Molecular cloning, codon-optimized gene expression, and bioactivity assessment of two novel fungal immunomodulatory proteins from Ganoderma applanatum in Pichia.

Fungal immunomodulatory proteins (FIPs) have been identified from a series of fungi, especially in Ganoderma species. However, little is known about the FIPs from G. applanatum. In this study, two novel FIP genes, termed as FIP-gap1 and FIP-gap2, were cloned from G. applanatum, characterized and functionally expressed after codon optimization in Pichia pastoris GS115. Results showed that FIP-gap1 and FIP-gap2 comprised 342-bp encoding peptides of 113 amino acids, which shared a high homology with other Ganoderma FIPs. The yield of recombinant FIP-gap1 and FIP-gap2 increased significantly after codon optimization and reached 247.4 and 197.5 mg/L, respectively. Bioactivity assay in vitro revealed that both rFIP-gap1 and rFIP-gap2 could agglutinate mouse, sheep, and human red blood cells. Besides, rFIP-gap1 and rFIP-gap2 obviously stimulated the proliferation of mouse splenocytes and enhanced IL-2 and IFN-γ release. Cytotoxicity detection indicated that IC50 of rFIP-gap1 towards A549 and HeLa cancer cells were 29.89 and 8.34 µg/mL, respectively, whereas IC50 of rFIP-gap2 to the same cancer cells were 60.92 and 41.05 µg/mL, respectively. Taken together, novel FIP gaps were cloned and functionally expressed in P. pastoris, which can serve as feasible and stable resources of rFIP gaps for further studies and potential applications.

First-line immunotherapy efficacy in advanced squamous non-small cell lung cancer with PD-L1 expression ≥50%: a network meta-analysis of randomized controlled trials.

Objective: The optimal first-line immunotherapy regimen for patients with PD-L1 expression ≥50% in squamous non-small cell lung cancer (Sq-NSCLC) remains uncertain. This study utilized net-work meta-analysis (NMA) to indirectly compare the efficacy of various first-line immuno-therapy regimens in this patient subset. Methods: Systematic searches were conducted across PubMed, the Cochrane Library, Web of Science, and Embase databases for randomized controlled trials reporting overall survival (OS) and progression-free survival (PFS) outcomes. The search spanned from database inception to November 3, 2023. Bayesian network meta-analysis was employed for a comprehen-sive analysis. To ensure scientific rigor and transparency, this study is registered in the Interna-tional Prospective Register of Systematic Reviews (PROSPERO) under the registration number CRD42022349712. Results: The NMA encompassed 9 randomized controlled trials (RCTs), involving 2170 patients and investigating 9 distinct immunotherapy regimens. For OS, the combination of camrelizumab and chemotherapy demonstrated the highest probability (36.68%) of efficacy, fol-lowed by cemiplimab (33.86%) and atezolizumab plus chemotherapy (23.87%). Regarding PFS, the camrelizumab and chemotherapy combination had the highest probability (39.70%) of efficacy, followed by pembrolizumab (22.88%) and pembrolizumab plus chemotherapy (17.69%). Compared to chemotherapy, first-line treatment with immune checkpoint inhibitors (ICIs) in Sq-NSCLC pa-tients exhibited significant improvements in OS (HR 0.59, 95% CI 0.47-0.75) and PFS (HR 0.44, 95% CI 0.37-0.52). Conclusion: This study suggests that, for Sq-NSCLC patients with PD-L1 expression ≥50%, the first-line immunotherapy regimen of camrelizumab plus chemotherapy provides superior OS and PFS outcomes. Furthermore, ICIs demonstrate enhanced efficacy compared to chemotherapy in this patient population. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier: CRD 42022349712.