A Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) for ovarian cancer.

BACKGROUND: Pronectins™ are a new class of fibronectin-3-domain 14th-derived (14Fn3) antibody mimics that can be engineered as bispecific T cell engager (BTCE) to redirect immune effector cells against cancer. We describe here the in vitro and in vivo activity of a Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) against Epithelial Ovarian Cancer (EOC). METHODS: pAXLxCD3ε T-cell mediated cytotoxicity was evaluated by flow cytometry and bioluminescence. pAXLxCD3ε mediated T-cell infiltration, activation and proliferation were assessed by immunofluorescence microscopy and by flow cytometry. Activity of pAXLxCD3ε was also investigated in combination with poly-ADP ribose polymerase inhibitors (PARPi). In vivo antitumor activity of pAXLxCD3ε was evaluated in immunocompromised (NSG) mice bearing intraperitoneal or subcutaneous EOC xenografts and immunologically reconstituted with human peripheral blood mononuclear cells (PBMC). RESULTS: pAXLxCD3ε induced dose-dependent cytotoxicity by activation of T lymphocytes against EOC cells, regardless of their histologic origin. The addition of PARPi to cell cultures enhanced pAXLxCD3ε cytotoxicity. Importantly, in vivo, pAXLxCD3ε was highly effective against EOC xenografts in two different NSG mouse models, by inhibiting the growth of tumor cells in ascites and subcutaneous xenografts. This effect translated into a significantly prolonged survival of treated animals. CONCLUSION: pAXLxCD3ε is an active therapeutics against EOC cells providing a rational for its development as a novel agent in this still incurable disease. The preclinical validation of a first-in-class agent opens the way to the development of a new 14Fn3-based scaffold platform for the generation of innovative immune therapeutics against cancer.

The First-In-Class Anti-AXL×CD3ε Pronectin™-Based Bispecific T-Cell Engager Is Active in Preclinical Models of Human Soft Tissue and Bone Sarcomas.

Sarcomas are heterogeneous malignancies with limited therapeutic options and a poor prognosis. We developed an innovative immunotherapeutic agent, a first-in-class Pronectin™-based Bispecific T-Cell Engager (pAXL×CD3ε), for the targeting of AXL, a TAM family tyrosine kinase receptor highly expressed in sarcomas. AXL expression was first analyzed by flow cytometry, qRT-PCR, and Western blot on a panel of sarcoma cell lines. The T-cell-mediated pAXL×CD3ε cytotoxicity against sarcoma cells was investigated by flow cytometry, luminescence assay, and fluorescent microscopy imaging. The activation and degranulation of T cells induced by pAXL×CD3ε were evaluated by flow cytometry. The antitumor activity induced by pAXL×CD3ε in combination with trabectedin was also investigated. In vivo activity studies of pAXL×CD3ε were performed in immunocompromised mice (NSG), engrafted with human sarcoma cells and reconstituted with human peripheral blood mononuclear cells from healthy donors. Most sarcoma cells showed high expression of AXL. pAXL×CD3ε triggered T-lymphocyte activation and induced dose-dependent T-cell-mediated cytotoxicity. The combination of pAXL×CD3ε with trabectedin increased cytotoxicity. pAXL×CD3ε inhibited the in vivo growth of human sarcoma xenografts, increasing the survival of treated mice. Our data demonstrate the antitumor efficacy of pAXL×CD3ε against sarcoma cells, providing a translational framework for the clinical development of pAXL×CD3ε in the treatment of human sarcomas, aggressive and still-incurable malignancies.

AXL-Receptor Targeted 14FN3 Based Single Domain Proteins (Pronectins™) from 3 Synthetic Human Libraries as Components for Exploring Novel Bispecific Constructs against Solid Tumors.

