas no clusters were observed in control cells. Moreover, the interaction of GXM with GXMR-CAR+ T cells was detected via flow cytometry by using a GXM-specific antibody, and the recognition of GXM by GXMR-CAR T cells triggered the secretion of granzyme and interferon gamma (IFN-γ). The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by fluorescent microscopy, but no binding was detected in mock-transduced control T cells (NoDNA T cells). Moreover, lung tissue sections were stained with Gomori Methenamine Silver and evaluated by NanoZoomer (Hamamatsu), revealing a significantly lower number of titan cells, with perimeters ranging from 50 to 130 µm and giant cells >130 µm in the CAR T-cell treated group when compared with other groups. Therefore, the authors validated the study's hypothesis by the redirection of GXMR-CAR+ T cells to target GXM, which induces the secretion of cytotoxic granules and IFN-γ that will aid in the control of cryptococcosis CONCLUSIONS: Thus, these findings reveal that GXMR-CAR+ T cells can target C. neoformans. Future studies will be focused on determining the therapeutic efficacy of GXMR-CAR+ T cells in an animal model of cryptococcosis.
Subject(s)
Cryptococcus neoformans , Polysaccharides , Receptors, Chimeric Antigen , Animals , CD8-Positive T-Lymphocytes , Cell- and Tissue-Based Therapy , HumansABSTRACT
Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.
Subject(s)
Interleukin-15/metabolism , Neoplasms, Experimental/therapy , Receptors, Antigen, T-Cell/metabolism , T-Lymphocyte Subsets/physiology , Animals , Antigens, CD19/metabolism , Humans , Immunotherapy, Adoptive , Lymphocyte Activation , Mice , Precursor Cells, T-Lymphoid/physiology , Recombinant Fusion Proteins/metabolism , Signal TransductionABSTRACT
Quantitative imaging of apoptosis in vivo could enable real-time monitoring of acute cell death pathologies such as traumatic brain injury, as well as the efficacy and safety of cancer therapy. Here, we describe the development and validation of F-18-labeled caspase-3 substrates for PET/CT imaging of apoptosis. Preliminary studies identified the O-benzylthreonine-containing substrate 2MP-TbD-AFC as a highly caspase 3-selective and cell-permeable fluorescent reporter. This lead compound was converted into the radiotracer [18F]-TBD, which was obtained at 10% decay-corrected yields with molar activities up to 149 GBq/µmol on an automated radiosynthesis platform. [18F]-TBD accumulated in ovarian cancer cells in a caspase- and cisplatin-dependent fashion. PET imaging of a Jo2-induced hepatotoxicity model showed a significant increase in [18F]-TBD signal in the livers of Jo2-treated mice compared to controls, driven through a reduction in hepatobiliary clearance. A chemical control tracer that could not be cleaved by caspase 3 showed no change in liver accumulation after induction of hepatocyte apoptosis. Our data demonstrate that [18F]-TBD provides an immediate pharmacodynamic readout of liver apoptosis in mice by dynamic PET/CT and suggest that [18F]-TBD could be used to interrogate apoptosis in other disease states.
Subject(s)
Apoptosis , Caspase 3/metabolism , Positron Emission Tomography Computed Tomography/methods , Animals , Cell Line, Tumor , Female , Mice , Mice, Nude , Substrate SpecificityABSTRACT
Adoptive therapy with regulatory T cells (Tregs) to prevent graft-versus-host disease (GVHD) would benefit from a strategy to improve homing to the sites of inflammation. We hypothesized that adding fucose to human Tregs, forming the Sialyl Lewis X moiety on P-selectin glycoprotein ligand-1, would improve their trafficking pattern. The selectin pathway recruiter, α-1,3-fucosyltransferase-VI enzyme, significantly increased Treg surface fucosylation (66% vs 8%). In a xenogenic GVHD mouse model, fucosylated Tregs showed prolonged periods of in vivo persistence. When given at a lower dose compared with the untreated Tregs, the murine recipients of fucosylated Tregs maintained weight, had ameliorated clinical GVHD, and improved survival (70% vs 30%; P < .0001). These preclinical data indicate that fucosylated human Tregs is an effective strategy for prevention of GVHD and, as such, warrants consideration for future clinical trials.
