The potential of bioengineered PD-L1 siRNA entities for NSCLC immunotherapy is demonstrated and their immunotherapeutic efficacy against non-small cell lung cancer (NSCLC) in vitro is defined.
Abstract
Background/Objectives: Recent advances in immunotherapy have revolutionized cancer treatment, as exemplified by multiple monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1). Nevertheless, immunotherapeutic antibodies exhibit certain limitations, which drives the development of alternative approaches, such as small interfering RNA (siRNA)-based therapeutics. The aim of this study was to design and produce new biological PD-L1 siRNA (BioRNA/PD-L1-siRNA) molecules and further define their immunotherapeutic efficacy against non-small cell lung cancer (NSCLC) in vitro. Methods: A novel RNA molecular bioengineering platform was employed to produce new BioRNA/PD-L1-siRNA agents. The functions of BioRNA/PD-L1-siRNAs were determined by quantitative PCR, Western blot, immunofluorescence confocal imaging, flow cytometry, and PD-1/PD-L1 blockade assays in human NSCLC cells, alone and co-cultured with human peripheral blood mononuclear cells (PBMCs). Results: After heterologous overexpression and purification of five BioRNA molecules, one siRNA named BioRNA/PD-L1-siRNA-1 was identified as the most effective to selectively suppress human PD-L1 mRNA and protein levels in H460 and H1975 cells. Disruption of PD-1/PD-L1 interactions by BioRNA/PD-L1-siRNA-1 was further demonstrated via a PD-1/PD-L1 blockade bioassay. In addition, the immunomodulatory effectiveness of BioRNA/PD-L1-siRNA-1 was established in co-culture models, as indicated by the induction of T-cell and natural killer cell populations and an increase in specific cytokines and cytotoxic granules, and subsequent enhancement of apoptosis and greater inhibition of NSCLC cell viability. Conclusions: Overall, these findings demonstrate the potential of bioengineered PD-L1 siRNA entities for NSCLC immunotherapy.
CP-1 exhibits balanced, dual-nanomolar inhibitory activity against PD-L1 and CD73 and displays potent immunomodulatory effects at the cellular level and serves as a promising lead candidate for developing novel bifunctional agents to advance tumor immunotherapy.
Jing-Jing Du, Sen Wu, Shiyun Cheng et al.· Frontiers in Immunology· 0 citations
Immune checkpoint inhibition has transformed the treatment of advanced non-small cell lung cancer (NSCLC), but primary and acquired resistance remain common. Vascular endothelial growth factor (VEGF) contributes to both tumor angiogenesis and immune suppression by promoting abnormal vasculature, limiting lymphocyte trafficking, and fostering an immunosuppressive tumor microenvironment. These complementary mechanisms provide a rationale for simultaneously targeting the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) and VEGF pathways. Bispecific antibodies integrating checkpoint blockade and VEGF neutralization within a single molecule have therefore emerged as a rapidly developing therapeutic strategy in NSCLC. Ivonescimab, a PD-1/VEGF bispecific antibody, currently has the most mature evidence, with randomized phase III trials demonstrating improvements in progression-free survival and, in selected settings, overall survival compared with established PD-1-based regimens. Other PD-1/VEGF agents, including PF-08634404, MK-2010, and JS207, have shown encouraging early activity but require randomized validation. Among PD-L1/VEGF bispecific antibodies, pumitamig, HB0025, and IMM2510 have demonstrated promising antitumor activity, although evidence is derived predominantly from early-phase or single-arm studies. Multiple phase III trials are evaluating these agents in first-line, post-immunotherapy, epidermal growth factor receptor (EGFR)-mutated, and stage III consolidation settings. VEGF-associated toxicities, including hypertension, proteinuria, hemorrhage, and thromboembolism, remain clinically relevant. Moreover, differences in checkpoint target, molecular architecture, target affinity, fragment crystallizable (Fc) engineering, pharmacokinetics, and VEGF-binding strategy preclude the assumption of a uniform class effect. The fixed bispecific configuration also prevents independent dose adjustment of the checkpoint and antiangiogenic components, which may complicate toxicity management. Mature randomized survival data, longer-term safety assessment, and biomarker development are needed to define optimal patient selection and the clinical role of PD-(L)1/VEGF bispecific antibodies in NSCLC.
