OBJECTIVE: Targeted radionuclide therapy (TRT) is a cancer treatment with relative therapeutic efficacy across various cancer types. We studied the therapeutic potential of TRT using fibroblast activation protein-α (FAP) targeting sdAbs (4AH29) labelled with 225Ac or 131I in immunocompetent mice in a human FAP (hFAP) expressing lung cancer mouse model. We further explored the combination of TRT with programmed cell death ligand 1 (PD-L1) immune checkpoint blockade (ICB).
METHODS: We studied the biodistribution and tumour uptake of [131I]I-GMIB-4AH29 and [225Ac]Ac-DOTA-4AH29 by ex vivo γ-counting. Therapeutic efficacy of [131I]I-GMIB-4AH29 and [225Ac]Ac-DOTA-4AH29 was evaluated in an immunocompetent mouse model. Flow cytometry analysis of tumours from [225Ac]Ac-DOTA-4AH29 treated mice was performed. Treatment with [225Ac]Ac-DOTA-4AH29 was repeated in combination with PD-L1 ICB.
RESULTS: The biodistribution showed high tumour uptake of [131I]I-GMIB-4AH29 with 3.5 ± 0.5% IA/g 1 h post-injection (p.i.) decreasing to 0.9 ± 0.1% IA/g after 24 h. Tumour uptake of [225Ac]Ac-DOTA-4AH29 was also relevant with 2.1 ± 0.5% IA/g 1 h p.i. with a less steep decrease to 1.7 ± 0.2% IA/g after 24 h. Survival was significantly improved after treatment with low and high doses [131I]I-GMIB-4AH29 or [225Ac]Ac-DOTA-4AH29 compared to vehicle solution. Moreover, we observed significantly higher PD-L1 expression in tumours of mice treated with [225Ac]Ac-DOTA-4AH29 compared to vehicle solution. Therefore, we combined high dose [225Ac]Ac-DOTA-4AH29 with PD-L1 ICB showing therapeutic synergy.
CONCLUSIONS: [225Ac]Ac-DOTA-4AH29 and [131I]I-GMIB-4AH29 exhibit high and persistent tumour targeting, translating into prolonged survival in mice bearing aggressive tumours. Moreover, we demonstrate that the combination of PD-L1 ICB with [225Ac]Ac-DOTA-4AH29 TRT enhances its therapeutic efficacy.

Although immune checkpoint inhibitors (ICIs) are effective in some patients with salivary gland carcinoma (SGC), biomarkers which predict the efficacy and prognosis of SGC patients treated with pembrolizumab have not been identified. We conducted a multi-institutional retrospective cohort study to evaluate the efficacy and safety of pembrolizumab monotherapy in patients with recurrent and/or metastatic SGC and to determine optimal cut-off values of the combined positive score (CPS) and tumor proportion score (TPS) as numerical expression levels of programmed death-ligand 1 (PD-L1), which predict the efficacy of pembrolizumab. Furthermore, we investigated the association of patient characteristics and hematological markers with clinical outcomes, including overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). From 2016 to 2021, 27 patients were included in the analysis. ORR of SGC was 25.9%. Optimal cut-off values of CPS and TPS were 15 and 25%, respectively. ORRs of CPS-high and TPS-high were 55.6 and 75.0%, respectively, and significantly higher than those of CPS-low and TPS-low. Furthermore, patients with a low platelet-lymphocyte ratio (PLR) had a significantly longer PFS. No grade 4 or greater adverse events were observed. This study demonstrated the efficacy and safety of pembrolizumab monotherapy and identified optimal cut-off values of CPS and TPS.

