Monitoring the Response of PD-L1 Expression to Epidermal Growth

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Monitoring the Response of PD-L1 Expression to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Nonsmall-Cell Lung Cancer Xenografts by Immuno-PET Imaging Dan Li,† Sijuan Zou,† Siyuan Cheng,† Shuang Song,† Pilin Wang,‡ and Xiaohua Zhu*,† †

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Department of Nuclear Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China ‡ Alphamab Co. Ltd., Suzhou 215000, China S Supporting Information *

ABSTRACT: Accumulating evidence has suggested that the tumor microenvironment of nonsmall-cell lung cancer (NSCLC) may be impacted by chemotherapy, radiotherapy, or epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). PD-L1 is an important biomarker in the tumor microenvironment that can predict patient response to immunotherapies. Therefore, it is highly desirable to achieve a real-time, noninvasive assessment of PD-L1 expression, which can provide critical information for recruiting patients as well as monitoring therapeutic efficacy. We herein studied the EGFR-TKI-induced effects on PD-L1 levels in NSCLC tumor models using immuno-PET imaging with 89Zr-Df-KN035, an imaging tracer previously established by our group. A549 human NSCLC xenografts were established in BALB/c nude mice and treated with different doses of an EGFR-TKI gefitinib. PET imaging with 89Zr-Df-KN035 was performed before and after the treatment to evaluate PD-L1 expression, which was further verified by immunohistochemical staining. Our results demonstrate that 89Zr-Df-KN035 can specifically evaluate PD-L1 levels in NSCLC tumor models. Compared to the untreated control, the high dose of gefitinib inhibited tumor growth and lowered the tumor uptake of 89Zr-Df-KN035. In comparison, the low dose of gefitinib did not affect tumor growth, although the extensive tumor necrosis also led to the lower uptake of 89Zr-Df-KN035. In conclusion, our results demonstrate that immunoPET imaging with 89Zr-Df-KN035 is a promising tool to noninvasively monitor PD-L1 expression in NSCLC treated with EGFR-TKIs and can be used to optimize treatment plans for immunotherapy. KEYWORDS: PD-L1, PET imaging, EGFR-TKIs, NSCLC

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INTRODUCTION Lung cancer is the leading cause of cancer mortality worldwide with an estimated 1.6 million deaths for the year 2017.1 Nonsmall-cell lung cancer (NSCLC) is one of the two major types of lung cancer, comprising approximately 85% of all lung cancer cases.2 More than half of the NSCLC patients present with metastatic disease upon diagnosis and are no longer fit for surgery.2 Platinum-based chemotherapy and radiotherapy are the standard-of-care options for these patients with NSCLC, yet only to provide limited improvements in survival.3 Recently, vital breakthroughs in immunotherapies, especially the discovery of immune checkpoint blockade, have revolutionized cancer treatment, with the potential to elicit durable response and ultimately improve long-term survival.4 Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) or programmed cell death ligand 1 (PD-L1) are now standard first-line treatments for patients with tumoral PD-L1 expression over 50% and second-line options for those with advanced NSCLC.5−11 However, less than 20% of patients benefit from immunotherapy.12 The benefit of immunotherapy can be © XXXX American Chemical Society

extended to a broader population of cancer patients by using adjuvant chemo- or targeted therapies, which can induce an immunogenic cell death, liberate neoantigens, and therefore further enhance the antitumor immune responses.13 Alternatively, the efficacy of immunotherapy can be maximized by preselection of patients who are candidates for immunotherapy. It is reported that high expression of PD-L1 correlates with worse prognosis of patients. Therefore, PD-L1 has received much attention as a predictive biomarker for patient selection, whereas quite a few PD-L1-targeting imaging probes have been developed, including a variety of monoclonal antibodies (mAbs), antibody fragments, or peptides labeled with various radionuclides.14−20 Our recent work on PET imaging using an 89 Zr-labeled anti-PD-L1 domain antibody has also shown promise in both mice bearing subcutaneous glioma xenografts Received: Revised: Accepted: Published: A

