Preliminary efficacy of [90Y]DOTA-biotin-avidin radiotherapy against non-muscle invasive bladder cancer.

PURPOSE: Bladder cancer represents 3% of all new cancer diagnoses per year. We propose intravesical radionuclide therapy using the ß-emitter 90Y linked to DOTA-biotin-avidin ([90Y]DBA) to deliver short-range radiation against non-muscle invasive bladder cancer (NMIBC). MATERIAL AND METHODS: Image-guided biodistribution of intravesical DBA was investigated in an animal model by radiolabeling DBA with the 68Ga and dynamic microPET imaging following intravesical infusion of [68Ga]DBA for up to 4 h and post-necropsy γ-counting of organs. The antitumor activity of [90Y]DBA was investigated using an orthotopic MB49 murine bladder cancer model. Mice were injected with luciferase-expressing MB49 cells and treated via intravesical administration with 9.2 MBq of [90Y]DBA or unlabeled DBA 3 days after the tumor implantation. Bioluminescence imaging was conducted after tumor implantation to monitor the bladder tumor growth. In addition, we investigated the effects of [90Y]DBA radiation on urothelial histology with immunohistochemistry analysis of bladder morphology. RESULTS: Our results demonstrated that DBA is contained in the bladder for up to 4 h after intravesical infusion. A single dose of [90Y]DBA radiation treatment significantly reduced growth of MB49 bladder carcinoma. Attaching 90Y-DOTA-biotin to avidin prevents its re-absorption into the blood and distribution throughout the rest of the body. Furthermore, immunohistochemistry demonstrated that [90Y]DBA radiation treatment did not cause short-term damage to urothelium at day 10, which appeared similar to the normal urothelium of healthy mice. CONCLUSION: Our data demonstrates the potential of intravesical [90Y]DBA as a treatment for non-muscle invasive bladder cancer.

Single-Dose Intra-Cartilage Delivery of Kartogenin Using a Cationic Multi-Arm Avidin Nanocarrier Suppresses Cytokine-Induced Osteoarthritis-Related Catabolism.

OBJECTIVE: Kartogenin (KGN) has proven as a both chondrogenic and chondroprotective drug for osteoarthritis (OA) therapy. However, being a small hydrophobic molecule, KGN suffers from rapid joint clearance and inability to penetrate cartilage to reach chondrocytes following intra-articular administration. As such multiple high doses are needed that can lead to off-target effects including stimulation and tissue outgrowth. Here we design charge-based cartilage targeting formulation of KGN by using a multi-arm cationic nano-construct of Avidin (mAv) that can rapidly penetrate into cartilage in high concentrations owing to weak-reversible electrostatic binding interactions with negatively charged aggrecan-glycosaminoglycans (GAGs) and form an extended-release drug depot such that its therapeutic benefit can be reaped in just a single dose. DESIGN: We synthesized 2 novel formulations, one with a releasable ester linker (mAv-OH-KGN, release half-life ~58 h) that enables sustained KGN release over 2 weeks and another with a non-releasable amide linker (mAv-NH-KGN) that relies on mAv's ability to be uptaken and endocytosed by chondrocytes for drug delivery. Their effectiveness in suppressing cytokine-induced catabolism was evaluated in vitro using cartilage explant culture model. RESULTS: A single 100 µM dose of cartilage homing mAv-KGN was significantly more effective in suppressing cytokine-induced GAG loss, cell death, inflammatory response and in rescuing cell metabolism than a single dose of free KGN; multiple doses of free KGN were needed to match this therapeutic response. CONCLUSION: mAv mediated delivery of KGN is promising and can facilitate clinical translation of KGN for OA treatment with only a single dose.

Anticancer drug-loaded mesenchymal stem cells for targeted cancer therapy.

Mesenchymal stem cells (MSCs) have a tumor-homing ability-they accumulate inside tumors after systemic injection, and may thus be useful as carriers for tumor-targeting therapy. To use MSCs effectively as an anti-cancer therapy, they must first be functionalized with a large amount of anti-cancer drugs without causing any significant changes to their tumor-tropism. In the present study, we attempted to modify the cell surface of MSCs with doxorubicin-loaded liposomes (DOX-Lips), using the avidin-biotin complex method, and evaluated delivery efficiency and anti-tumor efficacy of DOX-Lip-modified MSCs. The amount of DOX in DOX-Lip-modified C3H10T1/2 cells, a murine mesenchymal stem cell line, was approximately 21.5 pg per cell, with no significant changes to the tumor-tropism of C3H10T1/2 cells. Notably, DOX-Lip-modified C3H10T1/2 cells significantly suppressed the proliferation of firefly luciferase-expressing murine colon adenocarcinoma colon26/fluc cells, compared to DOX-Lips alone. Fluorescent DOX accumulated at the cell contact surface and inside green fluorescence protein-expressing colon26 (colon26/GFP) in co-cultures of DOX-Lip-modified C3H10T1/2 and colon26/GFP cells. This localized distribution was not observed when only DOX-Lips was added to colon26/GFP cells. These results suggest that DOX-Lips are efficiently delivered from DOX-Lip-modified C3H10T1/2 cells to the neighboring colon26 cells. Furthermore, DOX-Lip-modified C3H10T1/2 cells suppressed tumor growth in subcutaneous tumor-bearing mice, and in a lung metastasis mouse model. Taken together, these results indicate that the intercellular delivery of DOX may be enhanced using DOX-Lip-modified MSCs as an efficient carrier system for targeted tumor therapy.

