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Frontiers in Pharmacology 2022Alterations to the EGFR (epidermal growth factor receptor) gene, which primarily occur in the axon 18-21 position, have been linked to a variety of cancers, including...
Alterations to the EGFR (epidermal growth factor receptor) gene, which primarily occur in the axon 18-21 position, have been linked to a variety of cancers, including ovarian, breast, colon, and lung cancer. The use of TK inhibitors (gefitinib, erlotinib, lapatinib, and afatinib) and monoclonal antibodies (cetuximab, panitumumab, and matuzumab) in the treatment of advanced-stage cancer is very common. These drugs are becoming less effective in EGFR targeted cancer treatment and developing resistance to cancer cell eradication, which sometimes necessitates stopping treatment due to the side effects. One study has been conducted to identify EGFR antagonists using other compounds, databases without providing the toxicity profile, comparative analyses, or morphological cell death pattern. The goal of our study was to identify potential lead compounds, and we identified seven compounds based on the docking score and four compounds that were chosen for our study, utilizing toxicity analysis. Molecular docking, virtual screening, dynamic simulation, and screening indicated that these compounds' effects were superior to those of already marketed medication (gefitinib). The four compounds obtained, ZINC96937394, ZINC14611940, ZINC103239230, and ZINC96933670, demonstrated improved binding affinity (-9.9 kcal/mol, -9.6 kcal/mol, -9.5 kcal/mol, and -9.2 kcal/mol, respectively), interaction stability, and a lower toxicity profile. toxicity analysis showed that our compounds have a lower toxicity profile and a higher LD value. At the same time, a selected compound, i.e., ZINC103239230, was revealed to attach to a particular active site and bind more tightly to the protein, as well as show better esults when compared to our selected gefitinib medication. MTT assay, gene expression analysis (BAX, BCL-2, and β-catenin), apoptosis analysis, TEM, cell cycle assay, ELISA, and cell migration assays were conducted to perform the cell death analysis of lung cancer and breast cancer, compared to the marketed product. The MTT assay exhibited 80% cell death for 75 µM and 100µM; however, flow cytometry analysis with the IC value demonstrated that the selected compound induced higher apoptosis in MCF-7 (30.8%) than in A549.
PubMed: 36457709
DOI: 10.3389/fphar.2022.1027890 -
Clinical Infectious Diseases : An... Mar 2023The microbial etiology of prosthetic valve infective endocarditis (PVE) can be difficult to identify. Our aim was to investigate the benefit of molecular imaging...
BACKGROUND
The microbial etiology of prosthetic valve infective endocarditis (PVE) can be difficult to identify. Our aim was to investigate the benefit of molecular imaging technique fluorescence in situ hybridization (FISH) combined with 16S rRNA-gene polymerase chain reaction (PCR) and sequencing (FISHseq) for the analysis of infected prosthetic heart valves.
METHODS
We retrospectively evaluated the diagnostic outcome of 113 prosthetic valves from 105 patients with suspected PVE, treated in 2003-2013 in the Department of Cardiac Surgery, Charité University Medicine Berlin. Each prosthetic valve underwent cultural diagnostics and was routinely examined by FISH combined with 16S rRNA gene PCR and sequencing. We compared classical microbiological culture outcomes (blood and valve cultures) with FISHseq results and evaluated the diagnostic impact of the molecular imaging technique.
RESULTS
Conventional microbiological diagnostic alone turned out to be insufficient, as 67% of preoperative blood cultures were noninformative (negative, inconclusive, or not obtained) and 67% of valve cultures remained negative. FISHseq improved the conventional cultural diagnostic methods in PVE in 30% of the cases and increased diagnostic accuracy. Of the valve culture-negative PVE cases, FISHseq succeeded in identifying the causative pathogen in 35%.
