Experimental Study Of CIK Cells Combined With IL-12 Expressing Oncolytic Adenovirus For Hepatocellular Carcinoma Therapy

BackgroundsThe morbidity and mortality of malignant tumor remain high, which seriously harm to human health. The traditional treatments are very limited to the advanced cancer. Immune cell therapy and gene therapy of biological therapy have developed rapidly in recent years, and have achieved series breakthrough in the technique and efficacy.Adoptive cell therapy has been widely used in clinical and obtain a certain therapeutic effect. In recent years, a breakthrough of CAR-T technology in the clinical treatment of hematological malignancies obtained remarkable therapeutic effect. However due to the relatively complex of tumor microenvironment in solid tumor: 1)lack of tumor antigen and costimulatory signal for immune cell activation in immunosuppressive microenvironment, causes the immune response is difficult to initiate; 2) a large number of extracellular matrix so that immune cells difficult infiltration into tumor tissue; 3) in tumor tissues large amounts of the immunosuppressive cells and immune suppression signaling molecules, makes the infiltrating immune cells can not exert tumor-killing effect, and under these immune inhibitory signals the infiltrating effector cells die or become anergic. Therefore, how to break through the limitation of tumor microenvironment and improve the effect of immune therapy is becoming to the cutting edge research.Cancer gene therapy strategy is based on gene therapy, through efficient gene transfer vector, therapeutic gene transfers into tumor cells or tumor lesions, playing continues repair or anti-tumor effect to achieve the cancer treatment purpose. Oncolytic virus-mediated gene therapy( "virus- gene" therapy) is a new gene therapy strategy. Through modification of the virus to achieve tumor specific oncolytic virus, they only replicate and express gene in tumor cells, but does not affect the biological function of normal cells. Using oncolytic viruses in tumor tissue continuous replication and re infection effect, to extent the greatest infection of tumor cells, and maintain long-term gene expression.Study found that after infection and lysis of tumor cells by oncolytic virus can produce dangerous signal, release of type I interferon and exposure of the tumor antigen. Antigen presenting cells in tumor identify the signal by pattern recognition receptor(PRR), and present the tumor antigen, which can induce the immune response against the tumor by the host immune system. Using oncolytic viral vectors to express a large number of immune activation factor or immune inhibitory receptor blocker, further relieve of the tumor immunosuppression and enhance the antitumor immune responses.IL-12 is an important immune-activating factor, it can act directly on T lymphocytes and NK cells, and playing an important role in the innate immune response and adoptive immune responses. IL-12 in addition to the direct effects on the immune system, but also can induce upregulation of endothelial adhesion molecules in vascular endothelial cells, such as VCAM-1, etc., to guide the T lymphocytes infiltration. IL-12 is mainly derived from the activated antigen presenting cells, such as DC cells, macrophages, etc. However in the tumor microenvironment, tumor tissue resident dendritic cells and macrophages loss due immune activation function, and unable to release IL-12 and other immune activation factor, therefore IL-12 concentration in local tumor serious shortage, affecting the anti-tumor effect of effector cells.For these reasons, the first part of this paper wishes to explore the combination possibility of “cells adoptive immunotherapy" and "virus gene" therapies. On the one hand by the "virus- gene" therapy, direct inject oncolytic adenovirus expressing immune activation gene IL-12 to tumor site, using viral oncolysis and high levels of IL-12 factor expressed in tumor local, break tumor immune suppressive microenvironment, result in acute inflammatory environment. On the other hand by exogenous infusion adoptive immune cells CIK, the exogenous immune cells in acute inflammatory environment play a better anti-tumor effect, achieving "Cell- Virus- Gene" powerful combination treatment.Although the virus gene therapy has many advantages, it shows a wide range of therapeutic potential; however, it is restricted by the way of drug delivery, limiting its indications. 1)The tumor local injection of drug is injected directly into the tumor tissue or adjacent tissue. This drug delivery method is capable of reaching the maximum extent of drug delivery to the tumor tissue. However, in this way for some patients it brings a injury and risky by surgery, and mostly for diffuse growth malignant or widespread metastasis drug is difficult to achieve in situ direct injection. 