Subject(s)
Disease Models, Animal , Fucose/metabolism , Graft vs Host Disease/immunology , Graft vs Host Disease/prevention & control , T-Lymphocytes, Regulatory/immunology , Animals , Blotting, Western , Cells, Cultured , E-Selectin/metabolism , Female , Fetal Blood/cytology , Fetal Blood/immunology , Fetal Blood/metabolism , Flow Cytometry , Fucosyltransferases/metabolism , Graft vs Host Disease/mortality , Humans , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Oligosaccharides/metabolism , Sialyl Lewis X AntigenABSTRACT
Clinical-grade T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) to redirect their specificity to target tumor-associated antigens in vivo. We now have developed this molecular strategy to render cytotoxic T cells specific for fungi. We adapted the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-ζ (designated "D-CAR") upon binding with carbohydrate in the cell wall of Aspergillus germlings. T cells genetically modified with the Sleeping Beauty system to express D-CAR stably were propagated selectively on artificial activating and propagating cells using an approach similar to that approved by the Food and Drug Administration for manufacturing CD19-specific CAR(+) T cells for clinical trials. The D-CAR(+) T cells exhibited specificity for ß-glucan which led to damage and inhibition of hyphal growth of Aspergillus in vitro and in vivo. Treatment of D-CAR(+) T cells with steroids did not compromise antifungal activity significantly. These data support the targeting of carbohydrate antigens by CAR(+) T cells and provide a clinically appealing strategy to enhance immunity for opportunistic fungal infections using T-cell gene therapy.
Subject(s)
Aspergillosis/immunology , Aspergillosis/therapy , Bioengineering/methods , Carbohydrates/antagonists & inhibitors , Opportunistic Infections/immunology , Opportunistic Infections/therapy , T-Lymphocytes/immunology , Animals , Antigens, CD19/metabolism , Aspergillosis/microbiology , Aspergillosis/pathology , Aspergillus/drug effects , Aspergillus/physiology , Dexamethasone/pharmacology , Humans , Hyphae/drug effects , Hyphae/physiology , Immunophenotyping , Lectins, C-Type/metabolism , Lymphocyte Activation/drug effects , Mice , Opportunistic Infections/pathology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/drug effectsABSTRACT
BACKGROUND AIMS: Naturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graft-versus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1-5% of total nucleated cells in cord blood (CB) (<3 × 106 cells), and therefore novel methods of Treg expansion to generate clinically relevant numbers are needed. METHODS: Several methodologies are currently being used for ex vivo Treg expansion. We report a new approach to expand Treg from CB and demonstrate their efficacy in vitro by blunting allogeneic mixed lymphocyte reactions and in vivo by preventing GvHD through the use of a xenogenic GvHD mouse model. RESULTS: With the use of magnetic cell sorting, naturally occurring Treg were isolated from CB by the positive selection of CD25⺠cells. These were expanded to clinically relevant numbers by use of CD3/28 co-expressing Dynabeads and interleukin (IL)-2. Ex vivo-expanded Treg were CD4âº25⺠FOXP3âº127(lo) and expressed a polyclonal T-cell receptor, Vß repertoire. When compared with conventional T-lymphocytes (CD4âº25â» cells), Treg consistently showed demethylation of the FOXP3 TSDR promoter region and suppression of allogeneic proliferation responses in vitro. CONCLUSIONS: In our NOD-SCID IL-2Rγ(null) xenogeneic model of GvHD, prophylactic injection of third-party, CB-derived, ex vivo-expanded Treg led to the prevention of GvHD that translated into improved GvHD score, decreased circulating inflammatory cytokines and significantly superior overall survival. This model of xenogenic GvHD can be used to study the mechanism of action of CB Treg as well as other therapeutic interventions.
Subject(s)
Fetal Blood/transplantation , Graft vs Host Disease/therapy , T-Lymphocytes, Regulatory/cytology , Transplantation, Homologous/adverse effects , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/transplantation , Cell Proliferation , Cell- and Tissue-Based Therapy , Fetal Blood/cytology , Graft vs Host Disease/pathology , Hematopoietic Stem Cell Transplantation/adverse effects , Humans , Mice , T-Lymphocytes, Regulatory/transplantationABSTRACT
BACKGROUND AIMS: Advantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo. METHODS: CD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment. RESULTS: Both FTVI and FTVII fucosylated CB CD34⺠cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34⺠cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes. CONCLUSIONS: Although FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34⺠cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation.
