Transcriptional responses by alpha-emitting radioimmunoconjugate Bi-213-CD20-rituximab
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE7322 | GEO | 2007/07/01
SECONDARY ACCESSION(S): PRJNA98035
REPOSITORIES: GEO
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Project description:The possibility to deliver radioisotopes directly to tumor cells by using monoclonal antibodies has become a promising concept for the elimination of small tumor nodules or single disseminated tumor cells. The alpha-particle emitter Bismut-213 which has a high linear energy transfer and a very short path length appears to be able to kill cells by only few nuclear hits. Bismut-213 based alpha-immunotherapy treatment is currently proved in clinical trials for AML, NHL and preclinically for CLL and multiple myeloma. Because little is known about the biological effects of alpha radiation we examined the molecular effects of the alpha emitter Bismut-213 in malignant and normal B-cells. In this study, we used the cell line Karpas 422 (K422) which had been derived from a malignant B-cell Non-Hodgkin’s lymphoma as well as normal primary CD19+ B-cells which had been selected immunomagnetically from peripheral blood. For the delivery and binding of the alpha emitter we used the monoclonal antibody rituximab which targets the CD20 receptor of malignant and normal B-cells. The lymphoma cell line K422 was incubated in vitro with Bismut-213-CD20 with 100 µCi and 200 µCi respectively, for 46 min (1 half life time of the alpha emitter) or 24 h. Immunomagnetically isolated CD19+ cells from healthy donors were incubated in vitro with Bismut-213-CD20 with 100 µCi or 200 µCi, for 46 min. Gene expression profiles of irradiated cells were analysed using cDNA oligonucleotide arrays (Affymetrix Human Genome Focus Arrays) comprising 8,793 genes and compared with those from untreated control cells. Following normalization significantly altered genes were identified by using the variance stabilization normalization (VSN) method and the significance analysis of microarrays (SAM) algorithm. Differentially expressed genes were defined to exhibit a fold change of 1,4 and higher or 0,7 and lower and a q-value of 5% or lower compared to the genes of the untreated control cells. Irradiated K422 cells with 100 µCi showed 42 differential expressed genes after 46 minutes (one half life time) and 451 deregulated genes after exposure of 24 hours. A similar response could be observed at an exposure of 200 µCi (50 differentially expressed genes after 46 min; 485 genes after 24 h). Hence, a severe radiation induced response could not be detected before 24 hours. 200 µCi irradiated CD19+ cells showed 67 differentially expressed genes after 46 minutes compared to the untreated CD19+ cells (for 100 µCi: 42 differentially expressed genes). By performing cluster analyses the irradiated cells exhibited a distinct homogenous molecular phenotype in comparison to not irradiated cells. Alpha irradiated cells showed transcriptional activation of DNA repair genes. Furthermore, both examined cell types showed up-regulated genes which are known to play a role in general and oxidative stress response. Surprisingly, up-regulation of interferon-stimulated genes in K422 cells could be observed. Because of the transcriptional activation of genes regulating the G1-S- cell cycle transition the supposition came up that the irradiated K422 do progress into the S-phase of the cell cycle and arrest in the S- or G2/M-phase. In contrast to the K422 cells the primary CD19+ B-cells revealed transcriptional activation of cell cycle inhibitors which is an already known reaction following ionizing radiation but no apoptosis associated genes could be observed. In spite of the massive cytotoxic damages which are assumed referring to the gene expression data the K422 cells appear to be able to activate several defense mechanisms in a precise manner. Hence, the malignant K422 cells exhibit a larger amount as well as more various responses than the CD19+ cells. This might be due to the tumorigenic transformation of the malignant cells that exhibit probably more effective defense strategies than the CD19+ cells. The balance between the therapeutic effect of alpha radiation on malignant cells and the damaging effect on normal cells is an essential issue and it is of important clinical relevance concerning radioimmunotherapy with alpha radiation. Keywords: time course and dose dependency
