Modified Danggui Beimu Kushen Pills Inhibit Tumor Growth and Regulates T Cell Subsets in H22 Hepatocellular Carcinoma-bearing Mice / 中国实验方剂学杂志

ObjectiveTo explore the effects of modified Danggui Beimu Kushen pills on tumor growth and T-cell subsets in H22 hepatocellular carcinoma-bearing mice and to provide an experimental basis for the treatment of hepatocellular carcinoma with modified Danggui Beimu Kushen pills combined with immune checkpoint antibodies. MethodA H22 hepatocellular carcinoma-bearing mouse model was established. The modeled mice were randomized into model， cisplatin， low- （4 g·kg-1·d-1）， medium- （8 g·kg-1·d-1）， and high-dose （16 g·kg-1·d-1） modified Danggui Beimu Kushen pills groups. After continuous administration for 14 days， the mice were sacrificed on day 15. The tumor volume was measured on days 0， 4， 8， 12， 15 of drug administration. Tumors were weighed and thymus index and spleen index were calculated. Spleen lymphocytes were co-cultured with H22 hepatoma cells， and the tumor cell-killing rate was detected by the cell counting kit-8 （CCK-8）. Real-time polymerase chain reaction was carried to determine the mRNA levels of programmed cell death protein-1 （PD-1） and lymphocyte activation gene-3 （LAG-3） in spleen and tumor tissues. The number of CD4+ and CD8+ T cells and the expression of PD-1 and LAG-3 were detected by immunohistochemistry （IHC）. ResultOn day 8 of drug administration， tumor volumes in all treatment groups decreased compared with that in the model group. On day 15， both tumor volume and tumor weight were significantly lower in the treatment groups than in the model group （P<0.01）， with the cisplatin group showing the most pronounced reduction. Compared with the model and cisplatin groups， medium- and high-dose modified Danggui Beimu Kushen pills increased the thymus index （P<0.01）. Compared with the model group， all treatment groups showed increased spleen index （P<0.05， P<0.01）， with the cisplatin group showing the most significant increase. Compared with the model and cisplatin groups， all the groups of modified Danggui Beimu Kushen pills demonstrated increased number of CD4+ and CD8+ T cells and tumor cell-killing rate in the spleen and tumor tissues （P<0.01） and down-regulated mRNA and protein levels of LAG-3 （P<0.05， P<0.01）. The high-dose group of modified Danggui Beimu Kushen pills had lower mRNA level of PD-1 in the tumor tissue than the model and cisplatin groups （P<0.01）. ConclusionModified Danggui Beimu Kushen pills may promote the proliferation and tumor microenvironment infiltration of CD4+ and CD8+ T cells in H22 tumor-bearing mice by down-regulating LAG-3 expression， thereby improving T-cell immune activity and inhibiting tumor growth. This study provides an experimental basis for the combination of modified Danggui Beimu Kushen pills and immune checkpoint antibodies in the treatment of hepatocellular carcinoma.

Research Progress on Immunization and Targeted Therapy for Triple-negative Breast Cancer / 肿瘤防治研究

Triple-negative breast cancer (TNBC) accounts for approximately 15%–20% of all breast cancers. Patients with TNBC have a rapidly progressive clinical course, an earlier age of onset, faster distant recurrence, and more common visceral metastases as compared with other subtypes. However, treatment of TNBC is often limited to chemotherapy and has a poor prognosis. Therefore, developing the best treatment strategy for patients is essential to reduce the burden of disease caused by TNBC. Various potential available drug targets have been discovered, as well as precision treatment and classified treatment are changing the clinical practice of TNBC, thereby indicating a new therapeutic area for TNBC in addition to traditional chemotherapy. This article reviews the systemic treatment options for TNBC in recent years, including immunotherapy and targeted therapy.