Subject(s)
Fetal Blood/drug effects , Fucosyltransferases/administration & dosage , Graft vs Host Disease/therapy , Animals , Disease Models, Animal , Fetal Blood/cytology , Fetal Blood/transplantation , Graft vs Host Disease/pathology , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/drug effects , Humans , MiceABSTRACT
This study was conducted to compare the heat shock responses of cells grown in 2D and 3D culture environments as indicated by the level of heat shock protein 70 expression and the incidence of apoptosis and necrosis of prostate cancer cell lines in response to graded hyperthermia. PC3 cells were stably transduced with a dual reporter system composed of two tandem expression cassettes-a conditional heat shock protein promoter driving the expression of green fluorescent protein (HSPp-GFP) and a cytomegalovirus (CMV) promoter controlling the constitutive expression of a "beacon" red fluorescent protein (CMVp-RFP). Two-dimensional and three-dimensional cultures of PC3 prostate cancer cells were grown in 96-well plates for evaluation of their time-dependent response to supraphysiological temperature. To induce controlled hyperthermia, culture plates were placed on a flat copper surface of a circulating water manifold that maintained the specimens within ±0.1°C of a target temperature. Hyperthermia protocols included various combinations of temperature, ranging from 37°C to 57°C, and exposure times of up to 2 h. The majority of protocols were focused on temperature and time permutations, where the response gradient was greatest. Post-treatment analysis by flow cytometry analysis was used to measure the incidences of apoptosis (annexin V-FITC stain), necrosis (propidium iodide (PI) stain), and HSP70 transcription (GFP expression). Cells grown in 3D compared with 2D culture showed reduced incidence of apoptosis and necrosis and a higher level of HSP70 expression in response to heat shock at the temperatures tested. Cells responded differently to hyperthermia when grown in 2D and 3D cultures. Three-dimensional culture appears to enhance survival plausibly by activating protective processes related to enhanced-HSP70 expression. These differences highlight the importance of selecting physiologically relevant 3D models in assessing cellular responses to hyperthermia in experimental settings.
Subject(s)
Apoptosis , Cell Culture Techniques/methods , HSP70 Heat-Shock Proteins/genetics , Necrosis , Caspases/metabolism , Cell Line, Tumor , Cell Survival , Gene Expression , Heat-Shock Response , HumansABSTRACT
The importance of the EGF receptor (EGFR) signaling pathway in the development and progression of nonsmall cell lung carcinomas (NSCLC) is widely recognized. Gene sequencing studies revealed that a majority of tumors responding to EGFR kinase inhibitors harbor activating mutations in the EGFR kinase domain. This underscores the need for novel biomarkers and diagnostic imaging approaches to identify patients who may benefit from particular therapeutic agents and approaches with improved efficacy and safety profiles. To this goal, we developed 4-[(3-iodophenyl)amino]-7-{2-[2-{2-(2-[2-{2-([(18)F]fluoroethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}-ethoxy]-quinazoline-6-yl-acrylamide ([(18)F]F-PEG6-IPQA), a radiotracer with increased selectivity and irreversible binding to the active mutant L858R EGFR kinase. We show that PET with [(18)F]F-PEG6-IPQA in tumor-bearing mice discriminates H3255 NSCLC xenografts expressing L858R mutant EGFR from H441 and PC14 xenografts expressing EGFR or H1975 xenografts with L858R/T790M dual mutation in EGFR kinase domain, which confers resistance to EGFR inhibitors (i.e., gefitinib). The T790M mutation precludes the [(18)F]F-PEG6-IPQA from irreversible binding to EGFR. These results suggest that PET with [(18)F]F-PEG6-IPQA could be used for the selection of NSCLC patients for individualized therapy with small molecular inhibitors of EGFR kinase that are currently used in the clinic and have a similar structure (i.e., iressa, gefitinib, and erlotinib).
