Quality evaluation of anti-PD-1/CTLA-4 bispecific antibody / 中国生物制品学杂志

@#Objective Establish quality control methods for critical quality attribute of bispecific antibody against programmed cell death protein 1(PD-1)/cytotoxic T-lymphocyte-associated protein 4(CTLA-4).Methods The biological activity of PD-1 target was determined by reporter gene assay,and the competitive binding activity of CTLA-4 target was determined by flow cytometry;The antibody molecular size variants were controlled by reducing/non-reducing capillary electrophoresis-sodium dodecyl sulfonate(CE-SDS) and size exclusion chromatography-high performance liquid chromatography(SEC-HPLC);Charge heterogeneity was determined by imaging capillary isoelectric focusing electrophoresis(iCIEF);Bispecific anti-PD-1/CTLA-4 antibody was identified by peptide map analysis;Glycosylation was analysed by high performance liquid chromatography(HPLC)Results The concentration for 50% of maximal effect(EC_(50)) of PD-1target was(6.91±0.78) nmol/L,and the relative biological potency to the reference was(103.50±13.08)% with the RSD of 12.64%;The EC_(50) of CTLA-4 target activity was(0.35±0.28) nmol/L,and the relative biological potency was(99.30±9.15)% with the RSD of 8.32%.The percentage of peak area of light chain and heavy chain of reducing CE-SDS was(98.86±0.02)%.The main peak area percentage of non-reducing CE-SDS was(93.07±0.13)%,fragment percentage was(4.44±0.13)%,and polymer percentage was(2.49±0.15)%.The peak area percentage of SEC-HPLC monomer and polymer were(97.20±0.01)% and(2.68±0.01)%,respectively.The area percentage of peak A group,peak B group,peak C group and peak D group were(38.43±0.54)%,(43.26±0.32)%,(11.31±0.14)% and(7.00±0.17)%,respectively.Peptide mapping showed the specific spectrum of the bispecific anti-PD-1/CTLA-4 antibody,which could be adopted for identification test.The highest proportion of glycotype was GOF,with a content of(41.06±0.11)%,There were three types of glycan containing sialic acid,namely G2F+G1F-NANA,G2F-NANA and G2F-2NANA,with the content of(12.44±0.12)%,(12.00±0.05)% and(5.37±0.05)%,respectively.The total content of glycan containing sialic acid was(29.80±0.20)%.Conclusion The critical quality attributes of bispecific anti-PD-1/CTLA-4 antibody were studied and the corresponding quality control methods were established to ensure its safety,effectiveness and quality control,which provides a reference for the quality control methods and strategies of this type of monoclonal antibody products.

Immune Checkpoint Molecules in Cancer Cells: Not in Their Places but on Their Duty / 中国生物化学与分子生物学报

Immune checkpoints represent a group of inhibitory receptor molecules that are expresses in the surface of immune cells and play a pivotal role in maintaining immune homeostasis. In recent years, several key immune checkpoint molecules such as CTLA-4 and PD-1, has been found to exist in some kinds of tumor cells. These ectopic expressed checkpoint molecules are named as “cancer cell-intrinsic immune checkpoint molecules”. Although our understanding on cancer cell-intrinsic immune checkpoint molecules is quite limited, emerging evidence suggests that the expression and the biological functions of such molecules in different types of cancer cells are heterozygous and diverse. In particular, the discovery of “adaptive immune-independent” regulation on cancer cell behaviors would potentially benefit to design a customized cancer immune therapy, as well as to develop new therapeutic strategies for cancer. In this review, we will briefly describe the timeline of the studies, deeply discuss the complicated biological functions and the regulatory mechanisms (CTLA-4 and PD-1 as representative examples). And finally we put forward a research perspective on cancer cell-intrinsic immune checkpoint molecules. This review aims to present and promote the studies of cancer cell-intrinsic immune checkpoint molecules to the broad scientific community.

