Preparation of TH 227 -Labeled Radioimmunoconjugates, Assessment of Serum Stability and Antigen Binding Ability (original) (raw)
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Cancer research, 1993
A major factor that is critical to the potential effectiveness of alpha-emitter 212Bi radioimmunotherapy is the design of radiometal-chelated antibodies that will be stable in vivo. The chelate should bind the radiometal firmly to minimize release of the radionuclide from the monoclonal antibody-chelate complex. The present study examines a member of a new class of polyamine carboxylate chelating compounds, the DOTA ligands, for conjugating radiometal ions to antibody. Biocompatibility and stability are assessed with the anti-Tac monoclonal antibody that is directed against the human interleukin 2 receptors. The scientific basis for the clinical use of this antibody in radioimmunotherapy is that resting normal cells do not express the interleukin 2 receptor, whereas the receptor is expressed on the surface of certain neoplasms and by activated T-cells in select autoimmune diseases and in allograft rejection. First, we examined the impact of the labeling procedure and the presence of...
Cancer Science, 1998
Stability and immunoreactivity of 186 Re-labeled monoclonal antibody were examined, and its in vivo kinetics was investigated in tumor-bearing Balb/c nu/nu female mice to assess the feasibility of using it in radioimmunotherapy (RIT). A murine IgG 1 , A7, against a 45 kD glycoprotein in human colon cancer was radiolabeled with 186 Re by using a chelating method with a mercaptoacetyltriglycine (MAG3). 186 Re-MAG3 complex was conjugated to A7 after esterification of 186 Re-MAG3 with tetrafluorophenol (TFP). The efficiency of 186 Re-MAG3-TFP production and the labeling efficiency of A7 were 51-59% and 57-60%, respectively. Immunoreactivity of purified 186 Re-MAG3-A7 was 68.2% at infinite antigen excess. In 0.9% NaCl at 4°C, the radioactivity (12.7 MBq/mg, 3.55 MBq/ml) dissociated with time from 186 Re-MAG3-A7 as a small molecular weight moiety because of autoradiolysis. The addition of ascorbic acid, 5 mg/ml, as a radioprotectant or storage at − − − −80°C could effectively prevent the radiolysis of 186 Re-MAG3-A7 for 7 days. Immunoreactivity of 186 Re-MAG3-A7, 6.70 MBq/mg (6.66 MBq/ml), stored in the presence of ascorbic acid was well retained up to 8 days after the preparation. In colon cancer xenografted mice, 31.0% of the injected dose/g of 186 Re-MAG3-A7 had accumulated in the tumors at 24 h postinjection. Estimated radiation dose to tumors was 14.9 cGy/37 kBq up to 8 days postinjection which was 12-fold greater than the whole-body radiation dose. These in vivo characteristics were superior to those of A7 labeled with radioiodine, affording greater therapeutic ratios than 131 I-A7. Because of the better image quality of 186 Re-MAG3-A7 as well as more favorable dosimetry, 186 Re-MAG3-A7 would be a better choice for RIT of colon cancer than 131 I-A7. These results indicated the feasibility of RIT with 186 Re-MAG3-A7, though the prevention of radiolysis of the labeled antibody should be considered.
Cancer Research, 1991
The use of l88Refrom an alumina-based "*W/1MRegenerator has been investigated for antibody radiolabeling. It was found that, with simple labeling techniques, '""Re can be used immediately after elution. The direct radiolabeling of intact antibodies with >S8Reis described. Lyophilized antibody preparations have been reconstituted with 18*Retaken directly from the generator at specific activities of up to 15 mCi of IS8Re per mg of antibody. Radiolabeling yields of 90 to 98% have been obtained, with the incorporation rate being dependent upon time and the relative concentrations of the reagents. It was determined that the conjugates were immunoreactive and stable when challenged by serum in vitro, with l8*Re-immunoglobulin G showing adequate resistance to reoxidation with no transfer of 18*Reto serum protein. l8*Re-antibody conjugates were shown to clear from the blood faster than the corresponding I3ll-labeled antibody, giving rise to good tumor/nontumor ratios at 24 to 72 h postinjection, while serum samples taken from the animals have shown that the circulating l8*Re remained bound to immunoglobulin G. The combination of the technologies of the 188\V/l8*Regenerator, the direct labeling methodology, and the use of single-vial lyophilized antibody makes the use of l88Re-radiolabeled monoclonal antibodies a simple and convenient method of cancer radioimmunotherapy with a /3-emitting radionuclide.