A highly specific AXL-receptor targeted family of non-immunoglobulin, single domain protein binders (Pronectins™) have been isolated from three (3) synthetic libraries that employ the human scaffold of the 14th domain of Fibronectin III (14FN3) and evolutionary CDRs diversity of over 25 billion loop sequences. The three libraries, each containing diversity in two loops, were designed to expand upon a human database of more than 6000 natural scaffold sequences and approximately 3000 human loop sequences. We used a bioinformatic-based approach to maximize "human" amino acid loop diversity and minimize or prevent altogether CDR immunogenicity created by the use of mutagenesis processes to generate diversity. A combination of phage display and yeast display was used to isolate 59 AXL receptor targeted Pronectins with KD ranging between 2 and 100 nM. FACS analysis with tumor cells over-expressing AXL and the use of an AXL knock-out cell line allowed us to identify Pronectin candidates with exquisite specificity for AXL receptor. Based upon several in vitro cell-based tests, we selected the best candidate, AXL54, to further characterize its in vitro cancer cells killing activity. Finally, AXL54 was used to produce the first bi-specific T cell engager protein (AXL54 [Pronectin]-linker-scFV CD3), a "new in class" protein for further testing of its anti-tumor activity in vitro and in vivo.

Potential prevention and treatment of neurodegenerative disorders by olive polyphenols and hidrox.

Most chronic illnesses are caused by the biological reaction to an injury, rather than the initial injury or the injurious agent itselves as in neurodegeneration. With respect to this, notable attention is emerging on the therapeutic effects of dietary polyphenols for human health, able to counteract and neutralize oxidative stress and inflammatory processes involved in the etiopathogenesis of major neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. The acquired concept that cellular stress at low doses induces neuroprotective responses against degenerative processes is a frontier area of the neurobiological research focusing on the development of novel preventive and therapeutic interventions for neurodegenerative disorders. Notably, basal levels of prooxidant species are essential to promote adaptive redox cellular responses including vitagenes, tightly correlated to cell survival against age-related diseases. In this paper we discuss the concept of cellular stress response and hormesis and its applications to the field of neuroprotection and the potential therapeutic support provided by olive polyphenols, in particular hydroxytyrosol (HT)-rich aqueous olive pulp extract (Hidrox), as a pivotal activator of Nrf2 pathway and related vitagenes, and inhibitor of Keap1-Nrf2 interaction.Olive polyphenols are considered potential pharmacological modulators of neuroinflammation by upregulation of the Keap1/Nfr2/ARE pathway thus providing a strong rationale for treating neurodegenerative disorders.

Wnt/ß-Catenin Pathway in Experimental Model of Fibromyalgia: Role of Hidrox®.

Fibromyalgia (FM) is a chronic condition characterized by persistent widespread pain that negatively affects the quality of life of patients. The WNT/ß-catenin signaling pathway seems to be involved in central sensitization and different pain states. The objective of this study was to investigate the beneficial effects of a new compound called Hidrox® (HD), containing 40-50% hydroxytyrosol, in counteracting the pain associated with FM. An FM-like model was induced in rats by subcutaneous injections of reserpine (1 mg/kg) for three consecutive days. Later, HD (10 mg/kg) was administered orally to the animals for seven days. Reserpine injections induced WNT/ß-catenin pathway activation, release of pro-inflammatory mediators as well as a significant increase in oxidative stress. Daily treatment with HD was able to modulate the WNT/ß-catenin and Nrf2 pathways and consequently attenuate the behavioral deficits and microglia activation induced by reserpine injection. These results indicate that nutritional consumption of HD can be considered as a new therapeutic approach for human FM.

Redox modulation of vitagenes via plant polyphenols and vitamin D: Novel insights for chemoprevention and therapeutic interventions based on organoid technology.

Polyphenols are chemopreventive through the induction of nuclear factor erythroid 2 related factor 2 (Nrf2)-mediated proteins and anti-inflammatory pathways. These pathways, encoding cytoprotective vitagenes, include heat shock proteins, such as heat shock protein 70 (Hsp70) and heme oxygenase-1 (HO-1), as well as glutathione redox system to protect against cancer initiation and progression. Phytochemicals exhibit biphasic dose responses on cancer cells, activating at low dose, signaling pathways resulting in upregulation of vitagenes, as in the case of the Nrf2 pathway upregulated by hydroxytyrosol (HT) or curcumin and NAD/NADH-sirtuin-1 activated by resveratrol. Here, the importance of vitagenes in redox stress response and autophagy mechanisms, as well as the potential use of dietary antioxidants in the prevention and treatment of multiple types of cancer are discussed. We also discuss the possible relationship between SARS-CoV-2, inflammation and cancer, exploiting innovative therapeutic approaches with HT-rich aqueous olive pulp extract (Hidrox®), a natural polyphenolic formulation, as well as the rationale of Vitamin D supplementation. Finally, we describe innovative approaches with organoids technology to study human carcinogenesis in preclinical models from basic cancer research to clinical practice, suggesting patient-derived organoids as an innovative tool to test drug toxicity and drive personalized therapy.