2)Infusion through vascular system, the operation of the method is relatively simple, drugs can through the blood system transport to various tissues and organs, to solve the diffusion transfer problem. However, the virus is directly exposed to the blood system, the virus will be absorbed by a large number of vascular endothelial cells and blood cells, or neutralized by the antibody and the loss of infection. In addition, the virus delivery by blood system lacks tumor specificity, a large number of viruses will be transported to the non-tumor tissue and ultimately only limited virus can reach to the tumor site.The use of cells as a vehicle for gene therapy vectors delivery has the advantage of: 1) Loading gene therapy vector onto the carrier cell, which is possible to reduce the direct contact with the fluid environment, avoids antibody neutralization. 2) The selection of cell carrier with tumor tropism can help the loaded virus reach to the tumor lesions, reducing losses and side effects due to virus infection in healthy tissue. 3) Virus can further proliferate in the cell vehicle during delivery, for which the titer of virus releasing to the tumor site will be greatly improved.Adenovirus type 5 is one of the widely used vectors for gene therapy. However, immune cells do not express the receptors(Coxsackie virus and adenovirus receptor and integrin protein alpha v beta 5 and alpha v beta 3 which are necessary for Ad5 virus infection, making it difficult to delivery by immune cells. Ad5 / 11 chimeric virus is based on type 5 virus, which konb and shaft structure of fiber protein is replaced by Ad11 type konb and shaft structure. The Ad5 / 11 chimeric virus have the same infection characteristics of adenovirus type 11,which can recognize a wide range of CD46 expressing cells, including epithelial and mesenchymal originated tumor cells, and lymphoid immune cells, is ideal vector for immune cells delivery.For these reasons, the second part of this paper would like to explore the use of CIK cells as a viral vector delivery vehicle. By tumor-homing properties of CIK cells, the virus will be carried to the tumor site, then solve the drug administration problems of the mass growth or widespread metastasis patients.The first Part: Combined Therapy with Cytokine-induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor ModelResearch purposesThe aim of this study is to investigate the combined therapy with CIK cells and oncolytic adenovirus Expressing IL-12 in vivo antitumor effect and analysis mechanism of the combined anti-tumor effect, to provide the experimental basis for “cell- virus –gene” triple combination therapy.Experiment content and method1. The recombinant bacteria technology. In the project group double target to oncolytic virus Ad CN205 system based on construct the expression of IL-12 gene tumor specific oncolytic virus Ad CN205-IL12;Research content and methods1. Using bacterial recombinant techniques, to construct the IL-12 gene expressing tumor specific oncolytic virus( Ad CN205-IL12),based on the dual-targeted oncolytic virus Ad CN205 system constructed by our research group;2. To verify the tumor-specific of virus, viral replication capacity, IL-12 gene expression levels were detected after virus infected BEL7404, Hu H7, SMMC7721 and other tumor cell lines and normal liver cell AKN-1;3. To verify its tumor targeting kill ability, in vitro cytotoxicity of oncolytic virus on tumor cells BEL7404, Hu H-7, Hep3 B and normal liver cells of AKN-1 were detected;4. Human PBMCs were acquired from health donors in southwest hospital. PBMC cells were stimulated by IFN-γ, OKT3, IL-2, IL-1 cytokines then cultured with IL-2. After 14 days culture, cells were identified by CD3、CD4、CD8、CD56、NKG2D antibody in flow cytometry;5. By using luciferase expressing vector p CDH-EF1α-Luciferase- 2A-m Cherry, the engineering cell lines which stably expressing luciferase were constructed. Detection the residual luciferase activity of Hu H-7 and Hep3 B after co-cultured with CIK and/or oncolytic virus, to study the combined killing effect;6. Use Hu H-7 cell lines to establish in vitro xenograft model in SCID mice. Local injection of oncolytic tumor virus(2 × 108PFU) combined with intravenous injection of CIK cells(1 × 107), tumor growth curve and mice survival curve were drawn to study the effect of combined treatment,7. Expression of GFP was observed by immunofluorescence in liver tissue and tumor tissue. Detecteing The expression of IL-12 by Elisa and Hexon by immunohistochemistry to study the specificity of exogenous gene expression and viral replication;8. The infiltration of CIK cells was detected by immunohistochemistry labeling human CD3 molecule. The vascular density was measured through immunohistochemical