Construction of an immunotoxin specifically targeted towards pancreatic cancer cells

Abstract

Some types of cancers are more difficult to treat than others, and to date, there is no general accepted standard approach how to treat pancreatic cancer. The aim of this project was to construct an immunotoxin with an affinity towards pancreatic cancer cells. The idea is that the coupling of a scFv antibody to a toxin will improve tumor selectivity of a drug that is too toxic to be used on its own, as well as to confer cell killing power to the scFv antibody that is tumor-specific but not sufficiently cytotoxic.

A naïve phage scFv library was screened with the CBASTM method and the tumor cell line specific polyclonal mixture, obtained from the panning, was cloned into the cloning vector pHOG21 and expressed in E. coli. 6000 clones were randomly picked and after characterization and DNA fingerprinting, 96 promising clones where chosen for further characterization. Of these 96 clones, 85 % were obtained from panning at 37 °C, supporting the hypothesis that the CBASTM method preferentially selects for internalizing antibodies. Finally one pancreatic carcinoma specific scFv, B1-J21, acquired from panning at 37 °C, was selected to serve as a delivery vehicle for the toxin in the immunotoxin complex.

6-Alkenylpurines have been proven to show cytotoxic activity against some human cancer cells and were chosen to serve as a model for the toxin that was to be synthesized. In order to be able to couple the toxin to the scFv via the SPDP linker, a free amino group on the toxin was needed and it was therefore decided that 9-(4-aminobenzyl)-2-chloro-6-E-styryl-9Hpurine (7) would be employed as a toxin in these studies and was to be synthesized in 2 slightly different ways. After N-alkylation of 2,6-dichloropurine (2) with 2 different benzyl chlorides, 4-nitrobenzyl chloride (8) and 4-acetamidebenzyl chloride (9), the corresponding products 3a and 4a were subjected to Stille couplings in order to introduce a styryl group in the purine 6-position. Despite the fact that during the coupling reactions only half of the recommended amount of the tin reagent was used, (E)-N-{4-[(2-chloro-6-trans-styryl-9Hpurin-9-yl)methyl]phenyl}acetamide (5) was isolated and purified in 41 % yield.

Under the recommended conditions, the compound with an acetamide group para substituted in the benzyl group at the purine 9-position 4a, seems to be much more reactive in the applied cross-coupling reaction than the corresponding compound 3a with a nitro group in the para position of the benzyl group at the purine 9-position. With only step left in the synthesis of the toxin 7, the hydrolyzation of the acetamide group of compound 5 to the amino group, the time intended for this master thesis came to an end. If the conversion of compound 3a to (E)-2-chloro-9-(4-nitrobenzyl)-6-trans-styryl-9H-purine (6) had been successful, the plan was to reduce the nitro group in the purine 9-position to the required amino group.