The Safety And Immune Response Of Immunotherapy With Multiple Tumor Antigens Activated T Cells Treated Patients With HCC

Background&AimImmunotherapy is emerging as a new treatment stratage for tumors,especially for advanced malignant cancers,which has been cosidered as the fourth treatment option following surgery,chemotherapy and radiotherapy.Aaccording to different mechanisms,Immunotherapy can be divided into two kinds which are active immunotherapy like DC vaccine and adoptive celluar immunotherapy(ACT).ACT has proved to play an important role in the development of immunotherapy,which are initially characterized by tumor-specific T cells that are naturally activated and amplified,either from the autologous peripheral blood mononuclear cells(PBMCs)or tumor-infiltrating lymphocytes(TIL)or using genetically modified antigen-specific T cells(CAR-T)of the patient.CAR-T is effective for Leukemia and some kinds of solid tumors.A liver cancer may have both hepatocellular as well as cholangiolar differentiation,90%is the former called Hepatocellular Carcinoma(HCC),most HCC patients(80%)had reached BCLC C or D stage,only 15.3%is BCLC early stage when diagnosed.According to guidelines,Curative treatment options like resection or ablation only apply to BCLC early stage patients.while patients with advanced HCC did not have very much therapeutic options,only Chemoembolization and Sorafenib.Multifaceted immune-based approaches have been played to evaluate the efficiency of achieving disease regression.Our study present a novel adoptive cancer immunocelluar therapy,named MASCT TM(Multiple Antigen Stimulating Cellular Therapy),by preparing 14 kinds of tumor antigens activated T lymphocytes ex vivo from HCC patients' PBMCs to treat the same cancer patients.we launched the study to evaluate the safety and immune response stimulated by Immunotherapy with Multiple Tumor Antigens Activated T cells treated patients with hepatocellular carcinoma in HCC patients and Preliminary discusse whether could improve the clinical outcome of HCC patients compared with conventional therapy.Method:1.The safety of Immunotherapy with Multiple Tumor Antigens Activated T cells(MASCTTM)treated patients with HCCThe clinical datas,the causes of adverse reactions,Clinical manifestations,liver and renal functions,blood routine examinations of 45 patients with hepatocellular carcinoma after receiving Multiple Antigen Stimulating Cellular Therapy(MASCT TM)from January 2012 to December 2014 in the center of Liver Cancer were gathered and retrospectively analyzed.These patients did not receive any other immunotherapy,their latest treatments(surgery,radiotherapy,chemotherapy)before receiving MASCTTM should be finished at least one month or more.Peripheral blood mononuclear cells(PBMCs)were separated,and the adherent cells were induced into DCs.DCs were loaded withl4 kinds of antigen peptides to prepare mDCs,a small part mDCs were injected intracutaneously in day 8.the un-adherent cells of PBMCs co-cultured with most mDCs in day 7,induced into CTL and were injected intravenously in day 26.mDCs and CTL had been quality controled.Gathered the causes of adverse reactions,clinical manifestations,liver and renal function,blood routine examination of 45 patients with HCC before and after MASCTTM treatment.2?The immune responses in the patients' PBMCs induced by MASCT TM treatment.Retrospectively and prospectively analyzed the immune responses induced by MASCTTM in the HCC patients' PBMCs during treatment course.Experiments were designed as follows:1)T cell phenotype detection of PBMCAntibodies for cell surface staining were obtained from BD Biosciences,Flow cytometry staining solution is:NKT(CD3+CD56+);CD8+NKG2D+Granzyme B+CD107a+;Treg(CD4+CD25+FoxP3+);CD45RO-FITC CD27-Percp-Cy5.5 CD57-APC CCR7-PE CD3-APC-Cy7.Flow cytometry was performed using FACS Canto? flow cytometers,and then analyzed datas.2)Proliferation assay and IFN-y production of antigen-specific T cellsPBMCs from patients were prestimulated with peptides pool and cytokinesfor 3 days.To determine the proliferative percentage of specific T cells,FACS analysis was performed as described in the manufacturer's instructions of Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit.The IFN-y production of specific T cells was also detected by intracellular cytokine staining and FACS analysis.PBMCs incubated with irrelevant peptides at the same concentration were used as negative controls.The results were shown as fold changes,computed by the ratio of specific peptide stimulation to irrelevant peptide stimulation.3)Specific immune response of each kind of antigen peptide(ELISPOT