Development of anti-CD30 radioimmunoconstructs (RICs) for treatment of Hodgkin's lymphoma

2010 ◽  
Vol 49 (03) ◽  
pp. 97-105 ◽  
Author(s):  
S. M. Börner ◽  
T. Fischer ◽  
H. Hansen ◽  
R. Schnell ◽  
B. Zimmermanns ◽  
...  
Keyword(s):  
Binding Studies ◽  
High Background ◽  
Plot Analysis ◽  
Biodistribution Studies ◽  
Vitro Binding ◽  
Chloramine T ◽  
Radiochemical Yields ◽  
Tumour Blood

Summary Objectives: Comparison of the binding affinity to a CD30-positive Hodgkin lymphoma (HL) cell line and biodistribution in HL bearing mice of new anti-CD30 radioimmunoconjugates (RICs) of varying structure and labelling nuclides. Methods: The antibodies Ki-4 and 5F11 were radioiodinated by the chloramine T method or labelled with 111In via p-NCSBenzyl- DOTA. In addition, the Ki-4-dimer was investigated in the iodinated form. The RICs were analyzed for retained immunoreactivity by immunochromatography. In-vitro binding studies were performed on CD30-positive L540 cell lines. For in-vivo biodistribution studies, SCID mice bearing human HL xenografts were injected with the various radioimmunoconjugates. After 24 h, activities in the organs and tumour were measured for all 5 RICs. Tumour-free animals were studied in the same way with 131I- Ki-4 24 h p. i. The three RICs with the highest tumour/background ratios 24 h p.i. (131I-Ki-4, 131I–5F11, 111In-bz- DOTA-Ki-4) were analysed further at 48 h and 72 h. Results: All the RICs were successfully labelled with high specific activities (28–47 TBq/ mmol) and sufficient radiochemical yields (> 80%). Scatchard plot analysis proved high tumour affinity (KD = 20–220 nmol/l). In-vivo tumour accumulation in % of injected dose per g tissue (%ID/g) lay between 2.6 (131I-5F11) and 12.3 % ID/g (131I-Ki-4) with permanently high background in blood. Tumour/blood-ratios of all RICs were below one at all time points. Conclusions: In-vitro tumour cell affinities of all RICs were promising. However, in-vivo biokinetics tested in the mouse model did not meet expectations. This highlights the importance of developing and testing further new anti-CD30 conjugates.

scholarly journals Effect of ethanol and cocaine on [11C]MPC-6827 uptake in SH-SY5Y cells

2021 ◽  
Author(s):  
Naresh Damuka ◽  
Miranda Orr ◽  
Paul W. Czoty ◽  
Jeffrey L. Weiner ◽  
Thomas J. Martin ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Ex Vivo ◽  
Neuroblastoma Cells ◽  
Proper Function ◽  
Brain Functions ◽  
Biodistribution Studies ◽  
Positron Emission ◽  
Vitro Binding

AbstractMicrotubules (MTs) are structural units in the cytoskeleton. In brain cells they are responsible for axonal transport, information processing, and signaling mechanisms. Proper function of these processes is critical for healthy brain functions. Alcohol and substance use disorders (AUD/SUDs) affects the function and organization of MTs in the brain, making them a potential neuroimaging marker to study the resulting impairment of overall neurobehavioral and cognitive processes. Our lab reported the first brain-penetrant MT-tracking Positron Emission Tomography (PET) ligand [11C]MPC-6827 and demonstrated its in vivo utility in rodents and non-human primates. To further explore the in vivo imaging potential of [11C]MPC-6827, we need to investigate its mechanism of action. Here, we report preliminary in vitro binding results in SH-SY5Y neuroblastoma cells exposed to ethanol (EtOH) or cocaine in combination with multiple agents that alter MT stability. EtOH and cocaine treatments increased MT stability and decreased free tubulin monomers. Our initial cell-binding assay demonstrated that [11C]MPC-6827 may have high affinity to free/unbound tubulin units. Consistent with this mechanism of action, we observed lower [11C]MPC-6827 uptake in SH-SY5Y cells after EtOH and cocaine treatments (e.g., fewer free tubulin units). We are currently performing in vivo PET imaging and ex vivo biodistribution studies in rodent and nonhuman primate models of AUD and SUDs and Alzheimer's disease.

