Structure and molecular interaction analysis of monoclonal antibodies in complex with receptor tyrosine kinases

Misregulated receptor tyrosine kinases (RTKs), i.e. the epidermal growth factor receptor EGFR or the insulin-like growth factor receptor 1 (IGF-1R), can be involved in the development of cancer. Monoclonal antibodies specifically inhibit the RTKs in cancer therapy. The scope of this thesis is to investigate the molecular basis of the inhibition through the therapeutic antibodies matuzumab (EMD72000) against EGFR and EMD1159476 against IGF-1R. The 3D crystal structure of matuzumab in complex with the EGFR domain III shows an eptiope connected with a novel inhibition mechanism: a non-competitive, sterical inhibition of receptor acitivation. The anti-IGF-1R targeted monoclonal antibody EMD1159476 shows a reduced binding capacity to the receptor in the presence of ligand indicating a competitive inhibition mechanism. The epitope of EMD1159476 is within domain II of the receptor. The results of these molecular interaction studies are important for the clinical therapies with these monoclonal antibodies. The matuzumab-EGFR complex crystal structure shows that a simultaneous binding of matuzumab and cetuximab (Erbitux) is possible. The latter antibody is already in clinical use. A combination of several therapeutic antibodies in cancer treatment might show synergistic effects and benefits for the patients.Verschiedene Rezeptor-Tyrosinkinasen (RTKs), wie z.B. der Epidermale Wachstumsfaktor-rezeptor EGFR oder der Insulin-ähnliche Wachstumsfaktorrezeptor 1 IGF-1R, können durch Misregulation die Entstehung von neoplastischen Zellen hervorrufen. Ein Ansatz in der Krebstherapie ist die gezielte Inhibition von RTKs durch monoklonale Antikörper. Hier wird die molekulare Basis der Inhibition durch die beiden therapeutischen Antikörper Matuzumab (EMD72000) gegen EGFR und EMD1159476 gegen IGF-1R untersucht. Die 3D-Komplexstruktur von Matuzumab mit der EGFR Domäne III zeigt ein Epitop, das auf einen neuen Inhibitionsmechanismus für EGFR hinweist: eine nicht-kompetitive, rein sterische Blockierung der Rezeptoraktivierung. Der gegen IGF-1R gerichtete monoklonale Antikörper EMD1159476 dagegen zeigt eine eingeschränkte Bindung an den Rezeptor in Gegenwart des Liganden. Das Epitop von EMD1159476 liegt innerhalb der Domäne II des Rezeptors. Die Ergebnisse dieser molekularen Interaktionsstudien könnten Einfluss auf die klinischen Therapieansätze mit monoklonalen Antikörpern haben. Die Matuzumab - EGFR Ko-Kristallstruktur zeigt, dass eine simultane Bindung Matuzumabs und des bereits zugelassenen Antikörpers Cetuximab (Erbitux) an EGFR möglich ist. Eine Kombination von mehreren therapeutischen Antikörpern könnte in der Krebstherapie einen synergistischen Effekt zeigen

Vergleichende molekulare Charakterisierung ESBL-produzierender Enterobacteriaceae-Isolate von Menschen und Tieren

