Site-Specific Immuno-PET Tracer to Image PD-L1

Abstract

The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-PET) allows for sensitive and quantitative imaging of target levels and has promising potential for the noninvasive evaluation of immune checkpoint proteins. However, the advancement of immuno-PET is currently limited by available imaging tools, which heavily rely on full-length IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with chelators for imaging. Herein, we have developed a site-specific αPD-L1 Fab conjugate with the chelator 1,4,7-triazacyclononane- N, N', N″-triacetic acid (NOTA), enabling radiolabeling for PET imaging, using the amber suppression-mediated genetic incorporation of unnatural amino acid (UAA), p-azidophenylalanine. This Fab conjugate is homogeneous and demonstrated tight binding toward the PD-L1 antigen in vitro. The radiolabeled version, ⁶⁴Cu-NOTA-αPD-L1, has been employed in PET imaging to allow for effective visualization and mapping of the biodistribution of PD-L1 in two normal mouse models, including the capturing of different PD-L1 expression levels in the spleens of the different mouse types. Follow-up in vivo blocking studies and ex vivo fluorescent staining further validated specific tissue uptakes of the imaging agent. This approach illustrates the utility of UAA-based site-specific Fab conjugation as a general strategy for making sensitive PET imaging probes, which could facilitate the elucidation of the roles of a wide variety of immune checkpoint proteins in immunotherapy.

Keywords: PD-L1; antibody; immune checkpoint; immunotherapy; positron emission tomography; site-specific; unnatural amino acid.

Figure 1.

Synthesis and characterization of the site-specific ⁶⁴Cu-NOTA-anti-PD-L1 Fab conjugate. (A) In silico screening with Rosetta has been performed with 23 solvent-exposed sites to determine the optimal mutation site, followed by site-directed mutagenesis to generate the anti-PD-L1 Fab (HC K129X, X = pAzF). Conjugation of this mutant with linker compound 4 will afford site-specific NOTA-anti-PD-L1 Fab conjugate 5 and its radiolabeled version, 6. (B) Enzyme-linked immunosorbent assay (ELISA) analysis of the binding affinities of anti-PD-L1 Fab fragments. The EC₅₀ was calculated to be 7.8 × 10⁻¹⁰ M for wild type, 7.3 × 10⁻¹⁰ M for the mutant (HC K129X, X = pAzF), and 7.2 × 10⁻¹⁰ M for conjugate 5.

Figure 2.

In silico screening of mutation sites on αPD-L1 Fab. (A) Changes in stability ΔREU (Rosetta energy units) for single-residue mutations to alanine (red) and phenylalanine (blue). Error bars represent standard deviations in ΔREU across the 20 structures given by the RosettaBackrub algorithm. (B) Similar changes in stability were predicted for alanine and phenylalanine. (C) Cumulative histograms of the predicted score show that mutations at (heavy chain) H_K129 are predicted to be the most favorable of the 23 selected residues. Predictions for the chosen negative control (light chain) L_V202, in contrast, are not ranked as especially favorable.

Figure 3.

In vivo PET imaging studies with ⁶⁴Cu-NOTA-αHER2 and ⁶⁴Cu-NOTA-αPD-L1 in nude mice. (A) PET scans at 15 and 45 min pi of ⁶⁴Cu-NOTA-αHER2 (left), ⁶⁴Cu-NOTA-αPD-L1 (middle), or ⁶⁴Cu-NOTA-αPD-L1 with preblocking by αPD-L1 w.t. (right); the yellow arrowhead indicates brown fat, whereas the white arrowhead points to the spleen. (B) Tracer uptake (% ID/g) for the three imaging groups in brown fat based on quantitative region-of-interest (ROI) analysis of the PET images. (C) Tracer uptake (% ID/g) in the spleen. “*” represents p < 0.05. n = 3.

Figure 4.

Immunofluorescent staining of brown adipose tissue for PD-L1 expression (red) and spleen tissue for PD-L1 (cyan) and CD45 (green) expressions. Nuclei (blue) were stained as controls.

Scheme 1.

Synthetic Scheme of 1,4,7-Triazacyclononane-N,N′,N″-triacetic Acid (NOTA) Derivative with a (1R,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-yl-methylcarbamate (BCN) Linker