Ziyue Karen Jiang

Androgen-Independent Molecular Imaging Vectors to Detect Castration-Resistant and Metastatic Prostate Cancer

Prostate-specific promoters are frequently employed in gene-mediated molecular imaging and therapeutic vectors to diagnose and treat castration-resistant prostate cancer (CRPC) that emerges from hormone ablation therapy. Many of the conventional prostate-specific promoters rely on the androgen axis to drive gene expression. However, considering the cancer heterogeneity and varying androgen receptor status, we herein evaluated the utility of prostate-specific enhancing sequence (PSES), an androgen-independent promoter in CRPC. The PSES is a fused enhancer derived from the prostate-specific antigen (PSA) and prostate-specific membrane antigen gene regulatory region. We augmented the activity of PSES by the two-step transcriptional amplification (TSTA) system to drive the expression of imaging reporter genes for either bioluminescent or positron emission tomography (PET) imaging. The engineered PSES-TSTA system exhibits greatly elevated transcriptional activity, androgen independency, and strong prostate specificity, verified in cell culture and preclinical animal experimentations. These advantageous features of PSES-TSTA elicit superior gene expression capability for CRPC in comparison with the androgen-dependent PSA promoter-driven system. In preclinical settings, we showed robust PET imaging capacity of PSES-TSTA in a castrated prostate xenograft model. Moreover, intravenous administrated PSES-TSTA bioluminescent vector correctly identified tibial bone marrow metastases in 9 of 9 animals, whereas NaF- and FDG-PET was unable to detect the lesions. Taken together, this study showed the promising utility of a potent, androgen-independent, and prostate cancer-specific expression system in directing gene-based molecular imaging in CRPC, even in the context of androgen deprivation therapy.

Engineering polypeptide coatings to augment gene transduction and in vivo stability of adenoviruses.

We sought to modify adenoviral (Ad) particles by incorporating the advantageous characteristics of non-viral gene delivery vehicles to complement the viral vectors. α-Amino acid-N-carboxyanhydride chemistry was used to synthesize homopolypeptides and diblock copolypeptides that possess well-defined secondary structures. Using cryo-electron and fluorescence microscopy, we showed that these polypeptides can coat the surfaces of Ad particles in a non-covalent manner to modify their transduction properties. The coated Ad particles were found to bind to and be internalized by cells. In contrast to reports using covalently PEGylated Ad particles, we found that our physically coated Ad hybrid complexes facilitate gene transfer both in vitro and in vivo. We showed that our polypeptide coating was able to shield the Ad particles from the neutralizing effect of antibodies and mitigate the binding of blood coagulation factor (Factor X) in vitro. The coating also reduced the antigenicity of Ad in immunocompetent mice. The biodistribution of the systemically administered hybrid complexes mirrored the behavior of both viral and non-viral vectors, exhibiting liver tropism as well as enhanced lung transduction. These data demonstrated that our non-covalent modification was able to alter Ads interactions with cells and organs with retention of transduction efficiency. Advantages such as facile coating of the Ad vector, design flexibility and ease of attaching ligands to the polypeptides make this system potentially useful as a platform for adding functionalities to Ad to target cancer metastasis.

Chapter Five - The Development of Transcription-Regulated Adenoviral Vectors with High Cancer-Selective Imaging Capabilities

A clear benefit of molecular imaging is to enable noninvasive, repetitive monitoring of intrinsic signals within tumor cells as a means to identify the lesions as malignant or to assess the ability of treatment to perturb key pathways within the tumor cells. Due to the promising utility of molecular imaging in oncology, preclinical research to refine molecular imaging techniques in small animals is a blossoming field. We will first discuss the several imaging modalities such as fluorescent imaging, bioluminescence imaging, and positron emission tomography that are now commonly used in small animal settings. The indirect imaging approach, which can be adapted to a wide range of imaging reporter genes, is a useful platform to develop molecular imaging. In particular, reporter gene-based imaging is well suited for transcriptional-targeted imaging that can be delivered by recombinant adenoviral vectors. In this review, we will summarize transcription-regulated strategies used in adenoviral-mediated molecular imaging to visualize metastasis and monitor oncolytic therapy in preclinical models.

Rapamycin enhances adenovirus-mediated cancer imaging and therapy in pre-immunized murine hosts.

Tumor-specific adenoviral vectors comprise a fruitful gene-based diagnostic imaging and therapy research area for advanced stage of cancer, including metastatic disease. However, clinical translation of viral vectors has encountered considerable obstacles, largely due to host immune responses against the virus. Here, we explored the utilization of an immunosuppressant, rapamycin, to circumvent the anti-adenovirus immunity in immunocompetent murine prostate cancer models. Rapamycin diminished adenoviral-induced acute immune response by inhibiting NF-κB activation; it also reduced the scale and delayed the onset of inflammatory cytokine secretion. Further, we found that rapamycin abrogated anti-adenovirus antibody production and retarded the function of myeloid cells and lymphocytes that were activated upon viral administration in pre-immunized hosts. Thus, the co-administration of rapamycin prolonged and enhanced adenovirus-delivered transgene expression in vivo, and thereby augmented the imaging capability of adenoviral vectors in both bioluminescent and positron emission tomography modalities. Furthermore, we showed that despite an excellent response of cancer cells to a cytotoxic gene therapeutic vector in vitro, only minimal therapeutic effects were observed in vivo in pre-immunized mice. However, when we combined gene therapy with transient immunosuppression, complete tumor growth arrest was achieved. Overall, transient immunosuppression by rapamycin was able to boost the diagnostic utility and therapeutic potentials of adenoviral vectors.