March 15, 2019 June 23, 2019 June 25, 2019 June 25, 2019 DOI: 10.1021/acs.molpharmaceut.9b00307 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics and healthy nonhuman primates (NHPs).21 These reports demonstrated that it was plausible to image PD-L1-overexpressing tumors using a potent and selective, radiolabeled antibody. However, the therapeutic response within tumor immune microenvironment remains unclear, highlighting the urgency to investigate whether immuno-PET can monitor therapy-induced changes of PD-L1 expression over time. Recent advances on epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have substantially prolonged the survival of patients with NSCLC. Interestingly, many reports suggest that exposure to EGFR-TKIs (e.g., gefitinib) downregulates PD-L1 expression.3 On the basis of these findings, we supposed that the novel immuno-PET imaging agent which we reported previously could monitor the variation of PD-L1 expression in tumors treated with EGFRTKIs. In this work, we studied EGFR-TKI-induced PD-L1 downregulation in experimental animal models by immuno-PET using 89Zr-Df-KN035 previously reported.21 By PD-L1-targeted PET imaging pre- and post-EGFR-TKI therapy, we analyzed the effects of EGFR-TKI on PD-L1 expression in NSCLC xenografts, which would have great potential to optimize the individual immunotherapeutic strategy.

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was analyzed by radio-HPLC at different time points (1, 24, 48, and 96 h). Small Animal PET Imaging. Whole-body PET images were acquired on a trans-PET system (Raycan Technology Co., Ltd., Suzhou, China). Four mice with subcutaneous A549 xenografts were anesthetized with isoflurane/O2 with real-time respiratory monitoring. After an intravenous injection of approximately 3.7 MBq (100 μCi) of 89Zr-Df-KN035, images were acquired for 15 min at the 1, 6, and 24 h time point for 20 min at the 48 and 72 h time point and 30 min at the 120 h time point. Control animals (n = 4) were administrated with an excess of 2 mg unlabeled KN035 1 h in advance to block PD-L1 in vivo as a competition assay. After reconstruction, maximum intensity projections (MIPs) were computed using PMOD software (PMOD Technologies LLC, Switzerland). The tracer uptake quantified as percentage injected dose per gram (%ID/g) in major organs/ tissues and tumor was determined by regions of interest (ROI) analyses on the images. Ex vivo Biodistribution. Three groups (n = 4) of mice with subcutaneous A549 xenografts received an intravenous injection of 0.74 MBq (20 μCi) of 89Zr -Df-KN035. At 24, 48, and 120 h postinjection, mice were sacrificed, and the ex vivo biodistribution of the radiotracer was determined as described previously.21 Animal Therapy Studies with Gefitinib. Twenty-four mice bearing A549 tumors were randomly divided into four groups of six animals each. For high-dose treatment studies, 2 groups received gefitinib of 100 mg/kg body weight (AstraZeneca) and vehicle of 0.2 mL of sterile water via oral gavage once daily for 14 consecutive days. For low-dose treatment studies, two groups received gefitinib of 50 mg/kg body weight (AstraZeneca) and vehicle of 0.1 mL of sterile water via oral gavage once daily for 21 consecutive days. Tumor measurements by digital caliper, determinations of body weight, and assessments of clinical status were performed daily beginning at the day of gefitinib or vehicle administration. Tumor volume was calculated by the formula V = (π/6)LW2 (L = largest, W = shortest diameter of the tumor). 89 Zr-Df-KN035 (100 μCi, 3.7 MBq, 100 μg) was administered intravenously by tail vein before and after therapy. PET imaging was performed at 48 h postinjection of the tracer. Hematoxylin and Eosin Staining and Immunohistochemistry. Tumor samples from A549 tumor-bearing nude mice were cut open across their maximum dimension, fixed in 10% neutral buffered formalin, dehydrated in a series of ethanol concentrations, cleared in xylene, embedded in paraffin, and sectioned at 4 μm thick for further studies. Tumor sections were processed for histological observations using hematoxylin and eosin (H&E) staining. Immunostaining was performed with Bond Polymer Refine Detection System (Leica Biosystems, UK) as described previously.21 The rabbit monoclonal antibody clones (ZA-0629) against PD-L1 at 1:1 dilution were purchased from ZSGB-BIO (Beijing, China). Pictures taken at high magnification were viewed using Image-Pro Plus 6.0 professional image analysis software. For IHC analyses, five noncontiguous and nonoverlapping regions were blindly selected for each slide. The mean percentage of tumor cells expressing PDL1 from the five different areas was calculated after therapy and subsequently used for statistical purposes. Statistical Analysis. Data was analyzed using the unpaired, two-tailed t-test, and significance was assumed as p < 0.05. Results are presented as mean ± standard error (SD).