UNASSIGNED: Programmed Death-Ligand 1 is a well-known immune checkpoint molecule. Recent studies evaluated its expression in different canine cancer types through different laboratory techniques. The present study aims to evaluate the surface membrane protein expression (mPD-L1) by means of flow cytometry (FC) in different canine lymphoma immunophenotypes. Furthermore, in a subset of cases, mRNA and plasmatic soluble protein (sPD-L1) have been assessed in the same patient, and correlations among results from the three analyses investigated.
UNASSIGNED: Samples obtained for diagnostic purpose from untreated dogs with a confirmed lymphoma immunophenotype were included: surface protein was assessed via FC and quantified with median fluorescence index ratio (MFI ratio), gene expression was evaluated by real time quantitative polymerase chain reaction (RT-qPCR) and plasmatic concentration of soluble protein (sPD-L1) measured with ELISA. Statistical analyses were performed to investigate any difference among FC immunophenotypes, updated Kiel cytological classes, and in the presence of blood infiltration.
UNASSIGNED: Considering FC, most B-cell lymphomas (BCL) were positive, with higher MFI ratios than other subtypes (81%, median MFI ratio among positive samples = 1.50, IQR 1.21-2.03, range 1.01-3.47). Aggressive T-cell lymphomas had a lower percentage of positive samples (56%) and showed low expression (median MFI ratio in positive samples = 1.14, IQR 1.07-1.32, range 1.02-2.19), while T-zone lymphomas (TZL) were frequently positive (80%) but with low expression (median MFI ratio in positive samples = 1.19, IQR 1.03-1.46, range 1.02-6.03). Cellular transcript and sPD-L1 were detected in all samples, without differences among immunophenotypes. No correlation between results from different techniques was detected, but sPD-L1 resulted significantly increased in FC-negative lymphomas (p = 0.023).
UNASSIGNED: PD-L1 molecule is involved in canine lymphoma pathogenesis, with differences among immunophenotypes detected by FC. Specifically, BCL have the highest expression and aggressive T-cell lymphomas the lowest, whereas TZL need further investigations.

Anti-cluster of differentiation (CD) 3 × α programmed death-ligand 1 (PD-L1) bispecific T-cell engager (BsTE)-bound T-cells (BsTE:T) are a promising new cancer treatment agent. However, the mechanisms of action of bispecific antibody-armed activated T-cells are poorly understood. Therefore, this study aimed to investigate the anti-tumor mechanism and efficacy of BsTE:T. The BsTE:T migration was assessed in vivo and in vitro using syngeneic and xenogeneic tumor models, flow cytometry, immunofluorescence staining, transwell migration assays, microfluidic chips, Exo View R100, western blotting, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. In murine B16 melanoma, MC38 colon cancer, and human multiple myeloma cells, BsTE:T exhibited superior tumor elimination relative to that of T-cells or BsTE alone. Moreover, BsTE:T migration into tumors was significantly enhanced owing to the presence of PD-L1 in tumor cells and secretion of PD-L1-containing exosomes. Furthermore, increased infiltration of CD44highCD62Llow effector memory CD8+ T-cells into tumors was closely associated with the anti-tumor effect of BsTE:T. Therefore, BsTE:T is an innovative potential anti-tumor therapy, and exosomal PD-L1 plays a crucial role both in vitro and in vivo in the anti-tumor activity of BsTE:T.

OBJECTIVE: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of non-small cell lung cancer (NSCLC) and programmed cell death-ligand 1 (PD-L1) is a companion biomarker. This study aims to use baseline arterial-phase enhanced CT (APECT) to construct efficient radiomic models for predicting PD-L1 expression and immunotherapy prognosis in NSCLC.
METHODS: We extracted radiomics features from the baseline APECT images of 204 patients enrolled in a published multicenter clinical trial that commenced on August 23, 2018, and concluded on November 15, 2019 (ClinicalTrials.gov: NCT03607539). Of these patients, 146 patients from selected centers were assigned to the training cohort. The least absolute shrinkage and selection operator (LASSO) method was used to reduce dimensionality of radiomics features and calculate tumor scores. Models were created using naive bayes, decision trees, XGBoost, and random forest algorithms according to tumor scores. These models were then validated in an independent validation cohort comprising 58 patients from the remaining centers.
RESULTS: The random forest algorithm outperformed the other methods. In the three-classification scenario, the random forest model achieving the area under the curve (AUC) values of 0.98 and 0.94 in the training and validation cohorts, respectively. In the two-classification scenario, the random forest model achieved AUCs of 0.99 (95%CI: 0.97-1.0, P < 0.0001) and 0.93 (95%CI: 0.83-0.98, P < 0.0001) in the training and validation cohorts, respectively. Furthermore, patients classified as PD-L1 high-expression by this model can predict treatment response (AUC=0.859, 95%CI: 0.7-0.96, P < 0.001) and improved survival (HR=0.2, 95%CI: 0.08-0.53, P = 0.001) only in validation sintilimab arm.
CONCLUSIONS: Radiomics models based on APECT represent a potential non-invasive approach to robustly predict PD-L1 expression and ICI treatment outcomes in patients with NSCLC, which could significantly improve precision cancer immunotherapy.