CONCLUSIONS
FISHseq improves PVE diagnostics, complementing conventional cultural methods. In addition to species identification, FISH provides information about the severity of PVE and state of the pathogens (eg, stage of biofilm formation, activity, and localization on and within the prosthetic material). As a molecular imaging technique, FISHseq enables the unambiguous discrimination of skin flora as contaminant or infectious agent.
Topics: Humans; Endocarditis, Bacterial; Heart Valve Prosthesis; Retrospective Studies; In Situ Hybridization, Fluorescence; RNA, Ribosomal, 16S; Prosthesis-Related Infections; Endocarditis; Molecular Imaging
PubMed: 36318608
DOI: 10.1093/cid/ciac860 -
Pharmaceutics Sep 2022Matuzumab and nimotuzumab are anti-EGFR monoclonal antibodies that bind to different epitopes of domain III of EGFR. We developed Zr-matuzumab as a PET probe for...
Matuzumab and nimotuzumab are anti-EGFR monoclonal antibodies that bind to different epitopes of domain III of EGFR. We developed Zr-matuzumab as a PET probe for diagnosis/monitoring of response to treatment of a noncompeting anti-EGFR nimotuzumab antibody drug conjugate (ADC) using mouse colorectal cancer (CRC) xenografts. We developed Zr-matuzumab and performed quality control in EGFR-positive DLD-1 cells. The K of matuzumab, DFO-matuzumab and Zr-matuzumab in DLD-1 cells was 5.9, 6.2 and 3 nM, respectively. A competitive radioligand binding assay showed that Zr-matuzumab and nimotuzumab bound to noncompeting epitopes of EGFR. MicroPET/CT imaging and biodistribution of Zr-matuzumab in mice bearing EGFR-positive xenografts (HT29, DLD-1 and MDA-MB-231) showed high uptake that was blocked with pre-dosing with matuzumab but not with the noncompeting binder nimotuzumab. We evaluated nimotuzumab-PEG-DM1 ADC in CRC cells. IC of nimotuzumab-PEG-DM1 in SNU-C2B, DLD-1 and SW620 cells was dependent on EGFR density and was up to five-fold lower than that of naked nimotuzumab. Mice bearing the SNU-C2B xenograft were treated using three 15 mg/kg doses of nimotuzumab-PEG-DM1, and Zr-matuzumab microPET/CT was used to monitor the response to treatment. Treatment resulted in complete remission of the SNU-C2B tumor in 2/3 mice. Matuzumab and nimotuzumab are noncompeting and can be used simultaneously.
PubMed: 36145664
DOI: 10.3390/pharmaceutics14091917 -
Frontiers in Immunology 2021In this work, we have generated epidermal growth factor receptor (EGFR)-specific cattle-derived ultralong CDR-H3 antibodies by combining cattle immunization with yeast...
Milking the Cow: Cattle-Derived Chimeric Ultralong CDR-H3 Antibodies and Their Engineered CDR-H3-Only Knobbody Counterparts Targeting Epidermal Growth Factor Receptor Elicit Potent NK Cell-Mediated Cytotoxicity.
In this work, we have generated epidermal growth factor receptor (EGFR)-specific cattle-derived ultralong CDR-H3 antibodies by combining cattle immunization with yeast surface display. After immunization, ultralong CDR-H3 regions were specifically amplified and grafted onto an IGHV1-7 scaffold by homologous recombination to facilitate Fab display. Antigen-specific clones were readily obtained by fluorescence-activated cell sorting (FACS) and reformatted as chimeric antibodies. Binning experiments revealed epitope targeting of domains I, II, and IV of EGFR with none of the generated binders competing with Cetuximab, Matuzumab, or EGF for binding to EGFR. Cattle-derived chimeric antibodies were potent in inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against EGFR-overexpressing tumor cells with potencies (EC killing) in the picomolar range. Moreover, most of the antibodies were able to significantly inhibit EGFR-mediated downstream signaling. Furthermore, we demonstrate that a minor fraction of CDR-H3 knobs derived from generated antibodies was capable of independently functioning as a paratope facilitating EGFR binding when grafted onto the Fc part of human IgG1. Besides slightly to moderately diminished capacities, these engineered Knobbodies largely retained main properties of their parental antibodies such as cellular binding and triggering of ADCC. Hence, Knobbodies might emerge as promising tools for biotechnological applications upon further optimization.
