Expression and purification of a novel single-chain diabody (scDb-hERG1/β1) from Pichia pastoris transformants

Duranti, Claudia; Lastraioli, Elena; Iorio, Jessica; Capitani, Chiara; Carraresi, Laura; Gonnelli, Leonardo; Arcangeli, Annarosa

doi:10.1016/j.pep.2021.105879

Cited by 11 publications

(14 citation statements)

References 15 publications

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“…Finally, the occurrence of a hERG1/β1 integrin complex in primary PDAC was confirmed by IHC using a bispecific antibody, the scDb-hERG1-β1, which selectively recognizes the hERG1/β1 integrin complex [ 31 , 37 ]. In total, 132 primary PDAC samples were analysed and a high (>50% of the cells) scDb-hERG1-β1 staining was detected in 68.2% (90/132) of cases, with a statistically significant association with hERG1 staining ( p < 0.0001) ( Figure 2 ).…”

Section: Resultsmentioning

confidence: 99%

“…The scDb-hERG1-β1 antibody was expressed in yeast cells as described previously [ 31 , 37 ], by performing an induction with methanol and harvesting the supernatant in which the protein is secreted for subsequent purification. Purification of scDb-hERG1-β1 was performed by affinity chromatography, using an ÄKTA protein purification system (Cytiva, Marlborough, MA, USA) with a HisTrap HP 5 mL column as previously described [ 31 , 37 ]. Elution fractions in which the scDb-hERG1-β1 protein was detected were then collected, pooled and dialyzed into a PBS using a Slide-A-Lyzer™ dialysis cassette (Thermo Fisher, Waltham, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

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Combination Therapy with a Bispecific Antibody Targeting the hERG1/β1 Integrin Complex and Gemcitabine in Pancreatic Ductal Adenocarcinoma

Lottini

Duranti

Iorio

et al. 2023

Cancers

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Pancreatic ductal adenocarcinoma (PDAC) represents an unmet medical need. Difficult/late diagnosis as well as the poor efficacy and high toxicity of chemotherapeutic drugs result in dismal prognosis. With the aim of improving the treatment outcome of PDAC, we tested the effect of combining Gemcitabine with a novel single chain bispecific antibody (scDb) targeting the cancer-specific hERG1/β1 integrin complex. First, using the scDb (scDb-hERG1-β1) in immunohistochemistry (IHC), Western blot (WB) analysis and immunofluorescence (IF), we confirmed the presence of the hERG1/β1 integrin complex in primary PDAC samples and PDAC cell lines. Combining Gemcitabine with scDb-hERG1-β1 improved its cytotoxicity on all PDAC cells tested in vitro. We also tested the combination treatment in vivo, using an orthotopic xenograft mouse model involving ultrasound-guided injection of PDAC cells. We first demonstrated good penetration of the scDb-hERG1-β1 conjugated with indocyanine green (ICG) into tumour masses by photoacoustic (PA) imaging. Next, we tested the effects of the combination at either therapeutic or sub-optimal doses of Gemcitabine (25 or 5 mg/kg, respectively). The combination of scDb-hERG1-β1 and sub-optimal doses of Gemcitabine reduced the tumour masses to the same extent as the therapeutic doses of Gemcitabine administrated alone; yielded increased survival; and was accompanied by minimised side effects (toxicity). These data pave the way for a novel therapeutic approach to PDAC, based on the combination of low doses of a chemotherapeutic drug (to minimize adverse side effects and the onset of resistance) and the novel scDb-hERG1-β1 targeting the hERG1/β1 integrin complex as neoantigen.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Combination Therapy with a Bispecific Antibody Targeting the hERG1/β1 Integrin Complex and Gemcitabine in Pancreatic Ductal Adenocarcinoma

Lottini

Duranti

Iorio

et al. 2023

Cancers

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“…Moreover, in vivo tests will be a further step in order to better clarify the biological effects of US treatment, after a proper adaptation of the ultrasound set up. The system will also be useful to allow for the testing of different effects between LICUS and LIPUS efficacy using the same frequencies and exposition time, both alone and in combination with the use of small molecules and antibody fragments, which allow precise tumor penetration and can be easily administered both for therapeutic and diagnostic purposes [12,[19][20][21]. The latter opens the way for a combined pharmacological approach to PDAC treatment.…”