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , ErbB Receptors/metabolism , Lung Neoplasms/metabolism , Molecular Imaging/methods , Mutant Proteins/metabolism , Amino Acid Substitution , Animals , Binding, Competitive , Blotting, Western , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , ErbB Receptors/genetics , Fluorodeoxyglucose F18/metabolism , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Mice , Mice, Nude , Mutant Proteins/genetics , Mutation , Positron-Emission Tomography/methods , Protein Binding , Protein Kinase Inhibitors/pharmacology , Quinazolines/metabolism , Radiopharmaceuticals/metabolism , Tomography, X-Ray Computed/methods , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Chimeric antigen receptor (CAR)-T cell immunotherapy has modified the concept of treatment in hematological malignancies. In comparison with pediatric patients, where responses are maintained over many years, older patients, such as those with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), present lower persistence of CAR-T cells that might be due to decreased fitness of T cells acquired with aging. Moreover, cord blood derived-NK cells (CB-NKs) and CAR-NK cells derived from CB-NK can be used 'off-the-shelf' as immune cells with antitumor properties for the treatment of cancer patients. However, to date, clinical studies have only demonstrated the safety of these therapies but not optimal efficacy. To confront the shortcomings of each therapy, we devised a novel approach consisting of simultaneous (CAR-)NK cell and CAR-T cell administration. In this setting, NK cells demonstrate an important immunoregulation of T cells that could be exploited to enhance the efficacy of CAR-T cells. METHODS: A combinatorial treatment based on either CAR-T and CAR-NK cells or CB-NK and CAR-T cells in two models of NHL and MM was performed. Antitumor efficacy was analyzed in vitro and in vivo, and parameters related to early activation, exhaustion and senescence of T cells were analyzed. RESULTS: We show that CAR-NK cells derived from CB-NK are only effective at high doses (high E:T ratio) and that their activity rapidly decreases over time in comparison with CAR-T cells. In comparison and to exploit the potential of 'off-the-shelf' CB-NK, we demonstrate that a low number of CB-NK in the CAR-T cell product promotes an early activation of CAR-T cells and their migration to MM cells leading to enhanced anti-MM efficacy. Moreover, cytokines related to CRS development were not increased, and importantly, CB-NK enhanced the fitness of both CARpos and CARneg T cells, promoting lower levels of exhaustion and senescence. CONCLUSION: This study demonstrates a relevant immunoregulatory role of CB-NK collaborating with CAR-T cells to enhance their antitumor activity. A novel and different approach to consider in CAR-T cell immunotherapy studies is presented here with the goal to enhance the efficacy of the treatment.
Subject(s)
Hematologic Neoplasms/therapy , Immunotherapy, Adoptive/methods , Killer Cells, Natural/immunology , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , Animals , Cell Line, Tumor , Hematologic Neoplasms/immunology , Heterografts , Humans , K562 Cells , Killer Cells, Natural/transplantation , Mice , T-Lymphocytes/transplantationABSTRACT
BACKGROUND: The adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated robust efficacy in metastatic melanoma patients. Tumor antigen-loaded dendritic cells (DCs) are believed to optimally activate antigen-specific T lymphocytes. We hypothesized that the combined transfer of TIL, containing a melanoma antigen recognized by T cells 1 (MART-1) specific population, with MART-1-pulsed DC will result in enhanced proliferation and prolonged survival of transferred MART-1 specific T cells in vivo ultimately leading to improved clinical responses. DESIGN: We tested the combination of TIL and DC in a phase II clinical trial of patients with advanced stage IV melanoma. HLA-A0201 patients whose early TIL cultures demonstrated reactivity to MART-1 peptide were randomly assigned to receive TIL alone or TIL +DC pulsed with MART-1 peptide. The primary endpoint was to evaluate the persistence of MART-1 TIL in the two arms. Secondary endpoints were to evaluate clinical response and survival. RESULTS: Ten patients were given TIL alone while eight patients received TIL+DC vaccine. Infused MART-1 reactive CD8+ TIL were tracked in the blood over time by flow cytometry and results show good persistence in both arms, with no difference in the persistence of MART-1 between the two arms. The objective response rate was 30% (3/10) in the TIL arm and 50% (4/8) in the TIL+DC arm. All treatments were well tolerated. CONCLUSIONS: The combination of TIL +DC showed no difference in the persistence of MART-1 TIL compared with TIL therapy alone. Although more patients showed a clinical response to TIL+DC therapy, this study was not powered to resolve differences between groups. TRIAL REGISTRATION NUMBER: NCT00338377.