Clinical significance of LAG-3 on microvessel density in primary hepatocellular carcinoma

Aims: We aimed to determine whether lymphocyte activation gene 3 (LAG-3), also known as CD223, is associated with microvessel density (MVD) in primary hepatocellular carcinoma (HCC), as well as their clinical significance in predicting survival. Materials and methods: One hundred and twenty-seven patients were enrolled in the study. Samples were obtained on resection at the Department of Hepatobiliary Surgery of the Qingdao Municipal Hospital from June 2014 to June 2016. Immunohistochemistry was used to determine vessel density and LAG-3 abundance. Statistical analyses were performed to test for correlation of LAG-3 density and other clinicopathological variables with overall survival (OS). Results: High LAG-3 abundance was significantly correlated with increased MVD in primary HCC (P < 0.05). The ?2 test revealed a significant association of LAG-3 with preoperative AFP level, tumor diameter, N stage, and the presence of HBV infection (P < 0.05). Patients with high LAG-3 expression had shorter OS compared to those with low LAG-3 expression (P < 0.05). The Cox proportional hazards model showed that both higher LAG-3 and MVD density, age, the number of tumors, preoperative AFP level, tissue differentiation, Child–Pugh grade, and lymph node metastasis correlated with survival. Conclusions: High expression of LAG-3 is associated with angiogenesis and poor prognosis in HCC patients. With the deepening of research, LAG-3 is likely to become a novel biomarker for clinical diagnosis and prognosis and can even be a therapeutic target of HCC.

Anti-tumor activity of oncolytic adenovirus expressing LAG-3 antibody on glioblastoma / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the anti-tumor activity of oncolytic adenovirus co-expressing lymphocyte activation gene 3 (LAG-3) antibody (aLAG) against glioblastoma. Methods: aLAG sequence was inserted into the skeleton of oncolytic adenovirus Ad3 to obtain recombinant oncolytic adenovirus (Ad3-aLAG). The expression of aLAG in infected gliblastoma GL261 cells was detected by WB. The cytotoxicity of recombinant oncolytic adenovirus against glioblastoma was detected by MTT method. The tumor inhibitory activity of recombinant oncolytic adenovirus against glioblastoma in vivo was evaluated with mice subcutaneous xenograft model. Tumor infiltrating T cells were detected by immunohistochemical staining, and the levels of cytokines TNF-α and IFN-γ secreted by tumor infiltrating T cells were detected by Flow cytometry. Results: The recombinant oncolytic adenovirus was successfully constructed, which could effectively express aLAG and kill GL261 cells in vitro (all P<0.01). Experimental results of mice subcutaneous xenograft model showed that the tumor inhibition ability of recombinant oncolytic adenovirus Ad3-aLAG was stronger than that of Ad3 oncolytic adenovirus (P<0.01), and Ad3-aLAG could effectively enhance the infiltration of CD3+ T cells in tumor tissue (P<0.01) and enhance the IFN-γ secretion ability of infiltrating T cells (P<0.01). Conclusion: Ad3-aLAG recombinant oncolytic adenovirus can significantly inhibit the growth of glioblastoma cells in vivo and in vitro, and enhance the anti-tumor immune response in vivo, which is promising to provide a new scheme for the treatment of glioblastoma.

A novel cyclic peptide targeting LAG-3 for cancer immunotherapy by activating antigen-specific CD8 T cell responses

PD-1 and CTLA-4 antibodies offer great hope for cancer immunotherapy. However, many patients are incapable of responding to PD-1 and CTLA-4 blockade and show low response rates due to insufficient immune activation. The combination of checkpoint blockers has been proposed to increase the response rates. Besides, antibody drugs have disadvantages such as inclined to cause immune-related adverse events and infiltration problems. In this study, we developed a cyclic peptide C25 by using Ph.D.-C7C phage display technology targeting LAG-3. As a result, C25 showed a relative high affinity with human LAG-3 protein and could effectively interfere the binding between LAG-3 and HLA-DR (MHC-II). Additionally, C25 could significantly stimulate CD8 T cell activation in human PBMCs. The results also demonstrated that C25 could inhibit tumor growth of CT26, B16 and B16-OVA bearing mice, and the infiltration of CD8 T cells was significantly increased while FOXP3 Tregs significantly decreased in the tumor site. Furthermore, the secretion of IFN- by CD8 T cells in spleen, draining lymph nodes and especially in the tumors was promoted. Simultaneously, we exploited T cells depletion models to study the anti-tumor mechanisms for C25 peptide, and the results combined with MTT assay confirmed that C25 exerted anti-tumor effects CD8 T cells but not direct killing. In conclusion, cyclic peptide C25 provides a rationale for targeting the immune checkpoint, by blockade of LAG-3/HLA-DR interaction in order to enhance anti-tumor immunity, and C25 may provide an alternative for cancer immunotherapy besides antibody drugs.

