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.

Immunomodulatory activity of IR700-labelled affibody targeting HER2.

There is an urgent need to develop therapeutic approaches that can increase the response rate to immuno-oncology agents. Photoimmunotherapy has recently been shown to generate anti-tumour immunological responses by releasing tumour-associated antigens from ablated tumour cell residues, thereby enhancing antigenicity and adjuvanticity. Here, we investigate the feasibility of a novel HER2-targeted affibody-based conjugate (ZHER2:2395-IR700) selectively to induce cancer cell death in vitro and in vivo. The studies in vitro confirmed the specificity of ZHER2:2395-IR700 binding to HER2-positive cells and its ability to produce reactive oxygen species upon light irradiation. A conjugate concentration- and light irradiation-dependent decrease in cell viability was also demonstrated. Furthermore, light-activated ZHER2:2395-IR700 triggered all hallmarks of immunogenic cell death, as defined by the translocation of calreticulin to the cell surface, and the secretion of ATP, HSP70/90 and HMGB1 from dying cancer cells into the medium. Irradiating a co-culture of immature dendritic cells (DCs) and cancer cells exposed to light-activated ZHER2:2395-IR700 enhanced DC maturation, as indicated by augmented expression of CD86 and HLA-DR. In SKOV-3 xenografts, the ZHER2:2395-IR700-based phototherapy delayed tumour growth and increased median overall survival. Collectively, our results strongly suggest that ZHER2:2395-IR700 is a promising new therapeutic conjugate that has great potential to be applicable for photoimmunotherapy-based regimens.

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.

Effect of electrochemotherapy with betulinic acid or cisplatin on regulation of heat shock proteins in metastatic human carcinoma cells in vitro.

Betulinic acid (BTA) is naturally occurring triterpene that has received interest as a novel therapeutic substance with cytotoxicity towards a number of cancer cell lines. Despite the wide spectrum of biological and pharmacological effects, its effect may be limited its lipophobic properties. Therefore, strategies to improve the access of BTA to the cells are required to enhance the anticancer effects. Electroporation (EP) enables increased inflow of drugs into cancer cells, even at low doses, which may reduce the side effects caused by high doses of chemotherapy. The potential application of BTA in electrochemotherapy (ECT) in metastatic type of cancers was investigated in the present study. The efficacy of BTA with EP was estimated using a cell survival assay (MTT assay), microscopical morphology analysis and the immunocytochemical expression of heat shock proteins (HSPs). HSPs are molecules that protect the cell from harmful environmental, chemical and physical stresses, and ensure cell survival, recovery and proper functioning. HSP expression is induced various stress factors. Therefore, the expression of HSP27 and HSP70 was evaluated after cells were exposed to an external pulsed electric field and anticancer drugs. Facilitated drug delivery and the anticancer effect on metastatic tumor cells were evaluated in vitro. The effect of BTA was compared with cisplatin (CP), a standard cytostatic agent. Two different metastatic cancer cell lines were used, an ovary adenocarcinoma cell line (SW626) and melanoma cell line (Me45). BTA combined with EP exhibited similar efficacy to CP with EP after 24 and 48 h in SW626 and Me45 cancer cells. Me45 cells also had high HSP27 and low HSP70 immunosignals post­ECT treatment. ECT caused increased expression of HSP27 and HSP70 proteins in SW626 cells, which were less sensitive to ECT than the Me45 melanoma cell line. The results indicate that BTA may be efficiently applied instead of CP in ECT approaches, but its activity differs between tumor cell lines.

Electroporation with Cisplatin against Metastatic Pancreatic Cancer: In Vitro Study on Human Primary Cell Culture.