2007-07-01 | GSE7291 | GEO
Project description:The possibility to deliver radioisotopes directly to tumor cells by using monoclonal antibodies has become a promising concept for the elimination of small tumor nodules or single disseminated tumor cells. The alpha-particle emitter Bismut-213 which has a high linear energy transfer and a very short path length appears to be able to kill cells by only few nuclear hits. Bismut-213 based alpha-immunotherapy treatment is currently proved in clinical trials for AML, NHL and preclinically for CLL and multiple myeloma. Because little is known about the biological effects of alpha radiation we examined the molecular effects of the alpha emitter Bismut-213 in malignant and normal B-cells. In this study, we used the cell line Karpas 422 (K422) which had been derived from a malignant B-cell Non-Hodgkin’s lymphoma as well as normal primary CD19+ B-cells which had been selected immunomagnetically from peripheral blood. For the delivery and binding of the alpha emitter we used the monoclonal antibody rituximab which targets the CD20 receptor of malignant and normal B-cells. The lymphoma cell line K422 was incubated in vitro with Bismut-213-CD20 with 100 µCi and 200 µCi respectively, for 46 min (1 half life time of the alpha emitter) or 24 h. Immunomagnetically isolated CD19+ cells from healthy donors were incubated in vitro with Bismut-213-CD20 with 100 µCi or 200 µCi, for 46 min. Gene expression profiles of irradiated cells were analysed using cDNA oligonucleotide arrays (Affymetrix Human Genome Focus Arrays) comprising 8,793 genes and compared with those from untreated control cells. Following normalization significantly altered genes were identified by using the variance stabilization normalization (VSN) method and the significance analysis of microarrays (SAM) algorithm. Differentially expressed genes were defined to exhibit a fold change of 1,4 and higher or 0,7 and lower and a q-value of 5% or lower compared to the genes of the untreated control cells. Irradiated K422 cells with 100 µCi showed 42 differential expressed genes after 46 minutes (one half life time) and 451 deregulated genes after exposure of 24 hours. A similar response could be observed at an exposure of 200 µCi (50 differentially expressed genes after 46 min; 485 genes after 24 h). Hence, a severe radiation induced response could not be detected before 24 hours. 200 µCi irradiated CD19+ cells showed 67 differentially expressed genes after 46 minutes compared to the untreated CD19+ cells (for 100 µCi: 42 differentially expressed genes). By performing cluster analyses the irradiated cells exhibited a distinct homogenous molecular phenotype in comparison to not irradiated cells. Alpha irradiated cells showed transcriptional activation of DNA repair genes. Furthermore, both examined cell types showed up-regulated genes which are known to play a role in general and oxidative stress response. Surprisingly, up-regulation of interferon-stimulated genes in K422 cells could be observed. Because of the transcriptional activation of genes regulating the G1-S- cell cycle transition the supposition came up that the irradiated K422 do progress into the S-phase of the cell cycle and arrest in the S- or G2/M-phase. In contrast to the K422 cells the primary CD19+ B-cells revealed transcriptional activation of cell cycle inhibitors which is an already known reaction following ionizing radiation but no apoptosis associated genes could be observed. In spite of the massive cytotoxic damages which are assumed referring to the gene expression data the K422 cells appear to be able to activate several defense mechanisms in a precise manner. Hence, the malignant K422 cells exhibit a larger amount as well as more various responses than the CD19+ cells. This might be due to the tumorigenic transformation of the malignant cells that exhibit probably more effective defense strategies than the CD19+ cells. The balance between the therapeutic effect of alpha radiation on malignant cells and the damaging effect on normal cells is an essential issue and it is of important clinical relevance concerning radioimmunotherapy with alpha radiation. Keywords: time course and dose dependency
