Larissa Shenker

Monitoring the Efficacy of Adoptively Transferred Prostate Cancer–Targeted Human T Lymphocytes with PET and Bioluminescence Imaging

Noninvasive imaging technologies have the potential to enhance the monitoring and improvement of adoptive therapy with tumor-targeted T lymphocytes. We established an imaging methodology for the assessment of spatial and temporal distributions of adoptively transferred genetically modified human T cells in vivo for treatment monitoring and prediction of tumor response in a systemic prostate cancer model. Methods: RM1 murine prostate carcinoma tumors transduced with human prostate-specific membrane antigen (hPSMA) and a Renilla luciferase reporter gene were established in SCID/beige mice. Human T lymphocytes were transduced with chimeric antigen receptors (CAR) specific for either hPSMA or human carcinoembryonic antigen (hCEA) and with a fusion reporter gene for herpes simplex virus type 1 thymidine kinase (HSV1tk) and green fluorescent protein, with or without click beetle red luciferase. The localization of adoptively transferred T cells in tumor-bearing mice was monitored with 2′-18F-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-ethyluracil (18F-FEAU) small-animal PET and bioluminescence imaging (BLI). Results: Cotransduction of CAR-expressing T cells with the reporter gene did not affect CAR-mediated cytotoxicity. BLI of Renilla and click beetle red luciferase expression enabled concurrent imaging of adoptively transferred T cells and systemic tumors in the same animal. hPSMA-specific T lymphocytes persisted longer than control hCEA-targeted T cells in lung hPSMA-positive tumors, as indicated by both PET and BLI. Precise quantification of T-cell distributions at tumor sites by PET revealed that delayed tumor progression was positively correlated with the levels of 18F-FEAU accumulation in tumor foci in treated animals. Conclusion: Quantitative noninvasive monitoring of genetically engineered human T lymphocytes by PET provides spatial and temporal information on T-cell trafficking and persistence. PET may be useful for predicting tumor response and for guiding adoptive T-cell therapy.

Targeting the Internal Epitope of Prostate-Specific Membrane Antigen with 89Zr-7E11 Immuno-PET

The potential of the positron-emitting 89Zr has been recently investigated for the design of radioimmunoconjugates for immuno-PET. In this study, we report the preparation and in vivo evaluation of 89Zr-desferrioxamine B (DFO)-7E11, a novel 89Zr-labeled monoclonal antibody (mAb) construct for targeted imaging of prostate-specific membrane antigen (PSMA), a prototypical cell surface marker highly overexpressed in prostate cancer. The ability of 89Zr-DFO-7E11 to delineate tumor response to therapy was also investigated, because it binds to the intracellular epitope of PSMA, which becomes available only on membrane disruption in dead or dying cells. Methods: 7E11 as a marker of dying cells was studied by flow cytometry and microscopy of cells after antiandrogen-, radio-, and chemotherapy in LNCaP and PC3 PSMA–positive cells. The in vivo behavior of 89Zr-DFO-7E11 was characterized in mice bearing subcutaneous LNCaP (PSMA-positive) tumors by biodistribution studies and immuno-PET. The potential of assessing tumor response was evaluated in vivo after radiotherapy. Results: In vitro studies correlated 7E11 binding with markers of apoptosis (7-amino-actinomycin-D and caspase-3). In vivo biodistribution experiments revealed high, target-specific uptake of 89Zr-DFO-7E11 in LNCaP tumors after 24 h (20.35 ± 7.50 percentage injected dose per gram [%ID/g]), 48 h (22.82 ± 3.58 %ID/g), 96 h (36.94 ± 6.27 %ID/g), and 120 h (25.23 ± 4.82 %ID/g). Excellent image contrast was observed with immuno-PET. 7E11 uptake was statistically increased in irradiated versus control tumor as measured by immuno-PET and biodistribution studies. Binding specificity was assessed by effective blocking studies at 48 h. Conclusion: These findings suggest that 89Zr-DFO-7E11 displays high tumor–to–background tissue contrast in immuno-PET and can be used as a tool to monitor and quantify, with high specificity, tumor response in PSMA-positive prostate cancer.

A New Pyrimidine-Specific Reporter Gene: A Mutated Human Deoxycytidine Kinase Suitable for PET During Treatment with Acycloguanosine-Based Cytotoxic Drugs

In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET. Methods: Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2′-deoxy-2′-fluoroarabinofuranosylcytosine, 2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs. Results: Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals (18F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)–transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers (18F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine). Conclusion: The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with 18F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells.