Hidrox® and Chronic Cystitis: Biochemical Evaluation of Inflammation, Oxidative Stress, and Pain.

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic bladder condition characterized by frequent urination, inflammation, oxidative stress, and pain. The aim of the study was to evaluate the anti-inflammatory and antioxidant effects of an oral administration of Hidrox® (10 mg/kg) in the bladder and spinal cord in a rodent model of IC/BPS. The chronic animal model of cystitis was induced by repeated intraperitoneal injections of cyclophosphamide (CYP) for five consecutive days. Treatment with Hidrox® began on the third day of the CYP injection and continued until the 10th day. CYP administration caused macroscopic and histological bladder changes, inflammatory infiltrates, increased mast cell numbers, oxidative stress, decreased expression of the tight endothelial junction (e.g., zonula occludens-1 (ZO-1) and occludin), and bladder pain. Treatment with Hidrox® was able to improve CYP-induced inflammation and oxidative stress via the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway. It was also able to reduce bladder pain which was aggravated by the activation of neuroinflammation in the central nervous system. In particular, Hidrox® reduced the brain-derived neurotrophic factor (BDNF), as well as the activation of astrocytes and microglia, consequently reducing mechanical allodynia. These results indicate that nutritional consumption of Hidrox® can be considered as a new therapeutic approach for human cystitis, increasing the conceivable potential of a significant improvement in the quality of life associated with a lowering of symptom intensity in patients with IC/BPS.

Hidrox® and Endometriosis: Biochemical Evaluation of Oxidative Stress and Pain.

Endometriosis is a gynecological and painful condition affecting women of reproductive age. It is characterized by dysfunctional endometrium-like implants outside of the uterine cavity. The purpose of this study was to evaluate the effects of Hidrox®, an aqueous extract of olive pulp containing hydroxytyrosol, on endometriotic lesions associated with pro-oxidative alterations and pain-like behaviors. Endometriosis was induced by intraperitoneal injection of uterine fragments, and Hidrox® was administered daily. At the end of the 14-day treatment, behavioral alterations were assessed and hippocampal tissues were collected. Laparotomy was performed, and the endometrial implants were harvested for histological and biochemical analysis. Hidrox® treatment reduced endometriotic implant area, diameter and volumes. Vehicle-treated rats showed lesional fibrosis, epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation, angiogenesis and pro-oxidative alterations in the peritoneal cavity. Hidrox® treatment reduced the aniline blue-stained area, α-smooth muscle actin (α-sma) and CD34 positive expressions. Moreover, it reduced mast cell recruitment into the lesions, myeloperoxidase activity and lipid peroxidation and increased superoxide dismutase (SOD) activity and glutathione levels in the endometrial explants. In the peritoneal fluid, Hidrox® treatment reduced interleukin (IL)-1ß, IL2, IL6, tumor necrosis factor-α (TNF-α) and vascular endothelial grow factor (VEGF) levels increased by the disease. Hidrox® administration also reduced peripheral and visceral sensibility as shown by the behavioral tests (open field test, hot plate test, elevated plus maze test and acetic-acid-induced abdominal contractions). Animals treated with Hidrox® also showed reduced blood-brain barrier permeability and mast cell infiltration in the hippocampus, as well as astrocyte and microglia activation and brain oxidative status restoring brain-derived neurotrophic factor (BDNF) protein expression and increasing Nuclear factor erythroid 2-related factor 2 (Nfr2) nuclear translocation. In conclusion, Hidrox® displayed potential ameliorative effects on endometriotic implants and related pain-induced behaviors due to its potent antioxidative properties.

Hidrox® Counteracts Cyclophosphamide-Induced Male Infertility through NRF2 Pathways in a Mouse Model.

