Imaging PD-L1 Expression with ImmunoPET

Abstract

High sensitivity imaging tools could provide a more holistic view of target antigen expression to improve the identification of patients who might benefit from cancer immunotherapy. We developed for immunoPET a novel recombinant human IgG1 (termed C4) that potently binds an extracellular epitope on human and mouse PD-L1 and radiolabeled the antibody with zirconium-89. Small animal PET/CT studies showed that ⁸⁹Zr-C4 detected antigen levels on a patient derived xenograft (PDX) established from a non-small-cell lung cancer (NSCLC) patient before an 8-month response to anti-PD-1 and anti-CTLA4 therapy. Importantly, the concentration of antigen is beneath the detection limit of previously developed anti-PD-L1 radiotracers, including radiolabeled atezolizumab. We also show that ⁸⁹Zr-C4 can specifically detect antigen in human NSCLC and prostate cancer models endogenously expressing a broad range of PD-L1. ⁸⁹Zr-C4 detects mouse PD-L1 expression changes in immunocompetent mice, suggesting that endogenous PD-1/2 will not confound human imaging. Lastly, we found that ⁸⁹Zr-C4 could detect acute changes in tumor expression of PD-L1 due to standard of care chemotherapies. In summary, we present evidence that low levels of PD-L1 in clinically relevant cancer models can be imaged with immunoPET using a novel recombinant human antibody.

Figure 1

Defining the optimal time after injection to study PD-L1 expression levels in a xenograft model. (A) Representative coronal and transaxial PET/CT images of male nu/nu mice bearing subcutaneous H1975 tumors, a human model of NSCLC , show that peak tumor uptake of ⁸⁹Zr-C4 occurs 48 h after injection. (B) Biodistribution data also show peak tumor uptake of the radiotracer 48 h after injection. High uptake is also observed in PD-L1 positive tissues like the liver a spleen. (C) Representative data from a blocking study acquired 48 h after injection show the tumor specific uptake of ⁸⁹Zr-C4. Blocking was performed with 30-fold excess C4. Radiotracer uptake exceeded that observed in the blood pool and muscle: (∗) P < 0.01.

Figure 2

⁸⁹Zr-C4 detects PD-L1 expression levels in a PDX derived from a NSCLC patient that experienced a durable clinical response to anti-PD-1 and anti-CTLA4 therapies. (A) Transaxial CT slices showing a soft tissue lesion in the lung prior to the initiation of pembrolizumab and ipilimumab (left), and a smaller mass 3 months after the start of therapy (right). The position of the tumor  is indicated with a white arrow. This patient experienced a partial response for 8 months. The PDX was derived 7 months prior to the first CT scan. (B) Small animal PET/CT data showing the biodistribution of ⁸⁹Zr-C4 in mice bearing bilateral PDX tumors in the flank. The tumors can be clearly resolved, and radiotracer uptake in abdominal tissues like the liver is observed, as expected for a large biomolecule. Mice treated with ⁸⁹Zr-C4 that was heat denatured (HD) for 10 min prior to injection show no evidence of radiotracer uptake in the tumor. (C) Biodistribution data showing the uptake of ⁸⁹Zr-C4 in the PDX tissue 48 h after injection. The uptake is higher in the tumor compared to heat denatured ⁸⁹Zr-C4 (HD) and standard reference tissues like the blood and muscle. (D) Biodistribution data acquired 48 h after injection in mice bearing subcutaneous H1975, PC3, A549, and the PDX tumors show the different degree of ⁸⁹Zr-C4 uptake in the tumors.

Figure 3

⁸⁹Zr-C4 can specifically detect mouse PD-L1 in tumors established in an immunocompetent background. (A) Representative coronal and transaxial PET/CT images of male C57BL/6 mice bearing subcutaneous B16 F10 tumors, a mouse model of melanoma, show that peak tumor uptake of ⁸⁹Zr-C4 occurs 48 h after injection. (B) Biodistribution data also show peak tumor uptake of the radiotracer 48 h after injection. High uptake is also observed in PD-L1 positive tissues like the liver a spleen. (C) Representative data from a blocking study acquired 48 h after injection show the tumor specific uptake of ⁸⁹Zr-C4. Blocking was performed with 10-fold excess C4. Radiotracer uptake exceeded that observed in the blood pool and muscle: (∗) P < 0.01.

Figure 4

⁸⁹Zr-C4 can detect pharmacologically induced PD-L1 expression changes on the tumor cell. (A) Representative coronal and transverse PET images showing the distribution of ⁸⁹Zr-C4 48 h after injection in a cohort of male nu/nu mice bearing subcutaneous H1975 xenografts and treated with vehicle, paclitaxel (Taxol), or doxorubicine. The mice were treated with 20 mg/kg paclitaxel or 2 mg/kg doxorubicine for 2 days prior to radiotracer injection. (B) Representative biodistribution data in the tumor and selected normal tissues showing that paclitaxel increases tumor PD-L1 expression levels, while doxorubicin suppresses it compared to vehicle. No impact was observed on PD-L1 expressing normal tissues like liver and spleen. (∗) P < 0.01; (#) P < 0.05. (C) Representative coronal and transverse PET images showing the distribution of ⁸⁹Zr-C4 48 h after injection in a cohort of male C57BL/6 mice bearing subcutaneous B16 F10 xenografts and treated with vehicle, paclitaxel, or doxorubicine. The mice were treated with 20 mg/kg paclitaxel or 2 mg/kg doxorubicine for 2 days prior to radiotracer injection. (D) Representative biodistribution data in the tumor and selected normal tissues showing that paclitaxel increases tumor PD-L1 expression levels, while doxorubicin had no impact compared to vehicle. PD-L1 expression was upregulated in the spleen of mice treated with doxorubicin: (∗) P < 0.01.