scholarly journals The Use of a Non-Conventional Long-Lived Gallium Radioisotope 66Ga Improves Imaging Contrast of EGFR Expression in Malignant Tumours Using DFO-ZEGFR:2377 Affibody Molecule

Pharmaceutics ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 292 ◽  
Author(s):  
Maryam Oroujeni ◽  
Tianqi Xu ◽  
Katherine Gagnon ◽  
Sara S. Rinne ◽  
Jan Weis ◽  
...  
Keyword(s):  
Pet Imaging ◽  
Growth Factor Receptor ◽  
Whole Body ◽  
Malignant Tumours ◽  
Affibody Molecule ◽  
Egfr Expression ◽  
Biodistribution Data ◽  
Positron Emitter

Epidermal growth factor receptor (EGFR) is overexpressed in many malignancies. EGFR-targeted therapy extends survival of patients with disseminated cancers. Radionuclide molecular imaging of EGFR expression would make EGFR-directed treatment more personalized and therefore more efficient. A previous study demonstrated that affibody molecule [68Ga]Ga-DFO-ZEGFR:2377 permits specific positron-emission tomography (PET) imaging of EGFR expression in xenografts at 3 h after injection. We anticipated that imaging at 24 h after injection would provide higher contrast, but this is prevented by the short half-life of 68Ga (67.6 min). Here, we therefore tested the hypothesis that the use of the non-conventional long-lived positron emitter 66Ga (T1/2 = 9.49 h, β+ = 56.5%) would permit imaging with higher contrast. 66Ga was produced by the 66Zn(p,n)66Ga nuclear reaction and DFO-ZEGFR:2377 was efficiently labelled with 66Ga with preserved binding specificity in vitro and in vivo. At 24 h after injection, [66Ga]Ga-DFO-ZEGFR:2377 provided 3.9-fold higher tumor-to-blood ratio and 2.3-fold higher tumor-to-liver ratio than [68Ga]Ga-DFO-ZEGFR:2377 at 3 h after injection. At the same time point, [66Ga]Ga-DFO-ZEGFR:2377 provided 1.8-fold higher tumor-to-blood ratio, 3-fold higher tumor-to-liver ratio, 1.9-fold higher tumor-to-muscle ratio and 2.3-fold higher tumor-to-bone ratio than [89Zr]Zr-DFO-ZEGFR:2377. Biodistribution data were confirmed by whole body PET combined with magnetic resonance imaging (PET/MRI). The use of the positron emitter 66Ga for labelling of DFO-ZEGFR:2377 permits PET imaging of EGFR expression at 24 h after injection and improves imaging contrast.

Enhanced expression of EGF receptor in a model of salt-sensitive hypertension

2005 ◽  
Vol 289 (2) ◽  
pp. F314-F321 ◽  
Author(s):  
Wei-Zhong Ying ◽  
Paul W. Sanders
Keyword(s):  
Egf Receptor ◽  
Primary Cultures ◽  
Growth Factor Receptor ◽  
Sprague Dawley ◽  
Renal Vasculature ◽  
Downstream Signaling ◽  
Egfr Expression ◽  
Enhanced Expression

Chronic kidney disease in the Dahl/Rapp salt-sensitive (S) rat is related to an arteriolopathic process that occurs following the onset of hypertension and involves vascular smooth muscle cell (VSMC) hyperplasia and luminal constriction. Because previous studies have shown that activation of the epidermal growth factor receptor (EGFR) produces a mitogenic stimulus in VSMC and the EGFR participates integrally in the vasoconstrictor responses of renal arterioles, the present study analyzed the expression of EGFR in these animals. Compared with Sprague-Dawley (SD) rats, renal cortical expression of EGFR was increased in both prehypertensive and hypertensive S rats. Immunohistochemistry using a polyclonal antibody to EGFR demonstrated that EGFR expression was prominent in the renal vasculature, particularly in the media of afferent and efferent arterioles and the aorta of S rats. When examined, primary cultures of VSMC from S rats showed increased expression of EGFR, compared with VSMC from SD and Dahl/Rapp salt-resistant rats. Following addition of EGF, autophosphorylation of the EGFR was enhanced in cells from S rats, as was the downstream signaling events that included activation of p42/44 MAPK and Akt pathways. Thus in vivo and in vitro studies demonstrated augmented expression and functional activity of the EGFR in S rats.

scholarly journals Src Inhibitors Pyrazolo[3,4-d]pyrimidines, Si306 and Pro-Si306, Inhibit Focal Adhesion Kinase and Suppress Human Glioblastoma Invasion In Vitro and In Vivo