Topics: Animals; Antibodies; Antibody Affinity; Cattle; Complementarity Determining Regions; Cytotoxicity, Immunologic; ErbB Receptors; Humans; Killer Cells, Natural; Protein Engineering
PubMed: 34759924
DOI: 10.3389/fimmu.2021.742418 -
Scientific Reports Mar 2021Evaluation of treatment response is among the major challenges in modern oncology. We herein used a monoclonal antibody targeting the EGF receptor (EGFR) labelled with...
Evaluation of treatment response is among the major challenges in modern oncology. We herein used a monoclonal antibody targeting the EGF receptor (EGFR) labelled with the alpha emitter Bi (Bi-anti-EGFR-MAb). EJ28Luc (bladder) and LN18 (glioma) cancer cells, both overexpressing EGFR, were incubated for 3 h with the radioimmunoconjugate. To assess the responses in the core carbon metabolism upon this treatment, these cancer cell lines were subsequently cultivated for 18 h in the presence of [U-C]glucose. C-enrichment and isotopologue profiles of key amino acids were monitored by gas chromatography-mass spectrometry (GC/MS), in order to monitor the impacts of the radionuclide-treatment upon glucose metabolism. In comparison to untreated controls, treatment of EJ28Luc cells with Bi-anti-EGFR-MAb resulted in a significantly decreased incorporation of C from [U-C]glucose into alanine, aspartate, glutamate, glycine, proline and serine. In sharp contrast, the same amino acids did not display less C-enrichments during treatment of the LN18 cells. The data indicate early treatment response of the bladder cancer cells, but not of the glioma cells though cell lines were killed following Bi-anti-EGFR-MAb treatment. The pilot study shows that the C-labelling approach is a valid tool to assess the responsiveness of cancer cells upon radionuclide-treatment in considerable metabolic detail.
Topics: Amino Acids; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Biological Transport; Bismuth; Carbon Isotopes; Cell Line, Tumor; Dose-Response Relationship, Drug; Epithelial Cells; ErbB Receptors; Gas Chromatography-Mass Spectrometry; Glucose; Humans; Immunoconjugates; Neuroglia; Radioisotopes; Urinary Bladder
PubMed: 33737524
DOI: 10.1038/s41598-021-84421-4 -
Neuro-oncology Aug 2019Although epidermal growth factor receptor (EGFR) and its truncated, autoactive mutant EGFR variant (v)III are bona fide drivers of tumorigenesis in some gliomas,...
BACKGROUND
Although epidermal growth factor receptor (EGFR) and its truncated, autoactive mutant EGFR variant (v)III are bona fide drivers of tumorigenesis in some gliomas, therapeutic antibodies developed to neutralize this axis have not improved patient survival in a limited number of trials. Previous studies using cells transduced to exogenously express EGFRvIII may have compromised mechanistic studies of anti-EGFR therapeutics. Therefore, we re-assessed the activity of clinical EGFR antibodies in patient-derived gliomaspheres that endogenously express EGFRvIII.
METHODS
The antitumor efficacy of antibodies was assessed using in vitro proliferation assays and intracranial orthografts. Receptor activation status, antibody engagement, oncogenic signaling, and mechanism of action after antibody treatment were analyzed by immunoprecipitation and western blotting. Tracking of antibody receptor complexes was conducted using immunofluorescence.
