Efficacy And Safety Of A Novel Oncolytic Adenovirus BioTTT001 In The Treatment Of Human Esophageal Squamous Cell Carcinoma

Background and AimBecause of increasing incidence and mortality,esophageal cancer(EC)has attracted more and more attention,especially in China.There are about 572,000 new cases and 509,000 deaths each year,ranking as the sixth highest fatality rate in the world.Despite the continuous application of new therapies in clinical practice,the 5-year survival rate of patients is still less than 20%.Therefore,new treatment methods and efficacy evaluation systems are urgently needed to improve the survival rate of patients.The study of genomics and biological characteristics of tumors can provide evidence to guide the treatment of patients.Patient-derived tumor cells(PDCs)can well reflect the biological characteristics of tumors,maintain the genomic characteristics of original tumors,and have a higher tumorigenesis rate compared with subcutaneous transplanted tumors from patients(PDX).PDCs can be employed in a more accurate drug evaluation system.A drug evaluation system based on genomic and biological characteristics of PDCs will provide the important basis for clinical treatment.Among many new drugs,oncolytic viruses(OVs)have been gradually applied in the field of cancer treatment.OVs can selectively infect tumor cells,lyse and kill them,release virus particles,infect adjacent cancer cells,spread in tumor tissues,stimulate the host’s anti-virus and anti-tumor immunity,and prevent the metastasis and recurrence of tumors.Oncolytic adenoviruses(OAds)are the most commonly used viruses in anti-cancer research because of their high safety and efficacy.H101 is the first commercially produced oncolytic adenovirus in the world.It has been approved by the SFDA in China for the treatment of head and neck tumors.H101 has both E1B55 K gene and E3 B gene deleted,which greatly affects the replication ability of the virus in normal cells.Although H101 is often combined with conventional chemotherapeutic drugs to improve clinical efficacy,the therapeutic effect is not ideal.Bio TTT001(also known as Ad-TD-ns IL12)deleted three genes at the same time,including E1ACR2,E1B19 K and E3gp19 K,and retained the E3 B gene,which not only did not affect the replication ability of the virus,but also significantly improved the antitumor effect and safety in vivo.At the same time,the therapeutic gene human non-secretory IL12(ns IL12)delivered by the virus can produce an appropriately low level of IL-12 restricted to the tumor microenvironment to further enhance the antitumor effect.This study attempts to establish more Chinese esophageal cancer cell lines,and to evaluate the killing effect of conventional chemotherapy drugs and oncolytic adenovirus at the cell level by using a series of newly established esophageal cancer cell lines from patients.Furthermore,in vivo,the therapeutic effects of Bio TTT001,H101 and cisplatin were evaluated by establishing the immunodeficient Syrian hamster model bearing human esophageal squamous cell carcinoma.Part I Establishment and identification of biological characteristics of human esophageal squamous cell carcinoma cell lines1 ObjectiveTo establish patient-derived esophageal squamous carcinoma cell lines(ESCC),detect the biological activity of established cells,and analyze the genomic characteristics of the two cancer cell lines by exon sequencing.2 Methods2.1 Culture of esophageal squamous cell carcinoma cell linesTumor cells were obtained by collagenase digestion and fibroblasts were removed by differential digestion to purify tumor cells.2.2 Biological characterization of esophageal squamous carcinoma cell linesA panel of specific immunohistochemical markers and HE staining were used to detect the homology of esophageal squamous cell carcinoma cell lines and primary tumors.The cell growth curve was drawn.Monoclonal formation experiments,cell migration experiments,and chromosome karyotype analysis were carried out.2.3 Short tandem repeats analysisCells were sent to Jinweizhi Biotechnology for STR identification.Gene Mapper 4.0 software was used