Establishment of an animal model of immune-related adverse events induced by immune checkpoint inhibitors.

OBJECTIVE: Immunotherapy, specifically immune checkpoint inhibitors (ICIs), has revolutionized cancer treatment. However, it can also cause immune-related adverse events (irAEs). This study aimed to develop a clinically practical animal model of irAEs using BALB/c mice. METHODS: Subcutaneous tumors of mouse breast cancer 4T1 cells were generated in inbred BALB/c mice. The mice were treated with programmed death-1 (PD-1) and cytotoxic t-lymphocyte antigen 4 (CTLA-4) inhibitors once every 3 days for five consecutive administration cycles. Changes in tumor volume and body weight were recorded. Lung computed tomography (CT) scans were conducted. The liver, lungs, heart, and colon tissues of the mice were stained with hematoxylin-eosin (H&E) staining to observe inflammatory infiltration and were scored. Serum samples were collected, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ferritin, glutamic-pyruvic transaminase (ALT), tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and interleukin-6 (IL-6). Mouse liver and lung cell suspensions were prepared, and changes in macrophages, T cells, myeloid-derived suppressor cells (MDSCs), and regulatory (Treg) cells were detected by flow cytometry. RESULTS: Mice treated with PD-1 and CTLA-4 inhibitors showed significant reductions in tumor volume and body weight. The tissue inflammatory scores in the experimental group were significantly higher than those in the control group. Lung CT scans of mice in the experimental group showed obvious inflammatory spots. Serum levels of ferritin, IL-6, TNF-α, IFN-γ, and ALT were significantly elevated in the experimental group. Flow cytometry analysis revealed a substantial increase in CD3+T cells, Treg cells, and macrophages in the liver and lung tissues of mice in the experimental group compared with the control group, and the change trend of MDSCs was opposite. CONCLUSIONS: The irAE-related animal model was successfully established in BALB/c mice using a combination of PD-1 and CTLA-4 inhibitors through multiple administrations with clinical translational value and practical. This model offers valuable insights into irAE mechanisms for further investigation.

Anlotinib and anti-PD-1 mAbs perfected CIK cell therapy for lung adenocarcinoma in preclinical trials.

Murine cytokine-induced killer (CIK) cells are heterologous cells that kill various allogeneic and isogenic tumors and have functional and phenotypic characteristics of natural killer cells and T lymphocytes. However, the effect of CIK alone on solid tumor therapy is only limited. To enhance the therapeutic effect, it is vital to discover a mix of several therapy approaches. Immune cell function is inhibited by abnormal tumor vessels and the tumor microenvironment, which block lymphocyte entry into tumor tissue. To increase the effectiveness of CIK cells' anti-tumor activity, anti-vascular therapy and CIK cell therapy can be combined. Furthermore, anlotinib is a tiny drug with multi-target tyrosine kinase inhibitors (TKI) that can block cell migration, delay angiogenesis, and decrease blood vessel density. Compared with other anti-angiogenesis drugs, anlotinib stands out due to the wider target of action and lower effective dose. In this work, anlotinib and murine CIK cells were coupled to boost CD3+ T cell infiltration, CD3 + CD4+ T cell infiltration, and expression of granzyme B and IFN-γ from CD3 + CD8+ T cells, which increased the anti-tumor activity. Through the generation of cytotoxic cytokines by T lymphocytes, the therapeutic group using anti-PD-1 monoclonal antibodies (anti-PD-1 mAbs) in conjunction with anlotinib and CIK cells was more successful than the group receiving dual therapy. The preclinical study contributes to exploring the therapeutic alternatives for patients with lung adenocarcinoma, thus prolonging their lives.

Rational pemetrexed combined with CIK therapy plus anti-PD-1 mAbs administration sequence will effectively promote the efficacy of CIK therapy in non-small cell lung cancer.

Cytokine-induced killer (CIK) cells are heterogeneous cells composed mainly of CD3+CD56+ T cells. As an important treatment method of adoptive therapy, it has shown promising efficacy in many clinical trials, especially in combination with multidrug therapy. However, the maximal antitumor efficacy of CIK therapy in the combined administration of multidrug and CIK therapies and which administration scheme can maximize the antitumor efficacy of CIK therapy are still remain unclear. In this study, we observed that pemetrexed administration prior to the injection of CIK cells maximizes the efficacy of CIK therapy. Anti-PD-1 mAbs should be administered prior to CIK cell injection to maximize the efficacy of the therapy. However, administering anti-PD-1 mAbs after CIK cell injection significantly affects the binding rate of anti-PD-1 mAbs to the PD-1 receptor on CIK cells, affecting the efficacy of the antitumor therapy. In conclusion, our study observed that a rational administration sequence of pemetrexed combined with CIK therapy and anti-PD-1 mAbs significantly promotes the efficacy of CIK therapy, providing an experimental basis for the combination therapy mode and regimen of CIK therapy in clinical practice. We hope that this study can provide patients with lung adenocarcinoma with a prolonged survival time.

CD4+ T cells are required to improve the efficacy of CIK therapy in non-small cell lung cancer.

