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. 2024 May 10:15:1384733.
doi: 10.3389/fphar.2024.1384733. eCollection 2024.

Lysosomal degradation of PD-L1 is associated with immune-related adverse events during anti-PD-L1 immunotherapy in NSCLC patients

Takeru Kashiwada #  1 , Ryotaro Takano #  2   3 , Fumihiko Ando  2   3 , Shoko Kuroda  2 , Yoshishige Miyabe  2   4 , Ryuji Owada  2 , Akihiko Miyanaga  1 , Tomoko Asatsuma-Okumura  2 , Masaaki Hashiguchi  2 , Yoshikazu Kanazawa  3 , Hiroshi Yoshida  3 , Masahiro Seike  1 , Akihiko Gemma  1 , Yoshiko Iwai  2
Affiliations

Lysosomal degradation of PD-L1 is associated with immune-related adverse events during anti-PD-L1 immunotherapy in NSCLC patients

Takeru Kashiwada et al. Front Pharmacol. .

Abstract

Background: Immune checkpoint inhibitors (ICIs) can induce immune-related adverse events (irAEs). Liquid biomarkers to predict irAE occurrence are urgently needed. We previously developed an ELISA system to specifically detect soluble PD-L1 (sPD-L1) with PD-1-binding capacity (bsPD-L1). Here, we investigated the relationship between sPD-L1 and bsPD-L1 in gastric cancer (GC) and non-small cell lung cancer (NSCLC) treated with PD-1/PD-L1 blockade and their association with irAEs. Methods: We examined sPD-L1, bsPD-L1, matrix metalloproteinases (MMPs), and proinflammatory cytokine levels by ELISA in plasma samples from 117 GC patients prior to surgery and 72 NSCLC patients prior to and at 2 months after ICI treatment (anti-PD-1, n = 48; anti-PD-L1, n = 24). In mice treated with anti-PD-1/PD-L1 antibodies (Abs), sPD-L1 levels and localization of Abs were examined by ELISA and immunohistochemistry, respectively. Results:sPD-L1 was detected with higher frequency in GC patients than in NSCLC patients, whereas bsPD-L1 was detected with similar frequencies in GC and NSCLC patients. sPD-L1 levels were correlated with IL-1α, IL-1β, TNF-α, and IL-6 levels, while bsPD-L1 levels were correlated with MMP13, MMP3, and IFN-γ levels. In NSCLC patients, anti-PD-L1, but not anti-PD-1, treatment increased sPD-L1, which was associated with irAE development, but not with clinical outcomes. In mice, trafficking of anti-PD-L1 Abs to lysosomes in F4/80+ macrophages resulted in sPD-L1 production, which was suppressed by treatment with lysosomal degradation inhibitor chloroquine and macrophage depletion. Conclusion: Anti-PD-L1-mediated lysosomal degradation induces sPD-L1 production, which can serve as an indicator to predict irAE development during anti-PD-L1 treatment.

Keywords: immune-related adverse event; inflammation; lysosomal degradation; macrophage; soluble PD-L1.

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Conflict of interest statement

YI has patent applications for immunopotentiating compositions (WO/2009/0297518, 2011/0081341, 2014/0314714, 2015/0093380, 2015/0197572, 2016/0158356, 2016/0158355, 2017/0051060, and 2020/0062846) and an immune function evaluation method (WO/2019/049974). YI reports research grants from the Japan Society for the Promotion of Science (JP19K07783 and JP22K07262 to YI) and Sysmex Corporation. AG reports consulting fees from MSD, Nippon Kayaku, and Daiichi-Sankyo Company outside the submitted work. MS reports receiving research grants from Taiho Pharmaceutical, Chugai Pharmaceutical, Eli Lilly, Nippon Kayaku, and Kyowa Hakko Kirin and honoraria from AstraZeneca, MSD, Chugai Pharmaceutical, Taiho Pharmaceutical, Eli Lilly, Ono Pharmaceutical, Bristol-Myers Squibb, Nippon Boehringer Ingelheim, Pfizer, Novartis, Takeda Pharmaceutical, Kyowa Hakko Kirin, Nippon Kayaku, Daiichi-Sankyo Company, Merck Biopharma, and Amgen outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