MATERIALS AND METHODS

Cell Lines. A549 (human NSCLC) cell lines used in this study were obtained from Shanghai Institutes for Biological Sciences (Shanghai, China). A549 cells were maintained in RPMI1640 (Gibco, United States). Media was supplemented with 10% fetal bovine serum (FBS) (HyClone, United States) and 1% penicillin/streptomycin (P/S) (Shanghai Institutes for Biological Sciences, Shanghai, China) at 37 °C in a humidified atmosphere with 5% CO2. Animal Models. All animal studies were conducted in compliance with the principles laid out by the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, China). BALB/c nude mice (male, 4−5 weeks) purchased from JLC Biopharma Co., Ltd., (Nanjing, China) were inoculated with tumor cells (1 × 107 A549 suspended in 100 μL PBS) subcutaneously at the right flank. Synthesis of 89Zr-Df-KN035. KN035 was kindly provided by Alphamab Co. Ltd. (Suzhou, China). It was conjugated with p-isothiocyanatobenzyl-desferrioxamine B (DFO-Bz-NCS; Macrocyclics, United States) in 0.1 M Na2CO3, pH 9.0, at a 4fold molar excess of DFO-Bz-NCS for 1 h at room temperature (RT). Unconjugated DFO-Bz-NCS was removed using a PD10 column (GE Healthcare, United States).21 DFO-Bz-NCSconjugated KNO35 was subsequently radiolabeled with 89Zr (PerkinElmer Health Sciences, Netherland) as described previously and purified with PD-10 columns.21 Radiochemical Purity and Stability Testing. The final product was assessed by instant thin-layer chromatography (iTLC) (Bioscan, United States), as well as a 1500 highperformance liquid chromatography (HPLC) system (Alltech, United States) fitted with a B-FC-3600 detector (Bioscan, United States) and a TSK Gel G3000SWXL column (300 mm × 7.5 mm, Tosoh, Japan) for radiolabeling efficiency and radiochemical purity. As the mobile phases, 0.5 M of citric acid solution was used for iTLC and PBS (pH 7.0) was used at a flow rate of 0.8 mL/min for HPLC. The in vitro stability of the 89Zr-Df-KN035 was tested with acetate buffer (pH 7.4) at 37 °C. The tracer mixture (100 μL) B

DOI: 10.1021/acs.molpharmaceut.9b00307 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 1. Whole-body PET imaging of BALB/c nude mice bearing A549 xenografts after injection of 3.7 MBq of 89Zr-Df-KN035 into the right flank (white arrows). MIPs of representative mice are shown at several time points after injection (n = 4). Blocking was performed by injection of 2 mg of unlabeled KN035 1 h before PET tracer.