Abstract 111: In search of a lead fluorescent tracer for PSCA-expressing prostate cancer: A comprehensive analysis of imaging agents and dyes

Prostate stem cell antigen (PSCA) is over-expressed on the surface of many prostate cancer cells especially in castration-resistant and metastatic lesions. Thus, it is of great interest to develop PSCA-targeted imaging probes for cancer detection. To this end our group has generated anti-PSCA antibody fragments including minibodies and diabodies that can be fluorescently labeled via either site-specific or non-specific conjugations. We here report a detailed optimization of such labeling protocols and propose a lead imaging probe for PSCA+ prostate tumors. Male nude mice were bilaterally implanted with PSCA over-expressing and parental PSCAlow 22rv1 cells. All probes were injected intravenously and imaging was performed at 6 (diabody) or 48 (minibody) hours post injection. Anti-PSCA A2 diabody contains a cysteine tag in the linker region that is suitable for site-specific conjugation via maleimide chemistry. Two fluorescent dyes with maleimide moiety were tested: a far-red dye Cy5 (ex: 650 nm; em: 670 nm) and a near infrared IRDye800CW (ex: 774 nm; em: 789 nm). Our results revealed that although having a better tissue penetration rate, A2-800CW was mainly trapped in the kidney potentially due to the renal clearance of diabodies and thus failed to achieve PSCA-specific cancer imaging; whereas A2-Cy5 was able to delineate the PSCA-over-expressing tumor after skin removal (PSCA + to - tumor ratio: 1.58 ± 0.10). In vivo imaging in live animals was not feasible due to the interference of autofluorescence with Cy5 even though the mice were kept on an alfalfa-free diet. Such discrepancy in the behavior of the two probes might be attributed to the differential physical properties of the dyes, which could lead to altered 3-D structure and PSCA-binding affinity of the diabody after conjugation. Minibodies are larger molecules than diabodies and thus their binding affinity to PSCA is likely more resilient to non-specific labeling. We here used Cy5, IRDye800CW and an FDA-approved near infrared dye 4XPEG-ICG (ex: 774 nm; em: 805 nm) to label an anti-PSCA minibody A11. All three dyes were targeted to amines that are present mainly in lysine residues via NHS ester chemistry. Corroborating the diabody results, only the two near infrared dyes but not Cy5 permitted in vivo imaging in live animals. Further, A11-ICG enabled distinct tumor detection by achieving a PSCA + to - tumor ratio of 2.09 ± 0.37. However, heightened background in the liver and draining lymph nodes was observed. On the other hand, A11-800CW generated very impressive tumor contrasts with the average ratio of PSCA + to - tumors being 2.73 ±0.71. Overall, PSCA-targeted diabodies and minibodies are versatile imaging tools that can be linked to various fluorescent dyes. However, A11 minibody conjugated to IRDye800CW appeared to have reached the highest contrast in imaging and thus is a better candidate for translational imaging trials. Citation Format: Ziyue Karen Jiang, Anna Wu, Robert Reiter. In search of a lead fluorescent tracer for PSCA-expressing prostate cancer: A comprehensive analysis of imaging agents and dyes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 111. doi:10.1158/1538-7445.AM2014-111

Abstract 354: Monitor androgen blockade therapy with functional androgen receptor reporting system

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Hormonal manipulation remains the first line treatment for advanced prostate cancer. It includes surgical and medical means of androgen deprivation and androgen receptor (AR) blockade therapies. However, the majority of patients will develop castration resistant prostate cancer, often with metastasis that accounts for the lethality of this disease. Therefore, a reliable imaging modality capable of active surveillance of tumor recurrence and/or new incidence of metastasis would greatly benefit the timing and decision-making in adopting second line therapies. To this end, we recently constructed a prostate specific yet androgen independent promoter system, namely PSES-TSTA (Prostate Specific Enhancer Sequence coupled with Two-Step Transcriptional Amplification). We used this system to drive PET and optical imaging reporters and successfully visualized subcutaneous tumor and intra-tibial metastasis in castrated mice. The androgen independency of PSES-TSTA, which secured viability of this imaging approach when only castrated level of androgen is available, is attributed to the composition of the chimeric PSES sequence - an androgen inducible element derived from the prostate specific antigen (PSA) gene promoter and an androgen suppressible element from the prostate specific membrane antigen gene enhancer (PSME). Another challenge faced by patients after hormonal ablation therapy is the need for effective second-line therapies. Interestingly, a growing body of evidence points out that even at castration resistant stage, AR remains active and most prostate cancer still rely on AR signaling axis for proliferation and survival. Accordingly, much effort has been devoted to the development of second generation anti-androgen agents. Therefore, it would be greatly beneficial to establish a practical and reliable reporting system that reflects the functional status of the androgen-AR axis so that the efficacy of such new drugs can be monitored at the molecular level. Inspired by previous results, we separated the two components of PSES and generated two imaging reporters: PSA-driven Firefly Luciferase (PSA-FL) and PSME-driven Renilla Luciferase (PSME-RL). Based on their respective response to androgen signaling, we anticipated that the PSA-FL signal will decrease while the PSME-RL signal will increase in the face of an effective anti-androgen therapy. Indeed, we observed such pattern of reporter gene expression in multiple prostate cancer cell lines. Importantly, this system is able to differentiate the potency and efficacy between the first generation and the more potent second generation AR antagonists. Moreover, the simultaneous application of the PSME- and PSA-driven reporter system is advantageous over the single PSA promoter-based reporter in that it rules out the effect of tumor mass reduction on the readout because both up- and down-regulation of gene expression are being assessed and imaged. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 354. doi:1538-7445.AM2012-354