RESUMEN
Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 StarTM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC50 value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody−toxin conjugate activity in a time-efficient workflow without cell lysis or purification required.
Asunto(s)
Inmunotoxinas , Anticuerpos de Cadena Única , Animales , Cricetinae , Sistema Libre de Células , Inmunotoxinas/genética , Inmunotoxinas/farmacología , Escherichia coli/genética , Células CHO , Cricetulus , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/farmacología , EucariontesRESUMEN
Recent evidences indicating that cellular kinase signaling cascades are triggered by oligomers of N-acetylglucosamine (ChOS) and that condrocytes of human osteoarthritic cartilage secrete the inflammation associated chitolectin YKL-40, prompted us to study the binding affinity of partially acetylated ChOS to YKL-40 and their effect on primary chondrocytes in culture. Extensive chitinase digestion and filtration of partially deacetylated chitin yielded a mixture of ChOS (Oligomin™) and further ultrafiltration produced T-ChOS™, with substantially smaller fraction of the smallest sugars. YKL-40 binding affinity was determined for the different sized homologues, revealing micromolar affinities of the larger homologues to YKL-40. The response of osteoarthritic chondrocytes to Oligomin™ and T-ChOS™ was determined, revealing 2- to 3-fold increases in cell number. About 500 µg/ml was needed for Oligomin™ and around five times lower concentration for T-ChOS™, higher concentrations abolished this effect for both products. Addition of chitotriose inhibited cellular responses mediated by larger oligosaccharides. These results, and the fact that the partially acetylated T-ChOS™ homologues should resist hydrolysis, point towards a new therapeutic concept for treating inflammatory joint diseases.
Asunto(s)
Acetilglucosamina/metabolismo , Adipoquinas/metabolismo , Quitina/metabolismo , Condrocitos/patología , Lectinas/metabolismo , Acetilación , Adipoquinas/genética , Adipoquinas/farmacología , Recuento de Células , Supervivencia Celular , Células Cultivadas , Proteína 1 Similar a Quitinasa-3 , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Cromatografía en Gel , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Medios de Cultivo/metabolismo , Humanos , Lectinas/genética , Lectinas/farmacología , Oligosacáridos/metabolismo , Osteoartritis/metabolismo , Osteoartritis/patología , Polimerizacion , Cultivo Primario de Células , Unión Proteica , Trisacáridos/genética , Trisacáridos/metabolismoRESUMEN
Heterochitooligosaccharides possess interesting biological properties. Isobaric mixtures of such linear heterochitooligosaccharides can be obtained by chemical or enzymatic degradation of chitosan. However, the separation of such mixtures is a challenging analytical problem which is so far unresolved. It is shown that these isobaric mixtures can be sequenced and quantified simultaneously using standard derivatization and multistage tandem mass spectrometric techniques. A linear ion trap mass spectrometer equipped with a vacuum matrix-assisted laser desorption ionization (vMALDI) source is used to perform MS2 as well as MS3 experiments.
Asunto(s)
Quitosano/análisis , Oligosacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Carbohidratos , Cationes/química , Quitosano/análogos & derivados , Cromatografía por Intercambio Iónico/métodos , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Chitin/chitosan oligosaccharides composed of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and/or 2-amino-2-deoxy-D-glucopyranose (GlcN) were prepared by chemical degradation of chitin or chitosan and separated by gel permeation chromatography. Oligosaccharides obtained after enzymatic hydrolysis of chitosan [F(A) 0.19] with a fungal chitinase were derivatized by reductive amination with 2-aminoacridone and sequenced by matrix-assisted laser desorption ionization time-of-flight postsource decay (PSD) mass spectrometry (MS). The sequence of a trimer, D1A2, was established as D-A-A. The composition of a hexamer D3A3 was ca. 65% D-A-D-D-A-A and 35% D-D-A-D-A-A. The PSD MS of a nonamer D5A4-amac revealed four isobaric species D-X-Y-D-X-Y-D-A-A, where A is GlcNAc, D is GlcN, and X and Y (X not equal Y) are mutually either D or A. This structure motif was also observed in a dodecamer D7A5 which was composed of eight isobaric sequences of the general formula (D-X-Y)(3)-D-A-A.
