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Development of a radioimmunoassay for a pseudononapeptide bombesin/GRP antagonist with antitumor activity
K Groot1, J E Horvàth, R Z Cai
1Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, New Orleans, Louisiana, USA.
Abstract:
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.
Insights
A new radio-immunoassay (RIA) was developed to measure RC-3095, a bombesin/GRP receptor antagonist, in serum. This assay is crucial for evaluating the efficacy of RC-3095 in cancer treatment and monitoring patient therapy.
Area of Science:
- Oncology
- Endocrinology
- Biochemistry
Background:
- Bombesin-like peptides, including gastrin-releasing peptide (GRP), are implicated as autocrine growth factors in various cancers.
- Bombesin/GRP receptor antagonists, such as the pseudononapeptide [D-Tpi6,Leu13 psi(CH2NH)-Leu14]bombesin(6-14) (RC-3095) and its analogs, demonstrate significant tumor growth inhibition in preclinical cancer models.
Purpose of the Study:
- To develop a sensitive and specific radio-immunoassay (RIA) for the direct determination of RC-3095 and its related analogs in unextracted serum.
- To facilitate the experimental and clinical evaluation of RC-3095 as a therapeutic agent for bombesin/GRP receptor-mediated tumors.
Main Methods:
- Antibodies were generated against RC-3095 and Des-Tpi1-RC-3095 conjugated to bovine serum albumin.
- Antiserum JH-631b was characterized, showing high affinity binding to radiolabeled Tyr1-RC-3095 at a dilution of 1:189,000.
- Specificity was confirmed by lack of cross-reactivity with C-terminal fragments lacking the C-terminus, naturally occurring bombesin family peptides, and unrelated peptides.
Main Results:
- The developed RIA demonstrated a minimum detectable dose of 0.4 pg/tube for RC-3095.
- Intra-assay coefficients of variation ranged from 3.2% to 4.4%, and inter-assay coefficients ranged from 5.6% to 12.8%.
- The RIA is suitable for direct quantification of RC-3095 in serum without prior extraction.
Conclusions:
- A robust RIA has been established for the direct measurement of RC-3095 in serum.
- This assay is valuable for monitoring serum levels of RC-3095 during long-term therapy, aiding in clinical efficacy assessment and patient management.