(33f) Antibody Production Against Camptothecin- Derived Small Molecules :a Tool for Developing Pharmacokinetic Studies and Dose Management Chemotherapy
AIChE Annual Meeting
2022
2022 Annual Meeting
Pharmaceutical Discovery, Development and Manufacturing Forum
Predictive Scale-Up/Scale-Down for Production of Pharmaceuticals and Biopharmaceuticals I
Sunday, November 13, 2022 - 5:42pm to 6:04pm
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
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,