In vitro binding and in vivo biodistribution studies of the neuroprotective peptide humanin using [125I]humanin derivatives

Peptides ◽  
2009 ◽  
Vol 30 (12) ◽  
pp. 2409-2417 ◽  
Author(s):  
Alexandra Evangelou ◽  
Christos Zikos ◽  
Dimitra Benaki ◽  
Maria Pelecanou ◽  
Penelope Bouziotis ◽  
...  
Keyword(s):  
In Vitro Binding ◽  
In Vivo Biodistribution ◽  
Biodistribution Studies ◽  
Vitro Binding

Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation

1987 ◽  
Vol 7 (9) ◽  
pp. 3252-3259
Author(s):  
T Prezant ◽  
K Pfeifer ◽  
L Guarente
Keyword(s):  
Cytochrome C ◽  
Regulatory Region ◽  
Carbon Sources ◽  
Binding Studies ◽  
Multiple Factors ◽  
Vitro Binding ◽  
Genes Encoding ◽  
Nonfermentable Carbon Source

Regulation of the CYC7 gene of Saccharomyces cerevisiae, encoding iso-2-cytochrome c, was studied. Expression was induced about 20-fold by heme and derepressed 4- to 8-fold by a shift from glucose medium to one containing a nonfermentable carbon source. Deletion analysis showed that induction by heme depends upon sequences between -250 and -228 (from the coding sequence) and upon the HAP1 activator gene, previously shown to be required for CYC1 expression (L. Guarente et al., Cell 36:503-511, 1984). Thus, HAP1 coordinates expression of CYC7 and CYC1, the two genes encoding isologs of cytochrome c in S. cerevisiae. HAP1-18, a mutant allele of HAP1, which increased CYC7 expression more than 10-fold, also acted through sequences between -250 and -228. In vitro binding studies showed that the HAP1 product binds to these sequences (see also K. Pfeifer, T. Prezant, and L. Guarente, Cell 49:19-28, 1987) and an additional factor binds to distal sequences that lie between -201 and -165. This latter site augmented CYC7 expression in vivo. Derepression of CYC7 expression in a medium containing nonfermentable carbon sources depended upon sequences between -354 and -295. The interplay of these multiple sites and the factors that bind to them are discussed.

scholarly journals Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein.

1993 ◽  
Vol 13 (9) ◽  
pp. 5524-5537 ◽  
Author(s):  
R M Brazas ◽  
D J Stillman
Keyword(s):  
Zinc Finger Protein ◽  
Cooperative Interaction ◽  
Protein Fusion ◽  
Glutathione S Transferase ◽  
Binding Studies ◽  
Eukaryotic Transcription ◽  
Low Concentrations ◽  
Vitro Binding

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.

scholarly journals Physical and functional interactions between Stat5 and the tyrosine- phosphorylated receptors for erythropoietin and interleukin-3

Blood ◽  
1996 ◽  
Vol 88 (12) ◽  
pp. 4415-4425 ◽  
Author(s):  
H Chin ◽  
N Nakamura ◽  
R Kamiyama ◽  
N Miyasaka ◽  
JN Ihle ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Sh2 Domain ◽  
Binding Studies ◽  
Interleukin 3 ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Docking Sites ◽  
Carboxy Terminal

Erythropoietin (Epo) and interleukin-3 (IL-3) stimulate activation of the Jak2 tyrosine kinase and induce tyrosine phosphorylation and activation of Stat5. In the present study, we have shown that Epo or IL-3 stimulation induces binding of Stat5 to the tyrosine-phosphorylated Epo receptor (EpoR) or IL-3 receptor beta subunit (betaIL3), respectively, in IL-3-dependent 32D cells expressing the EpoR. The binding of Stat5 to these cytokine receptors was shown to be rapid and transient, occurring within 1 minute of stimulation of cells and significantly decreasing after 5 minutes of cell treatment. In vivo binding experiments in COS cells showed that binding of Stat5 to the EpoR was mediated through the Stat5 Src homology 2 (SH2) domain. In vitro binding studies further showed that Stat5, but not other Stats examined, bound specifically to tyrosine-phosphorylated recombinant EpoR fusion proteins. In these in vivo and in vitro binding studies, Stat5 bound, albeit to a lesser degree, to truncated EpoR mutants in which all the intracellular tyrosines except Y-343 were removed. Furthermore, EpoR-derived synthetic phosphotyrosine peptides corresponding to Y-343, Y-401, Y-431, and Y-479 inhibited the in vitro binding of Stat5. When expressed in 32D cells, a mutant EpoR in which all the intracellular tyrosines were removed by carboxy-terminal truncation showed a significantly impaired ability to induce tyrosine phosphorylation of Stat5, particularly at low concentrations of Epo, but exhibited an increased sensitivity to Epo for growth signaling as compared with the wild-type EpoR. These results indicate that Stat5 specifically and transiently binds to the EpoR through the interaction between the Stat5 SH2 domain and specific phosphorylated tyrosines, including Y-343, in the EpoR cytoplasmic domain. It was implied that betaIL3 may also have similar Stat5 docking sites. The Stat5 docking sites in the EpoR were shown to facilitate specific activation of Stat5, which, however, may not be required for the EpoR-mediated growth signaling.

scholarly journals Discrimination among potential activators of the beta-globin CACCC element by correlation of binding and transcriptional properties.

