Photodynamic tumor eradication with a novel targetable photosensitizer: strong vascular effects and dependence on treatment repetition versus potentiation.

A novel pyropheophorbide-a (PPa) derivative, Ac-sPPp, was developed in our lab for targeted photodynamic therapy (PDT) and combination therapies. Its versatile peptide moiety, high water-solubility, amphiphilicity, and micellar aggregation allow efficient coupling to targeting moieties and convenient mixing with other therapeutics. Photosensitizer immunoconjugate (PIC) targeted PDT, using Ac-sPPp conjugated to therapeutic anti-epidermal growth factor receptor (EGFR) antibody cetuximab, and PDT + chemotherapy combination treatment, using Ac-sPPp mixed with stealth liposomal doxorubicin (Doxil), were investigated as promising strategies for potentiating PDT and improving target specificity. Passively targeted PDT with Ac-sPPp only or surfactant-solubilized PPa was also investigated for comparison. The A-431 human vulvar squamous cell carcinoma, xenografted in nude mice, was chosen as a tumor model because of its high EGFR expression and sensitivity to liposomal doxorubicin in vitro. Fluorescence imaging and PDT experiments showed that Ac-sPPp formulations circulated far longer and provided superior tumor contrast and superior tumor control compared to PPa. Strong PDT vascular effects were observed by laser Doppler imaging regardless of whether Ac-sPPp was passively or actively targeted. Passively targeted Ac-sPPp PDT gave equivalent or better tumor control than PIC-targeted PDT or PDT + Doxil combination therapy, and when treatments were repeated, it also yielded the highest cure rate.

Biodistribution and imaging of fluorescently-tagged iron oxide nanoparticles in a breast cancer mouse model.

Iron oxide nanoparticle (IONP) hyperthermia is an emerging treatment that shows great potential as a cancer therapy both alone and in synergy with conventional modalities. Pre-clinical studies are attempting to elucidate the mechanisms of action and distributions of IONP in various in vitro and in vivo models, however these studies would greatly benefit from real-time imaging of IONP locations both in cellular and in mammalian systems. To this end, fluorescently-tagged IONP (fIONP) have been employed for real time tracking and co-registration of IONP with iron content. Starch-coated IONP were fluorescently-tagged, purified and analyzed for fluorescent signal at various concentrations. fIONP were incubated with MTGB cells for varying times and cellular uptake analyzed using confocal microscopy, flow cytometry and inductively-coupled plasma mass spectrometry (ICP-MS). fIONP were also injected into a bilateral mouse tumor model for radiation modification of tumor tissue and enhanced fIONP deposition assessed using a Xenogen IVIS fluorescent imager. Results demonstrated that fIONP concentrations in vitro correlated with ICPMS iron readings. fIONP could be tracked in vitro as well as in tissue samples from an in vivo model. Future work will employ whole animal fluorescent imaging to track the biodistribution of fIONP over time.

Epidermal growth factor receptor-targeted photosensitizer selectively inhibits EGFR signaling and induces targeted phototoxicity in ovarian cancer cells.

Targeted photosensitizer delivery to EGFR-expressing cells was achieved in the present study using a high purity, targeted photoimmunoconjugate (PIC). When the PDT agent, benzoporphyrin derivative monoacid ring A (BPD) was coupled to an EGFR-targeting antibody (cetuximab), we observed altered cellular localization and selective phototoxicity of EGFR-positive cells, but no phototoxicity of EGFR-negative cells. Cetuximab in the PIC formulation blocked EGF-induced activation of the EGFR and downstream signaling pathways. Our results suggest that photoimmunotargeting is a useful dual strategy for the selective destruction of cancer cells and also exerts the receptor-blocking biological function of the antibody.

Development of an ErbB-overexpressing A-431 optical reporting tumor xenograft model to assess targeted photodynamic therapy regimens.

To better assess the efficacy of erbB-targeted therapies, it would help to have optical reporting human tumor xenograft models that abundantly express erbB receptors. A-431 cells have frequently been used in erbB1-targeting studies, but a well-characterized optical reporting version of the cell line has not been readily available. In this study, optical reporting A-431 clones were developed that express both a fluorescent protein reporter (green, GFP; or red, RFP) and a bioluminescent reporter, firefly luciferase. Reporter genes were transduced into cells using commercial lentiviral vectors, and clonal selection was carried out using a series of procedures. A number of clones were isolated for further characterization. A GFP/luciferase clone, A-431/D4, and an RFP/luciferase clone, A-431/G4, were obtained that exhibit erbB1 expression levels and tumor growth kinetics similar to the parental cells. To demonstrate the utility of the optical reporting clones, A-431/G4 tumors were grown subcutaneously in nude mice and treated with vascular-targeted photodynamic therapy (PDT), which targets the angiogenic consequences of erbB signaling. The A-431/G4 tumor model permitted highly sensitive longitudinal monitoring of PDT treatment response using optical imaging. A-431/D4 and A-431/G4 optical reporting tumor models should also prove useful for assessing therapies that directly target the erbB1 receptor.

