Triggering anti-GBM immune response with EGFR-mediated photoimmunotherapy.

BACKGROUND: Surgical resection followed by chemo-radiation postpones glioblastoma (GBM) progression and extends patient survival, but these tumours eventually recur. Multimodal treatment plans combining intraoperative techniques that maximise tumour excision with therapies aiming to remodel the immunologically cold GBM microenvironment could improve patients' outcomes. Herein, we report that targeted photoimmunotherapy (PIT) not only helps to define tumour location and margins but additionally promotes activation of anti-GBM T cell response. METHODS: EGFR-specific affibody molecule (ZEGFR:03115) was conjugated to IR700. The response to ZEGFR:03115-IR700-PIT was investigated in vitro and in vivo in GBM cell lines and xenograft model. To determine the tumour-specific immune response post-PIT, a syngeneic GBM model was used. RESULTS: In vitro findings confirmed the ability of ZEGFR:03115-IR700 to produce reactive oxygen species upon light irradiation. ZEGFR:03115-IR700-PIT promoted immunogenic cell death that triggered the release of damage-associated molecular patterns (DAMPs) (calreticulin, ATP, HSP70/90, and HMGB1) into the medium, leading to dendritic cell maturation. In vivo, therapeutic response to light-activated conjugate was observed in brain tumours as early as 1 h post-irradiation. Staining of the brain sections showed reduced cell proliferation, tumour necrosis, and microhaemorrhage within PIT-treated tumours that corroborated MRI T2*w acquisitions. Additionally, enhanced immunological response post-PIT resulted in the attraction and activation of T cells in mice bearing murine GBM brain tumours. CONCLUSIONS: Our data underline the potential of ZEGFR:03115-IR700 to accurately visualise EGFR-positive brain tumours and to destroy tumour cells post-conjugate irradiation turning an immunosuppressive tumour environment into an immune-vulnerable one.

Thiol-Reactive PODS-Bearing Bifunctional Chelators for the Development of EGFR-Targeting [18F]AlF-Affibody Conjugates.

Site-selective bioconjugation of cysteine-containing peptides and proteins is currently achieved via a maleimide-thiol reaction (Michael addition). When maleimide-functionalized chelators are used and the resulting bioconjugates are subsequently radiolabeled, instability has been observed both during radiosynthesis and post-injection in vivo, reducing radiochemical yield and negatively impacting performance. Recently, a phenyloxadiazolyl methylsulfone derivative (PODS) was proposed as an alternative to maleimide for the site-selective conjugation and radiolabeling of proteins, demonstrating improved in vitro stability and in vivo performance. Therefore, we have synthesized two novel PODS-bearing bifunctional chelators (NOTA-PODS and NODAGA-PODS) and attached them to the EGFR-targeting affibody molecule ZEGFR:03115. After radiolabeling with the aluminum fluoride complex ([18F]AlF), both conjugates showed good stability in murine serum. When injected in high EGFR-expressing tumor-bearing mice, [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115 showed similar pharmacokinetics and a specific tumor uptake of 14.1 ± 5.3% and 16.7 ± 4.5% ID/g at 1 h post-injection, respectively. The current results are encouraging for using PODS as an alternative to maleimide-based thiol-selective bioconjugation reactions.

Assessment of phototoxicity in pigmented Long-Evans rat: sparfloxacin and 8-methoxypsoralen.

The aim of the present work was to evaluate the effects of photo-activated toxicity induced after administration of two known melanin-binding phototoxic compounds, sparfloxacin (SPX) and 8-methoxypsoralen (8-MOP), followed or not by UVA/Vis exposure, in pigmented rats (Long Evans: LE) and albino rats (Sprague Dawley: SD). Groups of three rats were treated with SPX or 8-MOP by oral gavage for six consecutive days. Irradiated animals were submitted to a UVA/Vis light dose standardized to 10 J/cm2 UVA daily. Clinical signs, cutaneous reactions and body weight were monitored throughout the study period. Ear biopsy weight, lymph node weight and lymph node cell count were determined at necropsy. Ophthalmologic examinations were performed before the first treatment and on the day of sacrifice. Microscopic examinations were performed on skin biopsies and eyes. Phototoxicity was demonstrated for both SPX and 8-MOP in the pigmented and albino strains, in terms of auricular irritation, lymph node weight and proliferation index, cutaneous reactions and ocular histopathology. LE rats were less sensitive than SD rats, especially at the ocular level, supporting the notion that pigmentation may provide protection against photo-activation. The pigmented rat may be a more relevant model than the albino rat for human safety evaluation.