Applied Radiation and Isotopes, 2001
188 Re is one of the radioisotopes expected to emerge as useful for therapy. Development of new radiopharmaceuticals based on 188 Re depends on the radiolabeling methods used, which would give stable complexes having prede®ned radiochemical properties and in vitro and in vivo stability. This paper has attempted to provide a perspective of 188 Re-labeled monoclonal antibodies, their radiolabeling characteristics, methods for quality control of radioimmunoconjugates and in vitro stability for radioimmunotherapy of solid tumors. The direct method of 188 Re radiolabeling of antibodies by reductive attachment of 188 Re in which free sulfhydryl groups have been generated by reduction of the intramolecular S±S disul®de bonds has been shown to be a promising approach in particular. Moreover, excellent methods have been developed to test the radionuclide, radiochemical purity and stability of 188 Re-radioimmunoconjugates using high performance liquid chromatography (HPLC) and paper chromatography. 7
Journal of Nuclear Medicine, 2008
and 2 Cyclotron and PET Unit, Rigshospitalet, Copenhagen, Denmark 211 At-labeled tumor-specific antibodies have long been considered for the treatment of disseminated cancer. However, the limited availability of the nuclide and the poor efficacy of labeling procedures at clinical activity levels present major obstacles to their use. This study evaluated a procedure for the direct astatination of antibodies for the production of clinical activity levels. Methods: The monoclonal antibody trastuzumab was conjugated with the reagent N-succinimidyl-3-(trimethylstannyl)benzoate, and the immunoconjugate was labeled with astatine. Before astatination of the conjugated antibody, the nuclide was activated with N-iodosuccinimide. The labeling reaction was evaluated in terms of reaction time, volume of reaction solvent, immunoconjugate concentration, and applied activity. The quality of the astatinated antibodies was determined by in vitro analysis and biodistribution studies in nude mice. Results: The reaction proceeded almost instantaneously, and the results indicated a low dependence on immunoconjugate concentration and applied activity. Radiochemical labeling yields were in the range of 68%281%, and a specific radioactivity of up to 1 GBq/mg could be achieved. Stability and radiochemical purity were equal to or better than those attained with a conventional 2-step procedure. Dissociation constants for directly astatinated, conventionally astatinated, and radioiodinated trastuzumab were 1.0 6 0.06 (mean 6 SD), 0.44 6 0.06, and 0.29 6 0.02 nM, respectively. The tissue distribution in non-tumor-bearing nude mice revealed only minor differences in organ uptake relative to that obtained with the conventional method. Conclusion: The direct astatination procedure enables the high-yield production of astatinated antibodies with radioactivity in the amounts required for clinical applications.
Preparation and preclinical evaluation of humanised A33 immunoconjugates for radioimmunotherapy
British journal of cancer, 1995
A humanised IgG1/k version of A33 (hA33) has been constructed and expressed with yields up to 700 mg l-1 in mouse myeloma NS0 cells in suspension culture. The equilibrium dissociation constant of hA33 (KD = 1.3 nM) was shown to be equivalent to that of the murine antibody in a cell-binding assay. hA33 labelled with yttrium-90 using the macrocyclic chelator 12N4 (DOTA) was shown to localise very effectively to human colon tumour xenografts in nude mice, with tumour levels increasing as blood concentration fell up to 144 h. A Fab' variant of hA33 with a single hinge thiol group to facilitate chemical cross-linking has also been constructed and expressed with yields of 500 mg l-1. Trimaleimide cross-linkers have been used to produce a trivalent Fab fragment (hA33 TFM) that binds antigen on tumour cells with greater avidity than hA33 IgG. Cross-linkers incorporating 12N4 or 9N3 macrocycles have been used to produce hA33 TFM labelled stably and site specifically with yttrium-90 or in...
