Background:hERG1 channels are aberrantly expressed in human cancers. The expression, functional role and clinical significance of hERG1 channels in pancreatic ductal adenocarcinoma (PDAC) is lacking.Methods:hERG1 expression was tested in PDAC primary samples assembled as tissue microarray by immunohistochemistry using an anti-hERG1 monoclonal antibody (α-hERG1-MoAb). The functional role of hERG1 was studied in PDAC cell lines and primary cultures. ERG1 expression during PDAC progression was studied in Pdx-1-Cre,LSL-KrasG12D/+,LSL-Trp53R175H/+ transgenic (KPC) mice. ERG1 expression in vivo was determined by optical imaging using Alexa-680-labelled α-hERG1-MoAb.Results:(i) hERG1 was expressed at high levels in 59% of primary PDAC; (ii) hERG1 blockade decreased PDAC cell growth and migration; (iii) hERG1 was physically and functionally linked to the Epidermal Growth Factor-Receptor pathway; (iv) in transgenic mice, ERG1 was expressed in PanIN lesions, reaching high expression levels in PDAC; (v) PDAC patients whose primary tumour showed high hERG1 expression had a worse prognosis; (vi) the α-hERG1-MoAb could detect PDAC in vivo.Conclusions:hERG1 regulates PDAC malignancy and its expression, once validated in a larger cohort also comprising of late-stage, non-surgically resected cases, may be exploited for diagnostic and prognostic purposes in PDAC either ex vivo or in vivo.
Checkpoint inhibitors (CPI) have revolutionized the treatment paradigm for advanced solid tumors; however, there remains an opportunity to improve response rates and outcomes. In preclinical models, 4-1BB costimulation synergizes with CPIs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis by activating cytotoxic T-cell–mediated antitumor immunity. DuoBody-PD-L1×4-1BB (GEN1046) is an investigational, first-in-class bispecific immunotherapy agent designed to act on both pathways by combining simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046 induced T-cell proliferation, cytokine production, and antigen-specific T-cell–mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures and exerted potent antitumor activity in transplantable mouse tumor models. In dose escalation of the ongoing first-in-human study in heavily pretreated patients with advanced refractory solid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheral blood consistent with its mechanism of action, manageable safety, and early clinical activity [disease control rate: 65.6% (40/61)], including patients resistant to prior PD-(L)1 immunotherapy. Significance: DuoBody-PD-L1×4-1BB (GEN1046) is a first-in-class bispecific immunotherapy with a manageable safety profile and encouraging preclinical and early clinical activity. With its ability to confer clinical benefit in tumors typically less sensitive to CPIs, GEN1046 may fill a clinical gap in CPI-relapsed or refractory disease or as a combination therapy with CPIs.
IntroductionWe previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis.MethodsViral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis.Results1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10).ConclusionsTargeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.
Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-à-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging.The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the VH domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the “native” anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo.In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.
Titanium dioxide (TiO2) has been widely used in many nanotechnology areas including nanomedicine, where it could be proposed for the photodynamic and sonodynamic cancer therapies. However, TiO2 nanoformulations have been shown to be toxic for living cells. In this article, we report the development of a new delivery system, based on nontoxic TiO2 nanoparticles, further conjugated with a monoclonal antibody against a novel and easily accessible tumor marker, e.g., the Kv 11.1 potassium channel. We synthesized, by simple solvothermal method, dicarboxylic acid-terminated PEG TiO2 nanocrystals (PEG–TiO2 NPs). Anti-Kv 11.1 monoclonal antibodies (Kv 11.1-Mab) were further linked to the terminal carboxylic acid groups. Proper conjugation was confirmed by X-ray photoelectron spectroscopy analysis. Kv 11.1-Mab-PEG–TiO2 NPs efficiently recognized the specific Kv 11.1 antigen, both in vitro and in pancreatic ductal adenocarcinoma (PDAC) cells, which express the Kv 11.1 channel onto the plasma membrane. Both PEG TiO2 and Kv 11.1-Mab-PEG–TiO2 NPs were not cytotoxic, but only Kv 11.1-Mab-PEG–TiO2 NPs were efficiently internalized into PDAC cells. Data gathered from this study may have further applications for the chemical design of nanostructures to be applied for therapeutic purposes in pancreatic cancer.Electronic supplementary materialThe online version of this article (doi:10.1007/s11051-013-2111-6) contains supplementary material, which is available to authorized users.
Background Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. Methods CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. Results CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. Conclusions CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.
Background and aimsThe aim of the present investigation was to examine the anti-wasting effects of theophylline (a methylxantine present in tea leaves) on a rat model of cancer cachexia.MethodsThe in vitro effects of the nutraceuticals on proteolysis were examined on muscle cell cultures submitted to hyperthermia. Individual muscle weights, muscle gene expression, body composition and cardiac function were measured in rats bearing the Yoshida AH-130 ascites hepatoma, following theophylline treatment.ResultsTheophylline treatment inhibited proteolysis in C2C12 cell line and resulted in an anti-proteolytic effect on muscle tissue (soleus and heart), which was associated with a decrease in circulating TNF-alpha levels and with a decreased proteolytic systems gene expression. Treatment with the nutraceutical also resulted in an improvement in body composition and cardiac function.ConclusionTheophylline - alone or in combination with drugs - may be a candidate molecule for the treatment of cancer cachexia.
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