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1570 ◽  
Author(s):  
Marija Nešović ◽  
Aleksandra Divac Rankov ◽  
Ana Podolski-Renić ◽  
Igor Nikolić ◽  
Goran Tasić ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Growth Factor Receptor ◽  
Similar Efficacy ◽  
Src Tyrosine Kinase ◽  
Downstream Signaling ◽  
Egfr Expression ◽  
Human Gbm

Glioblastoma (GBM), as the most aggressive brain tumor, displays a high expression of Src tyrosine kinase, which is involved in the survival, migration, and invasiveness of tumor cells. Thus, Src emerged as a potential target for GBM therapy. The effects of Src inhibitors pyrazolo[3,4-d]pyrimidines, Si306 and its prodrug pro-Si306 were investigated in human GBM cell lines (U87 and U87-TxR) and three primary GBM cell cultures. Primary GBM cells were more resistant to Si306 and pro-Si306 according to the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. However, the ability of all GBM cells to degrade the extracellular matrix was considerably compromised after Si306 and pro-Si306 applications. Besides reducing the phosphorylation of Src and its downstream signaling pathway components, both compounds decreased the phosphorylated form of focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) expression, showing the potential to suppress the aggressiveness of GBM. In vivo, Si306 and pro-Si306 displayed an anti-invasive effect against U87 xenografts in the zebrafish embryo model. Considering that Si306 and pro-Si306 are able to cross the blood–brain barrier and suppress the spread of GBM cells, we anticipate their clinical testing in the near future. Moreover, the prodrug showed similar efficacy to the drug, implying the rationality of its use in clinical settings.

scholarly journals BIOM-22. EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) AS A MOLECULAR DETERMINANT OF GLIOBLASTOMA RESPONSE TO DOPAMINE RECEPTOR 2 (DRD2) INHIBITORS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii6-ii6
Author(s):  
Yuyu He ◽  
Jie Li ◽  
Tomoyuki Koga ◽  
Jun Ma ◽  
Sanjay Dhawan ◽  
...  
Keyword(s):  
Dopamine Receptor ◽  
Ectopic Expression ◽  
Growth Factor Receptor ◽  
Inverse Correlation ◽  
Primary Brain Tumor ◽  
The Cancer Genome Atlas ◽  
Dopamine Synthesis ◽  
Egfr Expression

Abstract BACKGROUND There are ongoing clinical trials exploring the efficacy of dopamine receptor 2 (DRD2) inhibition against glioblastomas, the most common primary brain tumor. Here we examine potential molecular determinants of this efficacy. METHODS The Cancer Genome Atlas (TCGA) glioblastoma database and other published mRNA profiles were used to analyze the DRD2 and EGFR expression pattern. In vitro and in vivo responses to DRD2 inhibitors were determined using patient derived xenograft (PDX) glioblastoma models. Immunohistochemical studies were performed on clinically annotated glioblastoma samples derived from patients treated with ONC201, a DRD2 inhibitor. RESULTS Analysis of clinical glioblastoma specimens derived from independent patient cohorts revealed an inverse correlation between EGFR and DRD mRNA expression, with implication that signaling mediated by these proteins shares overlapping functions. In independent panels of PDX glioblastoma lines, high EGFR expression was associated with poor in vitro and in vivo response to DRD2 inhibitors, including haloperidol and ONC201. Moreover, ectopic expression of a constitutively active EGFR, EGFRvIII, suppressed glioblastoma sensitivity to ONC201. DRD2 expression positively correlated with expression of rate-limiting enzymes for dopamine synthesis as well as dopamine secretion, suggesting contribution of autocrine DRD2 signaling. Analysis of specimens from patients treated with ONC201 (n = 15) showed an inverse correlation between the intensity of EGFR staining and clinical response. The median overall survival for patients with high and low EGFR staining was 162 and 373 days, respectively (p = 0.037). CONCLUSIONS High EGFR expression is a determinant of poor glioblastoma response to DRD2. This finding should inform future clinical trial designs.

scholarly journals HER3 PET Imaging: 68Ga-Labeled Affibody Molecules Provide Superior HER3 Contrast to 89Zr-Labeled Antibody and Antibody-Fragment-Based Tracers

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 4791
Author(s):  
Sara S. Rinne ◽  
Charles Dahlsson Leitao ◽  
Ayman Abouzayed ◽  
Anzhelika Vorobyeva ◽  
Vladimir Tolmachev ◽  
...  
Keyword(s):  
Pet Imaging ◽  
Human Cancer ◽  
Growth Factor Receptor ◽  
Antibody Fragment ◽  
Preclinical Model ◽  
Human Cancer Cells ◽  
Her3 Expression ◽  
In Vivo Autoradiography

