ImmunoPET Imaging of αvß6 Expression Using an Engineered Anti-αvß6 Cys-diabody Site-Specifically Radiolabeled with Cu-64: Considerations for Optimal Imaging with Antibody Fragments.

PURPOSE: Increased expression of the αvß6 integrin correlates with advanced tumor grade and poor clinical outcome, identifying αvß6 as a prognostic indicator and an attractive target for molecular imaging. This work investigated the ability of a disulfide-stabilized [64Cu]NOTA-αvß6 cys-diabody to image αvß6 expression in vivo using a nu/nu mouse model bearing human melanoma xenografts and positron-emission tomography. PROCEDURES: Small-animal positron emission tomography (PET) imaging, quantitative ROI analysis, and ex vivo biodistribution were conducted to ascertain tumor uptake and organ distribution of the [64Cu]NOTA-αvß6 cys-diabody. Immunohistochemical staining of tumors and mouse organs and immunoreactivity assays were utilized to correlate in vivo and ex vivo observations. RESULTS: PET imaging of the [64Cu]NOTA-αvß6 cys-diabody revealed low tumor uptake at 24 h p.i. in DX3Puroß6 tumors (2.69 ± 0.45 %ID/g) with comparable results found in the DX3Puro tumors (2.24 ± 0.15 %ID/g). Quantitative biodistribution confirmed that DX3Puroß6 tumor uptake was highest at 24 h p.i. (4.63 ± 0.18 %ID/g); however, uptake was also observed in the stomach (4.84 ± 2.99 %ID/g), small intestines (4.50 ± 1.69 %ID/g), large intestines (4.73 ± 0.97 %ID/g), gallbladder (6.04 ± 1.88 %ID/g), and lungs (3.89 ± 0.69 %ID/g). CONCLUSIONS: Small-animal PET imaging was successful in visualizing αvß6-positive tumor uptake of the [64Cu]NOTA-αvß6 cys-diabody. Cys-diabody cross-reactivity was observed between human and murine αvß6 and immunohistochemical staining confirmed the presence of an endogenous αvß6 antigen sink, which led to suboptimal tumor contrast in this mouse model. Future investigations will focus on dose escalation studies to overcome the endogenous antigen sink while increasing DX3Puroß6 tumor uptake.

Development and characterization of an αvß6-specific diabody and a disulfide-stabilized αvß6-specific cys-diabody.

INTRODUCTION: This work describes the development and characterization of two antibody fragments that specifically target the α(v)ß(6) integrin, a non-covalent diabody and a disulfide-stabilized cys-diabody. The diabodies were analyzed for their ability to bind both immobilized and cell surface-bound α(v)ß(6). Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([(64)Cu]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [(18)F]-FB-α(v)ß(6) diabody, [(18)F]-FB-α(v)ß(6) cys-diabody, and the [(64)Cu]-NOTA-α(v)ß(6) cys-diabody. METHODS: Diabodies were constructed from the variable domains of the humanized 6.3G9 anti-α(v)ß(6) intact antibody. The anti-α(v(ß(6) cys-diabody was engineered with C-terminal cysteines to enable covalent dimerization and site-specific modification. Biochemical characterization included SDS-PAGE, Western blot, and electrospray ionization to confirm MW, and flow cytometry and ELISA experiments were used to determine binding affinity and specificity to α(v)ß(6). The diabodies were radiolabeled with [(18)F]-SFB and in addition, the anti-α(v)ß(6) cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [(64)Cu]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puroß(6) (α(v)ß(6)+) and DX3Puro (α(v)ß(6)-)cell lines. RESULTS: The diabodies were purified from cell culture supernatants with purities >98%. Subnanomolar binding affinity towards αvß6 was confirmed by ELISA (diabody IC(50)=0.8 nM, cys-diabody IC(50)=0.6 nM) and flow cytometry revealed high specificity only to the DX3Puroß(6) cell line for both diabodies. RCYs were 22.6%±3.6% for the [(18)F]-FB-α(v)ß(6) diabody, 8.3%±1.7% for the [(18)F]-FB-α(v)ß(6) cys-diabody and 43.5%±5.5% for the [(64)Cu]-NOTA-α(v)ß(6) cys-diabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([(18)F]-FB-α(v)ß(6) diabody=58.7%±6.7%, [(18)F]-FB-α(v)ß(6) cys-diabody=80.4%±4.4%, [(64)Cu]-NOTA-α(v)ß(6) cys-diabody=59.4%±0.6%) regardless of the radiolabeling method used. CONCLUSIONS: Two novel diabodies with excellent binding affinity and specificity for the α(v)ß(6) integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([(18)F]) and [(64)Cu] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach.

