Preparation of F (ab’) 2 trastuzumab fragment for Radioimmunoconjugate synthesis of 177Lu-DOTA-F (ab’)2- trastuzumab (original) (raw)
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Keywords: Trastuzumab F(ab 0) 2 Radioimmunoconjugate Lutetium-177 Breast cancer a b s t r a c t The use of trastuzumab as intact IgG labeling radionuclide for HER2 positive breast cancer theranostic agent is not ideal because it is slowly eliminated from the blood and normal tissues resulting in low tumor/blood (T/B) and tumor/normal tissue (T/NT) ratios. To overcome this limitation, we developed the trastuzumab F(ab 0) 2 fragments and radio-labeling of the fragments by b and g-particle of Lutetium-177. F(ab) 2 fragments were produced by digestion of trastuzumab IgG (Herceptin) with pepsin for 18 h at 37 C. The F(ab 0) 2 fragment fractionated in PD-10 column, followed by the conjugation with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) as a metal chelator and radiolabeling with 177 LuCl 3. Molecular weight of fragments was calculated by LCMS (Liquid Chromatography Mass Spectroscopy) and the radio-chemical purity was evaluated by ITLC-SG (Instan Thin Layer Chromatography). Our study showed that the purity of F(ab 0) 2 fragment generated by PD-10 fractions was >98% and the molecular weight of F(ab 0) 2 was 98.35 kDa. The average numbers of pSCN-Bn-DOTA che-lates per antibody fragment were 5.03 ± 1.5 and the optimum conjugation reactions was performed at molar ratio 20:1 (chelator to antibody). The stability test of the radio-immunoconjugate in the human serum albumin (HSA) at 37 C showed the radiochemical purity was 91.96 ± 0.26% after 96 h storage. This indicated that the radioimmunoconjugate is relatively stable when applied to the human body's physiological condition. ScienceDirect Journal of Radiation Research and Applied Sciences journal h omepage: http :/ / www .e lsev ie r. co m/ lo cate/ j rras J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s x x x (2 0 1 6) 1 e8
Nuclear Medicine and Biology, 2009
Aim: Trastuzumab is a monoclonal antibody that is used in treating breast cancer. We labeled this monoclonal antibody with lutetium-177 and performed in vitro quality control tests as a first step in the production of a new radiopharmaceutical. Material and Methods: Trastuzumab was labeled with lutetium-177 using DOTA as chelator. Radiochemical purity and stability in buffer and human blood serum were determined using thin layer chromatography. Immunoreactivity and toxicity of the complex were tested on MCF7 breast cancer cell line. Results: The radiochemical purity of the complex was 96±0.9%. The stabilities in phosphate buffer and in human blood serum at 96 h postpreparation were 93±1.2% and 85±3.5%, respectively. The immunoreactivity of the complex was 89±1.4%. At a concentration of 1 nM, the complex killed 70±3% of MCF7 cells. At 1.9 nM, 90±5% of the cells were killed.
Trastuzumab and its radioimmunoconjugates in treatment of cancer
2016
Monoclonal antibodies are new type of targeted anticancer therapy, which achieve specificity, selectivity and localization in tumor cells. There are many naked antibodies and immunoconjugates commercially approved for different types of cancer (Mehren et al., 2003). In order to improve specificity and selectivity of cytotoxic drugs and toxins, monoclonal antibodies are used for formulation of immunoconjugates. Many efforts are done to develop stable immunoconjugates of trastuzumab with various drugs, toxins and radioisotopes to improve the general conditions of the patients (Sharkey and Goldenberg, 2006). The aim of this paper is to focus on current achievements in the formulation of radioimmunoconjugates of HER2-targeting trastuzumab.
Preparation and examination of 177Lu- and 90Y-labeled immunoconjugates of Trastuzumab
2019
Trastuzumab is a monoclonal antibody for treatment of HER2 positive breast cancer. Labelled antibody with lutetium-177 and yttrium-90 has been investigated as potential radiopharmaceutical agent for use in radioimmunotherapy. In this study, the labeling was done via DOTA, DTPA and 1B4M-DTPA as a chelator in molar ratios of 1:20. A several chemical techniques have been used to characterize the stability and retained immunoreactivity of the antibody in the formulated immunoconjugates. A protein integrity and purity were proved by applying of reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Vibrational spectroscopy (Infra-red (IR) and Raman spectroscopy) provide molecular structure information and are convenient for verification of changes in the secondary structure. The number of chelating groups per one trastuzumab molecule was calculated by MALDI-TOF-MS. After conjugation, the freeze-drying process was performed in order to obtain stable immunoconjugate...
Journal of Drug Delivery Science and Technology, 2015
HER2/neu expressing cancers constitute 30% of all breast cancers. Trastuzumab, a monoclonal antibody, is widely used for the treatment of HER2/neu expressing breast cancers. The aim of this study was to conjugate trastuzumab with a bifunctional chelator, cyclic Diethylene triamine-pentaacetic anhydride (cDTPAA) for radiolabeling with Tc-99m to develop a radio-pharmaceutical for assessment of HER2/neu status for treatment planning and response monitoring post trastuzumab treatment. cDTPAA was conjugated to trastuzumab at various molar ratios (10:1e100:1). The conjugates were purified and characterized for the integrity and number of chelator molecules conjugated. Radiolabeling of cDTPAAtrastuzumab conjugates was done with Tc-99 m and labeling parameters were studied. The quality control of the radiolabeled preparation was carried out by radio thin layer chromatography (TLC). Endotoxin estimation and sterility test were performed. Biodistribution studies were carried out at different time intervals (1 h, 6 h and 12 h) post intravenous injections in normal rats. The average number of molecules conjugated varied from 0.75 to 7.5 for the different molar ratios experimented. Maximum radiolabeling yield obtained was 90%. Post purification radiochemical purity was >95%. The biodistribution studies showed high activity in liver, kidney and blood retention up to the period of 12 h.
