A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors

Abstract

Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [111In]PD-L1-mAb and near-infrared dye conjugated NIR-PD-L1-mAb imaging agents were developed using the mouse and human cross-reactive PD-L1 antibody MPDL3280A. We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors (CHO-PDL1, MDAMB231, H2444) but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings.

Keywords: MPDL3280A; immune escape; immunotherapy; molecular imaging; personalized medicine.

Figure 1. In vitro specificity of [¹¹¹In]PD-L1-mAb and NIR-PD-L1-mAb

Flow cytometry analysis of various cell lines for cell surface PD-L1 expression A. Representative mean fluorescence intensity (MFI) values for PE conjugated anti-human PD-L1 antibody binding to various cell lines B. In vitro uptake of [¹¹¹In]PD-L1-mAb in CHO-PDL1, CHO, MDAMB231, SUM149, H2444 and H1155 cells incubated with 37 kBq (1 μCi)/100 μL of [¹¹¹In]PD-L1-mAb at 37°C for 1h C. In vitro uptake of NIR-PD-L1-mAb in CHO-PDL1, CHO, MDAMB231, SUM149, H2444 and H1155 cells incubated with 1 μM NIR-PD-L1-mAb at 37°C for 1 h D. Data are represented as percentage of incubated dose (%ID) per million cells and represent mean values of three experiments ± SEM. The significance of the value is indicated by asterisks (*) and the comparative reference is the control or low PD-L1 expression cell line. **p < 0.01, ****p < 0.0001.

Figure 2. Imaging PD-L1 expression in subcutaneous CHO xenografts with [¹¹¹In]PD-L1-mAb and NIR-PD-L1-mAb

NSG mice with CHO and CHO-PDL1 xenografts were administered intravenously with 14.8 MBq (400 μCi) of [¹¹¹In]PD-L1-mAb or 22 μg of NIR-PD-L1-mAb and images were acquired at 24, 48, 72, 96 and 120 h after the injection of the mAbs. 3D volume rendered whole body SPECT/CT images demonstrate specific accumulation of activity in the CHO-PDL1 tumors A. Optical images acquired in the 800nm NIR channel B. Ex vivo biodistribution analysis representative image C. and semi-quantitative analysis of fluorescence intensity at 120 h after the injection of NIR-PD-L1-mAb (n = 5) D. Column numbers in panel D represent the tissue numbers in panel C. All the SPECT images were decay corrected and adjusted to the same maximum value to show the clearance of the imaging agent. The significance of the value is indicated by asterisk (*) and the comparative reference is the tumor with low PD-L1 expression. Arrows and circles depict tumors. ****p < 0.0001.

Figure 3. Imaging PD-L1 expression in orthotopic breast cancer xenografts with [¹¹¹In]PD-L1-mAb and NIR-PD-L1-mAb

NSG mice with orthotopic MDAMB231 and SUM149 xenografts were administered intravenously with 14.8 MBq (400 μCi) of [¹¹¹In]PD-L1-mAb or 22 μg of NIR-PD-L1-mAb and images were acquired at 24, 72, and 120 h after the injection of the mAbs. 3D volume rendered whole body SPECT/CT images demonstrating specific accumulation of activity in the MDAMB231 tumors A. Ex vivo biodistribution analysis of the [¹¹¹In]PD-L1-mAb at 24 h, 72 h and 120 h after injection, in the same tumor models B.. Immunohistochemical analysis for PD-L1 expression demonstrating intense immunoreactivity in MDAMB231 tumors compared to SUM149 tumors C. Optical images acquired in the 800 nm NIR channel show specific accumulation of fluorescence signal in the MDAMB231 tumors D. Ex vivo biodistribution analysis of fluorescence intensity in tissues (n = 4) E. and representative image at 120 h after the injection of NIR-PD-L1-mAb F. Column numbers in panel E represent the tissue numbers in panel F. SPECT images were decay corrected and adjusted to the same maximum value to show the clearance of the imaging agent.. The significance of the value is indicated by asterisk (*) and the comparative reference is the tumor with low PD-L1 expression. Arrows and circles depict tumors. *p < 0.05, ****p < 0.0001.

Figure 4. Imaging PD-L1 expression in subcutaneous and orthotopic lung cancer xenografts with [¹¹¹In]PD-L1-mAb and NIR-PD-L1-mAb

NSG mice with subcutaneous H2444 or H1155 xenograft were administered intravenously with 14.8 MBq (400 μCi) of [¹¹¹In]PD-L1-mAb or 22 μg of NIR-PD-L1-mAb and images were acquired at the specified time after the injection of the mAbs. 3D volume rendered whole body images demonstrating specific accumulation of activity in the H2444 tumors at 120 h and not in the H1155 tumors A. Ex vivo biodistribution analysis of the [¹¹¹In]PD-L1-mAb, at 144 h after injection, in the same tumor models B. Immunohistochemical analysis for PD-L1 expression demonstrating intense immunoreactivity in H2444 tumors compared to H1155 tumors C. NSG mice with orthotopic H2444 xenografts were administered with 14.8 MBq (400 μCi) of [¹¹¹In]PD-L1-mAb and SPECT/CT images were acquired. Transaxial SPECT/CT images showing specific accumulation of activity in orthotopic H2444 xenograft at 72 h and 120 h after injection of the [¹¹¹In]PD-L1-mAb and the corresponding histology D. Optical images of subcutaneous H2444 or H1155 xenograft acquired in the 800nm NIR channel E. and ex vivo biodistribution analysis of fluorescence intensity in tissues (n = 3) F. SPECT images were decay corrected and adjusted to the same maximum value to show the clearance of the imaging agent. The significance of the value is indicated by asterisk (*) and the comparative reference is the tumor with low PD-L1 expression. Arrows and circles depict tumors. *p < 0.05. **p < 0.01.