Figure 2. Quantitative PET analysis and ex vivo biodistribution of 89Zr-Df-KN035 in mice bearing A549 xenografts. (A) PET ROI analysis is shown as time−activity histograms of organs and tissues after intravenous injection of 89Zr-Df-KN035 (n = 4). (B) PET ROI analysis is shown as time−activity histograms of tumor-to-heart ratios (T/H), tumor-to-lung ratios (T/P), tumor-to-liver ratios (T/L), tumor-to-kidney ratios (T/K), tumor-to-muscle ratios (T/M), and tumor-to-brain ratios (T/B). (C) PET ROI analysis is shown as time−activity histograms of tumors of mice injected with (blocking) or without (nonblocking) 2 mg of unlabeled KN035 1 h before injection of 89Zr-Df-KN035 (n = 4). *p < 0.05, **p < 0.01. (D) Ex vivo biodistribution of 89Zr-Df-KN035 in the blood, organs, and tissues of mice at 24, 48, and 120 h postinjection of 0.74 MBq of 89Zr-Df-KN035 (n = 4). Blocking was performed by injection of 2 mg of unlabeled KN035 1 h before PET tracer.

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RESULTS

greater than 98% with specific activity of 37 MBq/mg. The radiochemical stability in acetate buffer was greater than 98% tested for up to 96 h (Figure S1). Small Animal PET Imaging and Biodistribution. Representative longitudinal PET images of mice bearing A549

Radiochemical Purity and Stability of 89Zr-Df-KN035 in Vitro. The overall labeling yields were greater than 70% after purification. The radiochemical purity of the final product was C

DOI: 10.1021/acs.molpharmaceut.9b00307 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics xenografts are displayed as MIPs in Figure 1. The blood pool radioactivity peaked at 1 h and decreased 50% from 6 to 120 h. A549 xenografts became visible at 6 h postinjection, and good delineation of the tumors persisted until the final imaging session. Preinjection of 20-fold unlabeled KN035 reduced the tumor uptake at different time points, while radioactivity in the liver and kidney increased. Signal intensity was also observed in the region of the thighbone at 24 h and onward, likely reflecting the bone seeker of 89Zr. Minimal uptake of the tracer was found in muscle and brain tissues. ROI analysis of PET imaging allowed for the in vivo biodistribution quantification. The concentration in the tumor was significantly greater than that in other organs at 48 h (10.78 ± 1.14%ID/g) and onward with favorable tumor-to-muscle ratios (Figure 2A and 2B). Lung accumulation remained relatively constant throughout the entire study with moderate tumor-to-lung ratios, the highest value of which was 3.58 ± 0.86 at 120 h (Figure 2A and 2B). Tumor uptakes were markedly lower in mice blocked with unlabeled KN035 than those injected with 89Zr-Df-KN035 alone (Figure 2C). Ex vivo tissue biodistribution was evaluated at 24, 48, and 120 h post administration of 89Zr-Df-KN035 (20 μCi, 0.74 MBq, 20 μg) (Figure 2D). Blood concentration was 14.40 ± 2.35%ID/g at 24 h postinjection, falling to 5.66 ± 0.96%ID/g by 120 h. Tumor and muscle uptake were 6.78 ± 1.33 and 1.73 ± 0.46% ID/g at 24 h, respectively. Organs with the highest accumulation of 89Zr-Df-KN035 at 48 h were the kidneys (11.27 ± 1.97%ID/ g), which showed slight removal after 120 h. The liver and lungs also showed relatively high accumulation. Tracer uptake was low in the spleen with the highest tracer uptake value of 3.53 ± 0.25%ID/g at 48 h. The brain, stomach, intestine, and pancreas accumulation remained steady throughout the study. Tissue uptake in the bone remained above 7%ID/g throughout the study. The tracer exhibited favorable tumor-to-muscle ratios, which were 3.99 ± 0.47, 3.79 ± 0.29, and 4.47 ± 0.83 at 24, 48, and 120 h postinjection, respectively. Analysis of EGFR-TKIs Treated A549 Tumor Xenografts. Given that exposure to EGFR-TKIs (e.g., gefitinib) leads to PD-L1 downregulation, we tested whether PET imaging agents could noninvasively assess the variations of PD-L1 levels in A549 tumor xenografts treated with EGFR-TKIs. For tumor growth, there were no significant differences in the volumes of A549 tumors for all groups before therapy. All groups had similar tumor sizes before treatment (Figure 4A). After therapy, the average tumor volume of the high-dose gefitinib group was 943.7 ± 132.8 mm3 on day 14, statistically smaller than that of the untreated control (1407.4 ± 98.6 mm3) (Figure 4A). The lowdose average tumor volume was 1831.9 ± 133.2 mm3 on day 21, which was not significantly different from that of the control (1672.8 ± 225.2 mm3) (Figure 4A). As demonstrated by PET imaging, subcutaneously injected xenograft tumors were readily visualized (Figure 3). All A549 tumors had similar uptake of 89 Zr-Df-KN035 before treatment. After treatment, however, the high-dose group had statistically lower uptake than its control group, according to the ROI analyses of imaging. The Δuptake in the tumor before and after therapy in high-dose gefitinib group and its control were 4.73 ± 1.58 and 0.73 ± 0.71%ID/g, respectively (Figure 4B). 89Zr-Df-KN035 uptake was quite low both in the tumors of mice treated with low-dose gefitinib and its control group after therapy with the Δuptake in the tumor of 3.39 ± 2.04 and 3.77 ± 0.83%ID/g, respectively (Figures 3 and 4B). PET studies revealed that as for high-dose gefitinib group, significant differences were obtained between the therapeutic