for data analysis and results were matched with reference databases such as ATCC,DSMZ and JCRB to identify the uniqueness of cell lines and the existence of cross-contamination.2.4 Exon sequencing of Cell LinesThe cells and their matched peripheral blood were transported to Shenzhen Huada gene for exon sequencing,and the sequencing platform was Illumina Hi Seq X-Ten.The results were analyzed by GATK(version 4.1.0)haplotype caller.2.5 Establishment of SBRC-EC01-Luc and SBRC-EC02-Luc cell linesThe lentiviral vector containing Luciferase(Luc)reporter gene was constructed by In-Fusion cloning.The virus was packaged with the third generation packaging system and infected into the cell lines.The Luc-expressing stable cells were established by blasticidin S selection.2.6 Tumor formation of esophageal squamous cells in immunodeficient miceThe cells of ESCC cell lines were inoculated subcutaneously into the right anterior scapula of immunodeficient B-NDG mice with the dose of 1 × 107 cell/mouse.The volume of the tumors was measured sequentially.The growth curve of the tumors was drawn,and the Luc assay was used in vivo for deep tissue tumor visualization.3 Results3.1 Establishment of human esophageal squamous cell carcinoma cell lines SBRC-EC01 and SBRC-EC02Two esophageal cancer cell lines were successfully established and can be passed on steadily to 60 generations.These two cell lines were named SBRC-EC01 and SBRC-EC02 respectively.3.2 Molecular characteristics of SBRC-EC01 and SBRC-EC02The morphological,pathological and molecular characteristics of the two cell lines were similar to the primary tumors.The two cell lines had high proliferative activity,strong clonogenic ability and the tendency of metastasis.Chromosomes were consistent with the characteristics of malignant tumors.3.3 Short tandem repeats of SBRC-EC01 and SBRC-EC02STR identification proved its uniqueness and no cross-contamination with other cells.They were newly cultured cancer cell lines.3.4 Exon sequencing of SBRC-EC01 and SBRC-EC02After exon sequencing,the cancer driver genes were screened,such as P53,NOTCH1,RB1,FAT1 and ZNF750,etc.These genes were closely related to the pathogenesis and prognosis of ESCC,and can be used in the pathogenicity study of ESCC,as well as in the screening of targeted drugs to guide the individualized treatment of patients.3.5 Establishment of SBRC-EC01-Luc and SBRC-EC01-Luc cell linesSBRC-EC01-Luc and SBRC-EC01-Luc cell lines were successfully established after antibiotic selection,and the Luc expression by cell lines was confirmed by chemiluminescence assays in vitro.3.6 Tumorigenicity of SBRC-EC01-Luc and SBRC-EC02 cells in immunodeficient miceTumor formation of two cell lines were observed in B-NDG mice post subcutaneously injection.4 Conclusion4.1 Two esophageal squamous cell carcinoma cell lines from patients were successfully established;4.2 The cancer driver genes were screened according to exon sequencing;4.3 The established cell line expressing luciferase gene can be used for dynamic observation of tumorigenesis,development and evaluation of new drugs;4.4 The established esophageal cancer cell line had the ability of subcutaneous tumorigenesis in immunodeficient mice.Part II The effect evaluation and mechanism of action for the killing of ESCCs with conventional chemotherapeutic drugs and Oncolytic Virus1 ObjectiveComparison of the newly established cell lines with the 14 conventional esophageal squamous cell carcinoma cell lines in virus sensitivity and response to chemotherapy drugs,to verify the reliability of the established cell lines.Screening out the drugs which have the obvious killing effect on ESCCs,studying the related mechanism,and thus guiding the clinical treatment of the patient.2 Methods2.1 The killing effect of conventional chemotherapeutic drugs on esophageal squamous carcinoma cell lines.Cisplatin,5-fluorouracil and paclitaxel,the first-line chemotherapeutic drugs,were selected for the killing assay of existing esophageal squamous cell carcinoma cell lines and newly established cell lines.MTS cell proliferation assay was used to evaluate the killing