As a widely studied adoptive treatment method, CIK (cytokine-induced killer cells) treatment has shown clinical benefits in many clinical trials on non-small cell lung cancer. As a heterogeneous cell population, however, CIK cells have a strong instability and individual differences in their efficacies, which are collaboratively regulated by the tumor microenvironment and CIK subpopulations. Among them, CD4+ T cells belong to a crucial subgroup of the CIK cell population, and their influence on CIK therapy is still unclear. Herein, we show how CD4+ T cells positively regulate the functions of CD3+CD56+ T and CD3+CD8+ T cells. During this process, we found that Th1/Th17 CD4+ subgroups can induce the phosphorylation of the AKT pathway by secreting IL-17A, and upregulate the expression of T-bet/Eomes transcription factors, thereby restoring the function of CD8+/CD3+CD56+ T cells and reversing the exhaustion of PD-1+Tim-3+ T cells. These findings will provide guidance for the clinical screening of suitable populations for CIK treatment and formulation of strategies for CIK therapy plus immune checkpoint treatment. Based on these findings, we are conducting an open-label phase II study (NCT04836728) is to evaluate the effects of autologous CIKs in combination with PD-1 inhibitor in the first-line treatment of IV NSCLC, and hope to observe patients' benefits in this clinical trial.

Prediction of immune-related adverse events in non-small cell lung cancer patients treated with immune checkpoint inhibitors based on clinical and hematological markers: Real-world evidence.

Clinical and hematological parameters can predict immune-related adverse events (irAEs) caused by immune checkpoint inhibitors (ICIs). However, the exact correlation between these parameters and irAEs is unclear. This study aimed to establish a prediction model for irAEs in patients with non-small cell lung cancer (NSCLC) treated with ICIs. This retrospective study included patients with NSCLC treated with a minimum of one dose of ICIs at the Tianjin Medical University Cancer Hospital and Shanxi Bethune Hospital from 2016 to 2020. Baseline characteristics, treatment details, and adverse events were evaluated. The Student's t-test, Chi-square test, and logistic regression were used to identify risk factors for irAEs to establish a prediction model. A total of 667 patients were included; the median age was 62.47 (range, 27-85) years. Most patients were men (74.5%) with stage IV cancer (93.1%). The incidence of any grade and grade 3 or higher irAEs was 21.74% (145/667) and 5.25% (35/667), respectively. A total of 145 patients experienced 220 irAEs; the incidence of endocrinopathies (35.91%, 79/220) was highest in all grade irAEs, while that of pneumonitis (7.73%, 17/220) was the highest in grade 3 or higher irAEs. A prediction model based on treatment lines, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), absolute lymphocyte count (ALC), and systemic immune inflammation index was established. The area under the receiver operator characteristic curve was 0.722 (95% confidence interval: 0.650-0.793), with a cut-off value of 0.247 and a sensitivity and specificity of 62.9% and 74.6%, respectively. The multivariate logistic regression analysis showed that the risk of irAEs was higher in patients undergoing second-line therapy than in those undergoing treatment with adjuvant therapy (odds ratio [OR] = 8.239, p = 0.011). AST (OR = 1.053, p = 0.007) and ALC (OR = 2.556, p = 0.001) showed a positive correlation with the risk of irAEs, while LDH showed a negative correlation with irAEs (OR = 0.994, p = 0.007). The model showed good prediction efficiency, whereas the treatment lines, AST, ALC, and LDH were independent risk factors for the onset of irAEs.

Gene augmented nuclear-targeting sonodynamic therapy via Nrf2 pathway-based redox balance adjustment boosts peptide-based anti-PD-L1 therapy on colorectal cancer.

BACKGROUND: Colorectal cancer is known to be resistant to immune checkpoint blockade (ICB) therapy. Sonodynamic therapy (SDT) has been reported to improve the efficacy of immunotherapy by inducing immunogenic cell death (ICD) of cancer. However, the SDT efficacy is extremely limited by Nrf2-based natural redox balance regulation pathway in cancer cells in response to the increased contents of reactive oxygen species (ROS). Nuclear-targeting strategy has shown unique advantages in tumor therapy by directly destroying the DNA. Thus it can be seen that Nrf2-siRNA augmented nuclear-targeting SDT could boost ICB therapy against colorectal cancer. RESULTS: The nuclear-targeting delivery system TIR@siRNA (TIR was the abbreviation of assembled TAT-IR780) with great gene carrier capacity and smaller diameter (< 60 nm) was designed to achieve the gene augmented nuclear-targeting SDT facilitating the anti-PD-L1 (programmed cell death-ligand-1) therapy against colorectal cancer. In CT26 cells, TIR@siRNA successfully delivered IR780 (the fluorescent dye used as sonosensitizer) into cell nucleus and Nrf2-siRNA into cytoplasm. Under US (utrasound) irradiation, TIR@siRNA notably increased the cytotoxicity and apoptosis-inducing activity of SDT through down-regulating the Nrf2, directly damaging the DNA, activating mitochondrial apoptotic pathway while remarkably inducing ICD of CT26 cells. In CT26 tumor-bearing mice, TIR@siRNA mediated gene enhanced nuclear-targeting SDT greatly inhibited tumor growth, noticeably increased the T cell infiltration and boosted DPPA-1 peptide-based anti-PD-L1 therapy to ablate the primary CT26 tumors and suppress the intestinal metastases. CONCLUSIONS: All results demonstrate that TIR@siRNA under US irradiation can efficiently inhibit the tumor progression toward colorectal CT26 cancer in vitro and in vivo by its mediated gene augmented nuclear-targeting sonodynamic therapy. Through fully relieving the immunosuppressive microenvironment of colorectal cancer by this treatment, this nanoplatform provides a new synergistic strategy for enhancing the anti-PD-L1 therapy to ablate colorectal cancer and inhibit its metastasis.