Disparity between prostate tumor interior versus peripheral vasculature in response to verteporfin-mediated vascular-targeting therapy.

Photodynamic therapy (PDT) is a light-based cancer treatment modality. Here we employed both in vivo and ex vivo fluorescence imaging to visualize vascular response and tumor cell survival after verteporfin-mediated PDT designed to target tumor vasculature. EGFP-MatLyLu prostate tumor cells, transduced with EGFP using lentivirus vectors, were implanted in athymic nude mice. Immediately after PDT with different doses of verteporfin, tumor-bearing animals were injected with a fluorochrome-labeled albumin. The extravasation of fluorescent albumin along with tumor EGFP fluorescence was monitored noninvasively with a whole-body fluorescence imaging system. Ex vivo fluorescence microscopy was performed on frozen sections of tumor tissues taken at different times after treatment. Both in vivo and ex vivo imaging demonstrated that vascular-targeting PDT with verteporfin significantly increased the extravasation of fluorochrome-labeled albumin in the tumor tissue, especially in the tumor periphery. Although PDT induced substantial vascular shutdown in interior blood vessels, some peripheral tumor vessels were able to maintain perfusion function up to 24 hr after treatment. As a result, viable tumor cells were typically detected in the tumor periphery in spite of extensive tumor cell death. Our results demonstrate that vascular-targeting PDT with verteporfin causes a dose- and time-dependent increase in vascular permeability and decrease in blood perfusion. However, compared to the interior blood vessels, peripheral tumor blood vessels were found less sensitive to PDT-induced vascular shutdown, which was associated with subsequent tumor recurrence in the tumor periphery.

Multiepitope HER2 targeting enhances photoimmunotherapy of HER2-overexpressing cancer cells with pyropheophorbide-a immunoconjugates.

Multi-targeting strategies improve the efficacy of antibody and immunotoxin therapies but have not yet been thoroughly explored for HER2-based cancer treatments. We investigated multi-epitope HER2 targeting to boost photosensitizer immunoconjugate uptake as a way of enhancing photoimmunotherapy. Photoimmunotherapy may allow targeted photodynamic destruction of malignancies and may also potentiate anticancer antibodies. However, one obstacle preventing its clinical use is the delivery of enough photosensitizer immunoconjugates to target cells. Anti-HER2 photosensitizer immunoconjugates were constructed from two monoclonal antibodies (mAb), HER50 and HER66, using a novel method originally developed to label photosensitizer immunoconjugates with the photosensitizer, benzoporphyrin derivative verteporfin. Photosensitizer immunoconjugates were labeled instead with a promising alternative photosensitizer, pyropheophorbide-a (PPa), which required only minor changes to the conjugation procedure. Uptake and phototoxicity experiments using human cancer cells were conducted with the photosensitizer immunoconjugates and, for comparison, with free PPa. SK-BR-3 and SK-OV-3 cells served as HER2-overexpressing target cells. MDA-MB-468 cells served as HER2-nonexpressing control cells. Photosensitizer immunoconjugates with PPa/mAb molar ratios up to approximately 10 specifically targeted and photodynamically killed HER2-overexpressing cells. On a per mole basis, photosensitizer immunoconjugates were less phototoxic than free PPa, but photosensitizer immunoconjugates were selective for target cells whereas free PPa was not. Multiepitope targeted photoimmunotherapy with a HER50 and HER66 photosensitizer immunoconjugate mixture was significantly more effective than single-epitope targeted photoimmunotherapy with a single anti-HER2 photosensitizer immunoconjugate, provided photosensitizer immunoconjugate binding was saturated. This study shows that multiepitope targeting enhances HER2-targeted photoimmunotherapy and maintains a high degree of specificity. Consequently, it seems that multitargeted photoimmunotherapy should also be useful against cancers that overexpress other receptors.

Photochemical targeting of epidermal growth factor receptor: a mechanistic study.

