Abstract 3448: A highly specific 18F-labelled peptide for non-invasive quantification of PD-L1 levels in tumors
Authors: Kumar Dhiraj , Mishra Akhilesh , Lisok Ala , Shelake Sagar , Wharram Bryan , Desilva Ravindra , Mease Ronnie , Nimmagadda Sridhar 
Published: September 2020

Introduction. Blockade of programmed death ligand-1 (PD-L1) and its receptor (PD-1) has emerged as a foundational treatment strategy for cancer. Immunohistochemical analysis (IHC) of PD-L1 expression is one of the biomarkers used to guide those treatments. However, total PD-L1 levels, dynamics and their relevance to treatment response are poorly understood. To facilitate non-invasive quantification of total PD-L1 levels and dynamics, here we report the synthesis of a highly specific PD-L1 binding radiolabeled peptide, [18F]DK222, and demonstrate its specificity for PD-L1 quantification in vivo in xenograft models of multiple cancer types.

Experimental procedures. A 14 amino acid macrocyclic peptide (DK222) was conjugated with a bifunctional chelator (NCS-MP-NODA, Chematech) and the corresponding fluorinated radioactive and non-radioactive analogs, [18/19F]DK222, were synthesized by aluminum fluoride method. In vitro competitive inhibition assays were used to establish peptide analog affinity for PD-L1. We then tested in vitro and in vivo specificity of [18F]DK222 for PD-L1 in cell binding, positron emission (PET) imaging and ex vivo biodistribution studies. Those studies were performed in xenograft models having varying levels of PD-L1 expression and derived from breast, bladder, lung and skin cancers. In vivo PD-L1 specificity was confirmed by co-injecting a blocking dose of 50 mg/kg of parent peptide. PD-L1 expression in cell lines and tumors was validated by flow cytometry and IHC, respectively.

Results. We observed half-maximal inhibitory concentrations of &lt; 25 nM for DK222 and its analogs to inhibit PD-L1 interaction with its receptor. We generated [18F]DK222 in 25% radiochemical yield (non-decay corrected) with high purity (&gt;95%) and with a specific activity of 256±18 mCi/micromole. In vitro studies showed that cell uptake of [18F]DK222 is reflective of PD-L1 expression detected by flow cytometry in the PD-L1high (MDAMB231, LOXIMVI, BFTC909) and PD-L1low (SUM149, MeWo, and T24) cells. PET imaging and biodistribution studies showed robust tracer uptake in PD-L1 high but not in PD-L1 low tumors. Blocking studies showed &gt;80% reduction in the uptake of [18F]DK222 in PD-L1high tumors (P&lt;0.0001). Imaging and biodistribution studies collectively demonstrated that [18F]DK222 binds rapidly and specifically to human PD-L1. PET imaging observations were consistent with high immunoreactivity observed in PD-L1 high tumors compared with PD-L1 low tumors.

Conclusions. The pharmacokinetics of [18F]DK222 indicate that quantification of PD-L1 levels is feasible within 60 min of radiotracer administration. The developed radiotracer [18F]DK222 could potentially enable non-invasive quantification of PD-L1 levels in vivo by PET imaging.

Citation Format: Dhiraj Kumar, Akhilesh Mishra, Ala Lisok, Sagar Shelake, Bryan Wharram, Ravindra Desilva, Ronnie Mease, Sridhar Nimmagadda. A highly specific 18F-labelled peptide for non-invasive quantification of PD-L1 levels in tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3448.