Barbara Leoni

Novel antitenascin antibody with increased tumour localisation for Pretargeted Antibody-Guided RadioImmunoTherapy (PAGRIT)

The Pretargeted Antibody-Guided RadioImmunoTherapy (PAGRIT) method is based on intravenous, sequential administration of a biotinylated antibody, avidin/streptavidin and 90Y-labelled biotin. The hybridoma clone producing the monoclonal antitenascin antibody BC4, previously used for clinical applications, was found not suitable for further development because of the production of an additional, nonfunctional light chain. In order to solve this problem, the new cST2146 hybridoma clone was generated. The monoclonal antibody ST2146, produced by this hybridoma, having the same specificity as BC4 but lacking the nonfunctional light chain, was characterised. ST2146 was found able to bind human tenascin at an epitope strictly related, if not identical, to the antigenic epitope of BC4. It showed, compared to BC4, higher affinity and immunoreactivity and similar selectivity by immunohistochemistry. Biodistribution studies of biotinylated ST2146 and three other monoclonal antitenascin antibodies showed for ST2146 the highest and more specific tumour localisation in HT29-grafted nude mice. On the overall, ST2146 appears to be a good alternative to BC4 for further clinical development of PAGRIT.

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.

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.

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 Schiffs 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 111In-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 Schiffs 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.

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.

In vitro activation of murine peritoneal exudate cells (PEC) and peritoneal macrophages by ST 789

ST 789 is a new synthetic compound characterized by an amino acidic group joined to the N9 position of the hypoxanthine ring, which has been shown recently to have immunomodulating properties and minimal toxicity. The drug has been reported to protect immunosuppressed mice from microbial infections and tumour growth, and to restore the mitogen-induced proliferation of splenocytes from immunosuppressed young mice. In this study, we show that in vitro addition of ST 789 is able to markedly augment the sheep red blood cells (SRBC) phagocytosis by PEC, and to potentiate the cytotoxic activity of peritoneal exudate (PE) macrophages (M phi) vs the L-M tumour cell line. We also found that ST 789 enhanced the rIFN-gamma-induced NO2- release from cultured PE M phi. Similarly, in vitro addition of ST 789 to the latter cultures significantly increased the production of interleukin 1 (IL-1) and tumour necrosis factor (TNF) induced by lipopolysaccharide (LPS). These studies demonstrate that ST 789 is a potent phagocyte activator for the induction of cytokine release, phagocytosis and cytotoxic activity against tumour cells in vitro.

AvidinOX-anchored biotinylated trastuzumab and pertuzumab induce down-modulation of ErbB2 and tumor cell death at concentrations order of magnitude lower than not-anchored antibodies

The oxidized version of Avidin, known as AvidinOX, was previously shown to link to tissue proteins upon injection or nebulization, thus becoming a stable receptor for biotinylated therapeutics. AvidinOX is currently under clinical investigation to target radioactive biotin to inoperable tumor lesions (ClinicalTrials.gov NCT02053324). Presently, we show that the anti-ErbB2 monoclonal antibodies Trastuzumab and Pertuzumab can be chemically biotinylated while maintaining their biochemical and biological properties. By using several and diverse experimental conditions, we show that when AvidinOX is conjugated to tumor cells, low antibody concentrations of biotinylated Trastuzumab (bTrast) or Pertuzumab (bPert) prevent internalization of ErbB2, induce endoplasmic reticulum stress, cell cycle arrest and apoptosis leading to inhibition of proliferation and ErbB2 signaling. Moreover, we found that the treatment is able to induce down-modulation of ErbB2 thus bypassing the known resistance of this receptor to degradation. Interestingly, we show that AvidinOX anchorage is a way to counteract agonistic activities of Trastuzumab and Pertuzumab. Present data are in agreement with previous observations from our group indicating that the engagement of the Epidermal Growth Factor Receptor (EGFR) by AvidinOX-bound biotinylated Cetuximab or Panitumumab, leads to potent tumor inhibition both in vitro and in animal models. All results taken together encourage further investigation of AvidinOX-based treatments with biotinylated antibodies directed to the members of the EGFR family.