Identification of an epitope of tumor-necrosis-factor (TNF) receptor-type-1 (p55) recognized by a TNF-alpha-antagonist monoclonal-antibody

The relationships between epitope topography and agonistic/antagonistic effects of anti-TNF receptor type 1 (TNF-R1) antibodies on TNF-alpha cytotoxic activity have been studied. To this purpose various monoclonal antibodies (mAbs) against the soluble form of TNF-R1 (sTNF-R1) have been generated and characterized. Epitope topography studies identified at least four distinct epitopes located outside (4E10) or within (or close to) the TNF-alpha binding site of urinary sTNF-R1 (7H3, 4C1, 9B11). mAbs 7H3 and 4C1 were able to neutralize the inhibition of human TNF-alpha cytotoxicity on L-M cells by sTNF-R1, while 4E10 was unable. Moreover, 7H3 and 4C1 were able to antagonize the TNF-a cytotoxicity on human U937 cells, while they were uneffective on mouse LM cells, suggesting that these antibodies recognize, in a species-specific mode, also the membrane form of the human receptor. No agonistic effects were observed when these antibodies were used in the absence of TNF-alpha. Epitope topography studies carried out using overlapping decapeptides covering most of the sTNF-R1 sequence showed that residues 143-148 of the fourth cysteine-rich domain of the receptor (FFLREN) contain antigenic determinants recognized by the antagonist antibody 7H3. These results suggest that at least part of residues 143-148 of sTNF-R1 are surface exposed on the soluble as well as on the membrane forms of TNF-R1 and are accessible to TNF-alpha antagonists.