HER3 (human epidermal growth factor receptor type 3) is a challenging target for diagnostic radionuclide molecular imaging due to the relatively modest overexpression in tumors and substantial expression in healthy organs. In this study, we compared four HER3-targeting PET tracers based on different types of targeting molecules in a preclinical model: the 89Zr-labeled therapeutic antibody seribantumab, a seribantumab-derived F(ab)2-fragment labeled with 89Zr and 68Ga, and the 68Ga-labeled affibody molecule [68Ga]Ga-ZHER3. The novel conjugates were radiolabeled and characterized in vitro using HER3-expressing BxPC-3 and DU145 human cancer cells. Biodistribution was studied using Balb/c nu/nu mice bearing BxPC-3 xenografts. HER3-negative RAMOS xenografts were used to demonstrate binding specificity in vivo. Autoradiography was conducted on the excised tumors. nanoPET/CT imaging was performed. New conjugates specifically bound to HER3 in vitro and in vivo. [68Ga]Ga-DFO-seribantumab-F(ab’)2 was considered unsuitable for imaging due to the low stability and high uptake in normal organs. The highest tumor-to-non-tumor contrast with [89Zr]Zr-DFO-seribantumab and [89Zr]Zr-DFO-seribantumab-F(ab’)2 was achieved at 96 h and 48 h pi, respectively. Despite lower tumor uptake, [68Ga]Ga-ZHER3 provided the best imaging contrast due to the fastest clearance from blood and normal organs. The results of our study suggest that affibody-based tracers are more suitable for PET imaging of HER3 expression than antibody- and antibody-fragment-based tracers.

scholarly journals Affibody Molecule for Positron Emission Tomography Imaging of Human Epidermal Growth Factor Receptor: Validation in Mouse Models and Human Cancer Tissues

2021 ◽  
Author(s):  
Weizhi Chen ◽  
Shuang Miao ◽  
Yao Sun ◽  
Yang Liu ◽  
Chunhe Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Radionuclide Imaging ◽  
Pet Imaging ◽  
Growth Factor Receptor ◽  
Affibody Molecule ◽  
Egfr Expression ◽  
Epidermal Growth

Abstract Background: Tumor heterogeneity and changes in epidermal growth factor receptor (EGFR) expression status over time post challenges for the design of strategies for effective anti-EGFR monoclonal antibodies in the treatment of non-small-cell lung cancer (NSCLC). Therefore, there is an urgent need to develop techniques for real-time and comprehensive tumor EGFR profiling especially in lung cancer precision medicine trials. Radionuclide imaging of EGFR expression in tumors may screen patients for EGFR-targeting therapies and predict response or resistance to certain treatments.Methods: EGFR-specific Affibody molecule (ZEGFR:1907) was radiolabeled with 68Ga. The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression: HCC827 (EGFR overexpression), H1975 (moderate-high), A549 (moderate), H358 (low), and H520 (negative). In vivo tumor targeting activity using PET imaging and biodistribution were conducted in tumor-bearing nude mice. Autoradiography, western blot, immunofluorescence, and immunohistochemistry were performed in human tumor samples. Statistical analyses were performed using GraphPad Prism 7.0. One-way or two-way analysis of variance (ANOVA) followed by the Bonferroni’s multiple comparisons test was used. Statistical significance was set at P < 0.05.Results: 68Ga-NOTA-ZEGFR:1907 showed higher uptake in high EGFR-expressing cells (HCC827, H1975) when compared to cells with moderate to low EGFR (A549, H358) or without EGFR (H520). Radionuclide imaging showed probe accumulation was preferential in EGFR-expressing tumors, particularly in HCC827, H1975 xenografts. A549 and H358 xenografts were mildly and indistinctly visualized. EGFR-negative H520 xenografts were barely visible at any time-point. Biodistribution showed a significantly higher accumulation in HCC827 tumors when compared to H520 tumors (3.20 ± 0.10 %ID/g vs. 0.81 ± 0.08 %ID/g at 2h, P< 0.05). Specific binding to EGFR could be competitively blocked by excess un-radiolabeled affibody molecules in cell uptake, PET imaging and biodistribution assays. Autoradiography showed the regions with high radiotracer uptake partly overlapped with the area of positive EGFR immunofluorescence and immunohistochemistry. Finally, the overall accumulation of autoradiography was positively correlated with immunohistochemistry score.Conclusion: Affibody-based radiotracer 68Ga-NOTA-ZEGFR:1907 is suitable for identification of EGFR expression, showing great potential for further applications and clinical translation.

