Spike mutation resilient scFv76 antibody counteracts SARS-CoV-2 lung damage upon aerosol delivery.

The uneven worldwide vaccination coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of variants escaping immunity call for broadly effective and easily deployable therapeutic agents. We have previously described the human single-chain scFv76 antibody, which recognizes SARS-CoV-2 Alpha, Beta, Gamma and Delta variants. We now show that scFv76 also neutralizes the infectivity and fusogenic activity of the Omicron BA.1 and BA.2 variants. Cryoelectron microscopy (cryo-EM) analysis reveals that scFv76 binds to a well-conserved SARS-CoV-2 spike epitope, providing the structural basis for its broad-spectrum activity. We demonstrate that nebulized scFv76 has therapeutic efficacy in a severe hACE2 transgenic mouse model of coronavirus disease 2019 (COVID-19) pneumonia, as shown by body weight and pulmonary viral load data. Counteraction of infection correlates with inhibition of lung inflammation, as observed by histopathology and expression of inflammatory cytokines and chemokines. Biomarkers of pulmonary endothelial damage were also significantly reduced in scFv76-treated mice. The results support use of nebulized scFv76 for COVID-19 induced by any SARS-CoV-2 variants that have emerged so far.

Synthesis and preliminary in vitro evaluation of DOTA-Tenatumomab conjugates for theranostic applications in tenascin expressing tumors.

Tenatumomab is an anti-tenascin murine monoclonal antibody previously used in clinical trials for delivering radionuclides to tumors by both pre-targeting (biotinylated Tenatumomab within PAGRIT) and direct 131Iodine labeling approaches. Here we present the synthesis and in vitro characterization of three Tenatumomab conjugates to bifunctional chelating agents (NHS-DOTA, NCS-DOTA and NCS-DTPA). Results indicate ST8198AA1 (Tenatumomab-DOTAMA, derived by conjugation of NHS-DOTA), as the most promising candidate in terms of conjugation rate and yield, stability, antigen immunoreactivity and affinity. Labeling efficiency of the different chelators was investigated with a panel of cold metals indicating DOTAMA as the best chelator. Labeling of Tenatumomab-DOTAMA was then optimized with several metals and stability performed confirms suitability of this conjugate for further development. ST8198AA1 represents an improvement of the previous antibody forms because the labeling with radionuclides like 177Lu or 64Cu would allow theranostic applications in patients bearing tenascin expressing tumors.

Radionuclide Therapy of Unresectable Tumors with AvidinOX and (90)Y-biotinDOTA: Tongue Cancer Paradigm.

Local treatment of unresectable tumors is challenging, particularly with radioactivity. Current practice relies on external beam irradiation or on a variety of medical devices for brachytherapy. Both approaches proved useful in controlling tumor growth, but are characterized by poor compliance of the patient, significant side-effects, high costs, and technological complexity, which hamper widespread use. The authors recently described a novel form of radionuclide therapy based on the oxidized form of avidin that, chemically reacting with tissue proteins, can secure radioactive biotin within the injected tissue, either when precomplexed or when taken from the blood stream after intravenous administration. AvidinOX-pretargeted (177)Lu-biotinDOTA ((177)Lu-ST2210) is currently under clinical investigation for the treatment of liver oligometastases from colorectal cancer (clinicaltrials.gov/NCT02053324). In the present work, the authors show that injected AvidinOX can link tissues of various natures such as prostate, kidney, breast, or brain and can react by contact with scraped tissues such as skin or urinary bladder. AvidinOX injected into human OSC19 tongue cancer masses orthotopically transplanted in nude mice takes up intravenously administered (90)Y-ST2210, which exerts significant antitumor activity, while preserving the integrity and functionality of the tongue. Present data confirm that AvidinOX-based radionuclide therapy is an innovative and promising approach for the local treatment of inoperable tumors.

Efficacy of aerosol therapy of lung cancer correlates with EGFR paralysis induced by AvidinOX-anchored biotinylated Cetuximab.