2007-07-01 | GSE7292 | GEO
Project description:Transcriptional responses by alpha-emitting radioimmunoconjugate Bi-213-CD20-rituximab
| PRJNA98035 | ENA
Project description:Background213Bismuth (213Bi, T1/2 = 45.6 min) is one of the most frequently used α-emitters in cancer research. High specific activity radioligands are required for peptide receptor radionuclide therapy. The use of generators containing less than 222 MBq 225Ac (actinium), due to limited availability and the high cost to produce large-scale 225Ac/213Bi generators, might complicate in vitro and in vivo applications though.Here we present optimized labelling conditions of a DOTA-peptide with an 225Ac/213Bi generator (< 222 MBq) for preclinical applications using DOTA-Tyr3-octreotate (DOTATATE), a somatostatin analogue. The following labelling conditions of DOTATATE with 213Bi were investigated; peptide mass was varied from 1.7 to 7.0 nmol, concentration of TRIS buffer from 0.15 mol.L-1 to 0.34 mol.L-1, and ascorbic acid from 0 to 71 mmol.L-1 in 800 μL. All reactions were performed at 95 °C for 5 min. After incubation, DTPA (50 nmol) was added to stop the labelling reaction. Besides optimizing the labelling conditions, incorporation yield was determined by ITLC-SG and radiochemical purity (RCP) was monitored by RP-HPLC up to 120 min after labelling. Dosimetry studies in the reaction vial were performed using Monte Carlo and in vitro clonogenic assay was performed with a rat pancreatic tumour cell line, CA20948.ResultsAt least 3.5 nmol DOTATATE was required to obtain incorporation ≥ 99 % with 100 MBq 213Bi (at optimized pH conditions, pH 8.3 with 0.15 mol.L-1 TRIS) in a reaction volume of 800 μL. The cumulative absorbed dose in the reaction vial was 230 Gy/100 MBq in 30 min. A minimal final concentration of 0.9 mmol.L-1 ascorbic acid was required for ~100 MBq (t = 0) to minimize radiation damage of DOTATATE. The osmolarity was decreased to 0.45 Osmol/L.Under optimized labelling conditions, 213Bi-DOTATATE remained stable up to 2 h after labelling, RCP was ≥ 85 %. In vitro showed a negative correlation between ascorbic acid concentration and cell survival.Conclusion213Bismuth-DOTA-peptide labelling conditions including peptide amount, quencher and pH were optimized to meet the requirements needed for preclinical applications in peptide receptor radionuclide therapy.
| S-EPMC5843812 | biostudies-literature
Project description:Since NGR-tripeptides (asparagine-glycine-arginine) selectively target neoangiogenesis-associated Aminopeptidase N (APN/CD13) on cancer cells, we aimed to evaluate the in vivo tumour targeting capability of radiolabelled, NGR-containing, ANP/CD13-selective [213Bi]Bi-DOTAGA-cKNGRE in CD13pos. HT1080 fibrosarcoma-bearing severe combined immunodeficient CB17 mice. 10 ± 1 days after cancer cell inoculation, positron emission tomography (PET) was performed applying [68Ga]Ga-DOTAGA-cKNGRE for tumour verification. On the 7th, 8th, 10th and 12th days the treated group of tumourous mice were intraperitoneally administered with 4.68 ± 0.10 MBq [213Bi]Bi-DOTAGA-cKNGRE, while the untreated tumour-bearing animals received 150 μL saline solution. In addition to body weight (BW) and tumour volume measurements, ex vivo biodistribution studies were conducted 30 and 90 min postinjection (pi.). The following quantitative standardised uptake values (SUV) confirmed the detectability of the HT1080 tumours: SUVmean and SUVmax: 0.37 ± 0.09 and 0.86 ± 0.14, respectively. Although no significant difference (p ≤ 0.05) was encountered between the BW of the treated and untreated mice, their tumour volumes measured on the 9th, 10th and 12th days differed significantly (p ≤ 0.01). Relatively higher [213Bi]Bi-DOTAGA-cKNGRE accumulation of the HT1080 neoplasms (%ID/g: 0.80 ± 0.16) compared with the other organs at 90 min time point yields better tumour-to-background ratios. Therefore, the therapeutic application of APN/CD13-affine [213Bi]Bi-DOTAGA- cKNGRE seems to be promising in receptor-positive fibrosarcoma treatment.