Figure 3. Representative coronal PET images (48 h postinjection) of 89 Zr-Df-KN035 in mice bearing subcutaneous A549 xenografts after injection of 3.7 MBq of 89Zr-Df-KN035 into the right flank (white arrows) pre- and post-treatment with high-dose and low-dose gefitinib, respectively. Sterile water groups were used as the controls.

and control group. In contrast, no significant differences were obtained between the low-dose gefitinib and its control group. Hematoxylin and Eosin Staining and Immunohistochemistry. The A549 tumors showed expression of PD-L1 on the membranes in the presence of the chromogen (brown) (Figure 5). After therapy, xenografts in high-dose gefitinib group revealed many areas of low-density PD-L1+ expression compared to its control (Figure 6), whereas the low-dose gefitinib group and its control group displayed a majority of the PD-L1-low expression (Figure 6) and extensive necrosis (Figure S2) in the treated A549 tumor xenografts. The high-dose gefitinib group showed a median percentage of tumor cells expressing PD-L1 of 4.33 ± 1.54%, whereas its control presented a median value of 10.8 ± 3.83%. The low-dose gefitinib group and its control group showed a median value of less than 1%, which did not differ significantly.

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DISCUSSION Recent studies have indicated that immune checkpoint blockade can be potentiated by combining with other treatment modalities. In clinical practice, it is also common to first treat patients with chemotherapy, radiotherapy, or EGFR-TKIs as the standard-of-care before eventually transferring them to the antiPD-1/anti-PD-L1 regimens.22−24 However, the PD-L1 expression can be altered by such pretreatment, which calls for a noninvasive, real-time assessment of PD-L1 expression instead of archival biopsy to qualify a patient for anti-PD-1/anti-PD-L1 therapy. KN035 is an anti-PD-L1 domain antibody with superior binding affinity to PD-L1 in vitro.21 In our recent study, its pisothiocyanatobenzyl-deferoxamine conjugate (Df-KN035) and 89 Zr-chelation (89Zr-Df-KN035) also showed high in vitro PDL1 binding affinity and in vivo specific targeting ability to PD-L1 positive-expressed tumors.21 In this work, we tested whether D

DOI: 10.1021/acs.molpharmaceut.9b00307 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 4. (A) Tumor growth curves are presented as tumor volume measurements after treatment. (B) PET ROI analysis of 89Zr-Df-KN035 is shown as histograms of the changes of tumor uptake in a cohort of male nude mice bearing subcutaneous A549 xenografts treated with high-dose and low-dose gefitinib, respectively. Sterile water groups were used as the controls. Δ%ID/g = %ID/gpretreatment − %ID/gpost‑treatment. *p < 0.05.