The functions of Th17/Treg cells and relevant studies on the treatment of periodontitis and atherosclerosis / 口腔疾病防治

@#Periodontitis is a chronic infectious disease in which periodontal tissue loss is caused by dental plaque biofilm. Atherosclerosis is a chronic inflammatory disease that occurs in the walls of arteries and is characterized by lipid accumulation. Recently, many studies have suggested that there is a certain relationship between periodontitis and atherosclerosis. From an epidemiological perspective, a previous literature review indicated that patients with periodontitis have a higher incidence of atherosclerosis. IL-17 secreted by Th17 cells may aggravate the progression of the two diseases by elevating the levels of matrix metalloproteinases, which may damage the connective tissue. Treg cells reduce the activation of T cells and limit the development of inflammation by secreting anti-inflammatory factors and expressing coinhibitory molecules. Periodontal intervention may contribute to the treatment of atherosclerosis by reducing inflammatory markers in atherosclerosis. Many studies have shown that periodontitis and atherosclerosis may interact with each other, but further studies are needed to explore the concrete mechanism of the interaction between periodontitis and atherosclerosis.

Immunology of cervical cancer

ABSTRACT Optimal function of the immune system allows the recognition and elimination of infected and tumor cells. However, these cells can develop mechanisms to evade the cellular immune response. In human papillomavirus (HPV) infection, dysregulation of major histocompatibility complex Class I molecules and other components of the innate immune system promote the survival of infected cells by allowing the infection to persist which, in turn, favors the development of cancer. Further, tumor cells possess inherent mechanisms designed to block the recognition and activation of cytotoxic lymphocytes: particularly, HPV proteins such as E1 and E2 and oncoproteins E5, E6, and E7 that inhibit immune mechanisms and/or stimulate the expression of immunosuppressive cytokines. These mechanisms include a decrease in receptor activation and costimulating molecules on the surface of immune cells, as well as the constitutive expression of molecules that inhibit their function, which allow HPV persistence and tumor progression. Immunotherapy-based therapeutic options are positioned as excellent candidates for the treatment of cervical cancer.

Immunotherapy treatment against cervical cancer

ABSTRACT Cervical cancer (CC) is one of the most common gynecological tumors and an important health problem, especially in developing countries. The vast majority of patients in early stages are cured of the disease with surgical treatment and with concomitant chemoradiotherapy in locally advanced stages. However, in patients with recurrent, persistent, or metastatic cervical CC, the effectiveness of treatment is limited, except for the combination of chemotherapy based on platinum doublets plus bevacizumab, the treatment that has achieved the best results to date. Programmed cell death-1/PD ligand-1 (PD-1/PD-L1) inhibitors could be a novel and cutting-edge therapeutic option to improve clinical outcomes in this group of patients. Thus far, there are a few Phase I/II clinical trials that have assessed the usefulness of pembrolizumab and nivolumab in this group of patients; these include the KEYNOTE 028, KEYNOTE 158, and CHECKMATE 358 trials, in which clinical benefit has been proven with PD-1/PD-L1 inhibitors in recurrent, persistent, or metastatic CC, as second-line treatment. There are also some ongoing trials that could provide further evidence on the PD-1/PD-L1 pathway as a therapeutic target in CC. In this review, we will focus on the usefulness of these PD-1/PDL1 inhibitors in CC, as well as on trials that are still in the recruitment phase, to confirm their effectiveness in this clinical setting.

Roles of Programmed Cell Death-1/Programmed Cell Death-ligand 1 and Cytotoxic T-lymphocyte-associated Protein 4 Signaling Pathways in Bladder Urothelial Carcinoma / 中国医学科学院学报

Bladder urothelial carcinoma(BUC)is a common malignant tumor in the urinary system.Pt-based chemotherapy has long been a standard therapeutic method for resectable or metastatic BUC,but with poor outcomes.Immune checkpoint inhibitors specific to programmed death 1(PD-1)/programmed death-ligand 1(PD-L1)and cytotoxic T lymphocyte-associated protein 4(CTLA-4)pathways have shown significant antitumor activities,safety,and enduring reactivity in clinical trials,thus creating a new epoch for the treatment of advanced-stage BUC.This article reviews the relationships of BUC with PD-1/PD-L1 and CTLA-4 pathways,as demonstrated in clinical trials.In particular,the authors elucidate the clinical studies on the application of immune checkpoint inhibitors in different BUC stages and their optimal combining strategies,with an attempt to improve the clinical use of immune inhibitors for BUC treatment.