Section: Discussionmentioning

confidence: 99%

Biophysical and Biomechanical Effect of Low Intensity US Treatments on Pancreatic Adenocarcinoma 3D Cultures

et al. 2022

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Current developments in medical technology have focused on therapeutic treatments that selectively and effectively address specific pathological areas, minimizing side effects on healthy tissues. In this regard, many procedures have been developed to provide non-invasive therapy, for example therapeutic ultrasound (US). In the medical field, in particular in cancer research, it has been observed how ultrasounds can cause cell death and inhibit cell proliferation of cancer cells, while preserving healthy ones with almost negligible side effects. Various studies have shown that low intensity pulse ultrasound (LIPUS) and low intensity continuous ultrasound (LICUS) regulate the proliferation, cell differentiation and cavitation phenomena. Nowadays, there are poorly known aspects of low intensity US treatment, in terms of biophysical and biomechanical effects on target cells. The aim of this study is to set up an innovative apparatus for US treatment of pancreatic ductal adenocarcinoma (PDAC) cells, monitoring parameters such as acoustic intensity, acoustic pressure, stimulation frequency and treatment protocol. To this purpose, we have developed a custom-made set up for the US stimulation at 1.2 and 3 MHz of tridimensional (3D) cultures of PDAC cells (PANC-1, Mia Paca-2 and BxPc3 cells). Images of the 3D cultures were acquired, and the Calcein/PI assay was applied to detect US-induced cell death. Overall, the setup we have presented paves the way to an innovative protocol for tumor treatment. The system can be used either alone or in combination with small molecules or recombinant antibodies in order to propose a novel combined therapeutic approach.

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“…Importantly, not all Kv11.1 blockers are arrhythmogenic ( Wallis, 2010 ), examples are represented by Verapamil and sertindole ( D’Amico et al, 2013 ). In order to overcome the cardiotoxicity induced by IC blockers different strategies can be applied ( D’Amico et al, 2013 ): i) using non torsadogenic blockers; ii) using state-specific blockers such as R -roscovitine ( Ganapathi et al, 2009 ); iii) using tumour-specific drugs such as CD 160130 ( Gasparoli et al, 2015 ); iv) using monoclonal antibodies ( Iorio et al, 2019 ) and v) using bispecific antibodies directed against tumour-specific macromolecular complexes such as the Kv11.1/β1 integrin complex ( Duranti et al, 2021a ; 2021b ; Iorio et al, 2022 ; Lottini et al, 2023 ).…”

Section: Ion Channels In Human Cancermentioning

confidence: 99%

Ion channels in lung cancer: biological and clinical relevance

Capitani,

Chioccioli Altadonna,

Santillo

et al. 2023

Front. Pharmacol.

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Despite improvements in treatment, lung cancer is still a major health problem worldwide. Among lung cancer subtypes, the most frequent is represented by adenocarcinoma (belonging to the Non-Small Cell Lung Cancer class) although the most challenging and harder to treat is represented by Small Cell Lung Cancer, that occurs at lower frequency but has the worst prognosis. For these reasons, the standard of care for these patients is represented by a combination of surgery, radiation therapy and chemotherapy. In this view, searching for novel biomarkers that might help both in diagnosis and therapy is mandatory. In the last 30 years it was demonstrated that different families of ion channels are overexpressed in both lung cancer cell lines and primary tumours. The altered ion channel profile may be advantageous for diagnostic and therapeutic purposes since most of them are localised on the plasma membrane thus their detection is quite easy, as well as their block with specific drugs and antibodies. This review focuses on ion channels (Potassium, Sodium, Calcium, Chloride, Anion and Nicotinic Acetylcholine receptors) in lung cancer (both Non-Small Cell Lung Cancer and Small Cell Lung Cancer) and recapitulate the up-to-date knowledge about their role and clinical relevance for a potential use in the clinical setting, for lung cancer diagnosis and therapy.

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Expression and purification of a novel single-chain diabody (scDb-hERG1/β1) from Pichia pastoris transformants

Cited by 11 publications

References 15 publications

Combination Therapy with a Bispecific Antibody Targeting the hERG1/β1 Integrin Complex and Gemcitabine in Pancreatic Ductal Adenocarcinoma

Combination Therapy with a Bispecific Antibody Targeting the hERG1/β1 Integrin Complex and Gemcitabine in Pancreatic Ductal Adenocarcinoma

Biophysical and Biomechanical Effect of Low Intensity US Treatments on Pancreatic Adenocarcinoma 3D Cultures

Ion channels in lung cancer: biological and clinical relevance

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