Aleena Kuriakose, Salem Gogah, E. Beleva· Journal of Clinical Question...· 0 citations
BACKGROUND
Recently, immune checkpoint inhibitors (ICIs), especially those that targets PD-1/PD-L1, have significantly altered the treatment approach for NSCLC. Nevertheless, many patients experience different levels of resistance after receiving treatment with ICIs, which restrict their broader use in clinical practice. Therefore, to enhance the overall efficacy of ICIs, there is an immediate necessity to further explain the processes of immune evasion in NSCLC, especially the modulatory mechanism of PD-L1.
METHODS
IHC was used to evaluate the protein expression level of PLIN3 and PD-L1 in NSCLC tissues. The impact of PLIN3 on PD-L1 was analyzed in NCI-H1975 and NCI-H157 cells using western blot, flow cytometry and quantitative PCR (qPCR). Immunofluorescence was performed to examine the effect of PLIN3 on the autophagy level. The levels of Granzyme B (GZMB) and interferon-gamma (IFN-γ) secreted by T cell were assessed. Bioinformatic analyses, including immune infiltration estimation and TIDE score, were performed using TCGA data.
RESULTS
In this research, we discovered that elevated PLIN3 level was linked to decreased infiltration of CD4+ and CD8+ T cell, a higher TIDE score, and poorer immunotherapy response. In NSCLC tissues, there was a positive correlation between PLIN3 and PD-L1. Besides, the level of PLIN3 protein is significantly reduced in patients who achieve pathological complete response. In addition, PLIN3 knockdown markedly reduced the level of PD-L1 protein. Mechanistically, PLIN3 knockdown activated autophagy, and promoted PD-L1 degradation via the autophagic-lysosomal pathway, which consequently shortened its protein half-life. Furthermore, PLIN3 knockdown enhanced T-cell-mediated tumor killing and resulted in an increased secretion of the effector molecules, including granzyme B and IFN-γ.
CONCLUSIONS
In summary, our study has shown that targeting PLIN3 can induce autophagy, which promotes the degradation of PD-L1, ultimately leading to enhanced activation of T cells. This research is the first to investigate the function of PLIN3 in the immune microenvironment, revealing its critical function in immune evasion and highlighting its promise as a treatment target. This offers an innovative approach to enhance the efficacy of immunotherapy in lung cancer.
Hanqiong Zhou, Songqing Fan, Ouyang Min et al.· International Immunopharmaco...· 0 citations
Background/aim
Targeted therapies with monoclonal antibodies provide cancer patients with better prognosis and disease-free survival. The blockade of immune checkpoints, including programmed cell death protein-1 (PD-1) and its ligand PD-L1, with monoclonal antibodies may boost immune responses against tumors and is regarded as an effective strategy in cancer immunotherapy. We describe the generation of anti-PD-L1 monoclonal antibodies with high affinity and specificity, and we assess their potential for therapeutic use in cancer.
Materials and methods
Hybridomas were selected for PD-L1 specificity and cross-reactivity with other immune checkpoint proteins and PD-L1 orthologs using indirect ELISA. Immunofluorescence and Western blotting assays were conducted for further characterization of the antibodies. The affinities of the antibodies for PD-L1 were determined using surface plasmon resonance. Receptor blocking activities were examined through competitive ELISA and cell-based luciferase reporter assays. Sequences of variable regions of the selected antibodies were determined by Sanger sequencing and subjected to BLAST analysis.
Results
A total of 25 PD-L1-specific monoclonal antibodies were generated. While most clones reacted with PD-L1 from cynomolgus monkeys, none of the antibodies displayed cross-reactivity with other checkpoint proteins. Immunofluorescence assays showed that the selected clones stained PD-L1-expressing cell membranes specifically, but not those of PD-L1-negative cells. Western blotting revealed that most of the clones recognized both glycosylated and nonglycosylated PD-L1, and a few reacted with the glycosylated form only. Only two clones with subnanomolar affinity for human PD-L1 were effective at blocking PD-1/PD-L1 and CD80/PD-L1 interactions. Sequence analysis of their variable regions revealed their unique specificity.
Conclusion
Of the 25 monoclonal antibodies produced in this study, only one was identified as a potential therapeutic drug candidate thanks to its high capacity for checkpoint blockade and affinity, as well as its unique sequence specificity. These properties are comparable to those of anti-PD-L1 antibodies currently used in clinical practice.
