DESIGN OF PEPTIDES WITH IMPROVED AFFINITIES FOR ANTI-HUMAN CHORIONIC-GONADOTROPIN MONOCLONAL-ANTIBODIES

Three monoclonal antibodies, LF-hCG-6, -26 and -28, raised against hol e-human chorionic gonadotropin and directed against its beta-subunit, have been used to design synthetic peptides with improved affinity. By PEPSCAN analyses with pin-bound, overlapping nonapeptides, it was sho wn that the peptide consisting of amino acids (125-133) of the beta-su bunit (i.e., PPSLPSPSR) reacted with monoclonal antibody LF-hCG-6; the sequence (135-143) (i.e., PGPSDTPIL), with LF-hCG-28; and the C-termi nal nonapeptide (137-145) (i.e., PSDTPILPQ), with LF-hCG-26. To determ ine amino acids essential for binding and those comprising the necessa ry amino acid core, and to establish the optimal length for binding re activity sets of replacement nonapeptides and overlapping octa- to dod ecapeptides derived from the beta-subunit sequences (122-134) (i.e., K APPPSLPSPSRL) and (132-145) (i.e., SRLPGPSDTPILPQ) were synthesized Am ino acid cores M,ere as follows: beta-hCG (125-131) (i.e., PPSLPSP) fo r monoclonal antibody LF-hCG-6, beta-hCG (135-142) for LF-hCG-28 and b eta-hCG (139-145) for LF-hCG-26. Based on the optimal length, pa,ent p eptide sequences [beta-hCG (125-133), (137-145) and (135-145)] M ere s ized by conventional solid-phase procedures. In addition, based on the results with the replacement peptides, peptide derivatives were produ ced in which specific single improvements had been combined. The affin ities of the monoclonal antibodies for the peptide derivatives, compar ed to the parent peptide sequences, were at least 700 times better for LF-hCG-6 reactive peptides (6.9 x 10(6) M(-1) and <10(4) M(-1), respe ctively) and 130 times better for LF-hCG-26 reactive peptides (1.3 x 1 0(6) M(-1) and <10(4) M(-1), respectively). Compared to its nonapeptid e derivative IRDTPIMVK, LF-hCG-26 had a similar affinity for the undec apeptide (135-145) (i.e., 5 x 10(6) M(-1)). This is close to the affin ity of this monoclonal antibody to holo-hCG as deter mined by a radioi mmunoassay (<10(7) M(-1)). Surprisingly, LF-hCG-28 had an affinity con stant of 1.1 x 10(8) M(-1) for undecapeptide (135-145), only 18-times (radioimmunoassay) to 40-times (enzyme-linked immunosorbent assay) low er than the affinity constant of this monoclonal antibody for holo-hCG .