Despite the rapid progression of cancer pharmacotherapy, the high drug resistance of pancreatic ductal adenocarcinoma (PDA) makes it one of the most lethal malignancies. Therefore, there are high expectations associated with experimental therapies, such as electrochemotherapy (ECT). This technique involves the application of short electric pulses to induce transitional permeabilization of the cellular membrane, thus enhancing drug molecules influx. The aim of the study was to investigate the influence of electroporation with cisplatin (CisEP) on the primary culture of human PDA cells from lung metastases-their survival and stress response. Considering the growing importance of various research models, two established human PDA cell lines, EPP85-181P (sensitive to daunorubicin) and EPP85-181RDB (resistant to daunorubicin), were utilized as a reference control. Cisplatin revealed higher cytotoxicity towards established cell lines. Following CisEP application, we observed a significant decrease of cells viability in the primary culture model. After CisEP therapy, an increased immunoreactivity with SOD-2 and Casp-3 antibodies was noticed. In conclusion, we discovered that electroporation can enhance the cytotoxic effect of cisplatin in pancreatic cancer cells in vitro. This effect was evident for cells from the primary culture. The obtained results confirm the importance of primary cells models in studies on the efficacy of experimental cancer therapies.

Near-infrared photoimmunotherapy targeting EGFR-Shedding new light on glioblastoma treatment.

Glioblastomas (GBMs) are high-grade brain tumors, differentially driven by alterations (amplification, deletion or missense mutations) in the epidermal growth factor receptor (EGFR), that carry a poor prognosis of just 12-15 months following standard therapy. A combination of interventions targeting tumor-specific cell surface regulators along with convergent downstream signaling pathways may enhance treatment efficacy. Against this background, we investigated a novel photoimmunotherapy approach combining the cytotoxicity of photodynamic therapy with the specificity of immunotherapy. An EGFR-specific affibody (ZEGFR:03115 ) was conjugated to the phthalocyanine dye, IR700DX, which when excited with near-infrared light produces a cytotoxic response. ZEGFR:03115 -IR700DX EGFR-specific binding was confirmed by flow cytometry and confocal microscopy. The conjugate showed effective targeting of EGFR positive GBM cells in the brain. The therapeutic potential of the conjugate was assessed both in vitro, in GBM cell lines and spheroids by the CellTiter-Glo® assay, and in vivo using subcutaneous U87-MGvIII xenografts. In addition, mice were imaged pre- and post-PIT using the IVIS/Spectrum/CT to monitor treatment response. Binding of the conjugate correlated to the level of EGFR expression in GBM cell lines. The cell proliferation assay revealed a receptor-dependent response between the tested cell lines. Inhibition of EGFRvIII+ve tumor growth was observed following administration of the immunoconjugate and irradiation. Importantly, this response was not seen in control tumors. In conclusion, the ZEGFR:03115 -IR700DX showed specific uptake in vitro and enabled imaging of EGFR expression in the orthotopic brain tumor model. Moreover, the proof-of-concept in vivo PIT study demonstrated therapeutic efficacy of the conjugate in subcutaneous glioma xenografts.

Estrogen Receptors in Cell Membranes: Regulation and Signaling.

Estrogens can stimulate the development, proliferation, migration, and survival of target cells. These biological effects are mediated through their action upon the plasma membrane estrogen receptors (ERs). ERs regulate transcriptional processes by nuclear translocation and binding to specific response elements, which leads to the regulation of gene expression. This effect is termed genomic or nuclear. However, estrogens may exert their biological activity also without direct binding to DNA and independently of gene transcription or protein synthesis. This action is called non-genomic or non-nuclear. Through non-genomic mechanisms, estrogens can modify regulatory cascades such as MAPK, P13K, and tyrosine cascade as well as membrane-associated molecules such as ion channels and G-protein-coupled receptors. The recent studies on the mechanisms of estrogen action provide an evidence that non-genomic and genomic effects converge. An example of such convergence is the potential possibility to modulate gene expression through these two independent pathways. The understanding of the plasma membrane estrogen receptors is crucial for the development of novel drugs and therapeutic protocols targeting specific receptor actions.