Comparative Analysis of T Cell Imaging with Human Nuclear Reporter Genes

Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging. Methods: Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodium-iodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 105 to 3 × 106 reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (background) was measured. Results: The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-18F-fluorobenzylguanidine (18F-MFBG) reporter system could detect less than 1 × 105 T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/124I-iodide reporter system could detect approximately 1 × 106 T cells; 124I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2′-18F-fluoro-5-ethyl-1-β-d-arabinofuranosyluracil (18F-FEAU) and hsvTK/18F-FEAU reporter systems detected approximately 3 × 105 T cells, respectively. 18F-FEAU was a more efficient probe (higher uptake, lower background) than 124I-1-(2-deoxy-2-fluoro-1-d-arabinofuranosyl)-5-iodouracil for both hdCKDM and hsvTK. Conclusion: A comparison of different reporter gene–reporter probe systems for imaging of T cell number was performed, and the hNET/18F-MFBG PET reporter system was found to be the most sensitive and capable of detecting approximately 35–40 × 103 T cells at the site of T cell injection in the animal model.

A New Acycloguanosine-Specific Supermutant of Herpes Simplex Virus Type 1 Thymidine Kinase Suitable for PET Imaging and Suicide Gene Therapy for Potential Use in Patients Treated with Pyrimidine-Based Cytotoxic Drugs

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene is widely used as a suicide gene in combination with ganciclovir (GCV) and as a nuclear imaging reporter gene with an appropriate reporter probe. Wild-type HSV1-tk recognizes a variety of pyrimidine and acycloguanosine nucleoside analogs, including clinically used antiviral drugs. PET of HSV1-tk reporter gene expression will be compromised in patients receiving nucleoside-based antiviral treatment. With the use of an acycloguanosine-specific mutant of the enzyme, PET of HSV1-tk reporter gene expression can be successfully performed with acycloguanosine-based radiotracers without interference from pyrimidine-based antiviral drugs. Methods: The levels of expression of wild-type HSV1-tk and HSV1-A167Ytk, HSV1-sr39tk, and HSV1-A167Ysr39tk mutants fused with green fluorescent protein (GFP) and transduced into U87 cells were normalized to the mean fluorescence of GFP measured by fluorescence-activated cell sorting. The levels of enzymatic activities of wild-type HSV1-tk and its mutants were compared by 2-h in vitro radiotracer uptake assays with 3H-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-ethyluracil (3H-FEAU), 3H-pencyclovir (3H-PCV), and 3H-GCV and by drug sensitivity assays. PET with 18F-FEAU and 18F-9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) was performed in mice with established subcutaneous tumors, expressing wild-type HSV1-tk and its mutants, followed by tissue sampling. Results: FEAU accumulation was not detected in HSV1-A167Ysr39tk–expressing cells and xenografts. Lack of conversion of pyrimidine derivatives by the HSV1-A167Ysr39tk supermutant was also confirmed by a drug sensitivity assay, in which the 50% inhibitory concentrations for thymine 1-β-d-arabinofuranoside and bromovinyldeoxyuridine were found to be similar to those in nontransduced cells. In contrast, we found that HSV1-A167Ysr39tk could readily phosphorylate 3H-GCV at levels similar to those of wild-type HSV1-tk and HSV1-A167Ytk but showed enhanced activity with 3H-PCV in vitro and with 18F-FHBG in vivo. Conclusion: We developed a new reporter gene, HSV1-A167Ysr39tk, which exhibits specificity and high phosphorylation activity for acycloguanosine derivatives. The resulting supermutant can be used for PET with 18F-FHBG and suicidal gene therapy protocols with GCV in patients treated with pyrimidine-based cytotoxic drugs.

PARP-1–Targeted Radiotherapy in Mouse Models of Glioblastoma

The DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1) is overexpressed in glioblastoma, with overall low expression in healthy brain tissue. Paired with the availability of specific small molecule inhibitors, PARP-1 is a near-ideal target to develop novel radiotherapeutics to induce DNA damage and apoptosis in cancer cells, while sparing healthy brain tissue. Methods: We synthesized an 131I-labeled PARP-1 therapeutic and investigated its pharmacology in vitro and in vivo. A subcutaneous tumor model was used to quantify retention times and therapeutic efficacy. A potential clinical scenario, intratumoral convection-enhanced delivery, was mimicked using an orthotopic glioblastoma model combined with an implanted osmotic pump system to study local administration of 131I-PARPi (PARPi is PARP inhibitor). Results: 131I-PARPi is a 1(2H)-phthalazinone, similar in structure to the Food and Drug Administration–approved PARP inhibitor AZD-2281. In vitro studies have shown that 131I-PARPi and AZD-2281 share similar pharmacologic profiles. 131I-PARPi delivered 134.1 cGy/MBq intratumoral injected activity. Doses to nontarget tissues, including liver and kidney, were significantly lower. Radiation damage and cell death in treated tumors were shown by p53 activation in U87-MG cells transfected with a p53-bioluminescent reporter. Treated mice showed significantly longer survival than mice receiving vehicle (29 vs. 22 d, P < 0.005) in a subcutaneous model. Convection-enhanced delivery demonstrated efficient retention of 131I-PARPi in orthotopic brain tumors, while quickly clearing from healthy brain tissue. Conclusion: Our results demonstrate 131I-PARPi’s high potential as a therapeutic and highlight PARP’s relevance as a target for radionuclide therapy. Radiation plays an integral role in brain tumor therapy, and radiolabeled PARP therapeutics could ultimately lead to improvements in the standard of care.

