WGP β-glucan was purified from the cell walls of S cerevisiae. Through a series of alkaline and acid extractions, cytoplasm and other cell wall polysaccharides such as mannose were removed. An intact hollow yeast cell wall remained, composed primarily of long β-1,3 glucose polymers, with 3% to 6% of the backbone glucose units possessing a b (1,6) branch (b-(1,3/1,6)-D-glucan).

Female C57BL/6 mice aged 6–8 weeks were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All mice were maintained under specific pathogen-free conditions in individually ventilated cages. All mouse experiments were performed in compliance with all relevant laws and institutional guidelines and were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.

A Lewis lung carcinoma (LLC) cell line derived from C57BL/6 mice was provided by the American Type Culture Collection. The LLC cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) in a humidified atmosphere and at 5% CO₂ in air. Culture flasks were used to reach the appropriate tumor cell concentrations for injection.

LLC cells (3 × 10⁵) suspended in 100 μL phosphate buffered saline (PBS; Thermo Fisher Scientific) were subcutaneously injected into the flanks of C57BL/6 mice. When the tumor masses were palpable (at approximately 6–7 days after injection), treatment was initiated. Mice were randomly assigned to 1 of 4 experimental groups: control (PBS), WGP, anti-PD1, or WGP + anti-PD1. The tumor masses were measured using a caliper every other day, and tumor volumes were calculated as follows: V = 1 2 ×the vertical length×the horizontal length 2

Mice with established subcutaneous LLC tumors were euthanized when the tumor length reached 15 mm (at approximately 20 days after injection).

Mice in the WGP and WGP + anti-PD1 groups received 1 mg WGP in 100 μL PBS daily via oral administration. Mice in the anti-PD1 and WGP + anti-PD1 groups received 20 μg anti-PD-1 antibody (clone 29F.1A12, Biolegend) in 100 μL PBS once every 3 days via intravenous injection into the tail. Mice in the control group received 100 μL PBS.

Surface staining and intracellular staining were performed as described previously. Briefly, tumor tissues were weighed and minced into small pieces. Then digested for 30 min at 37°C with rotation with a triple enzyme mixture containing collagenase type IV, hyaluronidase, and deoxyribonuclease (Sigma-Aldrich). Filtering was performed to remove insoluble fiber from the cells, and red blood cells were lysed with red blood cell lysis buffer (Beyotime Biotechnology). The cells were then washed twice with ice cold PBS.

For surface marker staining (Ning et al., 2016), single cell suspensions from mice tumors were stained with fluorescein-conjugated mouse-specific antibodies against CD11c, CD11b, F4/80, Gr-1, PD-1, PD-L1, CD45, CD3, CD4 or CD8 (Biolegend) at 4°C for 30 min in the dark. After the cells were washed twice with PBS, they were either directly measured or, for intracellular staining, fixated and permeabilized with Foxp3 (Biolegend) to stain nuclear factors. Intranuclear antibodies were then incubated at room temperature for 30 min in the dark. This was followed by washing and flow cytometry, which was performed on a FACS Canton II (BD Biosciences). Results were analyzed using FlowJo version 10 (Tree Star).

Immune cell subsets were characterized using the following sets of markers: DCs (CD11c⁺/CD8⁺), macrophages (CD11b⁺/F4-80⁺), myeloid-derived suppressor cells (MDSCs: CD11b⁺/Gr-1⁺), CD8⁺ T cells (CD45⁺/CD3⁺/CD4^-/CD8⁺), CD4⁺ T cells (CD45⁺/CD3⁺/CD8^-/CD4⁺), and Tregs (CD45⁺/CD3⁺/CD4⁺/Foxp3⁺).

Tumor samples were treated with TRIzol reagent (Invitrogen, Thermo Fisher Scientific), and total RNAs were isolated and reverse transcribed with TaqMan Reverse Transcription Reagents (Applied Biosystems). The indicated cytokine mRNA levels were quantified with quantitative RT-PCR amplification using the BioRad MyiQ single-color RT-PCR detection system. Briefly, complementary DNA was amplified in a 20-μL reaction mixture containing 10 μL of SYBR Green PCR supermix (Invitrogen, Thermo Fisher Scientific), 100 ng of complementary DNA template, and selected primers (200 nM) using the recommended cycling conditions. Data were acquired on an ABI ViiA 7 real-time PCR system. The primer sequences, designed with Primer Express Software Version 2.0 (Applied Biosystems), are summarized in the Supplementary Table S1.

This open label, one-arm pilot clinical study was registered with the Chinese Clinical Trial Registry (ChiCTR2100043913). Patients treated at Changzhou No. 2 People's Hospital affiliated to Nanjing Medical University were eligible for this study if they were aged 18 to 75 years; had a histopathological and/or cytological diagnosis of a distant metastases and advanced tumor; failed at least one-line regimens containing PD-1/PD-L1 inhibitor had ≥1 measurable lesion based on Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria; the expected survival ≥3 months; had no significant liver or renal impairment or severe cardiorespiratory disease; and had Eastern Cooperative Oncology Group (ECOG) performance status of 1 or 2. Patients were excluded from the study if they were enrolled in any clinical trials assessing other drugs within 4 weeks and if they had any factors affecting their ability to take oral medication (e.g., inability to swallow, chronic diarrhea, intestinal obstruction). Patients could leave the study at the midpoint of the trial period if they withdrew their informed consent. The investigator could exclude the cases that medication violate the treatment protocol and affected the judgment of drug efficacy, and the cases that incomplete data affected the judgment of efficacy and safety.