Modeling truncated AR expression in a natural androgen responsive environment and identification of RHOB as a direct transcriptional target.

Recent studies identifying putative truncated androgen receptor isoforms with ligand-independent activity have shed new light on the acquisition of androgen depletion independent (ADI) growth of prostate cancer. In this study, we present a model system in which a C-terminally truncated variant of androgen receptor (TC-AR) is inducibly expressed in LNCaP, an androgen-dependent cell line, which expresses little truncated receptor. We observed that when TC-AR is overexpressed, the endogenous full length receptor (FL-AR) is transcriptionally downmodulated. This in essence allows us to "replace" FL-AR with TC-AR and compare their individual properties in exactly the same genetic and cellular background, which has not been performed before. We show that the TC-AR translocates to the nucleus, activates transcription of AR target genes in the absence of DHT and is sufficient to confer ADI growth to the normally androgen dependent LNCaP line. We also show that while there is significant overlap in the genes regulated by FL- and TC-AR there are also differences in the respective suites of target genes with each AR form regulating genes that the other does not. Among the genes uniquely activated by TC-AR is RHOB which is shown to be involved in the increased migration and morphological changes observed in LN/TC-AR, suggesting a role of RHOB in the regulation of androgen-independent behavior of prostate cancer cells.

Establishment of clonal MIN-O transplant lines for molecular imaging via lentiviral transduction & in vitro culture.

As the field of molecular imaging evolves and increasingly is asked to fill the discovery and validation space between basic science and clinical applications, careful consideration should be given to the models in which studies are conducted. The MIN-O mouse model series is an established in vivo model of human mammary precancer ductal carcinoma in situ with progression to invasive carcinoma. This series of transplant lines is propagated in vivo and experiments utilizing this model can be completed in non-engineered immune intact FVB/n wild type mice thereby modeling the tumor microenvironment with biological relevance superior to traditional tumor cell xenografts. Unfortunately, the same qualities that make this and many other transplant lines more biologically relevant than standard cell lines for molecular imaging studies present a significant obstacle as somatic genetic re-engineering modifications common to many imaging applications can be technically challenging. Here, we describe a protocol for the efficient lentiviral transduction of cell slurries derived from precancerous MIN-O lesions, in vitro culture of "MIN-O-spheres" derived from single cell clones, and the subsequent transplantation of these spheres to produce transduced sublines suitable for optical imaging applications. These lines retain the physiologic and pathologic properties, including multilineage differentiation, and complex microanatomic interaction with the host stroma characteristic of the MIN-O model. We also present the in vivo imaging and immunohistochemical analysis of serial transplantation of one such subline and detail the progressive multifocal loss of the transgene in successive generations.

In vivo Cerenkov luminescence imaging: a new tool for molecular imaging.

Cerenkov radiation is a phenomenon where optical photons are emitted when a charged particle moves faster than the speed of light for the medium in which it travels. Recently, we and others have discovered that measurable visible light due to the Cerenkov effect is produced in vivo following the administration of ß-emitting radionuclides to small animals. Furthermore, the amounts of injected activity required to produce a detectable signal are consistent with small-animal molecular imaging applications. This surprising observation has led to the development of a new hybrid molecular imaging modality known as Cerenkov luminescence imaging (CLI), which allows the spatial distribution of biomolecules labelled with ß-emitting radionuclides to be imaged in vivo using sensitive charge-coupled device cameras. We review the physics of Cerenkov radiation as it relates to molecular imaging, present simulation results for light intensity and spatial distribution, and show an example of CLI in a mouse cancer model. CLI allows many common radiotracers to be imaged in widely available in vivo optical imaging systems, and, more importantly, provides a pathway for directly imaging ß(-)-emitting radionuclides that are being developed for therapeutic applications in cancer and that are not readily imaged by existing methods.