Lung cancer, as well as lung metastases from distal primary tumors, could benefit from aerosol treatment. Unfortunately, because of lung physiology, clearance of nebulized drugs is fast, paralleled by unwanted systemic exposure. Here we report that nebulized AvidinOX can act as an artificial receptor for biotinylated drugs. In nude and SCID mice with advanced human KRAS-mutated A549 metastatic lung cancer, pre-nebulization with AvidinOX enables biotinylated Cetuximab to control tumor growth at a dose lower than 1/25,000 the intravenous effective dose. This result correlates with a striking, specific and unpredictable effect of AvidinOX-anchored biotinylated Cetuximab, as well as Panitumumab, observed on a panel of tumor cell lines, leading to inhibition of dimerization and signalling, blockade of endocytosis, induction of massive lysosomal degradation and abrogation of nuclear translocation of EGFR. Excellent tolerability, together with availability of pharmaceutical-grade AvidinOX and antibodies, will allow rapid clinical translation of the proposed therapy.

Chemical linkage to injected tissues is a distinctive property of oxidized avidin.

We recently reported that the oxidized avidin, named AvidinOX®, resides for weeks within injected tissues as a consequence of the formation of Schiff's bases between its aldehyde groups and tissue protein amino groups. We also showed, in a mouse pre-clinical model, the usefulness of AvidinOX for the delivery of radiolabeled biotin to inoperable tumors. Taking into account that AvidinOX is the first oxidized glycoprotein known to chemically link to injected tissues, we tested in the mouse a panel of additional oxidized glycoproteins, with the aim of investigating the phenomenon. We produced oxidized ovalbumin and mannosylated streptavidin which share with avidin glycosylation pattern and tetrameric structure, respectively and found that neither of them linked significantly to cells in vitro nor to injected tissues in vivo, despite the presence of functional aldehyde groups. The study, extended to additional oxidized glycoproteins, showed that the in vivo chemical conjugation is a distinctive property of the oxidized avidin. Relevance of the high cationic charge of avidin into the stable linkage of AvidinOX to tissues is demonstrated as the oxidized acetylated avidin lost the property. Plasmon resonance on matrix proteins and cellular impedance analyses showed in vitro that avidin exhibits a peculiar interaction with proteins and cells that allows the formation of highly stable Schiff's bases, after oxidation.

Preclinical pharmacology and safety of a novel avidin derivative for tissue-targeted delivery of radiolabelled biotin.

We recently described an oxidized avidin variant, named AvidinOX(®) , which is a product that chemically links to tissue proteins while maintaining the capacity to uptake intravenously administered biotin. Such product proved to be successful in targeting radionuclide therapy in a mouse model of inoperable breast cancer. Here, we show that the uptake of a single or multiple doses of biotin (up to five times), by the tissue-bound AvidinOX(®) , is stable for 2 weeks. Taking into account that oxidized avidin is the first chemically reactive protein to be proposed for clinical use, we evaluated its tolerability, immunogenicity and mutagenicity. Present in vitro data indicate that AvidinOX(®) (up to 10 µg/5 × 10(5) cells) does not affect cell viability or proliferation of PC3 human prostate cancer or 3T3 mouse fibroblast cell lines as well as primary mouse spleen cells. Safety pharmacology and toxicology studies were conducted using AvidinOX(®) up to the highest concentration compatible with its solubility (about 12 mg/mL), representing four times the product concentration intended for human use, and in the maximum administrable volume compatible with each study system. The intramuscular administration in rat and monkey induced a moderate to strong inflammatory response particularly after a second administration and consistently with the induction of an immune response. Interestingly, the intramuscular administration of AvidinOX(®) to rodents and monkeys exhibiting very high anti-avidin antibody titres was well tolerated with no systemic symptoms of any kind. Intravenous administration of AvidinOX(®) , performed to mimic an accidental injection of the dose intended for a local administration (15 µL of 3.3 mg/mL solution), showed significant localization of the product into the spleen not associated with uptake of the radiolabelled biotin intravenously injected after 24 hr, thus suggesting rapid inactivation. No mutagenic activity was induced by oxidized avidin in prokaryotic and eukaryotic cells. Overall, the present data indicate that AvidinOX(®) is well tolerated in rodents and non-human primates, thus supporting its clinical use within protocols of radionuclide therapy of inoperable tumour lesions.

Therapeutic use of avidin is not hampered by antiavidin antibodies in humans.

Hen egg white avidin is increasingly used in the clinic as part of multifactor treatments such as pretargeted radionuclide therapy of cancer or as an antidote of biotinylated drugs. Taking into account that naturally occurring human antiavidin antibodies (HAVA) are common in humans, the present work investigates avidin immunogenicity as part of risk/benefit evaluations. Sera from 139 oncology patients naive to avidin were confirmed to exhibit HAVA with lognormally distributed titers. HAVA were boosted after avidin treatment, with no correlation with the avidin dose or with the basal titer. No antibody-related clinical symptoms were observed in 21 HAVA-positive patients treated with avidin. In mouse models, high mouse antiavidin antibody titers, induced to simulate the worst human condition, neither reduced the biotin uptake of intratissue-injected avidin nor affected the capacity of intravenously injected avidin to clear a biotinylated drug from circulation. In both models the avidin treatment was well tolerated. Results indicate that avidin immunogenicity does not affect its safety and efficacy, thus encouraging its further use in clinical applications.