effects of the drugs.2.2 Preparation of oncolytic virusFollowing the established production protocols,the adenovirus and vaccinia virus(VV)were produced and purified in batches.The virus titer was measured by the 50% tissue culture infection dose(TCID50)method.2.3 Evaluation of killing effect of oncolytic virus on esophageal squamous cell carcinoma cell linesThree Ad5 adenoviruses,one Ad11 adenovirus,and one ΔTKΔN1L-RFP(VV)were used for killing assays of esophageal squamous cell carcinoma cell lines.The MTS method was used to evaluate the killing effect of the virus.2.4 Measurement of Coxsackie virus and adenovirus receptor(CAR)m RNA levels among cell linesTotal RNA of the cell lines was extracted,the c DNA was reverse-transcribed,and the m RNA levels of CAR were detected by RT-q PCR.The relationship between the transcriptional levels of CAR and Ad5 sensitivity were studied.2.5 Detection of replication ability of oncolytic virus in esophageal squamous cell carcinoma cell linesThe replication ability of five selected replication oncolytic viruses in esophageal squamous cell carcinoma cells was tested by virus replication assay.2.6 Effects of chemotherapeutic drugs on cell killingThe cisplatin-resistant cell lines were developed by mimicking the cisplatin treatment.The MTS killing assay of Bio TTT001 and Quantitative proteomic analysis was performed with these cisplatin-resistant and their parental cell lines.3 Results3.1 Evaluation of the killing effect of conventional chemotherapeutic drugs by MTS on esophageal squamous cell carcinoma cell linesDrug killing results showed that the two newly established cell lines and the existing esophageal squamous cell carcinoma cell lines had similar response to conventional chemotherapy drugs.Most of them were sensitive to cisplatin.With the increase of drug concentration,all tumor cells can be killed.Although they were sensitive to paclitaxel and 5-fluorouracil,etc,there was a dose-related plateau in their ability to kill cancer cells.3.2 Preparation of oncolytic virusThe production of oncolytic virus at high titre can be achieved and they were free of bacterial and mycoplasma contamination.3.3 Killing effect of oncolytic virus on esophageal squamous cell carcinoma cell linesViral killing results showed that Bio TTT001 was superior to H101.Bio TTT001 and Ad11 were capable of selective killing of tumor cells,and VVΔTKΔN1L-RFP was generally susceptible to esophageal squamous cell carcinoma cell lines.3.4 Detection of CAR m RNA expression level in cellsRT-q PCR results showed that the expression levels of CAR in the cells of KYSE510 and KYSE150 were low.Thus,this may lead these cells to be less sensitive to virus infection.By contrast,the expression level of CAR in KYSE450 cells was lower than that of other cells,but CAR low-expressing cells were more sensitive to virus than other higher ones.These results indicated that the sensitivity of cell lines to Ad5 infection was not entirely dependent on the level of CAR.3.5 Replication ability of oncolytic virus in esophageal squamous cell carcinoma cell lineThe results of virus replication experiments showed that there was no significant difference in the replication ability between Bio TTT001 and Ad-TD-Luc,and the ability of H101 and VVΔTKΔN1L-RFP were both lower than Ad11 and Bio TTT001.3.6 Effect of cisplatin in the Bio TTT001 killing of cancer cellsThe sensitivity of esophageal squamous cell carcinoma cells to Bio TTT001 was significantly increased if these cells were cisplatin resistant,which may be caused by the cisplatin-induced elevated expression level of CAR or integrin.4 Conclusion4.1 The two newly established cell lines had high stability and reliability;4.2 Conventional chemotherapy drugs had selectivity and dose-related plateau in their killing ability of cancer cells;4.3 Bio TTT001 was superior to H101 in killing and replicating ability in esophageal squamous cell carcinoma cell lines;4.4 After the generation of cisplatin resistance,the sensitivity of esophageal cancer cells to the oncolytic adenovirus increased significantly,which was related to the elevated expression level