Figure 5. Histologic sections of PET-imaged mouse tumors stained with HE and immunohistochemistry for PD-L1 were viewed at 200× and 400× magnification. Brown color marked in white rows represents tumor PD-L1 expression. 89

proved PD-L1-targeted specificity of the probe; meanwhile, the tumor retention indicated KN035 as a promising therapeutic agent. The specificity of 89Zr-Df-KN035 was further validated in a blocking study, as the PET images of mice preinjected with excess cold KN035 displayed an obvious reduction of 89Zr-DfKN035 uptake in tumors. There was enhanced accumulation in liver and kidney tissue, possibly due to increased serum amounts of radiotracer after blocking as seen in previous study.21 The lung uptake in ex vivo biodistribution study was more than expected, which can be explained by the accumulation of the probe due to the compression of lungs ex vivo against the lower uptake distribution in the pneumatized lungs in vivo. The results

Zr-Df-KN035 PET can monitor the changes of PD-L1 expression due to EGFR-TKIs in vivo over extended time. We performed PET imaging and biodistribution studies in nude mice subcutaneously xenografted with A549 cell lines. Before this, we tested whether Df affected the affinity and immunoreactivity of KN035 in our previous study.21 The stability of 89Zr-Df-KN035 was detected to be >98% over 96 h, making it suitable for the next studies. Radiotracer uptake was similar in nude mice bearing NSCLC xenografts compared to glioma xenografts in a previous study.21 Uptake of 89Zr-DfKN035 could be observed in NSCLC xenografts as early as 6 h, persisted elevating until 48 h, and then reached a plateau and retained through 120 h postinjection. These observations E

DOI: 10.1021/acs.molpharmaceut.9b00307 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 6. Immunohistochemistry for PD-L1 was viewed at 200× magnification after treatment. The A549 tumors showed expression of PD-L1 on the membranes in the presence of the chromogen (brown). Many areas of low-density PD-L1+ expression were observed in A549 tumors of the high-dose gefitinib group compared to its control group. The low-dose gefitinib group and its control demonstrated a majority of the PD-L1-low expression.

Nowadays, PD-L1 IHC and immune checkpoint inhibitors are not used in NSCLC with defined EGFR mutation status. However, there exists an argument. Akbay et al.28 found that monoclonal blocking anti-PD-1 antibody resulted in tumor reduction and significantly increased overall survival in NSCLC mouse models in which lung adenocarcinomas are driven by EGFR mutation, which suggests that immune checkpoint blockade may be a promising therapeutic strategy to extend the duration of treatment response in EGFR-driven lung tumors. Recent studies29−31 showed that the PD-L1 status in patients with EGFR mutant NSCLC is controversial. The effect of immune checkpoint inhibitors in EGFR mutant NSCLC aroused a broad controversy. It is worth considering to extend the duration of immune checkpoint inhibitor responses with the help of potential biomarkers based on the preliminary findings which raise the opposite possibility in EGFR-driven lung tumors. The immune response plays a critical role in host protection against tumor development, progression, and metastasis, responsible for immunoediting and immunosurveillance. Although the PD-L1 PET imaging suggests a novel paradigm for monitoring immune response in treated tumors, ongoing investigations are required to utilize smaller molecules such as peptides, affibodies, or nanobodies to optimize imaging time and thus translate this paradigm for clinical application. In addition, the paradigm can be further evaluated with combination strategies using immunotherapy as a partner for radiotherapeutic, chemotherapeutic, targeted, and other immunotherapeutic agents to validate synergistic antitumor activity and therefore open new avenues in a broader patient population for the benefits of immunotherapy.

