Functional mapping and single chain construction of the anti-cytokeratin 8 monoclonal antibody TS1

Functional mapping of the anti-idiotypic antibody anti-TS1 scFv using site-directed mutagenesis and kinetic analysis

“…We identified the interacting surfaces of TS1 and CK8 and we showed mutual inhibition of CK8 and anti-TS1 to binding with TS1, although they do not show any obvious structural similarities. 4,10,33,34 In this study, we used site-directed…”

“…A detailed description of the interactions between an Id mAb paratope, its antigen and an anti-Id mAb may enable more effective engineering since the effect of mutations intended to improve the properties and attributes of mAbs may be more reliably predicted. [10][11][12][13][14] Many research groups have analyzed the structural basis of antibody-antigen recognition. 11,[15][16][17] It has been demonstrated that amino acid residues involved in establishing the complementary determining region (CDR) conformations are less frequently involved in antigenic interactions compared to residues in other positions in the CDR.…”

“…33 In addition to this geometric fit, both the groove in TS1 and the protruding parts of anti-TS1 are partly hydrophobic, which make hydrophobic interactions possible between these sites. 10 Using chemical modification, it was demonstrated that histidine was important for TS1 interaction with anti-TS1 and not CK8 and the only histidine found in the ACS is located in this groove. 33 Interestingly, despite the fact that anti-TS1 and CK8 compete for the TS1 binding, no resemblance could be seen between the ACS of anti-TS1 and the epitope of CK8.…”

Localization of Complexed Anticytokeratin 8 scFv TS1-218 to HeLa HEp-2 Multicellular Tumor Spheroids and Experimental Tumors

“…Frozen stocks of E. coli Rosetta (DE3) (Promega) strains containing TS1-218 14 and aTS1 scFv 29 in pET26b vector (Novagene), respectively, were cultured in LB media with 30 mg/mL kanamycin and 34 mg/mL chloramphenicol at 378C overnight. Expression with pET26b fuses the scFvs with a C-terminal hexahistidine-tag used for purification and detection.…”

“…2,[11][12][13] The TS1 localizes to the central necrotic part of the tumors, where it remains with a biological half-life of more than 600 h. 12 TS1-218 is an scFv derived from the mAb TS1, which retains the binding specificity of the intact TS1 and can thus be used for immunotargeting of carcinomas originating from epithelia expressing CK 8. 14 For ethical reasons and to exclude poor candidates it is important to screen and evaluate antibodies in vitro, before proceeding to in vivo animal and human studies. 15 Monolayer cultures of cell lines from a variety of human carcinomas have been widely used to study molecular and cellular mechanisms in their metabolism, proliferation, and apoptosis.…”