Preclinical development of novel PD-L1 tracers and first-in-human study of [68Ga]Ga-NOTA-RW102 in patients with lung cancers

Abstract

Background: The programmed cell death protein-1 (PD-1)/programmed death receptor ligand 1 (PD-L1) axis critically facilitates cancer cells' immune evasion. Antibody therapeutics targeting the PD-1/PD-L1 axis have shown remarkable efficacy in various tumors. Immuno-positron emission tomography (ImmunoPET) imaging of PD-L1 expression may help reshape solid tumors' immunotherapy landscape.

Methods: By immunizing an alpaca with recombinant human PD-L1, three clones of the variable domain of the heavy chain of heavy-chain only antibody (VHH) were screened, and RW102 with high binding affinity was selected for further studies. ABDRW102, a VHH derivative, was further engineered by fusing RW102 with the albumin binder ABD035. Based on the two targeting vectors, four PD-L1-specific tracers ([⁶⁸Ga]Ga-NOTA-RW102, [⁶⁸Ga]Ga-NOTA-ABDRW102, [⁶⁴Cu]Cu-NOTA-ABDRW102, and [⁸⁹Zr]Zr-DFO-ABDRW102) with different circulation times were developed. The diagnostic efficacies were thoroughly evaluated in preclinical solid tumor models, followed by a first-in-human translational investigation of [⁶⁸Ga]Ga-NOTA-RW102 in patients with non-small cell lung cancer (NSCLC).

Results: While RW102 has a high binding affinity to PD-L1 with an excellent K_D value of 15.29 pM, ABDRW102 simultaneously binds to human PD-L1 and human serum albumin with an excellent K_D value of 3.71 pM and 3.38 pM, respectively. Radiotracers derived from RW102 and ABDRW102 have different in vivo circulation times. In preclinical studies, [⁶⁸Ga]Ga-NOTA-RW102 immunoPET imaging allowed same-day annotation of differential PD-L1 expression with specificity, while [⁶⁴Cu]Cu-NOTA-ABDRW102 and [⁸⁹Zr]Zr-DFO-ABDRW102 enabled longitudinal visualization of PD-L1. More importantly, a pilot clinical trial shows the safety and diagnostic value of [⁶⁸Ga]Ga-NOTA-RW102 immunoPET imaging in patients with NSCLCs and its potential to predict immune-related adverse effects following PD-L1-targeted immunotherapies.

Conclusions: We developed and validated a series of PD-L1-targeted tracers. Initial preclinical and clinical evidence indicates that immunoPET imaging with [⁶⁸Ga]Ga-NOTA-RW102 holds promise in visualizing differential PD-L1 expression, selecting patients for PD-L1-targeted immunotherapies, and monitoring immune-related adverse effects in patients receiving PD-L1-targeted treatments.

Trial registration number: NCT06165874.

Keywords: Biomarker; Lung Cancer; Nuclear medicine; fMRI / PET.

Figure 1

Characterization of RW102 and ABDRW102. The purity of RW102 (A) and ABDRW102 (B) examined by sulfate-polyacrylamide gel electrophoresis was >95%. (C and D) The surface plasmon resonance studies showed the association and dissociation kinetics of RW102 (C) and ABDRW102 (D) interacting with recombinant human programmed death receptor ligand 1 protein. (E and F) ABDRW102 also showed high binding affinities to human serum albumin (HSA, E) and murine serum albumin (MSA, F). M, marker; N-R, non-reducing condition; R, reducing condition.

Figure 2

[⁶⁸Ga]Ga-NOTA-RW102 immunoPET imaging in subcutaneous tumor models. (A and C) [⁶⁸Ga]Ga-NOTA-RW102 immunoPET imaging in subcutaneous RKO (A, 7.64±1.42 MBq, n=3) and LM3-PD-L1 (C, 4.35±0.47 MBq, n=4) models 0.5 hours post-injection of the tracer. Maximum intensity projection (MIP) and coronal images showed clear delineation of the tumors (red arrowheads) and kidneys (yellow arrowheads), from which the tracer was excreted. (B and D) Quantitative data showed the tracer uptake and distribution patterns in RKO (B) and LM3-PD-L1 (D) models. immunoPET, immuno-positron emission tomography; PD-L1, programmed death receptor ligand 1.

