Preparation and examination of 177Lu- and 90Y-labeled immunoconjugates of Trastuzumab (original) (raw)
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Fourier Transform Infrared (FT-IR) and Raman spectroscopy were used to study the molecular structure of the recombinant monoclonal antibody and anti-CD20-conjugates which are intended to be used as anti-cancer therapeutic agents. We characterized the secondary structure of a therapeutic immunoconjugate of rituximab, formulated with three different bifunctional chelating agents (p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, and 1B4M-DTPA) and labeled with non-radioactive lutetium and yttrium. The secondary structure content of all three immunoconjugates was assessed to be similar to that of unlabeled antibody. In addition, no significant changes upon lyophilizing procedures were observed. The results demonstrate that amide bands could be used as analytical tool which provides a quick and reliable way for screening of protein pharmaceuticals during the development of lyophilized formulations.
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
Macedonian Journal of Chemistry and Chemical Engineering, 2015
Fourier Transform Infrared (FT-IR) and Raman spectroscopy were used to study the molecular structure of the recombinant monoclonal antibody and anti-CD20-conjugates which are intended to be used as anti-cancer therapeutic agents. We characterized the secondary structure of a therapeutic immunoconjugate of rituximab, formulated with three different bifunctional chelating agents (p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, and 1B4M-DTPA) and labeled with non-radioactive lutetium and yttrium. The secondary structure content of all three immunoconjugates was assessed to be similar to that of unlabeled antibody. In addition, no significant changes upon lyophilizing procedures were observed. The results demonstrate that amide bands could be used as analytical tool which provides a quick and reliable way for screening of protein pharmaceuticals during the development of lyophilized formulations.
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.
Radioimmunodiagnosis or radioimmunotherapy is a technique that has been developed and used in cancer management in recent years.The use of this technique which is based on the use of conjugated-monoclonal antibody (mAb) for imaging or therapy of cancer cells in the last three decades is due to the selectivity and specificity of monoclonal antibodies to the target. One of the antibodies are often used for breast cancer therapy is the anti-HER2 trastuzumab mAb. The use of trastuzumab however has been reported to have a few side effects. One of these effects has been considered to be life threatening, an increasing risk of lung dysfunctional when used was combined with other chemotherapy agent. Based on this fact, an alpha or beta particle emitter/ radionuclide-conjugated trastuzumab has been developed. The radioimmunoconjugate is expected to more be effective to be used either by itself or in its redueced dose. However, the use of radioimmunoconjugate based on a whole mAb for radioimmunotherapy of cancer has been reported to have some weaknesses due to its relatively large size molecule (approximately 150 kD). Thus, it results in a molecule is less effective in reaching the target cells (low pharmacokinetic) and lack of penetration to the target cells. A solution to this problem is by using a radiommonoconjugate derived from enzymatic fragmentation of mAb..This work is a preliminary study to a preparation of 177Lu-DOTA-F(ab’)2 trastuzumab. The aim of this work was to prepare trastuzumab F (ab')2 fragment which is going to be used for preparation the above mentioned radioimmunoconjugate. The results of this preliminary work to date were, the optimum time for completely digest of trastuzumab to form F(ab’)2 fragment was found to be 18 hrs, where trastuzumab F(ab’)2 fragment was able to be purified by using a PD10 column (G25 Sephadex), and purified trastuzumab F(ab’)2 fragment which was characterized by SE-HPLC equipped with a Bio-Suite SEC250 column and SDS-PAGE. The result showed a purity of F(ab’)2 fragment was carried out of 98% and a molecular weight of approximately 100 kDa respectively.
2015
Trastuzumab (Herceptin®) is a humanized IgG1 monoclonal antibody which is approved for therapy of HER2 positive breast cancer. Good clinical results and improvement of the patient’s general condition makes it interesting for further conjugation, in order to increase the therapeutic effect of trastuzumab. With the development of radiopharmacy many efforts are made for formulation of stable conjugates with various bifunctional chelators, further labelled with radioisotopes (α, β and γ emitters). According to the literature, successful conjugation of trastuzumab is achieved with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), TCMC (1,4,7,10-tetra-(2-carbamoyl methyl)-cyclododecane), HYNIC (succinimidyl-6-hydrazino-nicotinamide) and DTPA derivate 1B4M-DTPA (2-(4-isothiocyanatobenzyl)-6-methyl-diethylene-triaminepentaacetic acid). Significant radiopharmaceuticals based on peptide and antibody for diagnostic and therapeutical pur...
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.
Formulation and Characterization of "Ready to Use" 1B4M-DTPA-rituximab for Lu-177 Labeling
2014
Investigations for NHL treatment are oriented towards radiolabelled therapeutics. This research focuses on formulation and characterization of a new, ready to label immunoconjugate, 1B4M-DTPA-rituximab, which is suitable for labeling with Lu-177. The conjugation was performed using 20-fold molar excess of the bifunctional chelating agent, 1B4M-DTPA and subsequent lyophilization. The characterization of the (radio)immunoconjugate was performed using SE-HPLC, SDS-PAGE and MALDI-TOF-MS. The results show that the conjugation reaction yields immunoconjugates with average of 8.3 chelating groups per one rituximab molecule and the subsequent lyophilization did not affect the integrity, the structure, or radionuclide-binding properties. The radiolabelling with 555 GBq/mg Lu-177 resulted in over 95% radiochemical purity, which makes this agent a good candidate for further investigation of biological and pharmacological potency in NHL therapy.