AvidinOX for highly efficient tissue-pretargeted radionuclide therapy.

Avidin is widely used in vitro for its capacity to bind biotin. However, avidin's in vivo use is limited by its short residence in blood and tissues. An avidin variant, named AvidinOX, has been recently described. This product is obtained by 4-hydroxyazobenzene-2'-carboxylic acid-assisted sodium periodate oxidation of avidin. This method generates aldehyde groups from avidin carbohydrates, sparing biotin-binding sites from inactivation. AvidinOX binds cellular and interstitial protein amino groups through Schiff's bases, resulting in a tissue half-life of 2 weeks, compared with 2 hours of native avidin. Binding of AvidinOX occurs in normal and neoplastic tissues. Data show that AvidinOX, administered intranipple in the breast of transgenic BALB/neuT mice, is highly efficient for capturing (90)Y-biotinDOTA, intravenously injected after 48 hours, leading to eradication of multifocal cancer lesions. Efficacy data, together with good tolerability results, indicate that AvidinOX is a highly innovative reagent for tissue-pretargeted radionuclide therapy.

Biochemical and biological characterization of a new oxidized avidin with enhanced tissue binding properties.

Chicken avidin and bacterial streptavidin are widely employed in vitro for their capacity to bind biotin, but their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in medical treatments. Here we investigate the biochemical and biological properties of a new modified avidin, obtained by ligand-assisted sodium periodate oxidation of avidin. This method allows protection of biotin-binding sites of avidin from inactivation caused by the oxidation step and delay of avidin clearance from injected tissue by generation of aldehyde groups from avidin carbohydrate moieties. Oxidized avidin shows spectroscopic properties similar to that of native avidin, indicating that tryptophan residues are spared from oxidation damage. In strict agreement with these results, circular dichroism and isothermal titration calorimetry analyses confirm that the ligand-assisted oxidation preserves the avidin protein structure and its biotin binding capacity. In vitro cell binding and in vivo tissue residence experiments demonstrate that aldehyde groups provide oxidized avidin the property to bind cellular and interstitial protein amino groups through Schiff's base formation, resulting in a tissue half-life of 2 weeks, compared with 2 h of native avidin. In addition, the efficient uptake of the intravenously injected (111)In-BiotinDOTA (ST2210) in the site previously treated with modified avidin underlines that tissue-bound oxidized avidin retains its biotin binding capacity in vivo. The results presented here indicate that oxidized avidin could be employed to create a stable artificial receptor in diseased tissues for the targeting of biotinylated therapeutics.

OXavidin for tissue targeting biotinylated therapeutics.

Avidin is a glycoprotein from hen egg white that binds biotin with very high affinity. Here we describe OXavidin, a product containing aldehyde groups, obtained by ligand-assisted sugar oxidation of avidin by sodium periodate. OXavidin chemically reacts with cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks while preserving the biotin binding capacity. The long tissue residence of OXavidin as well as that of OXavidin/biotinylated agent complex occurs in normal and neoplastic tissues and immunohistochemistry shows a strong and homogenous stromal localization. Once localized in tissue/tumor, OXavidin becomes an "artificial receptor" for intravenous injected biotin allowing tumor targeting with biotinylated therapeutics like radioisotopes or toxins. Moreover, present data also suggest that OXavidin might be useful for the homing of biotinylated cells. Overall, OXavidin exhibits a remarkable potential for many different therapeutic applications.

Low and high tenascin-expressing tumors are efficiently targeted by ST2146 monoclonal antibody.