Research progress on the application of LAG-3 and its inhibitors in cancer immunotherapy / 中国肿瘤生物治疗杂志

@# Lymphocyte-activation gene 3 (LAG-3), also known as CD223, is a 498-amino-acid type I transmembrane protein encoded by LAG-3 gene, which consists of extracellular, transmembrane and intracellular regions.LAG-3 negatively regulates T lymphocyte by binding extracellular domain to ligand, thus avoiding autoimmunitycaused by T cell over-activation. Like programmed cell death 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3 is an important immune checkpoint in vivo and plays a balanced regulatory role in human immune system.Tumor cells escape the surveillance of the immune system by over-expressing LAG-3 ligand. With the development in research of immune checkpoints, LAG-3 has become a new generation of immunotherapy targets after PD-1 and CTLA-4. This article reviews the structure and function of LAG-3 and the application of its inhibitors in tumor immunotherapy, in order to provide reference for the further study of LAG-3.

Research progress on the application of immune checkpoint inhibitors in malignant lymphomas / 中国肿瘤临床

Immune checkpoint inhibitors are members of a class of immune-suppressive molecules that regulate the strength and range of immune responses to avoid normal tissue damage. However, immune checkpoint activity can be stimulated by tumors to es-cape immune surveillance. To elicit anti-tumor effects, immune checkpoint inhibitors can promote the activation of T cells by blocking immune checkpoint proteins. Therefore, these inhibitors can be efficiently and safely used to treat solid tumors. Although the clinical usage of these inhibitors is in the initial stage, they have exhibited good efficacy and safety in lymphoma treatment. This review sum-marizes the biological activities of CTLA-4, PD-1, and PD-L1 and the application of antibodies as drugs for lymphoma treatment.

Epigenetic regulatory effects of the methylation of CpG island shore on the expression of lag3 gene in various human T cell lines / 中华微生物学和免疫学杂志

Objective To investigate the effects of DNA methylation on the expression of lympho-cyte activation gene 3 (lag3) in different human T cell lines.Methods A quantitative PCR and a flow cy-tometry analysis were performed to measure the expression of lag3 gene in various T cell lines at mRNA and protein levels.The distribution of CpG sites within the promoter and body of lag3 gene was detected to locate the potential regulatory region(s) (CpG island and CpG island shore).The levels of DNA methylation in each cell line were analyzed.The T cell lines were demethylated with 5-Aza-2′-deoxycytidine (5-Aza-2′-dc) for further investigation on the changes of lag3 gene expression and DNA methylation.Results Jurkat E6-1 cells showed the highest expression level of lag3 gene as compared with J.CaM1.6 and CEM cells.Hyperm-ethylated CpG islands were detected in cells of each cell line.The methylation levels of CpG island shore in J.CaM1.6 and CEM cells were higher than that in Jurkat E6-1 cells.Treatment of J.CaM1.6 and CEM cells with 5-Aza-2′-dc significantly promoted the expression of lag3 gene at mRNA and protein levels as well as the demethylation of CpG island shore.No significant differences with the expression of lag3 gene and the methylation of CpG island were observed in Jurkat E6-1 cells with or without 5-Aza-2′-dc stimulation.Con-clusion Methylation and demethylation of CpG island shore played important roles in regulating the tran-scription of lag3 gene.