FIGURE 1
FIGURE 1
Detection of sPD-L1 and bsPD-L1 in the plasma of GC and NSCLC patients. (A) Baseline sPD-L1 and bsPD-L1 levels in plasma samples from GC patients (n = 117) and NSCLC patients (n = 72). (B) Correlation between baseline sPD-L1 and bsPD-L1 levels in GC and NSCLC patients. r indicates the correlation coefficient. (C) sPD-L1 levels and (D) bsPD-L1 levels in plasma samples from NSCLC patients prior to and at 2 months after ICI treatment. (E) Kinetic change of sPD-L1 and (F) bsPD-L1 levels in NSCLC patients prior to and at 2 months after anti-PD-1 (n = 48) or anti-PD-L1 (n = 24) treatment. r indicates the correlation coefficient. Horizontal lines indicate the mean. Statistical significance was calculated using the Mann–Whitney U test (A, E, and F). *p < 0.05; ****p < 0.0001; ns, not significant.
FIGURE 2
FIGURE 2
Correlation between baseline sPD-L1 and proinflammatory cytokine levels in NSCLC patients. (A) Correlation between baseline MMPs (MMP3, MMP9, and MMP13) and sPD-L1 or bsPD-L1 levels in NSCLC patients (n = 72). (B) Correlation between baseline proinflammatory cytokines (IL-1α, IL-1β, TNF-α, and IL-6) and sPD-L1 or bsPD-L1 levels in NSCLC patients (n = 72). (C) Correlation between baseline IL-10 and sPD-L1 or bsPD-L1 levels in NSCLC patients (n = 72). (D) Correlation between baseline IFN-γ and sPD-L1 or bsPD-L1 levels in NSCLC patients (n = 72). r indicates the correlation coefficient.
FIGURE 3
FIGURE 3
Intracellular trafficking of anti-PD-L1 mAb in mouse F4/80+ macrophages. (A) Plasma sPD-L1 and proinflammatory cytokine levels in mice after 2 h of treatment with Lipopolysaccharide (LPS). (B) Plasma sPD-L1 levels in mice at the indicated time points after administration of PBS, anti-PD-1, or anti-PD-L1 mAb. (C) Mice were injected with PBS, Alexa 488-labeled anti-PD-1, or anti-PD-L1 mAb (green), and then organs were harvested 24 h later. Spleen sections were stained with anti-F4/80 (red) and anti-B220 (blue) mAbs. Liver sections were stained with anti-F4/80 mAb (red) and DAPI (blue). Scale bars indicate 20 µm. (D) Mice were treated with clodronate-containing or control liposomes and then injected with anti-PD-L1 mAb. Blood samples were collected 24 h later, and sPD-L1 levels were analyzed by ELISA. (E) Mice were injected with Alexa 488-labeled anti-PD-L1 mAb (green), and then organs were harvested 24 h later. Liver sections were stained with anti-Rab7 or anti-LAMP1 Ab (red) in combination with anti-F4/80 mAb (blue). Scale bars indicate 20 µm. (F) Mice were treated with chloroquine (CQ) for 3 days and then injected with anti-PD-L1 mAb. Blood samples were collected 24 h later and sPD-L1 levels were analyzed by ELISA. (G) Mice were treated with CQ for 3 days and then injected with LPS. Blood samples were collected 2 h later and sPD-L1 levels were analyzed by ELISA. Horizontal lines indicate the mean. Statistical significance was calculated using the Student’s t-test (A, D, F, and G). *p < 0.05; **p < 0.01; ns, not significant.
FIGURE 4
FIGURE 4
Increased sPD-L1 levels in NSCLC patients with irAEs during anti-PD-L1 treatment. (A) Comparison of the sPD-L1 change between patients with disease control (DC, n = 33) and progressive disease (PD, n = 15) at 2 months of anti-PD-1 treatment (left), or between patients with DC (n = 17) and PD (n = 7) at 2 months of anti-PD-L1 treatment (right). (B) Comparison of the sPD-L1 change between patients with irAEs (n = 20) and without irAEs (n = 28) during anti-PD-1 treatment (left), or between patients with irAEs (n = 11) and without irAEs (n = 13) during anti-PD-L1 treatment (right). (C) ROC curve analysis of the sPD-L1 change to predict DC in patients with anti-PD-1 or anti-PD-L1 treatment. (D) ROC curve analysis of the sPD-L1 change to predict irAEs in patients with anti-PD-1 or anti-PD-L1 treatment. (E) Comparison of the sPD-L1 change between patients with acute irAEs (n = 10) and without acute irAEs (n = 14) during anti-PD-L1 treatment (left), or between patients with chronic irAEs (n = 4) and without chronic irAEs (n = 20) during anti-PD-L1 treatment (right). (F) ROC curve analysis of the sPD-L1 change to predict acute or chronic irAEs in patients with anti-PD-L1 treatment. (G) A schematic diagram of anti-PD-L1 treatment. (H) sPD-L1 levels prior to (left) and 2 months after (middle) treatment with atezolizumab (n = 16) or durvalumab (n = 8). sPD-L1 change (right) in patients during treatment with atezolizumab (n = 16) or durvalumab (n = 8). (I) Comparison of the sPD-L1 change between patients with acute irAEs (n = 4) and without acute irAEs (n = 12) during atezolizumab treatment (left), or between patients with acute irAEs (n = 6) and without acute irAEs (n = 2) during durvalumab treatment (right). (J) ROC curve analysis of the sPD-L1 change to predict acute irAEs in patients treated with atezolizumab or durvalumab. The Horizontal lines indicate the mean. Statistical significance was calculated using the Student’s t-test (A, B, E, H, and I). *p < 0.05; ns, not significant.
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