| S-EPMC9968005 | biostudies-literature
Project description:Here, we propose tailored lipid liquid-crystalline carriers (cubosomes), which incorporate an anticancer drug (doxorubicin) and complexed short-lived α-emitter (bismuth-213), as a strategy to obtain more effective action toward the cancer cells. Cubosomes were formulated with doxorubicin (DOX) and an amphiphilic ligand (DOTAGA-OA), which forms stable complexes with 213Bi radionuclide. The behavior of DOX incorporated into the carrier together with the chelating agent was investigated, and the drug liberation profile was determined. The experiments revealed that the presence of the DOTAGA-OA ligand affects the activity of DOX when they are incorporated into the same carrier. This unexpected influence was explained based on the results of release studies, which proved the contribution of electrostatics in molecular interactions between the positively charged DOX and negatively charged DOTAGA-OA in acidic and neutral solutions. A significant decrease in the viability of HeLa cancer cells was achieved using sequential cell exposure: first to the radiolabeled cubosomes containing 213Bi complex and next to DOX-doped cubosomes. Therefore, the sequential procedure for the delivery of both drugs encapsulated in cubosomes is suggested for further biological and in vivo studies.
| S-EPMC9346610 | biostudies-literature
Project description:Rituximab, a murine-human chimera, is the first monoclonal antibody (mAb) developed as a therapeutic agent to target CD20 protein. Its Fab domain and its interaction with CD20 have been extensively studied and high-resolution atomic models obtained by X-ray diffraction or cryo-electron microscopy are available. However, the structure of the full-length antibody is still missing as the inherent protein flexibility hampers the formation of well-diffracting crystals and the reconstruction of 3D microscope images. The global structure of rituximab from its dilute solution is here elucidated by small-angle X-ray scattering (SAXS). The limited data resolution achievable by this technique has been compensated by intensive computational modelling that led to develop a new and effective procedure to characterize the average mAb conformation as well as that of the single domains. SAXS data indicated that rituximab adopts an asymmetric average conformation in solution, with a radius of gyration and a maximum linear dimension of 52 Å and 197 Å, respectively. The asymmetry is mainly due to an uneven arrangement of the two Fab units with respect to the central stem (the Fc domain) and reflects in a different conformation of the individual units. As a result, the Fab elbow angle, which is a crucial determinant for antigen recognition and binding, was found to be larger (169°) in the more distant Fab unit than that in the less distant one (143°). The whole flexibility of the antibody has been found to strongly depend on the relative inter-domain orientations, with one of the Fab arms playing a major role. The average structure and the amount of flexibility has been studied in the presence of different buffers and additives, and monitored at increasing temperature, up to the complete unfolding of the antibody. Overall, the structural characterization of rituximab can help in designing next-generation anti-CD20 antibodies and finding more efficient routes for rituximab production at industrial level.
