Preclinical development and characterisation of 99mTc-NM-01 for SPECT/CT imaging of human PD-L1

Abstract

The level of expression of programmed cell death-1 (PD-1)/programmed death ligand-1 (PD-L1) is a predictive biomarker for cancer immunotherapy, however, its detection remains challenging due to tumour heterogeneity and the influence from the binding of therapeutic agents. We recently developed [99mTc]-NM-01 as a companion diagnostic imaging agent for non-invasive molecular imaging of PD-L1 by single-photon emission computed tomography (SPECT). The aim of the study was to evaluate the [99mTc] radiolabelling of GMP graded NM-01 and its pharmacology, pharmacokinetics and toxicology. NM-01 bound specifically to human PD-L1 (Kd=0.8 nM) and did not interfere with the binding of the anti-PD-L1 antibody atezolizumab. NM-01 can bind various PD-L1-positive cancer cell lines and only interact with PD-L1 expressed on the cell surface. In SPECT/CT imaging, high [99mTc]-NM-01 accumulation was observed in the HCC827 mouse xenografted tumour model (30-min: 1.50 ± 0.27 %ID/g; 90-min: 1.23 ± 0.18 %ID/g), demonstrated a predominantly renal elimination (high uptake in bladder and kidney), while activity in the blood pool and other major organs remained low. The tumour-to-muscle and tumour-to-blood ratios were comparable with/without atezolizumab (P<0.04) but were significantly lowered when co-injected with excess NM-01 (P=0.04 and P=0.01, respectively.) The blood clearance of [99mTc]-NM-01 is bi-phasic; consisting of an initial fast washout phase with half-life of 2.1 min and a slower clearance phase with half-life of 25.4 min. In an intravenous extended single-dose toxicity study, no treatment-related changes were observed and the maximum tolerated dose of [99mTc]-NM-01 was 2.58 mg/kg. [99mTc]-NM-01 has suitable properties as a potential candidate for SPECT/CT imaging of PD-L1 assessment in cancer patients.

Keywords: PD-L1; SPECT/CT imaging; radiolabelling; single domain antibody; tracer.

Conflict of interest statement

N.C.L.W., Y.C., L.K.M. and H.H.T. are employees of NanoMab Technology Ltd. H.J.B. is an advisor for NanoMab Technology Ltd. G.J.R.C. is a consultant and receive research support for NanoMab Technology Ltd, Theragnostics Ltd and Serac Healthcare Ltd. F. M. M is on the advisory board of Advanced Accelerator Applications GmbH and has received institutional grants from GE Healthcare, Siemens, and NanoMab Technology Ltd.

AJNMMI Copyright © 2021.

Figures

Figure 1

The characterisation of NM-01. A. The binding affinity of NM-01 to human and mouse and PD-L1 as determined by ELISA assays. B. The binding kinetics of NM-01 at different doses and with or without the presence of atezolizumab as determined by SPR. C. The impact of NM-01 on the inhibitory effect of atezolizumab as determined by a competitive ELISA assay. D. The binding ability of NM-01 to PD-L1 on different cancer cell lines as determined by flow cytometry.

Figure 2

The specificity of NM-01 as determined by microarray-based binding assays followed by anti-HA and anti-His detection, with CTLA4-Fc/His and PBS served as control slides.

Figure 3

The biodistribution of [99mTc]-NM-01 in mice. A. The representative image of [99mTc]-NM-01 in mice bearing subcutaneous, PD-L1-positive HCC827 xenografts injected with [99mTc]-NM-01 (Group 1); co-injection of excess NM-01 (Group 2); and pre-dosing with 2 mg atezolizumab (Group 3) as assessed by SPECT/CT imaging at 30-min and 90-min post-injection. B. Comparison of the image-derived uptake of [99mTc]-NM-01 in major organs at 30-min and 90-min post-injection.

Figure 4

Comparison of tumour-to-muscle or tumour-to-blood ratios at 30-min and 90-min post-injection. Significant difference in tumour-to-muscle ratios was found between Group 1 vs Group 2 and Group 2 vs Group 3 at 90-min post-injection (P=0.04 and P=0.01, respectively). Tumour-to-blood ratios at both time points were significantly higher in Groups A and C compared to Group 2 (P0.05).