Targeted cancer therapy with a novel low-dose rate -emitting radioimmunoconjugate
2007
␣-emitting radionuclides are highly cytotoxic and are of considerable interest in the treatment of cancer. A particularly interesting approach is in radioimmunotherapy. However, ␣-emitting antibody conjugates have been difficult to exploit clinically due to the short half-life of the radionuclides, low production capability, or limited source materials. We have developed a novel technology based on the low-dose rate ␣-particle-emitting nuclide 227 Th, exemplified here using the monoclonal antibody rituximab. In vitro, this radioimmunoconjugate killed lymphoma cells at Becquerel per milliliter (Bq/mL) levels. A single injection of 227 Thrituximab induced complete tumor regression in up to 60% of nude mice bearing macroscopic (32-256 mm 3 ) human B-lymphoma xenografts at Becquerel per gram (Bq/g) levels without apparent toxicity. Therapy with 227 Th-rituximab was sig-nificantly more effective than the control radioimmunoconjugate 227 Th-trastuzumab and the standard -emitting radioimmunoconjugate for CD20 ؉ lymphoma, 90 Ytiuxetan-ibritumomab. Thorium-227 based constructs may provide a novel approach for targeted therapy against a wide variety of cancers. (Blood. 2007;110: 2049-2056
Bioconjugate Chemistry, 2008
We are investigating the use of recombinant streptavidin (rSAv) as a carrier molecule for the shortlived α-particle emitting radionuclides 213 Bi (t 1/2 = 45.6 min) and 211 At (t 1/2 = 7.21 h) in cancer therapy. To utilize rSAv as a carrier, it must be modified in a manner that permits rapid chelation or bonding with these short-lived radionuclides, and also modified in a manner that diminishes its natural propensity for localization in kidney. Modification for labeling with 213 Bi was accomplished by conjugation of rSAv with the DTPA derivative p-isothiocyanato-benzyl-CHX-A″ (CHX-A″), 3a. Modification for direct labeling with 211 At was accomplished by conjugation of rSAv with an isothiocyanatophenyl derivative of a nido-carborane (nCB), 3b, or an isothiocyanatophenyl-dPEG ™ /decaborate(2-) derivative, 3c. After conjugation of the chelating or bonding moiety, rSAv was further modified by reaction with an excess (50-100 equivalents) of succinic anhydride. Succinylation of the lysine amines has previously been shown to greatly diminish kidney localization. rSAv modified by conjugation with 3a and succinylated radiolabeled rapidly with 213 Bi (< 5 min), providing a 72% isolated yield. 211 At labeling of modified rSAv was accomplished in aqueous solution using chloramine-T as the oxidant. Astatination of rSAv conjugated with 3b and succinylated occurred very rapidly (<1 min), providing a 50% isolated radiochemical yield. Astatination of rSAv conjugated with 3c and succinylated was also very rapid (<1 min) providing 66-71% isolated radiochemical yields. Astatination of succinylated rSAv, 2a, which did not have conjugated borane cage moieties, resulted in much lower radiolabeling yield (18%). The 213 Bi-or 211 At-labeled modified rSAv preparations were mixed with the corresponding 125 I-labeled rSAv, and dual-label in vivo distributions were obtained in athymic mice. The in vivo data show that 213 Bi-labeled succinylated rSAv [ 213 Bi]6a has tissue concentrations similar to 125 I-labeled modified rSAv [ 125 I] 6b, suggesting that 213 Bi is quite stable towards release from the chelate in vivo. In vivo data also indicate that the 211 At-labeled rSAv conjugated with 3b or 3c and succinylated are stable to in vivo deastatination, whereas succinylated rSAv lacking a boron cage moiety is subject to some deastatination. The modified rSAv conjugated with nido-carborane derivative 3b has a higher retention in many tissues than rSAv without the carborane conjugated. Interestingly, the rSAv conjugated with 3c, which also contains a m-dPEG 12 ™ moiety, has significantly decreased concentrations in blood and other tissues when compared with direct labeled rSAv, suggesting that it may be a good candidate for further study. In conclusion, rSAv that has been modified with CHX-A″ and succinylated (i.e. 5a) may be useful as a carrier of 213 Bi. The encouraging results obtained with the PEGylated decaborate(2-) derivative 3c and succinylated (i.e. 5c) suggests that its further study as a carrier of 211 At in pretargeting protocols is warranted.
Cancer Biotherapy & Radiopharmaceuticals, 2007
Th is currently under evaluation. 227 Th is conjugated to the chimeric anti-CD20 monoclonal antibody rituximab, using the chelator p-isothiocyanato-benzyl-DOTA. In this study, the binding of 227 Th-DOTA-p-benzyl-rituximab to three different CD-20-positive lymphoma cell lines, Raji, Rael, and Daudi, were evaluated. Equilibrium and kinetic binding experiments were used to determine binding parameters, including the association and dissociation rate constants, the equilibrium dissociation constants, and the total number of antigens for Raji, Rael, and Daudi cells. There were significant differences between the cell lines with respect to both K d and the total number of antigens. Rael cells had more than three times as many antigens as the other two cell lines, and the functional K d found for Rael cells was significantly higher than that found for Raji and Daudi cells. These results were confirmed using flow cytometry. Rituximab was found to be localized in patches on the cell membrane. The findings indicated that 227 Th-labeled rituximab has relevant antigen-targeting properties for radioimmunotherapy.