Abstract 1406: Involvement of DNA repair pathways in DAG-lactone radiosensitization of human LNCaP cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We previously demonstrated that pre-treatment of human androgen-dependent prostate cancer cell line LNCaP with either 12-0-tetradecanoylphorbol 13-acetate (TPA, a known protein kinase C activator), or diacylglycerol-lactone (DAG-lactone) radiosensitized these cells. This effect was mediated by down-regulation of ATM protein levels, thus de-repressing the enzyme ceramide synthase (CerS), ceramide generation and apoptosis. Here we assessed whether ATM down-regulation affected DNA repair pathways. LNCaP cells were pretreated for 16 hr with 10μM DAG-lactone and irradiated with 20 or 40Gy. Pre-treatment with DAG-lactone significantly enhanced apoptosis at 48 hours from 1.1% to 13.9% (20Gy) and 19.9% (40Gy) respectively. γ-H2AX and BRCA1 foci formation at 1 and 8 hours post-radiation were scored. Immunofluorescent staining for γ-H2AX demonstrated a dose-dependent increase in γ-H2AX foci formation at 20Gy and 40Gy for both 1hr and 8 hr time points. Our data also shows that 40Gy alone produces the same amount of DNA double strand breaks (dsbs; as measured by γ-H2AX at 8 hr) as 20Gy+DAG-lactone, yet 40Gy alone does not induce apoptosis in LNCaP cells, while 20Gy+DAG-lactone does, indicating that ATM down-regulation is necessary for the apoptotic response. Similarly there was a dose-dependent response in the generation of BRCA1 foci at 20Gy and 40 Gy at 8hr post-radiation. There was no difference in the BRCA1 foci between pre-treatment with DAG-lactone either at 20Gy or at 40 Gy at both 1 hr or at 8 hr post-radiation. While apoptotic degradation is associated with γ-H2AX phosphorylation, in LNCaP cells these events would be expected at 24-48 hr. Therefore the foci observed here are unlikely to be associated with apoptotic DNA degradation and most likely result from DNA dsbs. Our recent data showed that unrepaired DNA dsbs trigger post-transcriptional activation of CerS via a mechanism that remains unknown. Accordingly MRE 11, ATM or DNA-PKcs (SCID) deficiencies sensitize mice towards CerS-mediated apoptosis in crypt stem cells clonogens. Although these studies indicate that HR is active, further experiments need to be carried out to explore whether non-homologous end-joining (NHEJ) is or is not functional in LNCaP treated with DAG-lactone and radiation. Hence, the trigger for CerS activation still needs to be established. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1406.

763. Multimodality Imaging of Genetically Modified Lymphocytes In Vivo

Background: the lack of specific trafficking of genetically engineered T cells to tumor sites is one of the major limitations in adoptive immunotherapy. Multimodality imaging represents a powerful tool for repetitive non-invasive assessment of migration, activation and survival of immune effectors in living subjects in pre-clinical models and in patients. Methods: to establish a cancer model for adoptive immunotherapy we challenged immunodeficient mice with RM1 murine prostate carcinoma cells transduced with human prostate-specific membrane antigen (PSMA). The tumor cells were also co-transduced with dual fusion (red fluorescent protein and Renilla lusiferase) reporter gene for imaging of tumor progression. Human peripheral blood T-lymphocytes were transduced with chimeric receptors specific either for PSMA or CEA (control) and with reporter vector bearing HSV1-thymidine kinase (HSV1-tk), green fluorescent protein (GFP) and Click Beetle luciferase (CBR). GFP was used for assessment of transduction efficiency of T-lymphocytes and T-cell tumor infiltration. After ex vivo expansion genetically modified PSMA-specific or CEA-specific (control) lymphocytes were adoptively transferred into tumor bearing mice. Metastatic lung tumors were detected using in vivo bioluminescent imaging in all animals on the day of lymphocyte injection and thereafter. All mice underwent biolumincescent and microPET imaging with [18F]FEAU. Results: on day 1 after T-cell injection distinct luciferase and PET signals were detected in both PSMA-specific and CEA-specific (control) lymphocyte groups. On day 3 clear signal was obtained only in PSMA-specific lymphocyte group. PET signal was not detected on day 6 in either group. The %ID/g differed significantly on day 3 between two groups (Table 1). The bioluminescence data corroborated PET data. Tumor growth delay was observed using bioluminescent signal dynamic in the group, injected with PSMA-specific lymphocytes. Statistically significant survival advantage (p=0.01) was also observed in the same group. The presence of human genetically modified lymphocytes as well as PSMA antigen in lung tumor was confirmed by immunofluorescence and immunohistochemistry. Conclusion: multimodality imaging strategy is feasible and can be applied for longitudinal visualization of genetically modified immune cells and prediction of adoptive immunotherapy outcome.