Imaging, Biodistribution, and Dosimetry of Radionuclide-Labeled PD-L1 Antibody in an Immunocompetent Mouse Model of Breast Cancer

Abstract

The programmed cell death ligand 1 (PD-L1) participates in an immune checkpoint system involved in preventing autoimmunity. PD-L1 is expressed on tumor cells, tumor-associated macrophages, and other cells in the tumor microenvironment. Anti-PD-L1 antibodies are active against a variety of cancers, and combined anti-PD-L1 therapy with external beam radiotherapy has been shown to increase therapeutic efficacy. PD-L1 expression status is an important indicator of prognosis and therapy responsiveness, but methods to precisely capture the dynamics of PD-L1 expression in the tumor microenvironment are still limited. In this study, we developed a murine anti-PD-L1 antibody conjugated to the radionuclide Indium-111 ((111)In) for imaging and biodistribution studies in an immune-intact mouse model of breast cancer. The distribution of (111)In-DTPA-anti-PD-L1 in tumors as well as the spleen, liver, thymus, heart, and lungs peaked 72 hours after injection. Coinjection of labeled and 100-fold unlabeled antibody significantly reduced spleen uptake at 24 hours, indicating that an excess of unlabeled antibody effectively blocked PD-L1 sites in the spleen, thus shifting the concentration of (111)In-DTPA-anti-PD-L1 into the blood stream and potentially increasing tumor uptake. Clearance of (111)In-DTPA-anti-PD-L1 from all organs occurred at 144 hours. Moreover, dosimetry calculations revealed that radionuclide-labeled anti-PD-L1 antibody yielded tolerable projected marrow doses, further supporting its use for radiopharmaceutical therapy. Taken together, these studies demonstrate the feasibility of using anti-PD-L1 antibody for radionuclide imaging and radioimmunotherapy and highlight a new opportunity to optimize and monitor the efficacy of immune checkpoint inhibition therapy.

Conflict of interest statement

Conflict of interest: None

©2015 American Association for Cancer Research.

Figures

Figure 1

The real-time RT-qPCR was performed in EL4 (murine B7H1-positive cells), HBL100 (human breast cancer cells), 4T1 (murine breast tumor cells) and NT2.5 (mouse HER2 expressing mammary tumor cell line) cell lines with (w) and without (w/o) 200 ng/ml IFN-γ treatment for 24 hrs. The white bar shows the relative gene expression for the cell line w/o IFN-γ treatment normalized to 1. The black bar shows the relative gene expression of the cell lines with IFN-γ treatment relative the non-treated.

Figure 2

The flow cytometry for three different cell lines, EL4 (murine B7H1 cells that expresses PD-L1, positive control), 4T1 (murine breast tumor cells) and NT2.5 (mouse HER2 expressing mammary tumor cell line) with A: No staining with anti-PD-L1 Ab B: Staining with anti-PD-L1 Ab and without (w/o) INF-γ treatment and C: Staining with anti-PD-L1 Ab and treatment with (w) 200 ng/ml IFN-γ for 24 hrs. D: Shows the shift for no staining (NS, grey), staining with anti-PD-L1 Ab w/o INF-γ treatment (NT, blue), and staining with anti-PD-L1 Ab with INF-γ treatment (T, red).

Figure 3

Immunohistochemistry of PD-L1 expression (PD-L1) and negative control (NEG. CONTROL) for NT2.5 tumor, spleen, thymus, liver and kidney at 20× and 40× magnification.

Figure 4

Whole-body SPECT images (coronal view) in transgenic neu-N mice showing the uptake in normal tissues (e.g. spleen, thymus and liver) and tumors for anti-PD-L1 Ab labeled with 111In at 1, 24 and 72 hrs p.i.

Figure 5

A: Biodistribution in tumor bearing transgenic neu-N mice for the normal tissues blood, heart, lung, liver, kidneys, stomach (with content), intestine (with content), femur, thymus, muscle and tumor for anti-PD-L1 Ab at 1 h (black bar), 24 hrs (blue bar), 72 hrs (orange bar), and 144 hrs (red bar) p.i. B: Biodistribution in neu-N mice at 24 hrs p.i. for normal tissues and tumor of anti-PD-L1 Abs (blue bar) and co-injected for blocking with excess cold anti-PD-L1 Ab 30× (green bar) and 100× (yellow bar).