BACKGROUND: M2 macrophages are known to play a significant role in the progression of triple-negative breast cancer (TNBC) by creating an immunosuppressive microenvironment. The aim of this study is to investigate the impact of M2 macrophages on TNBC and their correlation with programmed death-ligand 1 (PD-L1) expression.
METHODS: We employed a co-culture system to analyze the role of the mutual regulation of M2 macrophages and TNBC cells. Employing a multifaceted approach, including bioinformatics analysis, Western blotting, flow cytometry analysis, ELISA, qRT-PCR, lentivirus infection, mouse models, and IHC, we aimed to elucidate the influence and mechanism of M2 macrophages on PD-L1 expression.
RESULTS: The results showed a substantial infiltration of M2 macrophages in TNBC tissue, which demonstrated a positive correlation with PD-L1 expression. CXCL1 exhibited abnormally high expression in M2 macrophages and enhanced the expression of PD-L1 in TNBC cells. Notably, silencing CXCL1 or its receptor CXCR2 inhibited M2 macrophages-induced expression of PD-L1. Mechanistically, CXCL1 derived from M2 macrophages binding to CXCR2 activated the PI3K/AKT/NF-κB signaling pathway, resulting in increased PD-L1 expression in TNBC.
CONCLUSIONS: Broadly speaking, these results provide evidence for the immunosuppressive role of M2 macrophages and CXCL1 in TNBC cells, indicating their potential as therapeutic biomarkers.

(1) Background: Exosomal PD-L1 has garnered attention owing to its role in instigating systemic immune suppression. The objective of this study is to elucidate whether bone and soft tissue sarcoma cells possess the capacity to secrete functionally active exosomal PD-L1 and whether radiotherapy (RT) induces the exosomal PD-L1 release. (2) Methods: Human osteosarcoma cell line 143B and human fibrosarcoma cell line HT1080 were utilized. Exosomes were isolated from the culture medium and blood via ultracentrifugation. The expression of PD-L1 on both tumor cells and exosomes was evaluated. The inhibitory effect on PBMC was employed to assess the activity of exosomal PD-L1. Post radiotherapy, changes in PD-L1 expression were compared. (3) Results: Exosomal PD-L1 was detected in the culture medium of tumor cells but was absent in the culture medium of PD-L1 knockout cells. Exosomal PD-L1 exhibited an inhibitory effect on PBMC activation. In tumor-bearing mice, human-derived exosomal PD-L1 was detected in the bloodstream. Following radiotherapy, tumor cells upregulated PD-L1, and human-derived exosomal PD-L1 were detected in the bloodstream. (4) Conclusions: Exosomal PD-L1 emanates from bone and soft tissue sarcoma cells and is disseminated into the circulatory system. The levels of PD-L1 in tumor cells and the release of exosomal PD-L1 were augmented after irradiation with RT.