Modular Synthesis of Semiconducting Graft Copolymers to Achieve "Clickable" Fluorescent Nanoparticles with Long Circulation and Specific Cancer Targeting.

Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9'-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically "clicked" onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics.

Near-InfraRed PhotoImmunoTherapy (NIR-PIT) for the local control of solid cancers: Challenges and potentials for human applications.

Near-InfraRed PhotoImmunoTherapy (NIR-PIT) is a novel cancer-targeted treatment effected by a chemical conjugation between a photosensitiser (e.g. the NIR phthalocyanine dye IRDye700DX) and a cancer-targeting moiety (e.g. a monoclonal antibody, moAb). Delivery of a conjugate in vivo leads to accumulation at the tumour cell surface by binding to cell surface receptors or antigens. Upon deployment of focal NIR-light, irradiation of the conjugate results in a rapid, targeted cell death. However, the mechanisms of action to produce the cytotoxic effects have yet to be fully understood. Herein, we bring together the current knowledge of NIR-PIT from preclinical and clinical studies in a variety of cancers highlighting the key unanswered research questions. Furthermore, we discuss how to enhance the local control of solid cancers using this novel treatment regimen.

18F-meta-fluorobenzylguanidine (18F-mFBG) to monitor changes in norepinephrine transporter expression in response to therapeutic intervention in neuroblastoma models.

Targeted radiotherapy with 131I-mIBG, a substrate of the human norepinephrine transporter (NET-1), shows promising responses in heavily pre-treated neuroblastoma (NB) patients. Combinatorial approaches that enhance 131I-mIBG tumour uptake are of substantial clinical interest but biomarkers of response are needed. Here, we investigate the potential of 18F-mFBG, a positron emission tomography (PET) analogue of the 123I-mIBG radiotracer, to quantify NET-1 expression levels in mouse models of NB following treatment with AZD2014, a dual mTOR inhibitor. The response to AZD2014 treatment was evaluated in MYCN amplified NB cell lines (Kelly and SK-N-BE(2)C) by Western blot (WB) and immunohistochemistry. PET quantification of 18F-mFBG uptake post-treatment in vivo was performed, and data correlated with NET-1 protein levels measured ex vivo. Following 72 h AZD2014 treatment, in vitro WB analysis indicated decreased mTOR signalling and enhanced NET-1 expression in both cell lines, and 18F-mFBG revealed a concentration-dependent increase in NET-1 function. AZD2014 treatment failed however to inhibit mTOR signalling in vivo and did not significantly modulate intratumoural NET-1 activity. Image analysis of 18F-mFBG PET data showed correlation to tumour NET-1 protein expression, while further studies are needed to elucidate whether NET-1 upregulation induced by blocking mTOR might be a useful adjunct to 131I-mIBG therapy.

Tribological design constraints of marine renewable energy systems.

Against the backdrop of increasing energy demands, the threat of climate change and dwindling fuel reserves, finding reliable, diverse, sustainable/renewable, affordable energy resources has become a priority for many countries. Marine energy conversion systems are at the forefront of providing such a resource. Most marine renewable energy conversion systems require tribological components to convert wind or tidal streams to rotational motion for generating electricity while wave machines typically use oscillating hinge or piston within cylinder geometries to promote reciprocating linear motion. This paper looks at the tribology of three green marine energy systems, offshore wind, tidal and wave machines. Areas covered include lubrication and contamination, bearing and gearbox issues, biofouling, cavitation erosion, tribocorrosion, condition monitoring as well as design trends and loading conditions associated with tribological components. Current research thrusts are highlighted along with areas needing research as well as addressing present-day issues related to the tribology of offshore energy conversion technologies.