Nurşah Ersezen, Maide Şeker, Arzu Aysan et al.· Turkish journal of biology =...· 0 citations
Abstract Objectives Although anti-PD-1 monoclonal antibodies represent a cornerstone in modern non-small cell lung cancer (NSCLC) management, sustained clinical efficacy remains restricted to a limited subset of patients. T-cell exhaustion and an immunosuppressive tumor microenvironment are major limiting factors. Some traditional Chinese medicine modalities have been developed to modulate the tumor immune microenvironment. Specifically, ginsenoside Rg3 serves as a potent dual-action agent, actively modulating the intratumoral niche while concurrently exerting strong inhibitory effects against tumor expansion. This study was designed to investigate whether Rg3 could augment the antitumor immunity elicited by anti-PD-1 blockade within non-small cell lung malignancies, while simultaneously deciphering the underlying molecular cascades. Methods We employed an in vitro tumor cell and tumor-specific cytotoxic T cell co-culture system, flow cytometry, quantitative PCR, Western blotting, whole-transcriptome sequencing (RNA-seq), and a mouse tumor xenograft model with adoptive CTL transfer to study the effects of Rg3 and PD-1 mAb combination therapy on cytotoxic T lymphocytes (CTLs) and the underlying molecular mechanism. The functions of key molecular mediators were validated by gene knockdown and overexpression approaches. Results Rg3 enhanced the cytotoxic activity of lung adenocarcinoma cell-specific CTLs, reducing A549 cell viability by 45.9 % at 10 μmol/L compared with the untreated control (p<0.05). Combination treatment with Rg3 and anti-PD-1 mAb showed significantly greater cytotoxic activity than either monotherapy, with CTL-mediated killing rates reaching 71.9 % compared with 50.7 % for anti-PD-1 mAb alone and 48.2 % for Rg3 alone (all p<0.05). Transcriptomic analysis and experimental validation revealed that the combination treatment induced upregulation of TBC1D15 in CTLs, which promoted mitophagy, improved mitochondrial integrity, and was associated with enhanced cytolytic activity of CTLs. Conclusions Co-administration of Rg3 with anti-PD-1 potentiates antitumor immunity by modulating CTL mitochondrial homeostasis via TBC1D15-mediated mitophagy, thereby enhancing CTL cytotoxic function. These findings support further investigation of Rg3 as an adjuvant to PD-1 blockade.
Gastric cancer (GC) continues to be a significant global contributor to cancer-related mortality, primarily due to late-stage diagnoses, tumor heterogeneity, and the frequent emergence of therapeutic resistance. Despite advancements in surgical techniques, chemotherapy, targeted therapies, and immunotherapy, long-term survival rates remain unsatisfactory, underscoring the need for innovative, molecularly driven approaches. SiRNA has emerged as a promising gene-silencing tool capable of selectively downregulating oncogenic drivers and pathways associated with resistance via RNA interference. Preclinical studies using GC models demonstrate promising biological activity following siRNA-mediated suppression of various molecular targets - including CD44v6, Rac1, and ZNRD1-leading to reduced cell proliferation, inhibited migration and invasion, enhanced apoptosis, and increased chemosensitivity. However, the successful translation of siRNA-based strategies into clinical practice is fundamentally reliant on the development of efficient and safe delivery systems. Recent advancements in nanotechnology have enabled the development of multifunctional nanocarriers, including lipid-based nanoparticles, peptide-based systems, chitosanderived platforms, exosome-mimetic vesicles, layer-by-layer architectures, and stimuli-responsive theranostic nanoparticles. These engineered platforms are designed to enhance siRNA stability, improve tumor targeting, facilitate intracellular trafficking, and promote endosomal escape while minimizing off-target effects and immune activation. Preclinical findings suggest significant biological potential; however, additional research is required to address issues concerning biodistribution, safety, scalability, and regulatory standardization before clinical application. Overall, siRNA-based strategies may play a crucial role in the future development of precision-oriented therapeutic frameworks in GC research. Importantly, this review emphasizes the integration of molecular target selection with delivery system design in a GC-specific context. By systematically linking validated siRNA targets with corresponding nanocarrier strategies, this work provides a more application-oriented and translationally relevant perspective that is not typically addressed in conventional RNA interference or nanocarrier-focused reviews.
Gheysar Seifollahnezhad, B. Erdağ· Experimental oncology· 0 citations