Secreted CTLA-4 fusion Plasmodium falciparum DNA vaccine combined with GM-CSF enhances immune response of mice / 基础医学与临床

Objective To study effects of secreted cytotoxic T-lymphocyte-associated protein-4(CTLA-4)fusion Plasmodium falciparum DNA vaccine combined with granulocyte-macrophage colony stimulating factor(GM-CSF) on humoral and cellular immune responses in mice.Methods The malaria antigen coding sequence fused with CTLA-4 extracellular region of mouse was constructed as eukaryotic secretory expression vector VR 1012-sES312-CTLA,recombinant protein in culture of transfected HEK 293 cells was detected by Western blot.Balb/c mice were co-administrated with VR1012-sES312-CTLA and GM-CSF expression vector.After immunization specific antibody IgG titers and cytokines IFN-γand IL-4 expression levels were evaluated by ELISA and ELISPOT respectively. Results The introduction of CTLA-4 into malaria DNA vaccine system and application of GM-CSF adjuvant signifi-cantly enhanced the specific immune response to the vaccine.Antibody titers in VR1012-sES312-CTLA and GM-CSF co-immunized mice showed a 190-fold increase compared with the simple designed VR1012-ES312 immunization(P<0.001).Conclusions Humoral and cellular immunity induced by malaria DNA vaccine are significantly en -hanced by both dendritic cell-targeting modification and the introduction of GM-CSF molecular adjuvant into the im-mune system.This result provides a new idea for effectively raising the immune response to malaria DNA vaccine.

Expression of E2A in Mid-secretory Endometrium of Women Suffering from Recurrent Miscarriage / 华中科技大学学报（医学）（英德文版）

E2A is involved in promoting forkhead box P3 (FOXP3) and retinoid-related orphan receptor gamma t (RORγt) gene transcription,which are pivotal transcription factors of T regulatory cells and Thl7 cells,respectively.Little is known about the involvement of E2A in pregnancy process.This study aimed to investigate the expression of E2A,cytotoxic T-lymphocyte-associated protein 4 (CTLA-4),and Foxp3 in luteal phase endometrium of women suffering recurrent miscarriage (RM) (n=21) and control group (n=11) by immunohistochemistry,with the Vectra(R) automated quantitative pathology imaging system for analysis.The percentage of E2A+ cells and CTLA-4+ cells was significantly higher in the endometrium of women with RM than in the controls.There was positive correlation between E2A and CTLA-4 (r=0.523,P=0.002),E2A and FOXP3 (r=0.380,P=0.032),and FOXP3 and CTLA-4 (r=0.625,P=0.000) in the mid-secretory phase of endometrium for all subjects.It was concluded that the abnormal expression of endometrial E2A existed in mid-secretory endometrium of women with RM,and there was a positive correlation between E2A and FOXP3,and E2A and CTLA-4,suggesting the possible regulation role of E2A involved in regulating endometrium receptivity.

Treatment of non-small cell lung cancer comes to the age of immunotherapy / 解放军医学杂志

Immune checkpoint inhibitors have become an important alternative for advanced non-small cell lung cancer (NSCLC) patients to surgery, chemotherapy, radiotherapy and targeted therapy. Monoclonal antibodies directed against immune checkpoint have shown better results in the application of first- or second-line treatment of NSCLC and for both squamous and non-squamous cell carcinoma patients, especially for those with positive PD-L1 tumor cells. Some comments will be made in present paper about the efficacy, biomarker, combined therapy and the resistant mechanism of immune checkpoint inhibitors.