BACKGROUND: Every year, men use cyclophosphamide to treat various cancers and autoimmune diseases. On the one hand, this chemotherapy often has the beneficial effect of regressing the tumor, but on the other hand, it leads to infertility due to excessive oxidative stress and apoptosis in the testes caused by its metabolite, acrolein. METHODS: The objective of this study was to evaluate the beneficial power of a new compound called Hidrox®, containing 40-50% hydroxytyrosol, in counteracting the damage related to fertility induced by cyclophosphamide. The study was conducted using a single intraperitoneal injection of cyclophosphamide at a dose of 200 mg/kg b.w, in distilled water at 10 mL/kg b.w. The treatment was administered via the oral administration of Hidrox® at a dose of 50 mg/kg. RESULTS: Our study confirms that the use of cyclophosphamide causes a series of sperm and histological alterations strongly connected with oxidative stress, lipid peroxidation, and apoptosis. CONCLUSION: Our results demonstrate for the first time that Hidrox® protects testes from CYP-induced alterations by the modulation of physiological antioxidant defenses.

Anti-inflammatory and Anti-oxidant Activity of Hidrox® in Rotenone-Induced Parkinson's Disease in Mice.

BACKGROUND: In developed countries, the extension of human life is increasingly accompanied by a progressive increase in neurodegenerative diseases, most of which do not yet have effective therapy but only symptomatic treatments. In recent years, plant polyphenols have aroused considerable interest in the scientific community. The mechanisms currently hypothesized for the pathogenesis of Parkinson's disease (PD) are neuroinflammation, oxidative stress and apoptosis. Hydroxytyrosol (HT), the main component of Hidrox® (HD), has been shown to have some of the highest free radical evacuation and anti-inflammatory activities. Here we wanted to study the role of HD on the neurobiological and behavioral alterations induced by rotenone. METHODS: A study was conducted in which mice received HD (10 mg/kg, i.p.) concomitantly with rotenone (5 mg/kg, o.s.) for 28 days. RESULTS: Locomotor activity, catalepsy, histological damage and several characteristic markers of the PD, such as the dopamine transporter (DAT) content, tyrosine hydroxylase (TH) and accumulation of α-synuclein, have been evaluated. Moreover, we observed the effects of HD on oxidative stress, neuroinflammation, apoptosis and inflammasomes. Taken together, the results obtained highlight HD's ability to reduce the loss of dopaminergic neurons and the damage associated with it by counteracting the three main mechanisms of PD pathogenesis. CONCLUSION: HD is subject to fewer regulations than traditional drugs to improve patients' brain health and could represent a promising nutraceutical choice to prevent PD.

Healthspan Enhancement by Olive Polyphenols in C. elegans Wild Type and Parkinson's Models.

Parkinson's disease (PD) is the second most prevalent late-age onset neurodegenerative disorder, affecting 1% of the population after the age of about 60 years old and 4% of those over 80 years old, causing motor impairments and cognitive dysfunction. Increasing evidence indicates that Mediterranean diet (MD) exerts beneficial effects in maintaining health, especially during ageing and by the prevention of neurodegenerative disorders. In this regard, olive oil and its biophenolic constituents like hydroxytyrosol (HT) have received growing attention in the past years. Thus, in the current study we test the health-promoting effects of two hydroxytyrosol preparations, pure HT and Hidrox® (HD), which is hydroxytyrosol in its "natural" environment, in the established invertebrate model organism Caenorhabditis elegans. HD exposure led to much stronger beneficial locomotion effects in wild type worms compared to HT in the same concentration. Consistent to this finding, in OW13 worms, a PD-model characterized by α-synuclein expression in muscles, HD exhibited a significant higher effect on α-synuclein accumulation and swim performance than HT, an effect partly confirmed also in swim assays with the UA44 strain, which features α-synuclein expression in DA-neurons. Interestingly, beneficial effects of HD and HT treatment with similar strength were detected in the lifespan and autofluorescence of wild-type nematodes, in the neuronal health of UA44 worms as well as in the locomotion of rotenone-induced PD-model. Thus, the hypothesis that HD features higher healthspan-promoting abilities than HT was at least partly confirmed. Our study demonstrates that HD polyphenolic extract treatment has the potential to partly prevent or even treat ageing-related neurodegenerative diseases and ageing itself. Future investigations including mammalian models and human clinical trials are needed to uncover the full potential of these olive compounds.