| S-EPMC9037831 | biostudies-literature
Project description:The radionuclide 213Bi can be applied for targeted α therapy (TAT): a type of nuclear medicine that harnesses α particles to eradicate cancer cells. To use this radionuclide for this application, a bifunctional chelator (BFC) is needed to attach it to a biological targeting vector that can deliver it selectively to cancer cells. Here, we investigated six macrocyclic ligands as potential BFCs, fully characterizing the Bi3+ complexes by NMR spectroscopy, mass spectrometry, and elemental analysis. Solid-state structures of three complexes revealed distorted coordination geometries about the Bi3+ center arising from the stereochemically active 6s2 lone pair. The kinetic properties of the Bi3+ complexes were assessed by challenging them with a 1000-fold excess of the chelating agent diethylenetriaminepentaacetic acid (DTPA). The most kinetically inert complexes contained the most basic pendent donors. Density functional theory (DFT) and quantum theory of atoms in molecules (QTAIM) calculations were employed to investigate this trend, suggesting that the kinetic inertness is not correlated with the extent of the 6s2 lone pair stereochemical activity, but with the extent of covalency between pendent donors. Lastly, radiolabeling studies of 213Bi (30-210 kBq) with three of the most promising ligands showed rapid formation of the radiolabeled complexes at room temperature within 8 min for ligand concentrations as low as 10-7 M, corresponding to radiochemical yields of >80%, thereby demonstrating the promise of this ligand class for use in 213Bi TAT.
| S-EPMC8491275 | biostudies-literature
Project description:Hepta- and octadentate bispidines (3,7-diazabicyclo[3.3.1]nonane, diaza-adamantane) with acetate, methyl-pyridine, and methyl-picolinate pendant groups at the amine donors of the bispidine platform have been prepared and used to investigate BiIII coordination chemistry. Crystal structure and solution spectroscopic data (NMR spectroscopy and mass spectrometry) confirm that the rigid and relatively large bispidine cavity with an axially distorted geometry is well suited for BiIII and in all cases forms nine-coordinate complexes; this is supported by an established hole size and shape analysis. It follows that nonadentate bispidines probably will be more suited as bifunctional chelators for 213 BiIII -based radiopharmaceuticals. However, two isomeric picolinate-/acetate-based heptadentate ligands already show very efficient complexation kinetics with 213 BiIII at ambient temperature and kinetic stability that is comparable with the standard ligands used in this field. The experimentally determined hydrophilicities (log D7.4 values) show that the BiIII complexes reported are relatively hydrophilic and well suited for medicinal applications. We also present a very efficient and relatively accurate method to compute charge distributions and hydrophilicities, and this will help to further optimize the systems reported here.
| S-EPMC7496608 | biostudies-literature
Project description:BackgroundQuantification of actinium-225 through gamma counter measurements, when there is no secular equilibrium between actinium-225 and its gamma emitting daughters bismuth-213 and/or francium-221, can provide valuable information regarding the possible relocation of recoiled daughters such that related radiotoxicity effects can be evaluated. This study proposes a multiple time-point protocol using the bismuth-213 photopeak with measurements before secular equilibrium between actinium-225 and bismuth-213, and a single time-point protocol using both the francium-221 and bismuth-213 photopeak while assuming secular equilibrium between actinium-225 and francium-221 but not between bismuth-213 and actinium-225.ResultsGood agreement (i.e. 3% accuracy) was obtained when relying on a multiple time-points measurement of bismuth-213 to quantify both actinium-225 and excess of bismuth-213. Following scatter correction, actinium-225 can be accurately quantified using the francium-221 in a single time-point measurement within 3% of accuracy. The analysis performed on the stability data of [225Ac]Ac-DEPA and [225Ac]Ac-DOTA complexes, before secular equilibrium between bismuth-213 and actinium-225 was formed, revealed considerable amounts of unbound bismuth-213 (i.e. more than 90%) after 24 h of the radiolabeling most likely due to the recoiled daughter effect.ConclusionBoth protocols were able to accurately estimate 225Ac-activities provided the francium-221 energy window was corrected for the down scatter of the higher-energy gamma-emissions by bismuth-213. This could prove beneficial to study the recoiled daughter effect and redistribution of free bismuth-213 by monitoring the accumulation or clearance of bismuth-213 in different tissues during biodistribution studies or in patient samples during clinical studies. On the other hand, the single gamma counter measurement protocol, although required a 30 min waiting time, is more time and cost efficient and therefore more appropriate for standardized quality control procedures of 225Ac-labeled radiopharmaceuticals.
| S-EPMC9588853 | biostudies-literature