New covalent capture probes for imaging and therapy, based on a combination of binding affinity and disulfide bond formation.

We describe the synthesis and development of new reactive DOTA-metal complexes for covalently targeting engineered receptors in vivo, which have superior tumor uptake and clearance properties for biomedical applications. These probes are found to clear efficiently through the kidneys and minimally through other routes, but bind persistently in the tumor target. We also explore the new technique of Cerenkov luminescence imaging to optically monitor radiolabeled probe distribution and kinetics in vivo. Cerenkov luminescence imaging uniquely enables sensitive noninvasive in vivo imaging of a ß(-) emitter such as (90)Y with an optical imager.

Male germ cell-associated kinase, a male-specific kinase regulated by androgen, is a coactivator of androgen receptor in prostate cancer cells.

Androgen receptor (AR) is a ligand-induced transcriptional factor, which plays an important role in the normal development of prostate as well as in the progression of prostate cancer. Numerous coactivators, which associate with AR and function to remodel chromatin and recruit RNA polymerase II to enhance the transcriptional potential of AR, have been identified. Among these coactivators, few are protein kinases. In this study, we describe the characterization of a novel protein kinase, male germ cell-associated kinase (MAK), which serves as a coactivator of AR. We present evidence, which indicates that (a) MAK physically associates with AR (MAK and AR are found to be coprecipitated from cell extracts, colocalized in nucleus, and corecruited to prostate-specific antigen promoter in LNCaP as well as in transfected cells); (b) MAK is able to enhance AR transactivation potential in an androgen- and kinase-dependent manner in several prostate cancer cells and synergize with ACTR/steroid receptor coactivator-3 coactivator; (c) small hairpin RNA (shRNA) knocks down MAK expression resulting in the reduction of AR transactivation ability; (d) MAK-shRNA or kinase-dead mutant, when introduced into LNCaP cells, reduces the growth of the cells; and (e) microarray analysis of LNCaP cells carrying kinase-dead MAK mutant showed a significant impediment of AR signaling, indicating that endogenous MAK plays a general role in AR function in prostate cancer cells and likely to be a general coactivator of AR in prostate tissues. The highly restricted expression of this kinase makes it a potentially useful target for intervention of androgen independence.

Characterization of a novel androgen receptor mutation in a relapsed CWR22 prostate cancer xenograft and cell line.

CWR22 has been a valuable xenograft model for the study of prostate cancer progression from an androgen-dependent tumor to one that grows in castrated animals. Herein, we report the identification and characterization of a novel androgen receptor (AR) mutation occurring in a relapsed tumor (CWR22R-2152) and in the CWR22Rv1 cell line established from it. The mutation was not detected in the original, hormone-dependent CWR22 xenograft, indicating that this change occurred during the progression to androgen independence. It is characterized by an in-frame tandem duplication of exon 3 that encodes the second zinc finger of the AR DNA-binding domain. Accordingly, immunoblot analyses demonstrated the expression of an AR species having an approximately 5-kDa increase in size relative to the LNCaP AR. This was accompanied by a COOH-terminally truncated AR species migrating with a relative mass of 75-80 kDa, referred to as ARDeltaLBD because it lacks the ligand-binding domain. By recreating the exon 3 duplication mutation in a wild-type AR expression construct, the generation of ARDeltaLBD could be recapitulated. Whereas ARDeltaLBD exhibited constitutive nuclear localization and DNA binding, these functions in the full-length AR remained androgen dependent. The CWR22Rv1 AR repertoire displayed dose-dependent, androgen-responsive transcriptional transactivation in reporter assays, albeit to a lesser extent in comparison with LNCaP. This cell line also expressed low levels of prostate-specific antigen mRNA and did not express or secrete detectable levels of prostate-specific antigen protein in androgen-depleted medium or in response to physiological androgenic stimulation. In summary, the CWR22Rv1 cell line displays both androgen-responsive and androgen-insensitive features due, at least in part, to a novel insertional mutation of the AR.