ST2146biot is a biotinylated anti-tenascin monoclonal antibody (mAb) to be used for Pretargeted Antibody Guided Radioimmunotherapy (PAGRIT) of solid tumors. In vivo biodistribution studies of (125)I-labeled ST2146biot were done in nude mice transplanted with human HT-29 colon carcinoma and/or human U-118MG glioblastoma cells characterized for low and high tenascin expression, respectively. In vitro results show that ST2146 retains immunoreactivity upon biotinylation, in contrast to other anti-tenascin mAbs. In vivo biodistribution of ST2146 shows specific tumor accumulation up to 10 days after the i.v. injection, with no relevant differences between biotinylated and nonbiotinylated ST2146. A dose of 4 microg/mouse saturates the low tenascin-expressing human colon carcinoma HT-29, whereas the high tenascin-expressing human glioblastoma U-118MG seems to be saturated at a ST2146biot dose between 320 and 640 microg/mouse. The percentage of injected dose per gram of tumor ranges from 10% to 30%, corresponding to an amount of ST2146biot/g of tumor of approximately 400 ng/g and >200 microg/g for HT-29 and U-118MG, respectively. Tumor to normal organs uptake ratios are between 15 and 60, confirming high tumor selectivity of ST2146biot despite its cross-reactivity with the tenascin expressed at low level in the normal mouse organs. The ST2146biot localization data are substantially confirmed even when both low and high tenascin-expressing tumors are implanted in the same animal. To our knowledge, the absolute amount of ST2146biot, specifically localized in xenotransplanted human tumors, is the highest thus far described and supports the clinical use of this mAb in PAGRIT(R).

Improved tumor targeting by combined use of two antitenascin antibodies.

PURPOSE: In the pretargeted antibody-guided radioimmunotherapy (PAGRIT) system, the combined use of two different antibodies directed against the same tumor antigen could represent a valid approach for improving tumor targeting and therapeutic efficacy. We developed a novel monoclonal antitenascin antibody, ST2485, and studied its biochemical and functional properties by in vitro and in vivo assays. We then investigated the first of the three-step therapy combining ST2485 with another antitenascin antibody, ST2146, previously described, to increase accumulation of biotinylated antibodies at the tumor site. EXPERIMENTAL DESIGN: Studies of immunoreactivity, affinity, immunohistochemistry, and biodistribution in xenograft model were carried out on ST2485. Analysis of the ST2485 and ST2146 combination was preliminary carried out by ELISA and BiaCore tests and then by in vivo distribution studies after administration of the radiolabeled biotinylated antibodies, followed by a chase with avidin as clearing agent. RESULTS: ST2485 was found to be a suitable antibody for therapeutic applications. Indeed, for its behavior in all tests, it was comparable with ST2146 and better than BC2, an antibody already used for clinical trials. The additivity of ST2146 and ST2485 in tenascin C binding, shown by in vitro tests, was confirmed by biodistribution studies in a xenograft model where tumor localization of the antibodies was near the sum of each antibody alone, with a tumor-to-blood ratio higher than 24. CONCLUSION: The results reported in this study suggest that a monoclonal antitenascin antibody mixture can improve tumor targeting. This strategy could represent progress for therapeutic approaches such as PAGRIT.

In vivo and in vitro cytokine modulatory activity of newly synthesised 2-aminotetraline derivatives.

In the present study, the protective effect of newly synthesised 2-aminotetralines was investigated in murine models of toxic shock. A few derivatives protected mice against lethality induced by lipopolysaccharide from different bacterial strains and shock induced by staphylococcal enterotoxin B in mice sensitized by D-Galactosamine (D-Galn). Notably, one derivative, S(-)-2-amino-6-fluoro-7-methoxy-1,2,3,4 tetrahydronaphthalene hydrochloride (ST1214), was also effective when administered orally (30 mg kg-1) in a therapeutic regimen. ST1214 markedly inhibited the production of the proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), Interleukin-1beta (IL-1beta), Interleukin-12 (IL-12), interferon-gamma (IFN-gamma), as well as the inflammatory mediator nitric oxide (NO), and concurrently enhanced the production of the anti-inflammatory cytokine IL-10. Moreover, ST1214 dose-dependently reduced TNF-alpha production by human peripheral blood mononuclear cells and promonocytic THP-1 cells in vitro. In the latter, ST1214 was found to inhibit lipopolysaccharide-induced TNF-alpha secretion but not cytokine mRNA accumulation. These results suggest that the mechanism of action of ST1214 involves blockade of posttranscriptional events of TNF-alpha production, apparently independent of p38 and ERK kinase activity. These results show beneficial effects of 2-aminotetralines in murine shock models and indicate a distinct counter-regulatory activity in down-regulating proinflammatory cytokine response, and upregulating IL-10. One derivative, i.e., ST1214, can be regarded as a lead compound in the development of novel drugs effective in anti-inflammatory strategies.