Multi-arm Avidin nano-construct for intra-cartilage delivery of small molecule drugs.

Targeted drug delivery to joint tissues like cartilage remains a challenge that has prevented clinical translation of promising osteoarthritis (OA) drugs. Local intra-articular (IA) injections of drugs suffer from rapid clearance from the joint space and slow diffusive transport through the dense, avascular cartilage matrix comprised of negatively charged glycosaminoglycans (GAGs). Here we apply drug carriers that leverage electrostatic interactions with the tissue's high negative fixed charge density (FCD) for delivering small molecule drugs to cartilage cell and matrix sites. We demonstrate that a multi-arm cationic nano-construct of Avidin (mAv) with 28 sites for covalent drug conjugation can rapidly penetrate through the full thickness of cartilage in high concentration and have long intra-cartilage residence time in both healthy and arthritic cartilage via weak-reversible binding with negatively charged aggrecans. mAv's intra-cartilage mean uptake was found to be 112× and 33× the equilibration bath concentration in healthy and arthritic (50% GAG depleted) cartilage, respectively. mAv was conjugated with Dexamethasone (mAv-Dex), a broad-spectrum glucocorticoid, using a combination of hydrolysable ester linkers derived from succinic anhydride (SA), 3,3-dimethylglutaric anhydride (GA) and phthalic anhydride (PA) in 2:1:1 M ratio that enabled 50% drug release within 38.5 h followed by sustained release in therapeutic doses over 2 weeks. A single 10 µM low dose of controlled release mAv-Dex (2:1:1) effectively suppressed IL-1α-induced GAG loss, cell death and inflammatory response significantly better than unmodified Dex over 2 weeks in cartilage explant culture models of OA. With this multi-arm design, <1 µM Avidin was needed - a concentration which has been shown to be safe, preventing further GAG loss and cytotoxicity. A charge-based cartilage homing drug delivery platform like this can elicit disease modifying effects as well as facilitate long-term symptomatic pain and inflammation relief by enhancing tissue specificity and prolonging intra-cartilage residence time of OA drugs. This nano-construct thus has high translational potential for enabling intra-cartilage delivery of a broad array of small molecule OA drugs and their combinations to chondrocytes, enabling OA treatment with a single injection of low drug doses and eliminating toxicity issues associated with multiple high dose injections.

Immobilization of bone morphogenetic protein-2 to gelatin/avidin-modified hydroxyapatite composite scaffolds for bone regeneration.

Bone scaffold surface characterization is important for improving cell adhesion, migration, and differentiation. In this study, bone morphogenetic protein-2 (BMP-2) was immobilized to the surface of the gelatin/hydroxyapatite composite using avidin-biotin binding system to produce a bone-tissue engineering scaffold. Firstly, hydroxyapatite particles reacted with hexamethylene diisocyanate and then the terminal group was converted into a primary amine group. Avidin was then immobilized on the surfaces of hydroxyapatite particles using N-ethyl-N'-(3-(dimethylamino)propyl) carbodiimide and N-hydroxysuccinimide as coupling agents. Gelatin was blended with avidin-modified hydroxyapatite and pure hydroxyapatite to obtain gelain/hydroxyapatite composite. The composite was then cross-linked with glutaraldehyde. Finally, biotin-conjugated BMP-2 was immobilized on the surface of the composite via avidin-biotin binding. In vitro study indicated that BMP-2-immobilized composite film had a higher ALP activity than that composite film without BMP-2. The composite scaffolds were then implanted into rabbit skulls to check bone-tissue regeneration. Ultrasound and micro-CT scans demonstrated that neovascularization and new bone formation in the BMP-2-immobilized composite scaffolds were higher than those in composite scaffolds without BMP-2. Histological evaluation result was similar to that of the micro-CT. Therefore, the surface immobilization of BMP-2 could effectively improve osteogenesis in the gelatin/hydroxyapatite composite scaffold.

The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis.