PURPOSE: Photoimmunotherapy may allow target-specific photodynamic destruction of malignancies and may also potentiate anticancer antibody therapies. However, clinical use of either of the two modalities is limited for different reasons. Antibody therapies suffer from being primarily cytostatic and the need for prolonged administration with consequent side effects. In the case of photoimmunotherapy, a major impediment has been the absence of well-characterized photosensitizer immunoconjugates (PIC). In this investigation, we suggest a strategy to overcome these limitations and present the successful targeting of epidermal growth factor receptor (EGFR) using a well-characterized PIC. EXPERIMENTAL DESIGN: The PIC consisted of the EGFR-recognizing chimeric monoclonal antibody, C225, conjugated with a two-branched polyethylene glycol and benzoporphyrin derivative (BPD, Verteporfin). Mechanistic studies included photophysics, phototoxicity, cellular uptake, and catabolism experiments to yield dosimetric parameters. Target cells included two EGFR-overexpressing human cancer cell lines, OVCAR-5 and A-431. Nontarget cells included an EGFR-negative fibroblast cell line, 3T3-NR6, and a monocyte-macrophage cell line, J774. RESULTS: BPD-C225 PICs targeted and photodynamically killed EGFR-overexpressing cells, whereas free BPD exhibited no specificity. On a per mole basis, PICs were less phototoxic than free BPD, but PICs were very selective for target cells, whereas free BPD was not. Phototoxicity of the PICs increased at prolonged incubations. Photodynamic dose calculations indicated that PIC photophysics, photochemistry, catabolism, and subcellular localization were important determinants of PIC phototoxic potency. CONCLUSIONS: This study shows the efficacy of EGFR targeting with PIC constructs and suggests approaches to improve PIC designs and targeting strategies for in vivo photoimmunotherapy. The approach offers the possibility of dual effects via antibody-mediated cytostasis and photoimmunotherapy-based cytotoxicity.

Targeting cells that overexpress the epidermal growth factor receptor with polyethylene glycolated BPD verteporfin photosensitizer immunoconjugates.

Photoimmunotherapy was introduced two decades ago but has been studied infrequently in vivo and is virtually untested clinically. Progress has been limited because high-quality, well-characterized photosensitizer immunoconjugates (PICs) have been difficult to make. Here, we describe the development of an innovative conjugation method for producing water-soluble PICs that are free of insoluble aggregates and free of unacceptable amounts of noncovalently associated photosensitizer impurities. The method exploits two procedures previously untried in this research area. First, a small number of antibody lysines (<3 per antibody) are polyethylene glycolated (PEGylated) using a 10 kDa branched polyethylene glycol (PEG), which dramatically enhances PIC solubility and reduces PIC aggregation. Second, a 50% dimethyl sulfoxide-50% aqueous two-solvent system is used to prevent photosensitizer aggregation and noncovalent interactions. These measures allow efficient covalent linkage of the photosensitizer BPD Verteporfin (BPD) to antibody lysines, thorough purification of the resulting PICs (verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), maintenance of PIC antigen-binding activity (verified by cellular binding-uptake assays) and reduction of nonspecific cellular uptake (e.g. macrophage capture) of the PICs. Loading levels could be varied controllably in the range < or = 11 BPD/antibody. PICs of the C225 anti-epidermal growth factor receptor (EGFR) chimeric monoclonal antibody killed EGFR-overexpressing A-431 cells photodynamically but did not significantly affect EGFR-negative NR6 cells. Although fluorescence measurements demonstrated that the PICs were quenched by as much as an order of magnitude compared with free BPD, an impressive 90% reduction in A-431 cell viability was achieved using 20 J/cm2 of 690 nm light after a 40 h incubation with the C225 PICs. The results suggest that PEGylated BPD-C225 PICs merit further investigation in animal models of EGFR-overexpressing cancers.

Steady-state and dynamic contrast MR imaging of human prostate cancer xenograft tumors: a comparative study.

Understanding tumor vascular physiology is critically important for developing non-invasive, molecularly targeted diagnostic agents and therapies. In this study, using three different human prostate cancer xenografts (MDA PCa 2b, PC3, and LnCap), structural and physiological parameters of neoplastic vasculature and interstitum were explored with a widely available magnetic resonance imaging (MRI) pulse sequence (3D SPGR: spoiled gradient echo). Using dual injection technique employing two T1 contrast agents of different molecular masses (Weissleder, R., Cheng, H. C., Marecos, E., Kwong, K. K., Bogdanov, A., Jr. Eur. J. Cancer 34, 1448-1454 (1998).), steady state (SS) MRI measurements and dynamic contrast agent enhancement (DCE) MRI measurements were simultaneously acquired and analyzed using a two-compartment model for calculating parameters reflecting tumoral architecture and physiology. In particular, interstitial volume and vascular permeability were independently quantified using these two different MRI techniques. Relative vascular water exchange rate, calculated by the flip angle (FA) dependence of measured blood volume using SS technique, and vascular permeability of contrast agent, extrapolated from DCE MRI, were compared. It was found that the SS and DCE techniques were comparable and yielded similar qualitative results for extravascular compartment (interstitial volume). However, the permeability (water exchange rate and contrast agent vascular permeability) values were in disagreement. The results of MR studies are important for interpreting optical imaging results obtained using long-circulating of tumor-associated enzymatic activity.