Subject(s)
Cancer Vaccines/therapeutic use , Dendritic Cells/transplantation , Immunotherapy, Adoptive , Lymphocyte Depletion , Lymphocytes, Tumor-Infiltrating/transplantation , Melanoma/therapy , Skin Neoplasms/therapy , T-Lymphocytes/transplantation , Adolescent , Adult , Cancer Vaccines/adverse effects , Combined Modality Therapy , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Humans , Immunotherapy, Adoptive/adverse effects , Lymphocyte Depletion/adverse effects , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , MART-1 Antigen/immunology , MART-1 Antigen/metabolism , Male , Melanoma/immunology , Melanoma/metabolism , Melanoma/secondary , Middle Aged , Neoplasm Staging , Skin Neoplasms/immunology , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Time Factors , Treatment Outcome , Young AdultABSTRACT
The use of umbilical cord blood (UCB) grafts for hematopoietic stem cell transplantation (HSCT) is a promising technique that permits a degree of human leukocyte antigen mismatch between the graft and the host without the concomitant higher rate of graft-versus-host disease that would be observed between an adult marrow graft and a mismatched host. A disadvantage to the use of UCB for HSCT is that immune reconstitution may be significantly delayed because of the low stem cell dose available in the graft. Ex vivo expansion of UCB CD34 cells would provide a greater number of stem cells; however, there are persistent concerns that ex vivo-expanded CD34 cells may lose pluripotency and the ability to contribute meaningfully to long-term engraftment. To address this issue, we transduced CD34-selected UCB cells with a lentiviral construct expressing luciferase, and determined homing and engraftment patterns in vivo by noninvasive bioluminescent imaging in sublethally irradiated NOD/SCID/IL-2Rgamma(-/-) (NSG) mice. Graft contribution to multilineage commitment was also confirmed by analysis of primary and secondary transplants by flow cytometry and immunohistochemistry. Our results demonstrate that, other than a mild delay at the onset of engraftment, there were no significant differences in lineage repopulation or in long-term or secondary engraftment between culture-expanded and unexpanded UCB CD34-selected cells. The results suggest that multipotent stem cells can be expanded ex vivo and can contribute meaningfully to long-term hematopoietic engraftment.
Subject(s)
Antigens, CD34/analysis , Cord Blood Stem Cell Transplantation/methods , Fetal Blood/cytology , Mesenchymal Stem Cells/cytology , Animals , Cell Lineage , Flow Cytometry , Humans , Immunohistochemistry , Luciferases, Firefly/chemistry , Luminescent Agents/chemistry , Luminescent Measurements/methods , Mice , Mice, Inbred NOD , Mice, SCID , Transduction, GeneticABSTRACT
Positron emission tomography (PET) using 89Zr is a clinically relevant imaging modality that enables long-term monitoring of adoptively transferred immune cells. This article describes a two-step radiometal labeling procedure utilizing the bifunctional siderophore p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS) that chelates 89Zr with high affinity and binds covalently to primary amines of cell-surface proteins via its isothiocyanate moiety. Cells labeled with 89Zr-DFO-Bz-NCS remain viable and retain the radiolabel, enabling repetitive PET imaging of adoptively transferred immune cells with high sensitivity and specificity for up to 2 weeks.
Subject(s)
Adoptive Transfer/methods , Isotope Labeling , Positron-Emission Tomography , Radiopharmaceuticals/chemistry , Animals , Cell Survival , Deferoxamine/chemistry , MiceABSTRACT