Avidin-biotin interaction is one of the strongest non-covalent interactions in the nature. Avidin and its analogues have therefore been extensively utilized as probes and affinity matrices for a wide variety of applications in biochemical assays, diagnosis, affinity purification, and drug delivery. Recently, there has been a growing interest in exploring this non-covalent interaction in nanoscale drug delivery systems for pharmaceutical agents, including small molecules, proteins, vaccines, monoclonal antibodies, and nucleic acids. Particularly, the ease of fabrication without losing the chemical and biological properties of the coupled moieties makes the avidin-biotin system a versatile platform for nanotechnology. In addition, avidin-based nanoparticles have been investigated as diagnostic systems for various tumors and surface antigens. In this review, we will highlight the various fabrication principles and biomedical applications of avidin-based nanoparticles in drug delivery and diagnosis. The structures and biochemical properties of avidin, biotin and their respective analogues will also be discussed.

VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever.

Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4(+) T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models.

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.

Targeted sonocatalytic cancer cell injury using avidin-conjugated titanium dioxide nanoparticles.

In this study, we applied sonodynamic therapy to cancer cells based on the delivery of titanium dioxide (TiO2) nanoparticles (NPs) modified with avidin protein, which preferentially discriminated cancerous cells from healthy cells. Subsequently, hydroxyl radicals were generated from the TiO2 NPs after activation by external ultrasound irradiation (TiO2/US treatment). Although 30% of the normal breast cells (human mammary epithelial cells) exhibited the uptake of avidin-modified TiO2 NPs, over 80% of the breast cancer cells (MCF-7) exhibited the uptake of avidin-TiO2 NPs. Next the effect of the TiO2/US treatment on MCF-7 cell growth was examined for up to 96 h after 1-MHz ultrasound was applied (0.1 W/cm(2), 30 s) to cells that incorporated the TiO2 NPs. No apparent cell injury was observed until 24h after the treatment, but the viable cell concentration declined to 68% compared with the control at 96 h.

Investigation of 90Y-avidin for prostate cancer brachytherapy: a dosimetric model for a phase I-II clinical study.

PURPOSE: A novel method for prostate irradiation is investigated. Similarly to (125)I or (103)Pd seed brachytherapy, (90)Y-avidin could be injected via the perineum under ultrasound image guidance. This study inspects the theoretical feasibility with a dosimetric model based on Monte Carlo simulation. METHODS: A geometrical model of the prostate, urethra and rectum was designed. The linear-quadratic model was applied to convert (125)I absorbed dose prescription/constraints into (90)Y dose through biological effective dose (BED) calculation. The optimal (90)Y-avidin injection strategy for the present model was obtained. Dose distribution was calculated by Monte Carlo simulation (PENELOPE,GEANT4). Dose volume histograms (DVH) for the prostate, urethra and rectum were compared to typical DVHs of (125)I seed brachytherapy, used routinely in our institute. RESULTS: With (90)Y-avidin, at least 95% of the prostate must receive more than 70 Gy. The absorbed dose to 10% of the urethra (D(10%_urethra)) and the maximum absorbed dose to the rectum (D(max_rectum)) must be lower than 122 Gy. For the present model, the optimum strategy consists in multiple injections of (90)Y-avidin 50 µl drops, for a total volume of 3.1 ml. The minimum activity to deliver the prescribed absorbed dose is 0.7 GBq, which also fully respects urethral and rectal constraints. The resulting dose map has a maximum in the central region with a sharp decrease towards the urethra and the prostate edge. Notably, D(10%_urethra) is 95 Gy and D(max_rectum) is below 2 Gy. Prostate absorbed dose is higher with (90)Y-avidin than (125)I seeds, although the total volume receiving the prescribed absorbed dose is 1-2% lower. Urethral DVH strictly depends on the (90)Y distribution, to be optimized according to prostate shape; in our model, BED(30%_urethra) is 90 Gy with (90)Y-avidin, whereas for patients receiving (125)I seeds it ranges between 150 and 230 Gy. The rectal DVH is always more favourable with (90)Y. CONCLUSION: The methodology is theoretically feasible and can deliver an effective treatment in T1-T2 prostate cancer. Pharmacokinetic and biodistribution studies in prostate cancer patients are needed for validation.

Comparison of 211At-PRIT and 211At-RIT of ovarian microtumors in a nude mouse model.

