Evaluation of an antibody-PNA conjugate as a clearing agent for antibody-based PNA-mediated radionuclide pretargeting

Anders Myrhammar,Takehiko Tsukahara,Shuichiro Yoneoka,Vladimir Tolmachev,Amelie Eriksson Karlström,Kristina Westerlund,Anzhelika Vorobyeva,Anna Orlova,Mohamed Altai

doi:10.1038/s41598-020-77523-y

Abstract

Radionuclide molecular imaging of cancer-specific targets is a promising method to identify patients for targeted antibody therapy. Radiolabeled full-length antibodies however suffer from slow clearance, resulting in high background radiation. To overcome this problem, a pretargeting system based on complementary peptide nucleic acid (PNA) probes has been investigated. The pretargeting relies on sequential injections of primary, PNA-tagged antibody and secondary, radiolabeled PNA probe, which are separated in time, to allow for clearance of non-bound primary agent. We now suggest to include a clearing agent (CA), designed for removal of primary tumor-targeting agent from the blood. The CA is based on the antibody cetuximab, which was conjugated to PNA and lactosaminated by reductive amination to improve hepatic clearance. The CA was evaluated in combination with PNA-labelled trastuzumab, T-ZHP1, for radionuclide HER2 pretargeting. Biodistribution studies in normal mice demonstrated that the CA cleared ca. 7 times more rapidly from blood than unmodified cetuximab. Injection of the CA 6 h post injection of the radiolabeled primary agent [131I]I-T-ZHP1 gave a moderate reduction of the radioactivity concentration in the blood after 1 h from 8.5 ± 1.8 to 6.0 ± 0.4%ID/g. These proof-of-principle results could guide future development of a more efficient CA.

Highlights

The use of monoclonal antibodies recognizing cancer-specific molecular abnormalities is one of the more promising ways to treat disseminated cancer
We have shown by SPR that peptide-nucleic acids (PNA)-conjugated trastuzumab retains sub-nanomolar affinity to HER2 after purification at pH 3 6
Pretargeting is a promising approach to reduce the background resulting from slow blood clearance of intact antibodies in immunoPET and radioimmunotherapy

Summary

Introduction

The use of monoclonal antibodies (mAbs) recognizing cancer-specific molecular abnormalities is one of the more promising ways to treat disseminated cancer. The most commonly used approach to molecular imaging is the labeling of therapeutic antibodies with positron-emitting nuclides (immunoPET) An accumulation of such antibodies in tumor sites can be visualized when a target is sufficiently expressed. The secondary agent is designed to have specific and high affinity binding to the recognition tag in the primary agent, and a rapid clearance from blood. The use of covalent photoconjugation ensured that the binding of ZHP1 to the antibody was irreversible[7] This trastuzumab-ZHP1 construct has been used together with 57Co-labeled HP2 in a proof-of-concept pretargeting imaging s tudy[6]. This approach has the potential for rapid conversion of any therapeutic antibody into a pretargeting primary probe for companion diagnostics and patient stratification. The CA should meet the following requirements: it should recognize the primary probe, efficiently remove the primary probe-CA complex from blood, have inefficient penetration of the CA into tumors to prevent interference with binding of the secondary probe to the recognition tag, and have a favorable safety profile

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Results

Conclusion