Figure 3

[⁶⁸Ga]Ga-NOTA-RW102 immunoPET imaging in A375-PD-L1 tumor models without or with ABDRW102 blocking. (A and C) [⁶⁸Ga]Ga-NOTA-RW102 immunoPET imaging in the control group (A) and ABDRW102 blocking group (C). (B) Quantitative regions of interest analysis results in the control group and ABDRW102 blocking group. (D) Ex vivo biodistribution data showed the detailed distribution and uptake patterns of [⁶⁸Ga]Ga-NOTA-RW102 in the control group and ABDRW102 blocking group. immunoPET, immuno-positron emission tomography; PD-L1, programmed death receptor ligand 1.

Figure 4

Serial [⁶⁸Ga]Ga-NOTA-ABDRW102 immunoPET imaging in RKO cancer models (5.87±0.27 MBq, n=6). (A) Representative PET images of [⁶⁸Ga]Ga-NOTA-ABDRW102 at multiple time points. The images showed clear delineation of the tumors (red arrowheads). (B) Regions of interest data of [⁶⁸Ga]Ga-NOTA-ABDRW102 immunoPET imaging at different time points. (C) Ex vivo biodistribution data showing the detailed uptake and distribution of [⁶⁸Ga]Ga-NOTA-ABDRW102 in the tumor, blood, major organs, and tissues. (D) Immunohistochemical verification of PD-L1 expression in tumors resected after immunoPET imaging. immunoPET, immuno-positron emission tomography; PD-L1, programmed death receptor ligand 1.

Figure 5

Serial [⁶⁴Cu]Cu-NOTA-ABDRW102 immunoPET imaging in RKO models. (A) Representative PET images of [⁶⁴Cu]Cu-NOTA-ABDRW102 at multiple time points. (B) Regions of interest data of [⁶⁴Cu]Cu-NOTA-ABDRW102 immunoPET imaging in tumors at different time points. (C) Ex vivo biodistribution data showed the detailed uptake and distribution of [⁶⁴Cu]Cu-NOTA-ABDRW102 in the tumor, blood, major organs, and tissues. immunoPET, immuno-positron emission tomography.

Figure 6

[⁸⁹Zr]Zr-DFO-ABDRW102 immuno-positron emission tomography imaging in LM3-PD-L1 models (2.66±0.74 MBq, n=8). (A) Maximum intensity projection PET/CT images of the control group (up panel) and ABDRW102 blocking group (down panel) at multi-time points. (B) Regions of interest data showed the uptake kinetics of [⁸⁹Zr]Zr-DFO-ABDRW102 in the control group and ABDRW102 blocking group at different time points. (C) Comparison of tumor uptake value between the control and ABDRW102 blocking groups at each time point. (D) Ex vivo biodistribution data showed the detailed uptake and distribution of [⁸⁹Zr]Zr-DFO-ABDRW102 in the control and ABDRW102 blocking groups. PD-L1, programmed death receptor ligand 1; PET, positron emission tomography.

Figure 7

[⁶⁸Ga]Ga-NOTA-RW102 immuno-positron emission tomography imaging of an adult patient with squamous lung cancer with 70% PD-L1 expression. (A–C) [⁶⁸Ga]Ga-NOTA-RW102 PET/CT images of two tumor lesions in this patient (yellow and red arrowheads). (D) The H&E staining of the subpleural lesion in the lower lobe of the patient’s right lung. (E) The PD-L1 staining of the biopsied squamous lung cancer tissue showed 70% positivity. PD-L1, programmed death receptor ligand 1; PET, positron emission tomography.