Programmed death-ligand 1 (PD-L1) has become a crucial focus in cancer immunotherapy considering it is found in many different cells. Cancer cells enhance the suppressive impact of programmed death receptor 1 (PD-1) through elevating PD-L1 expression, which allows them to escape immune detection. Although there have been significant improvements, the effectiveness of anti-PD-1/PD-L1 treatment is still limited to a specific group of patients. An important advancement in cancer immunotherapy involves improving the PD-L1 protein degradation. This review thoroughly examined the processes by which PD-L1 breaks down, including the intracellular pathways of ubiquitination-proteasome and autophagy-lysosome. In addition, the analysis revealed changes that affect PD-L1 stability, such as phosphorylation and glycosylation. The significant consequences of these procedures on cancer immunotherapy and their potential role in innovative therapeutic approaches are emphasised. Our future efforts will focus on understanding new ways in which PD-L1 degradation is controlled and developing innovative treatments, such as proteolysis-targeting chimeras designed specifically to degrade PD-L1. It is crucial to have a thorough comprehension of these pathways in order to improve cancer immunotherapy strategies and hopefully improve therapeutic effectiveness.

OBJECTIVE: Immunohistochemical staining of programmed death-ligand 1 (PD-L1) in tumor biopsies acquired through invasive procedures is routinely employed in clinical practice to identify patients who are most likely to benefit from anti-programmed cell death protein 1 (PD-1) therapy. Nevertheless, PD-L1 expression is observed in various cellular subsets within tumors and their microenvironments, including tumor cells, dendritic cells, and macrophages. The impact of PD-L1 expression across these different cell types on the responsiveness to anti-PD-1 treatment is yet to be fully understood.
METHODS: We synthesized polymer-based lysosome-targeting chimeras (LYTACs) that incorporate both PD-L1-targeting motifs and liver cell-specific asialoglycoprotein receptor (ASGPR) recognition elements. Small-animal positron emission tomography (PET) imaging of PD-L1 expression was also conducted using a PD-L1-specific radiotracer 89Zr-αPD-L1/Fab.
RESULTS: The PD-L1 LYTAC platform was capable of specifically degrading PD-L1 expressed on liver cancer cells through the lysosomal degradation pathway via ASGPR without impacting the PD-L1 expression on host cells. When coupled with whole-body PD-L1 PET imaging, our studies revealed that host cell PD-L1, rather than tumor cell PD-L1, is pivotal in the antitumor response to anti-PD-1 therapy in a mouse model of liver cancer.
CONCLUSIONS: The LYTAC strategy, enhanced by PET imaging, has the potential to surmount the limitations of knockout mouse models and to provide a versatile approach for the selective degradation of target proteins in vivo. This could significantly aid in the investigation of the roles and mechanisms of protein functions associated with specific cell subsets in living subjects.

UNASSIGNED: Chronic active Epstein-Barr virus disease (CAEBV) is a proliferative disease of EBV+ T or natural killer (NK) cells with an unclear pathogenesis. This study aimed to examine the frequency and exhaustion levels of lymphocyte subsets in patients with CAEBV to further investigate the pathogenesis.
UNASSIGNED: Using flow cytometry, we detected the frequency, expression levels of programmed cell death 1 (PD-1) and programmed death ligand 1 (PD-L1), and EBV infection status of peripheral T subsets and NK cells in patients with CAEBV and healthy individuals.
UNASSIGNED: 24 patients and 15 healthy individuals were enrolled in this study. Patients showed notably higher expression levels of PD-1 and PD-L1 in peripheral T subsets and NK cells compared to healthy individuals (P < 0.05). EBV+ lymphocytes exhibited significantly higher PD-L1 expression levels than EBV- lymphocytes. Additionally, the frequency of effector memory T (Tem) cells was significantly increased in patients, and the PD-L1 expression level was positively correlated with the EBV load. Besides, helper T cell 2 (Th2) immune bias, also favoring EBV amplification, was found in patients, including increased Th2 cell frequency, enhanced response capacity, and elevated serum levels of associated cytokines. The distribution and PD-1 expression levels of peripheral T subsets returned to normal in patients who responded to PD-1 blockade therapy.
UNASSIGNED: The up-regulation of the PD-1/PD-L1 pathway of peripheral T and NK cells and Th2 immune predominance jointly promoted EBV replication and the development of CAEBV. PD-1 blockade therapy reduced the PD-1 expression level of lymphocytes and helped normalize the distribution of the T subsets.