Randomized phase II trial of lymphodepletion plus adoptive cell transfer of tumor-infiltrating lymphocytes, with or without dendritic cell vaccination, in patients with metastatic melanoma.

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.

Glucuronoxylomannan in the Cryptococcus species capsule as a target for Chimeric Antigen Receptor T-cell therapy.

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.

Radiolabeling and Imaging of Adoptively Transferred Immune Cells by Positron Emission Tomography.

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.

The Emerging Role of Amino Acid PET in Neuro-Oncology.

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.

Caspase-3 Substrates for Noninvasive Pharmacodynamic Imaging of Apoptosis by PET/CT.

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.

Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells.

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.

Imaging of Sleeping Beauty-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography.

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.

Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection.

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.

Thermally induced apoptosis, necrosis, and heat shock protein expression in 3D culture.

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.

Imaging long-term fate of intramyocardially implanted mesenchymal stem cells in a porcine myocardial infarction model.

The long-term fate of stem cells after intramyocardial delivery is unknown. We used noninvasive, repetitive PET/CT imaging with [(18)F]FEAU to monitor the long-term (up to 5 months) spatial-temporal dynamics of MSCs retrovirally transduced with the sr39HSV1-tk gene (sr39HSV1-tk-MSC) and implanted intramyocardially in pigs with induced acute myocardial infarction. Repetitive [(18)F]FEAU PET/CT revealed a biphasic pattern of sr39HSV1-tk-MSC dynamics; cell proliferation peaked at 33-35 days after injection, in periinfarct regions and the major cardiac lymphatic vessels and lymph nodes. The sr39HSV1-tk-MSC-associated [(18)F]FEAU signals gradually decreased thereafter. Cardiac lymphography studies using PG-Gd-NIRF813 contrast for MRI and near-infrared fluorescence imaging showed rapid clearance of the contrast from the site of intramyocardial injection through the subepicardial lymphatic network into the lymphatic vessels and periaortic lymph nodes. Immunohistochemical analysis of cardiac tissue obtained at 35 and 150 days demonstrated several types of sr39HSV1-tk expressing cells, including fibro-myoblasts, lymphovascular cells, and microvascular and arterial endothelium. In summary, this study demonstrated the feasibility and sensitivity of [(18)F]FEAU PET/CT imaging for long-term, in-vivo monitoring (up to 5 months) of the fate of intramyocardially injected sr39HSV1-tk-MSC cells. Intramyocardially transplanted MSCs appear to integrate into the lymphatic endothelium and may help improve myocardial lymphatic system function after MI.

Molecular imaging of active mutant L858R EGF receptor (EGFR) kinase-expressing nonsmall cell lung carcinomas using PET/CT.

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).

Molecular-genetic PET imaging using an HSV1-tk mutant reporter gene with enhanced specificity to acycloguanosine nucleoside analogs.

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.

Fatty acid synthase inhibition results in a magnetic resonance-detectable drop in phosphocholine.

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.

Targeting SRC family kinases inhibits growth and lymph node metastases of prostate cancer in an orthotopic nude mouse model.

Aberrant expression and/or activity of members of the Src family of nonreceptor protein tyrosine kinases (SFK) are commonly observed in progressive stages of human tumors. In prostate cancer, two SFKs (Src and Lyn) have been specifically implicated in tumor growth and progression. However, there are no data in preclinical models demonstrating potential efficacy of Src inhibitors against prostate cancer growth and/or metastasis. In this study, we used the small molecule SFK/Abl kinase inhibitor dasatinib, currently in clinical trials for solid tumors, to examine in vitro and in vivo effects of inhibiting SFKs in prostate tumor cells. In vitro, dasatinib inhibits both Src and Lyn activity, resulting in decreased cellular proliferation, migration, and invasion. In orthotopic nude mouse models, dasatinib treatment effectively inhibits expression of activated SFKs, resulting in inhibition of both tumor growth and development of lymph node metastases in both androgen-sensitive and androgen-resistant tumors. In primary tumors, SFK inhibition leads to decreased cellular proliferation (determined by immunohistochemistry for proliferating cell nuclear antigen). In vitro, small interfering RNA (siRNA)-mediated inhibition of Lyn affects cellular proliferation; siRNA inhibition of Src affects primarily cellular migration. Therefore, we conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer and that Src and Lyn activities affect different cellular functions required for prostate tumor growth and progression.

Fusion of circulating blood cells with solid-organ tissue cells in clinical stem cell transplants: a potential therapeutic model?

AIM: In female patients who have undergone sex-mismatched peripheral blood stem cell transplantation, solid-organ tissue cells have been identified that carry the Y-chromosome. How genetic material from circulating cells is acquired by solid-organ tissue cells is debated. The purpose of this study was to provide clinical evidence for cell fusion between circulating cells and solid-organ tissue cells. MATERIAL & METHODS: The clinical model was a male-into-female blood stem cell transplantation setting using the Y-chromosome as a blood-derived cell marker and the patient's BCR/ABL fusion gene. Endometrial cells were chosen as the target cells because of their uniquely female genotype. RESULTS: The Y-chromosome and the BCR/ABL fusion gene were identified by fluorescence in situ hybridization and were colocalized with estrogen receptor-staining endometrial cells. Both donor-derived Y-chromosome and patient-derived fusion gene were identified in the same endometrial cells, thereby indicating cell fusion as the mechanism for genetic material transfer in a clinical setting. CONCLUSION: These findings contribute to our understanding of how blood-derived cells interact with solid-organ tissue cells.