Preparation and quality control and biodistribution studies of [90Y]-DOTA-cetuximab for radioimmunotherapy

Yttrium-90 is a useful radionuclide for radioimmunotherapy (RIT) and the anti-epidermal growth factor receptor (anti-EGFR) antibody cetuximab is clinicsally approved for the treatment of EGFR-expressing metastatic colorectal cancer and advanced head and neck cancer. Thus in this work radiolabeling of monoclonal anti-EGFR with ⁹⁰Y for radioimmunotherapy (RIT) is targetted. Cetuximab was successively labeled with [⁹⁰Y] chloride (74 MBq) 2 mCi after conjugation with macrocyclics bifunctional chelating agent, 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid mono-(N-hydroxysuccinimidyl) ester (DOTA-NHS), purified and concentrated by centrifugation using an Amicon Ultra-15 filter (Millipore, MWCo, 30000). ⁹⁰Y chloride was obtained by ⁹⁰Sr/⁹⁰Y generator. Radiolabeling was completed in 2 h by the addition of DOTA-cetuximab conjugate at 42 °C. The stability of radiolabeled was studied in human serum. Biodistribution studies in normal rats were carried out to determine the radioimmunoconjugate distribution up to 96 h. Radiochemical purity of 92 % (using ITLC) was obtained for final radioimmunoconjugate (Specific activity = 0.55 GBq/mg). Stability of radiolabeled protein in presence of human serum was tested at 37 °C for up to 24 h. Biodistribution studies demonstrated the highest ID/g % in the blood (2.62 ± 0.005 at 24 h) and the liver (2.19 ± 0.001). This study demonstrated that ⁹⁰Y-DOTA-cetuximab is a potential compound for the treatment of EGFR-expressing cancers.     

Production of a humanized Fab fragment of a neutralizing antibody against rabies virus

This paper describes the production of a functional humanized Fab fragment of a neutralizing antibody against the rabies virus. It is a prototype of a therapeutic agent that could be an alternative to equine anti-rabies immunoglobulins and anti-rabies serum immunoglobulins obtained from the blood of vaccinated human donors. Variable fragments of the high-affinity light and heavy chains neutralizing an antibody against the rabies virus were cloned and sequenced. Mouse constant regions were replaced by human constant regions followed by expression of the humanized Fab fragments in a yeast expression system. The immunochemical properties of the Fab fragments were evaluated using ELISA, polyacrylamide gel electrophoresis, and the Western blot assay. The binding capability of the produced humanized Fab fragments surpasses that of the parental antibody. A high degree of humanization was confirmed using sera against human immunoglobulins. The yield of the humanized Fab fragments was 21 mg per liter of culture medium. The purified Fab fragment preparation did not contain traces of the isolated light and heavy chains of the humanized Fab fragments.     

C-substituted macrocycles as candidates for radioimmunotherapy

The reaction between aryl aldehydes, the macrocyclic ligand 6-methyl-1,4,8,11-tetraazacyclotetradecane-6-amine (L1), and NaBH3CN produces the corresponding benzyl-substituted ligands in good yield. Copper(II) complexes of the ligands derived from salicylaldehyde (L2), p-hydroxybenzaldehyde (L4), and p-carboxybenzaldehyde (L5) were structurally characterized: [CuL2](ClO4)2.3H2O (monoclinic, P2(1)/c, a = 11.915(6) A, b = 13.861(2) A, c = 17.065(8) A, beta = 102.14(2) degrees, Z = 4); [CuL4](ClO4)2 (monoclinic, P2(1)/n, a = 9.550(3) A, b = 17.977(2) A, c = 14.612(4) A, beta 96.76(1) degrees, Z = 4), and [CuL4](ClO4)2 (monoclinic, P2(1)/n, a = 9.286(2) A, b = 11.294(1) A, c = 23.609(8) A, beta 93.68(1) degrees, Z = 4). Conjugation of several CuII complexes to a protein (bovine serum albumin) has been pursued with a view to the application of these macrocycles as bifunctional chelating agents in radioimmunotherapy.     

Development of <Superscript>177</Superscript>Lu-DOTA-anti-CD20 for radioimmunotherapy