1993 ◽  
Vol 13 (1) ◽  
pp. 44-56 ◽  
Author(s):  
G A Hartzog ◽  
R M Myers
Keyword(s):  
Binding Site ◽  
Site Selection ◽  
Selection Procedure ◽  
Beta Globin ◽  
Binding Studies ◽  
Cis Acting ◽  
Vitro Binding ◽  
Relative Potential

Adult beta-globin-like promoters contain a cis-acting element, CCACACCC, that is conserved across species and is required for wild-type levels of transcription. We have studied the contribution of this element and proteins that interact with it to activate beta-globin transcription. We found that an erythroid-like cell line, MEL, contains several proteins that specifically bind the CACCC element. By comparing the DNA-binding properties of promoters with mutations in the CACCC element with the transcriptional activities of these mutant promoters, we found that two CACCC-binding proteins did not bind to mutant promoters that direct decreased levels of transcription. One of these proteins is the transcriptional activator Sp1, and the other we have designated CACD (CACCC-binding species D). We subjected CACD to a binding site selection procedure and obtained high-affinity CACD binding sites that are identical to that of the beta-globin CACCC element. This result, combined with our finding that CACD binds the CACCC element with a higher affinity than does Sp1, argues that the CACCC element is a target of CACD rather than Sp1. The strategy of correlating the results of a binding site selection experiment with those of in vivo expression and in vitro binding studies may allow evaluation of the relative potential of different proteins to activate transcription through a single cis-acting site.

scholarly journals Microwave-assisted radiolabelling of 52Mn-porphyrins

2021 ◽  
Author(s):  
Peter J. Gawne ◽  
Sara M. A. Pinto ◽  
Karin M. Nielsen ◽  
Mariette M. Pereira ◽  
Rafael T. M. de Rosales
Keyword(s):  
Lipid Bilayers ◽  
Mri Contrast Agents ◽  
Mri Contrast ◽  
Manganese Porphyrins ◽  
Biodistribution Studies ◽  
Positron Emission ◽  
Radiochemical Yields

Manganese porphyrins have several therapeutic/imaging applications; including their use as radioprotectants (in clinical trials), and as paramagnetic MRI contrast agents. The affinity of porphyrins for lipid bilayers also makes them candidates for cell/liposome labelling. We hypothesised that metalation with the positron emission tomography (PET) radionuclide 52Mn (t1/2 = 5.6 d) would allow long-term in vivo biodistribution studies of Mn-porphyrins as well as a method to label and track cells/liposomes, but methods for fast and efficient radiolabelling are lacking. Several porphyrins were produced and radiolabelled by addition to neutralised [52Mn]MnCl2 and heated at 165 oC for 1 h using a microwave (MW) synthesiser at a ligand concentration of 0.6 – 0.7 mM. These conditions were compared with non-MW heating at 70oC. MW radiosynthesis allowed >95 % radiochemical yields (RCY) in just 1 h. Conversely, non-MW heating at 70 oC for 1 h resulted in low RCY (0 – 25 % RCY) and most porphyrins did not reach completion after 24h. Formation of the 52Mn-complexes were confirmed with radio-HPLC by comparison with their non-radioactive 55Mn counterparts. Following this, several 52Mn-porphyrins were used to radiolabel liposomes by incubation at 50 oC for 30 min resulting in 75 – 86 % labelling efficiency (LE). Two lead 52Mn-porphyrins were taken forward to label MDA-MB-231 cancer cells in vitro, achieving ca. 11 % LE. After 24 h, 32 – 45 % of the 52Mn-porphyrin was retained in cells. In contrast to standard methods, MW heating allows fast synthesis of 52Mn-porphyrins with >95% radiochemical yields that avoid purification. 52Mn-porphyrins also show promising cell/liposome labelling properties. This technique can potentially be exploited for the in vivo imaging of Mn-porphyrin therapeutics, as well as for the accurate in vivo quantification of Mn-porphyrin MRI agents.

Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein

1993 ◽  
Vol 13 (9) ◽  
pp. 5524-5537
Author(s):  
R M Brazas ◽  
D J Stillman
Keyword(s):  
Zinc Finger Protein ◽  
Cooperative Interaction ◽  
Protein Fusion ◽  
Glutathione S Transferase ◽  
Binding Studies ◽  
Eukaryotic Transcription ◽  
Low Concentrations ◽  
Vitro Binding

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.

scholarly journals Immuno-PET Imaging of the Programmed Cell Death-1 Ligand (PD-L1) Using a Zirconium-89 Labeled Therapeutic Antibody, Avelumab

2019 ◽  
Vol 18 ◽  
pp. 153601211982998 ◽  
Author(s):  
Elaine M. Jagoda ◽  
Olga Vasalatiy ◽  
Falguni Basuli ◽  
Ana Christina L. Opina ◽  
Mark R. Williams ◽  
...  
Keyword(s):  
Target Tissue ◽  
Dependent Manner ◽  
Binding Studies ◽  
Expression Levels ◽  
High Affinity ◽  
Biodistribution Studies ◽  
Programmed Cell Death 1 ◽  
Spleen And Lymph Nodes

Objective: The goal is to evaluate avelumab, an anti-PD-L1 monoclonal immunoglobulin G antibody labeled with zirconium-89 in human PD-L1-expressing cancer cells and mouse xenografts for clinical translation. Methods: [89Zr]Zr-DFO-PD-L1 monoclonal antibody (mAb) was synthesized using avelumab conjugated to desferrioxamine. In vitro binding studies and biodistribution studies were performed with PD-L1+MDA-MB231 cells and MDA-MB231 xenograft mouse models, respectively. Biodistributions were determined at 1, 2, 3, 5, and 7 days post coinjection of [89Zr]Zr-DFO-PD-L1 mAb without or with unlabeled avelumab (10, 20, 40, and 400 µg). Results: [89Zr]Zr-DFO-PD-L1 mAb exhibited high affinity (Kd ∼ 0.3 nM) and detected moderate PD-L1 expression levels in MDA-MB231 cells. The spleen and lymph nodes exhibited the highest [89Zr]Zr-DFO-PD-L1 mAb uptakes in all time points, while MDA-MB231 tumor uptakes were lower but highly retained. In the unlabeled avelumab dose escalation studies, spleen tissue–muscle ratios decreased in a dose-dependent manner indicating specific [89Zr]Zr-DFO-PD-L1 mAb binding to PD-L1. In contrast, lymph node and tumor tissue–muscle ratios increased 4- to 5-fold at 20 and 40 µg avelumab doses. Conclusions: [89Zr]Zr-DFO-PD-L1 mAb exhibited specific and high affinity for PD-L1 in vitro and had target tissue uptakes correlating with PD-L1 expression levels in vivo. [89Zr]Zr-DFO-PD-L1 mAb uptake in PD-L1+tumors increased with escalating doses of avelumab.

scholarly journals Dynamics of Ankyrin-containing Complexes in Chicken Embryonic Erythroid Cells: Role of Phosphorylation

2001 ◽  
Vol 12 (12) ◽  
pp. 3864-3874 ◽  
Author(s):  
Sourav Ghosh ◽  
John V. Cox
Keyword(s):  
Complex Formation ◽  
Anion Exchanger ◽  
Erythroid Cells ◽  
Binding Studies ◽  
Phosphatase Inhibitors ◽  
In Vitro Binding ◽  
Vitro Binding

Chicken erythroid ankyrin undergoes a fairly rapid cycle of cytoskeletal association, dissociation, and turnover. In addition, the cytoskeletal association of ankyrin is regulated by phosphorylation. Treatment of erythroid cells with serine and threonine phosphatase inhibitors stimulated the hyperphosphorylation of the 225- and 205-kDa ankyrin isoforms, and dissociated the bulk of these isoforms from cytoskeletal spectrin. In vitro binding studies have shown that this dissociation of ankyrin from spectrin in vivo can be attributed to a reduced ability of hyperphosphorylated ankyrin to bind spectrin. Interestingly, a significant fraction of detergent insoluble ankyrin accumulates in a spectrin-independent pool. At least some of this spectrin-independent pool of ankyrin is complexed with the AE1 anion exchanger, and the solubility properties of this pool are also regulated by phosphorylation. Treatment of cells with serine and threonine phosphatase inhibitors had no effect on ankyrin/AE1 complex formation. However, these inhibitors were sufficient to shift ankyrin/AE1 complexes from the detergent insoluble to the soluble pool. These analyses, which are the first to document the in vivo consequences of ankyrin phosphorylation, indicate that erythroid ankyrin-containing complexes can undergo dynamic rearrangements in response to changes in phosphorylation.

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