(2hm) Antibody Production Against Camptothecin-Derived Small Molecules: A Tool Fordeveloping Pharmacokinetic Studies and Dose Management of Chemotherapy

Antibody production against camptothecin-derived small molecules: a tool for developing pharmacokinetic studies and dose management of chemotherapy

Tahere Zarnoosheh Farahany¹, Mohammad- Reza Nejadmoghaddam²

¹Department of Cellular and Molecular Biology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran;

²Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran, Email: mrnejadmoghaddam@gmail.com

Introduction

Camptothecin compounds (such as irinotecan or topotecan) are one of the commonly used and most operative chemotherapeutic drugs for cancer therapy. The active form of all camptothecin derivatives is SN38 released by liver esterase enzyme as the super-cytotoxic form of camptothecin families. Furthermore, in recent years, SN38 itself is also used as a payload in new generation of targeted drug delivery systems termed ADCs technology. Pharmacokinetic studies (PK) and side effect management of the aforesaid cancer medicines have been remained a challenge for optimization of patient safety and improving medical practice. To overcome this challenge, developing simple analytical method, non-invasive and amenable to routine clinical laboratory is the main goal of our present study.

Method

For this purpose, The SN38–20-O-glycine and SN38–20-O-glycine-NHS was synthesized and confirmed by mass, 1^H-NMR, and 13^C-NMR spectroscopic techniques. Then two different immunogens were prepared and characterized, including SN38 conjugated with keyhole limpet hemocyanin (KLH) protein based on linking of SN38 either via its amine-containing SN38–20-O-glycine and SN38–20-O-glycine-NHS derivatives with the tyrosine amino acid residues of KLH by classical glutaraldehyde coupling reaction. The pertinence and efficacy of the coupling reactions of haptens (SN38-KLH and SN38NHS-KLH) were confirmed and characterized by ultraviolet (UV) spectrophotometry and then used for the immunization of animals. Antibodies production and isotypes determination were done by enzyme-linked immunosorbent assay (ELISA) and then purified either using handmade SN38 affinity or HiTrapby Protein G prepacked columns.

Results

Immunization of animals with the SN38NHS-KLH generated extraordinarily high anti-SN38 titers that were up to-fold higher than those immunized by SN38-KLH. The animal’s antiserum that showed the highest affinity was selected. The collected antiserum (polyclonal and monoclonal antibodies) had very high affinity to its cognate ligand with high affinity and high specificity among other irrelevant drug controls.

Conclusion

The produced polyclonal and monoclonal antibodies are valuable for the development of highly sensitive and selective immunoassays for camptothecin- containing cancer medicines. The sensitivity and specificity of the ELISA should provide a useful tool for developing pharmacokinetic studies, dose monitoring and side effect management of camptothecin-derived small molecules.

Key words

SN38, cancer chemotherapy, pharmacokinetic studies, anti-SN38 antibodies, ELISA, immunoassay,v