Rituximab was successively labeled with ¹⁷⁷Lu-lutetium chloride. ¹⁷⁷Lu chloride was obtained by thermal neutron flux (4 × 10¹³ n cm⁻² s⁻¹) of natural Lu₂O₃ sample with a specific activity of 2.6–3 GBq/mg. The macrocyclic bifunctional chelating agent, N-succinimidyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-NHS) was prepared at 25 °C using DOTA, N-hydroxy succinimide (NHS) in CH₂Cl₂. DOTA-rituximab was obtained by the addition of 1 mL of a rituximab pharmaceutical solution (5 mg/mL, in phosphate buffer, pH 7.8) to a glass tube pre-coated with DOTA-NHS (0.01–0.1 mg) at 25 °C with continuous mild stirring for 15 h. Radiolabeling was performed at 37 °C in 24 h. Radio-thin layer chromatography showed an overall radiochemical purity of >98% at optimized conditions (specific activity = 444 MBq/mg, labeling efficacy; 82%). The final isotonic ¹⁷⁷Lu-DOTA-rituximab complex was checked by gel electrophoresis for structure integrity control. Radio-TLC was performed to ensure that only one species was present after filtration through a 0.22 μm filter. Preliminary biodistribution studies in normal rats were carried out to determine complex distribution of the radioimmunoconjugate up to 168 h. The biodistribution data were in accordance with other antiCD20 radioimmunoconjugates already reported.     

Highly sensitive and rapid tandem bioluminescent immunoassay using aequorin labeled Fab fragment and biotinylated firefly luciferase

We established a simultaneous bioluminescent assay utilizing aequorin (Aq) and biotinylated firefly luciferase (b-Luc); furthermore, we developed a highly sensitive and rapid tandem bioluminescent immunoassay (BLIA) involving the Aq-labeled Fab fragment and b-Luc-streptavidin complex. Minimum detection limits of Aq and b-Luc were 9.4 × 10⁻²¹ mol assay⁻¹ (blank + 3S.D.) and 3.6 × 10⁻¹⁹ mol assay⁻¹ (blank + 3S.D.), respectively. Measurements of two luminescent proteins were completed in 4 s with a single assay medium. In this study, prostatic acid phosphatase (PAP) and prostate specific antigen (PSA), which served as analytes, were measured in the tandem BLIA. PAP and PSA were detected by the Aq-labeled anti-Dig Fab fragment and b-Luc-streptavidin complex, respectively. The measurable ranges of PAP and PSA were 0.04-100 and 0.2-200 ng mL⁻¹, respectively. This technique was also applied to the simultaneous measurement of PSA and α-fetoprotein (AFP). Measurable ranges of PSA and AFP were 0.2-200 and 1.95-1000 ng mL⁻¹, respectively. Levels of PAP and PSA or PSA and AFP in human serum could be accurately determined with the proposed BLIA. Satisfactory correlations were observed between results obtained from the proposed BLIA and those derived from commercial kits.     

Fluorimetric enzyme immunoassay for serum β2-microglobulin based on the horseradish peroxidase-labeled Fab′ fragment of immunoglobulin

A sensitive sandwich-type enzyme immunoassay for human β₂-microglobulin in serum is described. Rabbit anti-human β₂-microglobulin IgG is coated on a styrene-maleic anhydride copolymer bead, which is incubated with β₂-microglobulin in serum sample at 30°C for 1 h, and again incubated at 30°C for 1 h with a conjugate of anti-β₂-microglobulin Fab′ (a fragment of rabbit anti-human β₂-microglobulin IgC) and horseradish peroxidase to form a sandwich-type immunocomplex. The conjugate is prepared with a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate. The peroxidase activity of the immunocomplex is measured spectrofluorimetrically by use of 3-(p-hydroxyphenyl)propionic acid. The method permits precise assay of 0.05–10 ng of β₂-microglobulin in serum, with calibration linearity up to 1.0 ng of protein.     

A highly effective bifunctional ligand for radioimmunotherapy applications

A novel bifunctional ligand (3p-C-NETA) for antibody-targeted radioimmunotherapy (RIT) of β-emitting radioisotopes (90)Y and (177)Lu was efficiently synthesized via an unexpected regiospecific ring opening of an aziridinium ion. 3p-C-NETA instantly formed a very stable complex with (90)Y or (177)Lu. 3p-C-NETA is an excellent bifunctional ligand for RIT.     

Preparation of 90Y by the 90Sr-90Y Generator for Medical Purpose

The production of ⁹⁰Y by ⁹⁰Sr-⁹⁰Y generator was studied. ⁹⁰Sr was adsorbed on a column with Aminex A-5 resin. The daughter radionuclide ⁹⁰Y was eluted with 0.7M -hydroxyisobutyrate (-HIB, pH 5.4). Radionuclidic, radiochemical and chemical purities were >98% and yield >85%. After converting into chloride form ⁹⁰YCl₃-solution (pH:1) was used for preparing injectable yttrium citrate and labeling some other radiopharmaceuticals. Furthermore, a fast ITLC-method for the determination of ⁹⁰Sr in ⁹⁰Y-eluate was developed.     