UNLABELLED: Abstract Purpose: Pretargeted radioimmunotherapy (PRIT) against intraperitoneal (i.p.) ovarian microtumors using avidin-conjugated monoclonal antibody MX35 (avidin-MX35) and (211)At-labeled, biotinylated, succinylated poly-l-lysine ((211)At-B-PLsuc) was compared with conventional radioimmunotherapy (RIT) using (211)At-labeled MX35 in a nude mouse model. METHODS: Mice were inoculated i.p. with 1×10(7) NIH:OVCAR-3 cells. After 3 weeks, they received PRIT (1.0 or 1.5 MBq), RIT (0.9 MBq), or no treatment. Concurrently, 10 additional animals were sacrificed and examined to determine disease progression at the start of therapy. Treated animals were analyzed with regard to presence of tumors and ascites (tumor-free fraction; TFF), 8 weeks after therapy. RESULTS: Tumor status at baseline was advanced: 70% of sacrificed animals exhibited ascites. The TFFs were 0.35 (PRIT 1.0 MBq), 0.45 (PRIT 1.5 MBq), and 0.45 (RIT). The 1.5-MBq PRIT group exhibited lower incidence of ascites and fewer tumors >1 mm than RIT-treated animals. CONCLUSIONS: PRIT was as effective as RIT with regard to TFF; however, the size distribution of tumors and presence of ascites indicated that 1.5-MBq PRIT was more efficient. Despite advanced disease in many animals at the time of treatment, PRIT demonstrated good potential to treat disseminated ovarian cancer.

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.

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.

Biocompatibility of a novel avidin-agarose adsorbent for extracorporeal removal of redundant radiopharmaceutical from the blood.

The use of monoclonal antibodies (MAbs) in cytotoxic conjugates (radionuclides, toxins, or drugs) for targeting tumor cells is restricted due to toxicity in vital organs. Through improved tumor targeting, it is possible to administer larger amounts of such labeled MAbs, thus improving the ability to eradicate tumor cells without increased normal organ toxicity. Extracorporeal affinity adsorption treatment (ECAT) has therefore been developed using an avidin-agarose (AA) adsorbent with high binding affinity for the biotinylated radiolabeled MAb, rituximab. During ECAT, excess radioimmunoconjugates, not bound to the tumor cells, can be removed improving tumor targeting. The present study was performed to estimate the biocompatibility of the AA adsorber. Seven patients with B-cell lymphoma not responding to conventional treatment were studied. During the ECAT procedure, blood (B) components, plasma (P) complement fragments C3a, C5a, and P-bradykinin were analyzed, and other laboratory tests were carried out. Slight decreases in B-hemoglobin (8.3%), B-thrombocytes (11.4%), and P-albumin (14.3%) were observed, and could be explained by the dilution of the blood with normal saline and acid citrate dextrose. The AA adsorbent had no effect on the blood cells, immunological status or P-bradykinin level. The AA adsorber demonstrated good hemocompatibility and biocompatibility, without any side effects in the patients.

Recent advances in pretargeted radioimmunotherapy.

Pretargeted delivery of radionuclides is based upon bispecific immunoconjugates that bind a target tumor antigen and a small molecule carrying the active payload. This strategy is supposed to combine the advantage of antibodies to track tumor cells in vivo and of small radiolabeled molecules that clear rapidly from normal organs and minimize toxicity. Many pretargeting approaches have been proposed, but only those using the biotin/avidin recognition system and those using bispecific anti-tumor x anti-hapten antibodies have been tested in the clinic for both immunoscintigraphy and radioimmunotherapy. Their respective advantages and drawbacks, as well as hurdles in the way of an effective therapy against solid tumors, are discussed. In the light of the encouraging results obtained so far in the clinic, pretargeting remains a most promising challenge for chemistry and biotechnology.

Tumor pretargeting with avidin improves the therapeutic index of biotinylated tumor necrosis factor alpha in mouse models.

The clinical use of tumor necrosis factor alpha (TNF) as an anticancer drug is limited to local or locoregional administration because of dose-limiting systemic toxicity. We investigated in animal models whether the therapeutic index of systemically administered human or murine TNF can be increased by tumor pretargeting strategies based on the biotin-avidin system. Pretargeting of s.c. mouse WEHI-164 fibrosarcoma and RMA lymphoma genetically engineered to express the Thy 1.1 antigen on the cell membrane was achieved by i.p. injection of a biotinylated anti-Thy 1.1 antibody and avidin. This pretreatment increased the antitumor activity of systemically administered biotin-TNF conjugates by at least 5-fold. In contrast, pretargeting did not increase the toxicity of biotin-TNF, as judged by animal survival and weight loss after treatment. Ex vivo analysis of tumor cells 24 h after treatment showed that biotin-TNF persisted for several hours on the surface of pretargeted tumors, but not when avidin was omitted. The potentiation of the antitumor effects was related primarily to indirect mechanisms, involving a host-mediated response. The results indicate that tumor pretargeting improves the antitumor activity of TNF. Tumor pretargeting with avidin, which is currently used to increase the uptake of radioactive-labeled biotin in patients, could represent a new strategy for improving the therapeutic index of TNF.