β-glucan 500 mg was administered orally twice daily combined with the previous protocol (containing PD-1/PD-L1 inhibitor); 1 course was administered every 3 weeks. Treatment was continued until the onset of unacceptable toxicity, disease progression, or death.

Efficacy of the combination therapy was evaluated every 3 cycles according to RECIST 1.1. Complete response (CR) was defined as complete disappearance of the tumor with no new lesions. Partial response (PR) was defined as ≥30% decrease in the longest diameter of the target lesions and no progression in new lesions. Stable disease (SD) was defined as <30% decrease or <20% increase in the target lesions and no progression in new lesions. Progressive disease (PD) was defined as ≥20% increase in the longest diameter of the target lesions or the appearance of new lesions.

The primary study endpoint was the effect of PD-1/PD-L1 inhibitor plus β-glucan on median progression-free survival (mPFS). The secondary endpoint was the effect of this treatment on median overall survival (mOS) and disease control rate.

The cytokines in patients’ blood serum samples were examined with the LEGENDplex Human Essential Immune Response Panel (13-plex) (BD Biosciences), which is a bead-based multiplex assay panel that uses fluorescence-encoded beads suitable for use on various flow cytometers. For this analysis, we collected blood from patients, allowed it to clot for ≥30 min, and centrifuged the blood for 20 min at 1000 × g to obtain serum samples. The samples were stored at –20°C. We then added 25 μL of mixed beads, detection antibodies, and SA-PE to each serum sample and standard samples. The samples were shaken at approximately 500 rpm for hours at room temperature. The samples were washed, and 13 key targets were quantified with flow cytometry: IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17a, TNF-α, TGF-β1, IFN-γ, CXCL10 (IP-10), CCL2 (MCP-1), and CXCL8 (IL-8). Data analysis was then performed using LEGENDplex Data Analysis Software.

Data are expressed as means and standard errors of the mean (SEM). Comparisons between multiple groups with independent samples were performed using ANOVA, whereas paired t-tests were used to compare patients’ samples before WGP treatment and after WGP treatment. Survival data were plotted as PFS or OS curves. mOS and mPFS values were examined and a pooled effect size and 95% CI were calculated. The thresholds for statistical significance were defined as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Data were analyzed using IBM SPSS statistics (SPSS, 22.0) and GraphPad Prism software (Graphpad, V8.0).

To investigate the in vivo antitumor effects of WGP combined with anti-PD-1 antibody in C57BL/6 mice bearing murine lung LLC, we separated the mice into 4 treatment groups. The WGP and WGP + anti-PD1 groups received 1 mg WGP in 100 μL PBS daily via oral administration. The mice in the anti-PD1 and WGP + anti-PD1 groups received 20 μg anti-PD-1 antibody in 100 μL PBS once every 3 days via intravenous injection into the tail. The mice in the control group received 100 μL PBS (Figure 1A). We found that tumor-bearing mice treated with combination therapy had a significantly decreased tumor burden when compared with mice treated with single therapy or PBS only. Single therapy also suppressed tumor growth significantly compared with PBS only, but there was no significant difference between WGP only and anti-PD-1 antibody only (Figures 1B,C).

We then examined the tumor-infiltrating cells in the TME and found that with both combination therapy and WGP single therapy, the infiltration of DCs (CD11c⁺/CD8⁺) and macrophages (CD11b⁺/F4-80⁺) was significantly increased, whereas the frequency of MDSCs (CD11b⁺/Gr-1⁺) was significantly decreased (Figure 2A). In addition, combination therapy significantly decreased the proliferation of CD4⁺T cells and Tregs in the tumor (Figure 2A).

To further demonstrate the effects of checkpoint blockade anti-PD-1 antibody therapy in vivo, we examined the expression of PD-L1 or PD-1 levels. WGP was found to decrease PD-L1 expression in macrophages but increase PD-L1 expression in DCs. The PD-L1 levels in MDSCs and the PD-1 expression in CD4⁺ and CD8⁺T cells were not significantly altered with single or combination therapy (Figure 2B and Supplementary Figure S1). As shown in Figure 3, the mRNA levels of Foxp3 were significantly decreased in TME after single or combination therapy, with Tregs decreasing in TME (Figure 2A). However, the mRNA levels of IL-6, IL-1β, TNFα, and TGFβ were not significantly changed with or without treatment. These data indicate that WGP treatment expands the influence of anti-PD-1 antibody and is associated with regulating the tumor-infiltrating cells, modulating the suppressive TME, and decreasing Tregs differentiation, thus leading to a delay in tumor progression.

The study consisted of 13 patients (9 men, 4 women; mean age, 60 years; range, 47–79 years), all of whom were treated with PD-1/PD-L1 inhibitor plus β-glucan (Supplementary Table S2). Baseline patient characteristics are shown in Table 1.

Patients treated with combination therapy demonstrated a disease control rate of 69.2%. SD was seen in 9 of the 13 patients (69.2%), and PD was seen in the remaining 4 patients (30.8%). there was no change in the longest diameter of target lesions in 5 of the patients (38.5%), and the tumor size decreased in 2 patients (15.4%) after combination therapy (Figure 4). Cytokine analysis demonstrated that the concentrations of IL-1β, IL-6, and IL-8 were increased in serum samples after combination treatment (Figure 5 and Supplementary Figure S2). The mPFS for the 13 patients was 3.67 months, and the mOS was 8 months (Figure 6). As of 15 December 2021, 4 of the patients were still alive and undergoing continued follow-up.

Immune-related adverse events associated with both the first and rechallenged ICB are shown in Supplementary Table S3. No severe immune-related adverse events occurred in study patients.