Die Ergebnisse aus dieser Studie untermauern das alarmierende Auftreten und die zunehmende Verbreitung von verschiedenen ESBL-produzierenden, multiresistenten Enterobacteriaceae-Stämmen in der Human- und Tierpopulation in der geografischen Region Mittelhessen in Deutschland. Dieser Umstand zeigt sich unter anderem in sehr ähnlichen Resistenzmustern und in beiden Gruppen gefundenen beta-Laktamase- und PMQR-Genen. Die häufigsten Bakterienarten in dieser Studie waren E. coli (73,8 %) und K. pneumoniae (17,7 %). Die Untersuchung der phylogenetischen Gruppen der E. coli-Isolate ergab eine Unterrepräsentation der Gruppe B2 innerhalb der Tierisolate. In dieser Studie wurden 83,6 % der Humanisolate (n = 183) und 90,3 % der tierischen Isolate (n = 207) als Träger von ESBL-Genen durch PCR und anschließende Sequenzierung der Amplikons bestätigt. Vorherrschende ESBL-Subtypen in beiden Populationen waren CTX-M-15 (49,5 %) und CTX-M-1 (25,4 %) Der Subtyp blaCTX-M-2 wurde fast ausschließlich in Pferde- und nicht in Humanisolaten gefunden. Die Carbapenemase OXA-48 wurde in dieser Studie in 23 Ertapenem-resistenten K. pneumoniae und Enterobacter cloacae Tierisolaten nachgewiesen. Mit einer Nachweisrate von 27,9 % der Ciprofloxacin-resistenten Humanisolate und 35,5 % der Ciprofloxacin-resistenten Tierisolate war aac(´6) Ib cr das am häufigsten identifizierte PMQR-Gen. Kombinationen von 2 bis zu 6 verschiedenen Resistenzgenen (Penicillinasen, ESBL und PMQR) wurden in 70 % aller untersuchten Isolate nachgewiesen. Plasmide wurden bei 95 % der eingehender charakterisierten Isolate (n = 20) nachgewiesen und die Inc-Gruppen FIA, FIB, FIC, FII, I1 und N identifiziert, wobei das Vorkommen von IncN auf die Humanisolate beschränkt war. Insgesamt konnten bei den untersuchten Plasmid-assoziierten Sequenzen acht verschiedene Resistenzgenklassen detektiert und eine Assoziation der Gene blaCTX-M-15, blaOXA-1 und aac(´6)-Ib-cr festgestellt werden. Zwölf der 20 Isolate wurden als extraintestinal-pathogene E. coli (ExPEC) klassifiziert, wobei eine Kumulation von Virulenz- und Virulenz-assoziierten Faktoren bei den phylogenetischen Gruppen B2 und D zu beobachten war. Die Kombination von Genomsequenzierung und einem in-vivo Virulenzmodell (G. mellonella) konnte einige Risikofaktoren für einen einen höheren larviziden Effekt identifizieren (Präsenz eines Toxingens, Präsenz der Genkombination sitA/iucA/iss, Präsenz der Gene lpfA und traJ)

Multiresistant extended-spectrum beta-lactamase-producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany

BACKGROUND:Multiresistant Gram-negative bacteria producing extended-spectrum beta-lactamases (ESBLs) are an emerging problem in human and veterinary medicine. This study focused on comparative molecular characterization of beta-lactamase and ESBL-producing Enterobacteriaceae isolates from central Hesse in Germany. Isolates originated from humans, companion animals (dogs and cats) and horses. RESULTS:In this study 153 (83.6%) of the human isolates (n=183) and 163 (91.6%) of the animal isolates (n=178) were confirmed as ESBL producers by PCR and subsequent sequencing of the PCR amplicons. Predominant ESBL subtypes in human and animal samples were CTX-M-15 (49.3%) and CTX-M-1 (25.8%) respectively. Subtype blaCTX-M-2 was found almost exclusively in equine and was absent from human isolates. The carbapenemase OXA-48 was detected in 19 ertapenem-resistant companion animal isolates in this study. The Plasmid-encoded quinolone resistance (PMQR) gene aac(´6)-Ib-cr was the most frequently detected antibiotic- resistance gene present in 27.9% of the human and 36.9% of the animal ciprofloxacin-resistant isolates. Combinations of two or up to six different resistance genes (penicillinases, ESBLs and PMQR) were detected in 70% of all isolates investigated. The most frequent species in this study was Escherichia coli (74%), followed by Klebsiella pneumoniae (17.5%), and Enterobacter cloacae (4.2%). Investigation of Escherichia coli phylogenetic groups revealed underrepresentation of group B2 within the animal isolates. CONCLUSIONS:Isolates from human, companion animals and horses shared several characteristics regarding presence of ESBL, PMQR and combination of different resistance genes. The results indicate active transmission and dissemination of multi-resistant Enterobacteriaceae among human and animal populations

Multidrug-Resistant and Clinically Relevant Gram-Negative Bacteria Are Present in German Surface Waters

Water is considered to play a role in the dissemination of antibiotic-resistant Gram-negative bacteria including those encoding Extended-spectrum beta-lactamases (ESBL) and carbapenemases. To investigate the role of water for their spread in more detail, we characterized ESBL/Carbapenemase-producing bacteria from surface water and sediment samples using phenotypic and genotypic approaches. ESBL/Carbapenemase-producing isolates were obtained from water/sediment samples. Species and antibiotic resistance were determined. A subset of these isolates (n = 33) was whole-genome-sequenced and analyzed for the presence of antibiotic resistance genes and virulence determinants. Their relatedness to isolates associated with human infections was investigated using multilocus sequence type and cgMLST-based analysis. Eighty-nine percent of the isolates comprised of clinically relevant species. Fifty-eight percent exhibited a multidrug-resistance phenotype. Two isolates harbored the mobile colistin resistance gene mcr-1. One carbapenemase-producing isolate identified as Enterobacter kobei harbored bla(VIM-)(1). Two Escherichia coli isolates had sequence types (ST) associated with human infections (ST131 and ST1485) and a Klebsiella pneumoniae isolate was classified as hypervirulent. A multidrug-resistant (MDR) Pseudomonas aeruginosa isolate encoding known virulence genes associated with severe lung infections in cystic fibrosis patients was also detected. The presence of MDR and clinically relevant isolates in recreational and surface water underlines the role of aquatic environments as both reservoirs and hot spots for MDR bacteria. Future assessment of water quality should include the examination of the multidrug resistance of clinically relevant bacterial species and thus provide an important link regarding the spread of MDR bacteria in a One Health context.Peer reviewe