of IHC staining showed PD-L1 expression in A549 xenografts, which was consistent with imaging results. We then tested the 89Zr-Df-KN035 PET probe in the same tumor xenograft models of NSCLC treated with either gefitinib in different doses or sterile water as control groups. After EGFRTKIs therapy, PET studies revealed that 89Zr-Df-KN035 was substantially lower in the tumors of mice administrated with high-dose gefitinib (100 mg/kg/d) compared to the control arm, which was confirmed by IHC results. The tumor volume also grew slower in the high-dose gefitinib group. There were no substantial differences of tumor growth in low-dose gefitinib (50 mg/kg/d) treated mice relative to controls by day 21. However, the %ID/g decreased markedly in the mice treated with gefitinib (50 mg/kg/d) and its control (0.1 mL/per mouse), which can be explained with the extensive necrosis caused by the long period of unsuccessful therapy. It is reported that EGFR-TKIs drive tumor regression via inhibiting proliferation and inducing apoptosis of tumor cells.3 EGFR pathway activation has been found to be correlated with a signature of immunosuppression triggered by upregulation of PD-1, PD-L1, and proinflammatory cytokines through multiple signaling pathways, including mitogen-activated protein kinases (MAPKs), phosphoinositide 3-kinase (PI3K), as well as signal transducer and activator of transcription 3 (STAT3) pathways.3,25−27 However, EGFRTKIs may reverse the EGFR pathway driven immunosuppression in the tumor microenvironment and down-regulate the expression of PD-L1,3 which can be explained for the reduced tumor uptake of 89Zr-Df-KN035 in high-dose gefitinib group after therapy. The different treatment doses in this study were employed to identity the optimal dosage and low toxicity profile. These findings indicate that the changes of PD-L1 PET uptake in the EGFR-TKI treated mice are correlated with PD-L1 status and provide evidence that PD-L1 PET imaging can be used to evaluate PD-L1 expression for therapy management.

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CONCLUSIONS In this study, the expression of PD-L1 in NSCLC xenografts varied after gefitinib therapy, which was detected using immunoF

DOI: 10.1021/acs.molpharmaceut.9b00307 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics PET imaging with 89Zr-Df-KN035. This supports that the immuno-PET probe may be feasible to monitor the changes of PD-L1 levels in patients with NSCLC post EGFR-TKIs treatment, which has great potential for optimizing the individual immunotherapeutic strategy.