Structure-based fragment hopping for lead optimization using predocked fragment database

In this work, we describe a structure-based de novo optimization process, called "LeadOp" (short for lead optimization), that decomposes a compound into fragments of different molecular components either by chemical or user-defined rules. Each fragment is evaluated through a predocked fragment database that ranks fragments according to specific fragment-receptor binding interactions, replacing fragments that contribution the least to binding and finally reassembling the fragments to form a new ligand. The fundamental idea is to replace "bad" fragments of a ligand with "good" fragments while leaving the core of the ligand intact, thus improving the compound's activity. The molecular fragments were selected from a collection of 27,417 conformers that are the fragments of compounds in the DrugBank database. The collection of molecular fragments are docked to the target's binding site and evaluated using group efficiency (calculated binding affinity divided by the number of heavy atoms), and the "strongest" binder is selected. The LeadOp method was tested with two biomolecular systems: mutant B-Raf kinase and human 5-lipoxygenase. The LeadOp methodology was able to optimize the query molecules and systematically developed improved analogs for each of our example systems.     

Design of compound libraries for fragment screening

Approaches to the design of libraries for fragment screening are illustrated with reference to a 20 k generic fragment screening library and a 1.2 k generic NMR screening library. Tools and methods for library design that have been developed within AstraZeneca are described, including Foyfi fingerprints and the Flush program for neighborhood characterization. It will be shown how Flush and the BigPicker, which selects maximally diverse sets of compounds, are used to apply the Core and Layer method for library design. Approaches to partitioning libraries into cocktails are also described.     

211At labeling of a monoclonal antibody and its Fab fragment: Cytotoxicity on human gastric cancer cells and biodistribution in nude mice with tumor xenografts

An antigastric cancer monoclonal antibody, 3H11 and its Fab fragment, were labeled with #-emitter ²¹¹At using p-[²¹¹At] astatobenzoic acid (PAtBA) intermediate. The astatinated antibodies had conspicuous cytotoxic effect on human gastric cancer cell M85 in vitro. Tissue distribution of the astatinated antibodies were investigated in nude mice with subcutaneous tumor xenografts by i.v. injection. The astatinated Fab fragment was better suitable for 7.2-hour half life of ²¹¹At, since its tumor uptake remained higher (9.48–8.42 I.D%/g) than the astatinated intact antibody (~4.0 I.D%/g) from 3 to 14-hour post injection. However, the undesired high ²¹¹At uptake of the astatinated antibodies in some normal tissues, such as stomach, kidney and lung, suggested that the ²¹¹At labeled antibodies should be further explored.     

A configuration analysis for fragment interaction

We propose a modified version of configuration analysis (CA) for the fragment interaction in conjunction with Kitaura’s fragment molecular orbital (FMO) scheme. The proposal is abbreviated as CAFI. The MO sets of fragments are merged and then orthonormalized by the use of a weighted Löwdin orthonormalization. The energy calculation is performed with the concurrent electron relaxation functional (CERF). The relaxation energy is obtained in an orbital-wise fashion and is distinguished as the charge-transfer and the polarization. The utility of CAFI is demonstrated through test calculations on hydrogen-bonding systems.     

188Re-labeled hyperbranched polysulfonamine as a robust tool for targeted cancer diagnosis and radioimmunotherapy

Hyperbranched polysulfonamine (HPSA) is a promising biomaterial due to its highly branched spherical architecture and efficient intracellular translocation. To realize the functionalization of HPSA, both N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) for tethering the human-mouse chimeric monoclonal antibody CH12 and N-hydroxy succinimidyl S-acetylmercaptoacetyltriglycinate (NHS-MAG₃) for labeling ¹⁸⁸Re were sequentially grafted onto the primary amine terminals of HPSA via covalent linkages, attaining the SPDP-HPSA-MAG₃ intermediate. In order to reserve the structural integrity of CH12, the fragment crystallizable (Fc) region was also processed by oxidation of oligosaccharide moieties with sodium periodate and then reacted with N-(κ-maleimidoundecanoic acid) hydrazide (KMUH). After chelating ¹⁸⁸Re with MAG₃ group, the SPDP was reduced to PDP and connected onto the maleinimide group at the Fc region. As a result, both the epidermal growth factor receptor vIII (EGFRvIII) targeted monoclonal antibody CH12 and the radionuclide ¹⁸⁸Re were conjugated to the HPSA-based vehicles, forming the ¹⁸⁸Re-labeled and CH12-tethered HPSA (CH12-HPSA-¹⁸⁸Re). The molecular weight and in vitro stability of CH12-HPSA-¹⁸⁸Re were evaluated by gel electrophoresis and paper chromatography. On one hand, the CH12-HPSA-¹⁸⁸Re could specifically bind to the EGFRvIII-positive human hepatocarcinoma cells in vitro. On the other hand, it could also target at the tumor tissue of nude mice in vivo. Hence, the CH12-HPSA-¹⁸⁸Re could effectively target at the human hepatocarcinoma and facilitate the tumor detection and targeted radioimmunotherapy.