Purpose: Programmed death ligand-1 (PDL1) expression has been linked to tumor resistance and upregulated by tumor cells following radiation therapy to avoid immune clearance. Anti-PDL1 immunotherapy has proven successful in multiple cancers, however within similar tumors heterogenous outcomes are seen among patients necessitating the need for PDL1 expression monitoring prior, during, and post-treatment. To this end, we have developed an MR contrast agent using polysiloxane gadolinium nanoparticles (AGuIX) surface modified with a highly specific single-domain antibody (VHH-A12) against PDL1. AGuIX are currently in clinical trials as novel theranostic nanoparticles for MR tumor contrast and radiation dose amplification.
Methods: Two separate chemistries were developed to attach a novel single-domain PDL1 nanobody (VHH-A12) to the surface of AGuIX. A sortase-catalyzed litigation was developed with a heptamutant sortase followed by purification via Ni-NTA beads and a vivaspin collector. A second azide-DBCO click chemistry was developed in a two-step process: 1) AGuIX were first surface modified with DBCO-(PEG)-NHS and A12 nanobody was modified with NHS-(PEG)-azide 2) followed by a click reaction between DBCO-azide. Final product purity and conjugation success were characterized via HPLC-UV. Binding of the conjugate was verified via competition ELISA comparing free A12 against AGuIX-A12 and AGuIX.
Results: The sortagging chemistry reaction yielded a final product of 186 Gd/VHH while the DBCO-azide-click chemistry yielded a final product of 8.5 Gd/VHH, confirming a higher ratio of VHH to AGuIX with the latter. ELISA binding to PDL1 revealed a strong binding affinity of 16nM AGuIX-A12 compared to 5.957 nM free A12. No binding affinity was able to be determined for non-targeted AGuIX.
Conclusion: A novel MR diagnostic tool for PDL1 expression has been developed using a single-domain antibody with high specificity for PDL1 in conjugation with theranostic AGuIX nanoparticles.
Funding Support, Disclosures, and Conflict of Interest: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Viewray, Inc. Some authors are employed by NH Theraguix.