scholarly journals Epidermal growth factor receptor as a molecular determinant of glioblastoma response to dopamine receptor D2 inhibitors

2020 ◽  
Author(s):  
Yuyu He ◽  
Jie Li ◽  
Tomoyuki Koga ◽  
Jun Ma ◽  
Sanjay Dhawan ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Dopamine Receptor ◽  
Growth Factor Receptor ◽  
Inverse Correlation ◽  
Egfr Expression ◽  
Epidermal Growth

Abstract Background There are ongoing clinical trials exploring the efficacy of dopamine receptor D2 (DRD2) inhibition against glioblastomas, the most common primary brain tumor. Here we examine potential molecular determinants of this efficacy. Methods The Cancer Genome Atlas glioblastoma database and other published mRNA profiles were used to analyze the DRD2 and epidermal growth factor receptor (EGFR) expression pattern. In vitro and in vivo responses to DRD2 inhibitors were determined using patient-derived xenograft (PDX) glioblastoma models. Immunohistochemical studies were performed on clinically annotated glioblastoma samples derived from patients treated with ONC201. Results Analysis of clinical glioblastoma specimens derived from independent patient cohorts revealed an inverse correlation between EGFR and DRD2 mRNA expression, with implication that signaling mediated by these proteins shares overlapping functions. In independent panels of PDX glioblastoma lines, high EGFR expression was associated with poor in vitro and in vivo response to DRD2 inhibitors, including haloperidol and ONC201. Moreover, ectopic expression of a constitutively active EGFR, variant (v)III, suppressed glioblastoma sensitivity to ONC201. DRD2 expression positively correlated with expression of rate-limiting enzymes for dopamine synthesis as well as dopamine secretion, suggesting contribution of autocrine DRD2 signaling. Analysis of specimens from patients treated with ONC201 (n = 15) showed an inverse correlation between the intensity of EGFR staining and clinical response. The median overall survival for patients with high and low EGFR staining was 162 and 373 days, respectively (0.037). Conclusions High EGFR expression is a determinant of poor glioblastoma response to DRD2. This finding should inform future clinical trial designs.

scholarly journals Immunogenicity Risk Profile of Nanobodies

2021 ◽  
Vol 12 ◽  
Author(s):  
Chloé Ackaert ◽  
Natalia Smiejkowska ◽  
Catarina Xavier ◽  
Yann G. J. Sterckx ◽  
Sofie Denies ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Heavy Chain ◽  
Pet Imaging ◽  
Growth Factor Receptor ◽  
Mannose Receptor ◽  
Risk Profile ◽  
Negative Effects ◽  
Diagnostic Applications

Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or in vivo imaging reagents entering the clinic. They possess unique characteristics, including a minimal size, providing fast pharmacokinetics, high-target specificity, and an affinity in the (sub-)nanomolar range in conjunction with an easy selection and production, which allow them to outperform conventional antibodies for imaging and radiotherapeutic purposes. As for all protein theranostics, extended safety assessment and investigation of their possible immunogenicity in particular are required. In this study, we assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) for PET imaging of tumor-associated macrophages. For the anti-HER2 Nb, we show that only one out of 20 patients had a low amount of pre-existing anti-drug antibodies (ADAs), which only marginally increased 3 months after administering the Nb, and without negative effects of safety and pharmacokinetics. Further in vitro immunogenicity assessment assays showed that both non-humanized Nbs were taken up by human dendritic cells but exhibited no or only a marginal capacity to activate dendritic cells or to induce T cell proliferation. From our data, we conclude that monomeric Nbs present a low immunogenicity risk profile, which is encouraging for their future development toward potential clinical applications.One Sentence SummaryNanobodies, the recombinant single domain affinity reagents derived from heavy chain-only antibodies in camelids, are proven to possess a low immunogenicity risk profile, which will facilitate a growing number of Nanobodies to enter the clinic for therapeutic or in vivo diagnostic applications.

scholarly journals Preclinical Evaluation of [68Ga]Ga-DFO-ZEGFR:2377: A Promising Affibody-Based Probe for Noninvasive PET Imaging of EGFR Expression in Tumors

Cells ◽  
2018 ◽  
Vol 7 (9) ◽  
pp. 141 ◽  
Author(s):  
Maryam Oroujeni ◽  
Javad Garousi ◽  
Ken Andersson ◽  
John Löfblom ◽  
Bogdan Mitran ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
In Vivo Studies ◽  
Desferrioxamine B ◽  
Egfr Expression ◽  
Specific Accumulation ◽  
Epidermal Growth ◽  
Specificity Of Binding ◽  
Saturation Experiment