UNLABELLED: Imaging 2 different molecular-genetic events in a single subject by PET is essential in a variety of in vivo applications. Using herpes simplex virus-1 thymidine kinase (HSV1-tk) mutants with narrower substrate specificities in combination with wild-type HSV1-tk (wtHSV1-tk) would enable differential imaging with corresponding radiotracers, namely 2'-deoxy-2'-(18)F-fluoro-5-ethyl-1-beta-d-arabinofuranosyl-uracil ((18)F-FEAU) and the acycloguanosine derivative 9-(4-(18)F-fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG). In this study, we evaluated wtHSV1-tk and the A168H mutant, which has been reported to exhibit enhanced acycloguanosine substrate catalytic activity and diminished pyrimidine phosphorylating activity, as PET reporter genes. METHODS: Computational analysis was performed to assess the binding mode of FHBG and FEAU to wtHSV1-tk and the A168H variant. U87 cells were stably transduced with wtHSV1-tk or HSV1-tk(A168H) fused with green fluorescent protein and sorted to obtain equivalent transgene expression. In vitro uptake studies were performed to determine rates of substrate accumulation and retention. Nude mice bearing tumors expressing HSV1-tk variants were subsequently imaged using (18)F-FHBG and (18)F-FEAU. RESULTS: Docking results indicate that binding of FHBG to the A168H variant is unaffected whereas the binding of FEAU is hindered because of a steric clash with the bulkier mutant residues. U87 cells expressing HSV1-tk(A168H) accumulated (18)F-FHBG in in vitro uptake studies at a 3-fold higher rate than did cells expressing wtHSV1-tk without any detectable accumulation of (3)H-FEAU. Furthermore, HSV1-tk(A168H) demonstrated no thymidine phosphorylation activity. In contrast, U87 cells expressing wtHSV1-tk preferentially accumulated (3)H-FEAU at an 18-fold higher rate than they did (18)F-FHBG. Tumors expressing wtHSV1-tk or HSV1-tk(A168H) were distinctly imaged with (18)F-FEAU or (18)F-FHBG, respectively. Hence, tumors expressing HSV1-tk(A168H) accumulated 8.4-fold more (18)F-FHBG than did tumors expressing wtHSV1-tk. In addition, wtHSV1-tk tumors, compared with HSV1-tk(A168H)-expressing tumors (which retained baseline levels of the radiotracer), preferentially accumulated (18)F-FEAU. CONCLUSION: The FEAU and FHBG substrate discrimination capacity of the wtHSV1-tk and HSV1-tk(A168H) reporter enzymes was validated in vivo by PET of mice with tumor xenografts established from U87 cells expressing these different reporters. Thus, HSV1-tk(A168H) may potentially be used as a second reporter gene in combination with wtHSV1-tk to achieve differential PET.
Subject(s)
Arabinofuranosyluracil/analogs & derivatives , Fluorouracil/analogs & derivatives , Guanine/analogs & derivatives , Herpesvirus 1, Human/enzymology , Radiopharmaceuticals , Thymidine Kinase/metabolism , Animals , Arabinofuranosyluracil/chemistry , Cell Line, Tumor , Feasibility Studies , Fluorine Radioisotopes , Fluorouracil/chemistry , Genes, Reporter , Guanine/chemistry , Humans , Mice , Mice, Nude , Models, Molecular , Mutation , Neoplasm Transplantation , Positron-Emission Tomography , Protein Binding , Radiopharmaceuticals/chemistry , Structure-Activity Relationship , Thymidine Kinase/chemistry , Thymidine Kinase/genetics , Transplantation, HeterologousABSTRACT
Expression of fatty acid synthase (FASN), the key enzyme in de novo synthesis of long-chain fatty acids, is normally low but increases in cancer. Consequently, FASN is a novel target for cancer therapy. However, because FASN inhibitors can lead to tumor stasis rather than shrinkage, noninvasive methods for assessing FASN inhibition are needed. To this end, we combined (1)H, (31)P, and (13)C magnetic resonance spectroscopy (MRS) (a) to monitor the metabolic consequences of FASN inhibition and (b) to identify MRS-detectable metabolic biomarkers of response. Treatment of PC-3 cells with the FASN inhibitor Orlistat for up to 48 h resulted in inhibition of FASN activity by 70%, correlating with 74% inhibition of fatty acid synthesis. Furthermore, we have determined that FASN inhibition results not only in lower phosphatidylcholine levels but also in a 59% drop in the phospholipid precursor phosphocholine (PCho). This drop resulted from inhibition in PCho synthesis as a result of a reduction in the cellular activity of its synthetic enzyme choline kinase. The drop in PCho levels following FASN inhibition was confirmed in SKOV-3 ovarian cancer cells treated with Orlistat and in MCF-7 breast cancer cells treated with Orlistat as well as cerulenin. Combining data from all treated cells, the drop in PCho significantly correlated with the drop in de novo synthesized fatty acid levels, identifying PCho as a potential noninvasive MRS-detectable biomarker of FASN inhibition in vivo.