NF-κß signaling and chronic inflammatory diseases: exploring the potential of natural products to drive new therapeutic opportunities.

Chronic inflammation has a key role in the pathogenesis of multiple diseases that represent major public health and financial concerns, including heart failure (HF), Alzheimer's disease (AD) and arthritis. Nuclear factor kappa ß (NF-κß) is a central component of inflammation; owing to its upstream signaling position, it is considered an attractive target for new anti-inflammatory therapeutics. Hydroxytyrosol is an orally bioavailable polyphenol, obtained from olives, which inhibits NF-κß activity and has elicited promising efficacy signals in several inflammatory diseases. Here, we further examine the role of NF-κß in inflammation, provide an introduction to natural products and their anti-inflammatory effects and explore the potential of hydroxytyrosol as a new approach to combating the burden of chronic inflammatory diseases.

Olive phenolics increase glutathione levels in healthy volunteers.

Several in vitro and in vivo studies have shown that olive phenols exert potent biological activities including, but not limited to, antioxidant actions. These activities are shared by phenols found in olives, olive oil, and olive mill wastewater (OMWW). The aim of this study was to investigate whether a commercially available OMWW preparation could influence some parameters of oxidative status in healthy human volunteers. Ninety-eight healthy subjects with normal body weight were recruited, and 5 mL of blood was drawn from their antecubital vein after an overnight fast of at least 12 h. After this, subjects were asked to ingest 2 mL of a commercially available OMWW preparation. Another 5 mL of blood was drawn 1 h after ingestion of the preparation. Plasma antioxidant capacity and total and reduced glutathione were measured. No difference in plasma antioxidant capacity was observed between baseline and 1 h after the ingestion of the extract. Conversely, a significant increase in total plasma glutathione concentration was measured. This increase involved both the reduced and oxidized forms of glutathione; hence, their ratio was unaffected by the treatment. The observed effects of OMWW on glutathione levels might be governed by the antioxidant response element (ARE)-mediated increase in phase II enzyme expression, including that of gamma-glutamylcysteine ligase and glutathione synthetase. Future studies on groups of individuals who may benefit from an increase in their glutathione levels, for example, the elderly, will further elucidate the biological activities of this formulation.

A general method for greatly improving the affinity of antibodies by using combinatorial libraries.

Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-alpha antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (K(d) = 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and/or three CDRs for each V(H) and V(L) domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-alpha. These beneficial mutations in both V(H) and V(L) were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500- and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15- to 30-fold improvement in in vitro TNF-alpha neutralization in an L929 bioassay. Thus, this LTM/combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins.

Hydrolyzed olive vegetation water in mice has anti-inflammatory activity.

Fruit and vegetable simple and polyphenols are potent antioxidants. One of the most effective in terms of free radical scavenging is 3,4-dihydroxyphenyl ethanol or hydroxytyrosol (HT), a simple phenol found predominantly in Olea europea, or the olive plant. HT is most abundant in the aqueous fraction of olive pulp with trace amounts in the olive oil fraction and in the leaves. For these experiments, we evaluated the anti-inflammatory activity of olive vegetation water (OVW), which we showed previously to have potent antioxidant activity. Because some simple phenols and polyphenols with antioxidant activity have shown varying anti-inflammatory activities, we tested OVW and HT for their ability to inhibit the production of tumor necrosis factor-alpha (TNF-alpha), a pivotal cytokine in inflammation. In lipopolysaccharide (LPS)-treated BALB/c mice, a model system of inflammation, OVW at a dose of 125 mg/mouse (500 mg/kg) reduced serum TNF-alpha levels by 95%. In the human monocyte cell line, THP-1, OVW reduced LPS-induced TNF-alpha production by 50% at a concentration of 0.5 g/L (equivalent to approximately 0.03 g/L simple and polyphenols). OVW had no toxic effects in vitro or in vivo. When OVW was combined with glucosamine, a component of proteoglycans and glycoproteins that was shown to decrease inducible nitric oxide synthase production in cultured macrophage cells, the 2 compounds acted synergistically to reduce serum TNF-alpha levels in LPS-treated mice. These findings suggest that a combination of OVW and glucosamine may be an effective therapy for a variety of inflammatory processes, including rheumatoid and osteoarthritis.