Radionuclide imaging of epidermal growth factor receptor (EGFR) expression in tumors may stratify patients for EGFR-targeting therapies and predict response or resistance to certain treatments. Affibody molecules, which are nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging. In this study, maleimido derivative of desferrioxamine B (DFO) chelator was site-specifically coupled to the C-terminal cysteine of the anti-EGFR affibody molecule ZEGFR:2377, and the DFO-ZEGFR:2377 conjugate was labeled with the generator-produced positron-emitting radionuclide 68Ga. Stability, specificity of binding to EGFR-expressing cells, and processing of [68Ga]Ga-DFO-ZEGFR:2377 by cancer cells after binding were evaluated in vitro. In vivo studies were performed in nude mice bearing human EGFR-expressing A431 epidermoid cancer xenografts. The biodistribution of [68Ga]Ga-DFO-ZEGFR:2377 was directly compared with the biodistribution of [89Zr]Zr-DFO-ZEGFR:2377. DFO-ZEGFR:2377 was efficiently (isolated yield of 73 ± 3%) and stably labeled with 68Ga. Binding of [68Ga]Ga-DFO-ZEGFR:2377 to EGFR-expressing cells in vitro was receptor-specific and proportional to the EGFR expression level. In vivo saturation experiment demonstrated EGFR-specific accumulation of [68Ga]Ga-DFO-ZEGFR:2377 in A431 xenografts. Compared to [89Zr]Zr-DFO-ZEGFR:2377, [68Ga]Ga-DFO-ZEGFR:2377 demonstrated significantly (p < 0.05) higher uptake in tumors and lower uptake in spleen and bones. This resulted in significantly higher tumor-to-organ ratios for [68Ga]Ga-DFO-ZEGFR:2377. In conclusion, [68Ga]Ga-DFO-ZEGFR:2377 is a promising probe for imaging of EGFR expression.

scholarly journals Preclinical and Exploratory Clinical Studies of Novel 68Ga-labeled ᴅ-Peptide Antagonist for PET Imaging of TIGIT Expression in Cancers

2021 ◽  
Author(s):  
Xiaobo Wang ◽  
Ming Zhou ◽  
Bei Chen ◽  
Huanhuan Liu ◽  
Jianyang Fang ◽  
...  
Keyword(s):  
Pilot Study ◽  
Pet Imaging ◽  
Ex Vivo ◽  
Ct Imaging ◽  
Whole Body ◽  
Binding Assays ◽  
Pet Ct ◽  
Peptide Antagonist

Abstract Purpose While TIGIT has been propelled under the spotlight as a next-generation target in cancer immunotherapy, anti-TIGIT therapy seems to be promising for a fraction of patients in clinical trials. Therefore, patient stratification is critical for this therapy, which could benefit from a whole-body, non-invasive and quantitative evaluation of TIGIT expression in cancers. In this study, a 68Ga-labeled ᴅ-peptide antagonist, 68Ga-GP12, was developed and validated for PET imaging of TIGIT expression in vitro, in vivo, and first-in-human pilot study. Methods The ᴅ-enantiomer peptide antagonists were modified and radiolabeled with 68Ga. In vitro binding assays were performed in human peripheral blood mononuclear cells (PBMCs) to assess their affinity and specificity. The imaging capacity, biodistribution, pharmacokinetics, and radiation dosimetry were investigated in vivo. Flow cytometry, autoradiography, and immunohistochemical staining were used to confirm the expression of TIGIT ex vivo. The safety and potential of 68Ga-GP12 for PET/CT imaging of TIGIT expression were further evaluated in a first-in-human pilot study with advanced NSCLC. Results 68Ga-labeled ᴅ-peptides were conveniently produced with high radiochemical yields,radiochemical purity and molar activities. In vitro binding assays demonstrated 68Ga-GP12 has favorable affinity and specificity for TIGIT with a KD of 37.28 nM. In vivo and ex vivo studies demonstrated the favorable pharmacokinetics of 68Ga-GP12 for PET imaging of TIGIT expression with high tumor uptake of 4.22 ± 0.68 %ID/g and the tumor-to-muscle ratio of 12.94 ± 2.64 at 60 min post-injection. The primary and metastatic lesions found in the first-in-human studies of 68Ga-GP12 PET/CT imaging were comparable to that in 18F-FDG PET/CT imaging. Moreover, the inhomogenous intra-and-inter-tumoral uptake of 68Ga-GP12 was presented, reflecting the heterogeneity of TIGIT expression levels. Conclusion 68Ga-GP12 is a promising radiotracer for PET imaging of TIGIT expression in cancers, indicating its potential as a potential companion diagnostic for anti-TIGIT therapies.

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