Development of a radioimmunoassay for a pseudononapeptide bombesin/GRP antagonist with antitumor activity.

Bombesin-like and GRP-like peptides may act as autocrine growth factors in the proliferation of some cancers. A pseudononapeptide bombesin antagonist, [D-Tpi6,Leu13 psi(CH2NH)-Leu14]bombesin(6-14), and related analogs synthesized in our laboratory significantly inhibit tumor growth in various cancer models. A radio-immunoassay (RIA), suitable for determination of RC-3095 and its congeners in unextracted serum, was developed in order to facilitate further experimental and clinical evaluation of this bombesin/GRP receptor antagonist for the treatment of various tumors. Antibodies were generated against RC-3095 and Des-Tpi1-RC-3095, conjugated to bovine serum albumin with glutaraldehyde. Antiserum JH-631b was selected for further experiments based on the antibody characterization. At an antiserum dilution of 1:189,000, this antibody bound approximately 50% of 7 fmol of added radiolabeled Tyr1-RC-3095. The antibody crossreacted with C-terminal fragments of RC-3095. Fragments without the C-terminus and naturally existing peptides of the bombesin family or structurally unrelated peptides did not cross-react. The minimum detectable dose of RC-3095 was 0.4 pg/tube. Intra- and interassay coefficients of variation ranged from 3.2 to 4.4% and from 5.6 to 12.8%, respectively. The RIA is suitable for direct determination of RC-3095 in serum. The RIA should be of value for monitoring levels of this analog in serum during long-term therapy.