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cancer (OAK): a phase 3, open-label, multicentre randomised controlled trial. Lancet 2017, 389, 255−265. (12) Cheng, M.; Durm, G.; Hanna, N.; Einhorn, L. H.; Kong, F. S. Can radiotherapy potentiate the effectiveness of immune checkpoint inhibitors in lung cancer? Future Oncol. 2017, 13, 2503−2505. (13) Pilotto, S.; Molina-Vila, M.; Karachaliou, N.; et al. Integrating the molecular background of targeted therapy and immunotherapy in lung cancer: a way to explore the impact of mutational landscape on tumor immunogenicity. Transl Lung Cancer Res. 2015, 4, 721−727. (14) Heskamp, S.; Hobo, W.; Molkenboer-Kuenen, J. D.; Olive, D.; Oyen, W. J.; Dolstra, H.; Boerman, O. C. Non-invasive imaging of tumor PD-L1 expression using radiolabeled anti-PD-L1 antibodies. Cancer Res. 2015, 75, 2928−2936. (15) Chatterjee, S.; Lesniak, W. G.; Miller, M. S.; et al. Rapid PD-L1 detection in tumors with PET using a highly specific peptide. Biochem. Biophys. Res. Commun. 2017, 483, 258−263. (16) Maute, R. L.; Gordon, S. R.; Mayer, A. T.; McCracken, M. N.; Natarajan, A.; Ring, N. G.; Kimura, R.; Tsai, J. M.; Manglik, A.; Kruse, A. C.; Gambhir, S. S.; Weissman, I. L.; Ring, A. M. Engineering highaffinity PD-1 variants for optimized immunotherapy and immuno-PET imaging. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E6506−E6514. (17) Hettich, M.; Braun, F.; Bartholomä, M. D.; Schirmbeck, R.; Niedermann, G. High-Resolution PET Imaging with Therapeutic Antibody-based PD-1/PD-L1 Checkpoint Tracers. Theranostics 2016, 6, 1629−1640. (18) Josefsson, A.; Nedrow, J. R.; Park, S.; Banerjee, S. R.; Rittenbach, A.; Jammes, F.; Tsui, B.; Sgouros, G. Imaging, biodistribution, and dosimetry of radionuclide- labeled PD-L1 antibody in an immunocompetent mouse model of breast cancer. Cancer Res. 2016, 76, 472− 479. (19) Lesniak, W. G.; Chatterjee, S.; Gabrielson, M.; Lisok, A.; Wharram, B.; Pomper, M. G.; Nimmagadda, S. PD-L1 Detection in Tumors Using [64Cu]Atezolizumab with PET. Bioconjugate Chem. 2016, 27, 2103−2110. (20) Gonzalez Trotter, D. E.; Meng, X.; McQuade, P.; et al. In vivo Imaging of the Programmed Death Ligand 1 by 18F PET. J. Nucl. Med. 2017, 58, 1852−1857. (21) Li, D.; Cheng, S.; Zou, S.; et al. Immuno-PET Imaging of 89Zr Labeled Anti-PD-L1 Domain Antibody. Mol. Pharmaceutics 2018, 15, 1674−1681. (22) Zhang, P.; Su, D. M.; Liang, M.; Fu, J. Chemopreventive agents induce programmed death-1-ligand 1 (PD-L1) surface expression in breast cancer cells and promote PD-L1-mediated T cell apoptosis. Mol. Immunol. 2008, 45, 1470−1476. (23) Deng, L.; Liang, H.; Burnette, B.; et al. Irradiation and anti-PDL1 treatment synergistically promote antitumor immunity in mice. J. Clin. Invest. 2014, 124, 687−695. (24) Dovedi, S. J.; Adlard, A. L.; Lipowska-Bhalla, G.; et al. Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade. Cancer Res. 2014, 74, 5458−5468. (25) Zhang, N.; Zeng, Y.; Du, W.; et al. The EGFR pathway is involved in the regulation of PD-L1 expression via the IL-6/JAK/STAT3 signaling pathway in EGFR-mutated non-small cell lung cancer. Int. J. Oncol. 2016, 49, 1360−1368. (26) Jiang, X.; Zhou, J.; Giobbie-Hurder, A.; Wargo, J.; Hodi, F. S. The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition. Clin. Cancer Res. 2013, 19, 598−609. (27) Abdelhamed, S.; Ogura, K.; Yokoyama, S.; Saiki, I.; Hayakawa, Y. AKT-STAT3 Pathway as a Downstream Target of EGFR Signaling to Regulate PD-L1 Expression on NSCLC cells. J. Cancer 2016, 7, 1579− 1586. (28) Akbay, E. A.; Koyama, S.; Carretero, J.; et al. Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer Discovery 2013, 3, 1355−1363. (29) Soo, R. A.; Kim, H. R.; Asuncion, B. R.; et al. Significance of immune checkpoint proteins in EGFR-mutant non-small cell lung cancer. Lung Cancer. 2017, 105, 17−22.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.9b00307. Stability of 89Zr-Df-KN035 and HE staining of A549 xenografts after treatment of gefitinib (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-27-83663446; E-mail: [email protected]. ORCID

Xiaohua Zhu: 0000-0003-0495-9510 Funding

This work was supported by the National Natural Science Foundation of China (NSFC) (Grants 81671718 and 81873903). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge Alphamab Co. Ltd. (Suzhou, China) for providing KN035. REFERENCES

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DOI: 10.1021/acs.molpharmaceut.9b00307 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.molpharmaceut.9b00307 Mol. Pharmaceutics XXXX, XXX, XXX−XXX