Development of 90 Y-DOTA-nimotuzumab Fab fragment for radioimmunotherapy
Yttrium-90-( 90 Y) labeled monoclonal antibodies prepared with a chelating agent, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), have been used for radioimmunotherapy of cancer. In the present work, the Fab fragment of anti-EGFR monoclonal antibody nimotuzumab was prepared with high purity, integrity and biological activity. The Fab fragment with high specific recognition of EGFR in NCI-H125 human lung adenocarcinoma cells was derivatized with DOTA-NHS applying a simple procedure. DOTA-nimotuzumab Fab fragment was successfully radiolabeled with 90 Y with high radiochemical yield. The in vitro stability of labeled product was optimal over 24 h in buffered solution at 37°C. Biodistribution and pharmacokinetic studies correctly evaluated the in vivo non-tumor uptake, dosage regimen and excretion pathway in normal Wistar rats.
2017
Intact monoclonal antibodies with a high molecular weight tend to have a poor pharmacokinetic profile and tumor penetration, and potential for eliciting host antibody responses. F(ab’)2 fragments smaller than intact monoclonal antibodies that still maintain antigen binding could solve this problem. The objective of this study was to optimize the digestion process of nimotuzumab, an anti-EGFR monoclonal antibody, into its F(ab’)2 fragment and investigate its potential as a therapeutic radioimmunoconjugate. Optimal conditions for digestion of nimotuzumab to its F(ab’)2 fragment were found to be 6 hours of digestion time with a pH of 3.5 and 1:100 mol ratio of pepsin to nimotuzumab. The purity of the F(ab’)2-nimotuzumab was confirmed by SDSPAGE and HPLC analysis. Prior to its labeling with lutetium-177 radionuclide, the nimotuzumab-F(ab’)2 was conjugated to DOTA-PAMAM dendrimer [DOTA denotes 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, PAMAM denotes poly(amidoamine)] to fo...
Journal of Radioanalytical and Nuclear Chemistry, 2013
Breast cancer radioimmunoscintigraphy targeting HER2/neu expression is a growing field of work in nuclear medicine research. Trastuzumab is a monoclonal antibody that binds with high affinity to HER2/neu, which is over expressed on breast and other tumors. Developing new tracers for the detection of this cancer is of great interest. In this study, trastuzumab was successively labeled with [ 64 Cu]CuCl 2 after conjugation with DOTA-NHS-ester. The conjugate was purified by molecular filtration, the average number of DOTA conjugated per mAb was calculated and total concentration was determined by spectrophotometric method. DOTA-trastuzumab was labeled with 64 Cu produced by 68 Zn(p,an) 64 Cu nuclear reaction (30 MeV protons at 180 lA). Radiochemical purity, integrity of protein after radiolabeling and immunoreactivity of radiolabeled mAb trastuzumab with HER2/ neu antigen and SkBr3 cell line were performed by RIA. In vitro stability of radiolabeled mAb in human serum was determined by thin layer chromatography. In vitro internalization studies were performed with the SkBr3 cell line and the tissue biodistribution of the 64 Cu-DOTA-trastuzumab was evaluated in wild-type rat (90 ± 5.5 lCi, 2, 6, 12, 24 h p.i.). The radioimmunoconjugate was prepared with a radiochemical purity of higher than 96 ± 0.5 % (ITLC) and specific activity as high as 5.3 lCi/lg. The average number of chelators per antibody for the conjugate used in this study was 5.8/1. The sample was showed to have similar patterns of migration in the gel electrophoresis. The 64 Cu-DOTA-trastuzumab showed high immunoreactivity towards HER2/neu antigen and SkBr3 cell line. In vitro stability of the labeled product was found to be more than 94 % in PBS and 82 ± 0.5 % in human serum over 48 h. In vitro internalization studies of the 64 Cu-DOTA-trastuzumab showed that up to 11.5 % of the radioimmunoconjugate internalized after 10 h. The accumulation of the radiolabeled mAb in liver, skin, intestine, lung, spleen, kidney and other tissues demonstrates a similar pattern to the other radiolabeled anti-HER2 immunoconjugates. 64 Cu-DOTA-trastuzumab is a potential compound for molecular imaging of PET for diagnosis and treatment studies and follow-up of HER2 expression in oncology.
EJNMMI research, 2011
The aim of the present study was to explore the biodistribution, normal tissue toxicity, and therapeutic efficacy of the internalizing low-dose rate alpha-particle-emitting radioimmunoconjugate 227Th-trastuzumab in mice with HER2-expressing breast cancer xenografts. Biodistribution of 227Th-trastuzumab and 227Th-rituximab in nude mice bearing SKBR-3 xenografts were determined at different time points after injection. Tumor growth was measured after administration of 227Th-trastuzumab, 227Th-rituximab, cold trastuzumab, and saline. The toxicity of 227Th-trastuzumab was evaluated by measurements of body weight, blood cell, and clinical chemistry parameters, as well as histological examination of tissue specimens. The tumor uptake reached peak levels of 34% ID/g (4.6 kBq/g) 3 days after injection of 400 kBq/kg of 227Th-trastuzumab. The absorbed radiation dose to tumor was 2.9 Gy, while it was 2.4 Gy to femur due to uptake of the daughter nuclide 223Ra in bone; the latter already explor...