RESULTS
The EGFR domain III-targeting antibodies cetuximab, necitumumab, nimotuzumab, and matuzumab did not neutralize EGFRvIII activation. Chimeric monoclonal antibody 806 (ch806) neutralized EGFRvIII, but not wild-type (wt)EGFR activation. Panitumumab was the only antibody that neutralized both EGFRvIII and wtEGFR, leading to reduction of p-S6 signaling and superior in vitro and in vivo antitumor activity. Mechanistically, panitumumab induced recycling of receptor but not degradation as previously described. Panitumumab, via its unique avidity, stably cross-linked EGFRvIII to prevent its activation, while ch806 induced a marked reduction in the active EGFRvIII disulphide-bonded dimer.
CONCLUSIONS
We discovered a previously unknown major resistance mechanism in glioma in that most EGFR domain III-targeting antibodies do not neutralize EGFRvIII. The superior in vitro and in vivo antitumor activity of panitumumab supports further clinical testing of this antibody against EGFRvIII-stratified glioma.
Topics: Antibodies, Monoclonal; Cell Line, Tumor; ErbB Receptors; Glioma; Humans; Signal Transduction
PubMed: 31002307
DOI: 10.1093/neuonc/noz073 -
Scientific Reports Aug 2017Anti-idiotypic binders which specifically recognize the variable region of monoclonal antibodies have proven to be robust tools for pharmacokinetic studies of antibody...
Anti-idiotypic binders which specifically recognize the variable region of monoclonal antibodies have proven to be robust tools for pharmacokinetic studies of antibody therapeutics and for the development of cancer vaccines. In the present investigation, we focused on the identification of anti-idiotypic, shark-derived IgNAR antibody variable domains (vNARs) targeting the therapeutic antibodies matuzumab and cetuximab for the purpose of developing specific capturing ligands. Using yeast surface display and semi-synthetic, CDR3-randomized libraries, we identified several highly specific binders targeting both therapeutic antibodies in their corresponding variable region, without applying any counter selections during screening. Importantly, anti-idiotypic vNAR binders were not cross-reactive towards cetuximab or matuzumab, respectively, and comprised good target recognition in the presence of human and mouse serum. When coupled to magnetic beads, anti-idiotypic vNAR variants could be used as efficient capturing tools. Moreover, a two-step procedure involving vNAR-functionalized beads was employed for the enrichment of potentially bispecific cetuximab × matuzumab antibody constructs. In conclusion, semi-synthetic and CDR3-randomized vNAR libraries in combination with yeast display enable the fast and facile identification of anti-idiotypic vNAR domains targeting monoclonal antibodies primarily in an anti-idiotypic manner.
Topics: Antibodies, Anti-Idiotypic; Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Immunological; Cell Surface Display Techniques; Cetuximab; Immunomagnetic Separation; Protein Binding; Saccharomyces cerevisiae; Single-Domain Antibodies
PubMed: 28852148
DOI: 10.1038/s41598-017-10513-9 -
Structure (London, England : 1993) Jul 2013The epidermal growth factor receptor (EGFR) is implicated in human cancers and is the target of several classes of therapeutic agents, including antibody-based drugs....
The epidermal growth factor receptor (EGFR) is implicated in human cancers and is the target of several classes of therapeutic agents, including antibody-based drugs. Here, we describe X-ray crystal structures of the extracellular region of EGFR in complex with three inhibitory nanobodies, the variable domains of heavy chain only antibodies (VHH). VHH domains, the smallest natural antigen-binding modules, are readily engineered for diagnostic and therapeutic applications. All three VHH domains prevent ligand-induced EGFR activation, but use two distinct mechanisms. 7D12 sterically blocks ligand binding to EGFR in a manner similar to that of cetuximab. EgA1 and 9G8 bind an epitope near the EGFR domain II/III junction, preventing receptor conformational changes required for high-affinity ligand binding and dimerization. This epitope is accessible to the convex VHH paratope but inaccessible to the flatter paratope of monoclonal antibodies. Appreciating the modes of binding and inhibition of these VHH domains will aid in developing them for tumor imaging and/or cancer therapy.
Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Binding Sites; Binding, Competitive; Cetuximab; Crystallography, X-Ray; Cystine; ErbB Receptors; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Protein Binding; Protein Structure, Tertiary; Single-Domain Antibodies
PubMed: 23791944
DOI: 10.1016/j.str.2013.05.008 -
PloS One 2013Hypoxia is a central problem in tumor treatment because hypoxic cells are less sensitive to chemo- and radiotherapy than normoxic cells. Radioresistance of hypoxic tumor...
Hypoxia is a central problem in tumor treatment because hypoxic cells are less sensitive to chemo- and radiotherapy than normoxic cells. Radioresistance of hypoxic tumor cells is due to reduced sensitivity towards low Linear Energy Transfer (LET) radiation. High LET α-emitters are thought to eradicate tumor cells independent of cellular oxygenation. Therefore, the aim of this study was to demonstrate that cell-bound α-particle emitting (213)Bi immunoconjugates kill hypoxic and normoxic CAL33 tumor cells with identical efficiency. For that purpose CAL33 cells were incubated with (213)Bi-anti-EGFR-MAb or irradiated with photons with a nominal energy of 6 MeV both under hypoxic and normoxic conditions. Oxygenation of cells was checked via the hypoxia-associated marker HIF-1α. Survival of cells was analysed using the clonogenic assay. Cell viability was monitored with the WST colorimetric assay. Results were evaluated statistically using a t-test and a Generalized Linear Mixed Model (GLMM). Survival and viability of CAL33 cells decreased both after incubation with increasing (213)Bi-anti-EGFR-MAb activity concentrations (9.25 kBq/ml-1.48 MBq/ml) and irradiation with increasing doses of photons (0.5-12 Gy). Following photon irradiation survival and viability of normoxic cells were significantly lower than those of hypoxic cells at all doses analysed. In contrast, cell death induced by (213)Bi-anti-EGFR-MAb turned out to be independent of cellular oxygenation. These results demonstrate that α-particle emitting (213)Bi-immunoconjugates eradicate hypoxic tumor cells as effective as normoxic cells. Therefore, (213)Bi-radioimmunotherapy seems to be an appropriate strategy for treatment of hypoxic tumors.
Topics: Alpha Particles; Antibodies, Monoclonal, Humanized; Bismuth; Blotting, Western; Cell Hypoxia; Cell Line, Tumor; Cell Survival; ErbB Receptors; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoconjugates; Neoplasms; Oxygen; Particle Accelerators; Photons; Radioisotopes
PubMed: 23724085
DOI: 10.1371/journal.pone.0064730 -
The Korean Journal of Gastroenterology... Mar 2013Colorectal cancer is the third most common malignant disease in incidence according to a report in 2009 from Korea. The 5-fluorouracil (5-FU) remains to be a major... (Review)
Review
Colorectal cancer is the third most common malignant disease in incidence according to a report in 2009 from Korea. The 5-fluorouracil (5-FU) remains to be a major chemotherapeutic agents. But, over the last 10-15 years, the treatment pattern for metastatic colorectal cancer changed significantly. Irinotecan and oxaliplatin are cytotoxic drugs, or bevacizumab and cetuximab are monoclonal antibodies against molecular targets. The introduction of novel agents targeting specific molecular features of cancer cells promises more options and marked improvements in efficacy for the treatment of metastatic colon cancer. Bevacizumab has been shown to extend survival in colorectal cancer when used in combination with irinotecan and 5-FU-based chemotherapy, and the addition of cetuximab to irinotecan and 5-FU-based chemotherapy eliminates irinotecan resistance. Better understanding of the tumor biology and the molecular pathway and mechanisms of tumorigenesis has led to the discovery of novel agents with improved outcomes.
Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bevacizumab; Cetuximab; Colonic Neoplasms; ErbB Receptors; Humans; Molecular Targeted Therapy; Panitumumab; Vascular Endothelial Growth Factor A
PubMed: 23575231
DOI: 10.4166/kjg.2013.61.3.128