Subject(s)
Enzyme Inhibitors/pharmacology , Fatty Acid Synthases/antagonists & inhibitors , Phosphorylcholine/metabolism , Apoptosis , Base Sequence , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Primers , Humans , Lactones/pharmacology , Magnetic Resonance Spectroscopy , OrlistatABSTRACT
Imaging plays a critical role in the management of the highly complex and widely diverse central nervous system (CNS) malignancies in providing an accurate diagnosis, treatment planning, response assessment, prognosis, and surveillance. Contrast-enhanced magnetic resonance imaging (MRI) is the primary modality for CNS disease management due to its high contrast resolution, reasonable spatial resolution, and relatively low cost and risk. However, defining tumor response to radiation treatment and chemotherapy by contrast-enhanced MRI is often difficult due to various factors that can influence contrast agent distribution and perfusion, such as edema, necrosis, vascular alterations, and inflammation, leading to pseudoprogression and pseudoresponse assessments. Amino acid positron emission tomography (PET) is emerging as the method of resolving such equivocal lesion interpretations. Amino acid radiotracers can more specifically differentiate true tumor boundaries from equivocal lesions based on their specific and active uptake by the highly metabolic cellular component of CNS tumors. These therapy-induced metabolic changes detected by amino acid PET facilitate early treatment response assessments. Integrating amino acid PET in the management of CNS malignancies to complement MRI will significantly improve early therapy response assessment, treatment planning, and clinical trial design.
ABSTRACT
PURPOSE: We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells. PROCEDURES: We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19+ artificial antigen-presenting cells. RESULTS: After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19+ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2'-deoxy-2'-[18F]fluoro-5-ethyl-1-ß-D-arabinofuranosyl-uracil ([18F]FEAU). CONCLUSION: This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.
Subject(s)
Herpesvirus 1, Human/enzymology , Positron-Emission Tomography/methods , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , Thymidine Kinase/metabolism , Transposases/metabolism , Animals , Arabinofuranosyluracil/analogs & derivatives , Arabinofuranosyluracil/chemistry , Cell Line , Ganciclovir/pharmacology , Gene Transfer Techniques , Humans , Luciferases/metabolism , Mice , Radiopharmaceuticals/chemistry , Transgenes , XenopusABSTRACT
Mismatch of human leukocyte antigens (HLA) adversely impacts the outcome of patients after allogeneic hematopoietic stem-cell transplantation (alloHSCT). This translates into the clinical requirement to timely identify suitable HLA-matched donors which in turn curtails the chances of recipients, especially those from a racial minority, to successfully undergo alloHSCT. We thus sought to broaden the existing pool of registered unrelated donors based on analysis that eliminating the expression of the HLA-A increases the chance for finding a donor matched at HLA-B, -C, and -DRB1 regardless of a patient's race. Elimination of HLA-A expression in HSC was achieved using artificial zinc finger nucleases designed to target HLA-A alleles. Significantly, these engineered HSCs maintain their ability to engraft and reconstitute hematopoiesis in immunocompromised mice. This introduced loss of HLA-A expression decreases the need to recruit large number of donors to match with potential recipients and has particular importance for patients whose HLA repertoire is under-represented in the current donor pool. Furthermore, the genetic engineering of stem cells provides a translational approach to HLA-match a limited number of third-party donors with a wide number of recipients.
Subject(s)
Deoxyribonucleases/genetics , Gene Deletion , HLA-A Antigens/genetics , Hematopoietic Stem Cell Transplantation/ethnology , Hematopoietic Stem Cells/immunology , Alleles , Animals , Deoxyribonucleases/metabolism , Donor Selection/ethics , Gene Expression , Genetic Engineering/methods , HLA-A Antigens/immunology , HLA-B Antigens/genetics , HLA-B Antigens/immunology , HLA-C Antigens/genetics , HLA-C Antigens/immunology , HLA-DRB1 Chains/genetics , HLA-DRB1 Chains/immunology , Health Services Accessibility/ethics , Hematopoietic Stem Cell Transplantation/ethics , Hematopoietic Stem Cells/cytology , Histocompatibility Testing , Humans , Mice , Racial Groups , Transplantation, Heterologous , Transplantation, Homologous , Unrelated Donors , Zinc FingersABSTRACT
Many tumors overexpress tumor-associated antigens relative to normal tissue, such as EGFR. This limits targeting by human T cells modified to express chimeric antigen receptors (CAR) due to potential for deleterious recognition of normal cells. We sought to generate CAR(+) T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies that differ in affinity. T cells with low-affinity nimotuzumab-CAR selectively targeted cells overexpressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high-affinity cetuximab-CAR was not affected by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from nonmalignant cells.