Herstellung und Charakterisierung von rekombinanten, ETA-basierten CD64-Immuntoxinen zur Bekämpfung von dysregulierten Makrophagen bei chronischen Hautentzündungen

Abstract

Dysregulated, activated macrophages play a decisive role in different chronically inflammatory cutaneous diseases. The fact that a selective elimination of the high CD64 expressing macrophages, through CD64 immunotoxin (full length antibody with a toxin), results in a resolution of chronic inflammations, confirms this. The immunotoxins of the first generation cannot be produced (however) in homogeneous quality and their biological and physicochemical characteristics cannot be optimized adequately. The goal of this dissertation work therefore was to produce recombinant ETA´-based single-chain-fragments against the target molecule CD64 and to evaluate them as a possible therapy form in chronic (cutaneous)-inflammations. Both the monovalent H22(scFv)-ETA´ and the bivalent H22(scFv)2-ETA´ immunotoxin should be produced in E. coli, purified and characterized for their biological characteristics both in-vitro and in an transgenic animal model. The focus of this work is the comparison of the monovalent with the bivalent immunotoxin in-vitro and in-vivo. The uptake of the bivalent immunotoxin by dysregulated macrophages should be superior to that of the monovalent due to its CD64 crosslinking capability and should result in an improved specific cytotoxicity. The immunotoxins were purified successfully out of the E. coli periplasm through the combination of different chromatographical procedures such as (Metal Ion Affinity Chromatography) IMAC, (Size Exclusion Chromatography) SEC and (Hydroxy-Apatite Chromatography) HAPC. The HAPC ideally removed not only bacterial endotoxins (equal to or less than 0.5 EU/ml), but also degradation products of both immunotoxins. Throughout this work fermentation, cleaning and storage protocols were successfully established, providing high quantities of highly pure immunotoxin for the analyses over a longer time period without activity loss. The immunotoxin quantities after purification were for H22(scFv)-ETA´ about 2 mg/L and for H22(scFv)2-ETA´ about 0.5 mg/L. The specific binding of the pure immunotoxins could be confirmed by means of flow cytometry and ELISA. The receptor mediated endocytosis led to a specific elimination of monocytic U937 target cells and could be visualized in-vitro by use of confocal microscopy. The determined IC50-value of the bivalent construct proved to be 10-times lower (14 pM) than the IC50-value of the monovalent immunotoxin (140 pM). In addition the designated specific elimination of the target cell via apoptosis induction could be verified for both immunotoxins in an AnnexinV Assay. To further consolidate the outstanding in-vitro toxicity data both immunotoxins were tested in an mouse model transgenic for hCD64. Both immunotoxins induced a drastic reduction of the local CD64 positive macrophage populations in chronically inflamed mouse skin. Again the bivalent immunotoxin appeared to be more effective: with H22(scFv)ETA´ the amount of the CD64 positive macrophages was reduced to 21% in contrast to H22(scFv)2ETA´ where the amount of the CD64 positive macrophages was drastically reduced to 4.8% (in comparison to the PBS injected control). The amount at surviving cells after immunotoxin application was significant (*p>0,001). Therewith the animal data are statistically unambiguous. In summary both recombinant immunotoxins have low IC50-values, rendering them well suited for a treatment of diseases with dysregulated, activated macrophages. These are the first data that clearly demonstrate that a bivalent immunotoxin exhibits a stronger cytotoxicity in comparison with the monovalent construct both in-vitro and in-vivo. It could be pointed out in this work that an increased effectiveness unambiguously correlates with an increased valency