assay)PBMCs from patients were plated in 48-well plate and were further stimulated with irrelevant peptides,MASCT antigen peptides pool,and each kind of antigen peptide respectively.The PBMCs were then transferred onto a 96-well ELISPOT assay plate(U-CyTech Biosciences)for IFN-? detection.The PBMCs were stimulated again with peptides.The ELISPOT assay was performed and analyzed according to the manufacturer's instructions.The number of spot-forming units was determined with computer-assisted image analysis software(ChampSpot;Saizhi).Results were demonstrated as an IFN-y-producing fold index,computed by the ratio of specific peptide stimulation to irrelevant peptide stimulation.3?The clinical benefits of Immunotherapy with Multiple Tumor Antigens Activated T cells(MASCTTM)treated patients with HCCDuring retrospective analysis,the medical records of patients were used from a computerized database in the Centre of Liver Diseases,Nanfang Hospital of Southern Medical University.This database recorded the clinical pathologic information of all patients,including age,gender,tumor characteristics,stages,treatments,and RECIST evaluation.We analyzed the DCR of patients who were diagnosed as BCLC stage B HCC after 2012,and were continuously treated and regularly followed-up in this centre for at least one year.4?Statistical analysesStatistical analysis was carried out by using SPSS 19.0 statistical software.The results are expressed as mean±SD.ANOVA and Chi-squared test or Fisher's Exact Test are perform for analysis.GraphPad Prism software was used to draw figures.Differences were considered significantly at P<0.05 level.Results:1?The MASCTTM successfully induce mDC?CTL and is safe for patients with HCCIn mDCs,the percentage of CD80+was(98.5±5)%?CD83+ was(88±10)%?CD86+was(98.4±3)%,HLA-DR+was(98.8±2)%.mDCs secreted high level of IL-12(985±312 pg/ml)and low level of IL-10(53±10 pg/ml).In CTL,the percentage of CD3+CD8+was(83±10)%,CD3+CD56+was(24±5)%.CTL secreted high level of IFN-?(1222±650 pg/ml),low level of IL-10(6.8±5.0 pg/ml).All the 45 patients' condition were improved with varying degree after MASCT TM treatment,The main adverse events after infusion of active immunocytes were moderate fever(2 cases,4.44%),and there was no significant difference in renal function or blood routine examination before and after treatmentand there was no death related to MASCTTM treatment.But ATL?AST?TBIL were increased a little.2?The MASCTTM treatment improved immunosuppressive state and stimulate specific immune responses in patients with HCCWe have observed that,after repeating treatment of MASCTTM treatments,the resulting amplified CTL cells have restored immune function and MASCTTM,the frequency of regulatory T cells in the patients' PBMCs was significantly decreased,the specific immune responses against tumor antigens in different HCC patients by both the T cell proliferation assay and the IFN-y stimulation assay,as well as the decrease of Treg in patients' PBMCs after multiple treatments of MASCTTM.And these immune responses were gradually enhanced during multiple treatment of MASCTTM,indicating that repeating treatment may correlate with a better clinical outcome.3?The DCR of BCLC B stage HCC patients in one year with Multiple MASCTTM and conventional treatments is 80%We have retrospectively studied the disease control rate(DCR)of patients with BCLC stage B HCC,who were diagnosed after 2012 and were continuously treated(with or without MASCTTM)and regularly followed-up for longer than one year.Through reviewing of medical records,we found seventeen patients had received conventional treatments(Group Con),such as resection,TACE,and RFA(radiofrequency ablation)..The one-year DCR was significantly increased in the group of patients treated with MASCTTM every 2-3 months combined with conventional therapies(Group Con+MASCT TM),compared to the conventional group(80%vs 17.65%).Conclusion:MASCTTM induced high quality mDCs and CTL successfully,it is safe for patients with hepatocellular carcinoma,After repeating treatment of MASCT TM,the frequency of regulatory T cells in the patients' PBMCs was significantly decreased,and the proliferation and function of tumor antigen specific T cells were enhanced.The specific immune responses against each tumor antigens were also increased in patients with individual patterns.Moreover,the disease control rate(DCR)in one year was raised to 80%in stage B HCC patients receiving MASCTTM combined with conventional therapies.Our study has demonstrated that MASCTTM is a well tolerant immunotherapy that improves both the immunologic function and disease control of patients with HCC.