Comparison of approaches for source attribution of ESBL-producing Escherichia coli in Germany

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia (E.) coli have been widely described as the cause of treatment failures in humans around the world. The origin of human infections with these microorganisms is discussed controversially and in most cases hard to identify. Since they pose a relevant risk to human health, it becomes crucial to understand their sources and the transmission pathways. In this study, we analyzed data from different studies in Germany and grouped ESBL-producing E. coli from different sources and human cases into subtypes based on their phenotypic and genotypic characteristics (ESBL-genotype, E. coli phylogenetic group and phenotypic antimicrobial resistance pattern). Then, a source attribution model was developed in order to attribute the human cases to the considered sources. The sources were from different animal species (cattle, pig, chicken, dog and horse) and also from patients with nosocomial infections. The human isolates were gathered from community cases which showed to be colonized with ESBL-producing E. coli. We used the attribution model first with only the animal sources (Approach A) and then additionally with the nosocomial infections (Approach B). We observed that all sources contributed to the human cases, nevertheless, isolates from nosocomial infections were more related to those from human cases than any of the other sources. We identified subtypes that were only detected in the considered animal species and others that were observed only in the human population. Some subtypes from the human cases could not be allocated to any of the sources from this study and were attributed to an unknown source. Our study emphasizes the importance of human-to-human transmission of ESBL-producing E. coli and the different role that pets, livestock and healthcare facilities may play in the transmission of these resistant bacteria. The developed source attribution model can be further used to monitor future trends. A One Health approach is necessary to develop source attribution models further to integrate also wildlife, environmental as well as food sources in addition to human and animal data.Peer Reviewe

Molecular Characterization of Borrelia persica, the Agent of Tick Borne Relapsing Fever in Israel and the Palestinian Authority

The identification of the Tick Borne Relapsing Fever (TBRF) agent in Israel and the Palestinian Authority relies on the morphology and the association of Borrelia persica with its vector Ornithodoros tholozani. Molecular based data on B. persica are very scarce as the organism is still non-cultivable. In this study, we were able to sequence three complete 16S rRNA genes, 12 partial flaB genes, 18 partial glpQ genes, 16 rrs-ileT intergenic spacers (IGS) from nine ticks and ten human blood samples originating from the West Bank and Israel. In one sample we sequenced 7231 contiguous base pairs that covered completely the region from the 5′end of the 16S rRNA gene to the 5′end of the 23S rRNA gene comprising the whole 16S rRNA (rrs), and the following genes: Ala tRNA (alaT), Ile tRNA (ileT), adenylosuccinate lyase (purB), adenylosuccinate synthetase (purA), methylpurine-DNA glycosylase (mag), hypoxanthine-guanine phosphoribosyltransferase (hpt), an hydrolase (HAD superfamily) and a 135 bp 5′ fragment of the 23S rRNA (rrlA) genes. Phylogenic sequence analysis defined all the Borrelia isolates from O. tholozani and from human TBRF cases in Israel and the West Bank as B. persica that clustered between the African and the New World TBRF species. Gene organization of the intergenic spacer between the 16S rRNA and the 23S rRNA was similar to that of other TBRF Borrelia species and different from the Lyme disease Borrelia species. Variants of B. persica were found among the different genes of the different isolates even in the same sampling area

Resistance plasmids in ESBL-encoding Escherichia coli isolates from humans, dogs and cats

We characterized ESBL-producing Escherichia coli isolates from diseased dog, cat and human sources for their plasmid content. Plasmids with different Inc groups and combinations of resistance genes were detected in these isolates. The pan-genome of the plasmid-associated genes was found to be large, indicating diversity of the gene pool among the plasmids. No commonly occurring plasmids with similar gene content in isolates from dog, cats and humans were detected

Chemo-enzymatic synthesis of poly-N-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces

Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures and subsequent binding of ECM glycoproteins. First, we synthesized \u3b2-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human \u3b21,4-galactosyltransferase-1 and \u3b21,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model galectin His6CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment.Peer reviewed: YesNRC publication: Ye