of CAR or integrin.Part III The therapeutic effect and potential mechanisms of action of oncolytic adenovirus Bio TTT001,H101 and cisplatin in an immunodeficient Syrian hamster model bearing human esophageal squamous cell carcinoma1 ObjectiveTo evaluate the therapeutic effect of oncolytic adenovirus Bio TTT001,cisplatin and H101 in vivo,and further study the related mechanism.2 Methods2.1 Detection of IL-12 level in supernatant and lysate of infected cells with Bio TTT001Cell supernatants and lysates of tumor cells infected with Bio TTT001 at different time were collected,and IL-12 levels were detected by ELISA.2.2 Tumor formation by subcutaneous transplantation in immunodeficient miceThree types of immunodeficient animal,B-NDG,BALB/c Nude mice and ZZU-001 hamster,were selected for the comparison of tumorigenesis experiments.2.3 Therapeutic effect of Bio TTT001,H101 and cisplatin in an immunodeficient Syrian hamster model bearing human esophageal squamous cell carcinomaThe SBRC-EC01 immunodeficient Syrian hamster subcutaneous transplantation model was established to evaluate the therapeutic effects of Bio TTT001,H101 and cisplatin.2.4 Therapeutic mechanism of Bio TTT001,H101 and cisplatin in an immunodeficient Syrian hamster model bearing human esophageal squamous cell carcinomaThe SBRC-EC01 immunodeficient Syrian hamster subcutaneous transplantation model was established.Bio TTT001,H101 and cisplatin were given.On the 11 th,21st and 31 st days after the first treatment,three mice were killed in each group.Subcutaneous tumors were taken for immunohistochemistry to detect the expression level of viral protein in tumors,the proliferation of tumor cells and the density of tumor interstitial blood vessels.3 Results3.1 IL-12 level in supernatant and lysate of infected cells with Bio TTT001In the early stage of virus infection(24 hours),the level of IL-12 in the supernatant of cell culture was very low.After 48 hours,the level of IL-12 in the cell supernatant slowly increased,which was slightly lower but not significantly than that in the cell lysate(P > 0.05).3.2 Tumorigenicity comparison of SBRC-EC01 in subcutaneous transplantation of three types of immunodeficient miceThe tumorigenicity and speed of growth of SBRC-EC01 in ZZU-001 hamster subcutaneous transplantation were significantly higher than those of B-NDG and BALB/c Nude mice(P < 0.0001).3.3 Therapeutic effect of Bio TTT001,H101 and cisplatin in an immunodeficient Syrian hamster model bearing human esophageal squamous cell carcinomaAmong the three treatment groups,the effect of Bio TTT001 was better than that of cisplatin and H101.For the cisplatin group,recurrence of tumors occurred.The animals were in poor condition,and 28% of animals died within 30 days after treatment.3.4 Therapeutic mechanism of Bio TTT001,H101 and cisplatin in an immunodeficient Syrian hamster model bearing human esophageal squamous cell carcinomaOn the 11 th day after the first treatment,high levels of viral protein expression were detected in the tumor tissues of the Bio TTT001 treatment group.On the 21 st day,a high level of viral protein could still be detected,and on the 31 st day,no viral protein was detected.On the 11 th day after the first treatment of H101 virus,a large number of viral protein was detected in the tumor tissues,but no viral protein was detected on the 21 st day and 31 st days.These results indicated that the virus clearance rate of H101 group was faster than that of Bio TTT001 group.The microvessel density of Bio TTT001 group was significantly reduced compared with that of other treatment groups,and the difference was statistically significant(P < 0.01).4 Conclusion4.1 Low levels of IL-12 in cell supernatant and lysate were detected after infection of Bio TTT001;4.2 ZZU-001 hamster was an ideal animal model to evaluate the treatment of SBRC-EC01 subcutaneous transplanted tumor;4.3 Bio TTT001 was superior to H101 and cisplatin in the treatment of SBRC-EC01 subcutaneous transplanted tumors,which was related to the slow elimination rate of the virus in tumors and the inhibition of tumor interstitial angiogenesis by IL-12.