by labeling CD34 molecule. Then discuss the anti-tumor mechanism of the combined therapy with CIK and IL-12 expressing oncolytic adenovirus.Results1. We developed a double-controlled oncolytic adenovirus expressing IL-12 gene, Ad CN205-IL12, in which h TERT promoter was used to control the expression of CR2 24 bp deleted E1 A region and the 6.7K/gp19 K of E3 region were substituted by the exogenous genes IL-12.2. The replication of virus in SMMC7721, BEL7404, MHCC97 H, Hu H-7, Hep3 B cells and AKN-1 associated their telomerase expression, indicating that viral replication was regulated by telomerase promoter. In the normal liver-derived cell lines ANK-1 no replication was detected, indicating viral replication was tumor specific.3. By observing Ad CN205-GFP infected BEL7404 hepatoma cell lines and normal liver-derived cell lines AKN-1 found GFP gene expression in tumor cells was significantly higher than that in normal liver-derived cells; By ELSIA detecting IL-12 the expression of IL-12 in Ad CN205-IL12 infected of liver cancer cell lines BEL7404 and normal liver-derived cell lines AKN-1, the expression of IL-12 gene was significantly higher than normal liver-derived cell lines, suggesting that exogenous gene expression was tumor specific;4. After 14 days culture, the results showed CD3-positive T cells were 94.05% ± 6.74% of the total cell population, CD3 / CD4 Th cells double positive cells were 12.13 ± 1.58% of the total population, CD3 / CD8-positive CTL cells were 69.2% ± 4.75% of the total cell population, CD3 / CD56 double positive NKT cells in the total cell population were 25.48 ± 4.77%, CD3- / CD56 + NK cells were 5.33% ± 4.32%, a total of 83.5 ± 3.87% cells expressed non-specific anti-tumor associated NKG2 D molecules. This result is consistent with previous reports of CIK cell phenotype.5. Ad CN205-IL12 and Ad CN205-GFP have a strong ability to kill tumor cells, but there was no difference between them. IL-12 gene cannot improve in vitro cytotoxicity ability of oncolytic virus. Non-replicating virus Ad IL-12 had no killing effect on tumor cells in vitro. These results showed that IL-12 alone had no direct effect on tumor cells.6. In the case of 20: 1 efficiency target ratio, IL-12 increased the in vitro anti-tumor effect of CIK cells, increased from 14.37% ± 2.24% to 34.68% ± 1.78%, P = 0,000553.In 40: 1 case anti-tumor effect increased from 51.50% ± 1.19% to 70.23% ± 0.96%, p = 0.001979, suggesting that IL-12 can significantly enhance CIK cell inhibitory effect on liver cancer cells;7. In the efficiency target ratio was 10: 1, and virus infection coefficient was 10 MOI case. For hepatoma cell lines Hu H7, at the time point of 48 h, The tumor cell viability decreased in CIK cells combined Ad CN205-GFP group than single CIK group, the difference was significant(n = 3, p = 0.0260), The tumor cell viability decreased in CIK cells combined Ad CN205-IL12 group than single CIK tumor cell, the difference was significant(n = 3, p = 0.0018); At the time point 72 h, The tumor cell viability decreased in CIK combined Ad CN205-IL12 group comparing CIK combined Ad CN205-GFP group, the difference was significant(n = 3, p = 0.0135);8. In the efficiency target ratio was 10: 1, and virus infection coefficient was 10 MOI case. For hepatoma cell lines Hep3 B, at the time point of 48 h, the tumor cell viability decreased in CIK cells combined Ad CN205-IL-12 group than single CIK tumor cell group(the difference was significant p = 0.0001); than alone Ad CN205-GFP group(the difference was significant p = 0.00026); than Ad CN205-IL12 group,(the difference was significant p = 0.00032); than CIK combined Ad CN205-GFP group,( the difference was significant p = 0.00027); At the time point of 72 h, the tumor cell viability decreased in CIK cells combined Ad CN205-IL12 virus group than the other groups(P <0.001). Suggesting that CIK cells combined with oncolytic viruses can enhance the tumor cells in vitro, the expression of IL-12 gene may further enhance the anti-tumor effect.9. The single treatment group(Ad CN205-GFP, Ad CN205-IL12, CIK) relative to the PBS group has a certain tumor suppressive effect, and the difference is very significant(n=8, p<0.001), but there is no significant difference between each single treatment groups. CIK cells combined with Ad CN205-GFP group compared with the single treatment group Ad CN205-GFP(n=8, p=0.02) and CIK(n=8, p=0.008) with significant difference. CIK cells combined with Ad CN205-IL12 group has the strongest anti-tumor effect, compared with all single treatment group with significant difference(n = 8, P < 0.001), CIK combined Ad CN205-GFP group(n = 8, P = 0.047). During the treatment, we found that the CIK cells combined with Ad CN205-IL12 group has 2 mice tumor completely disappeared.10. For survival time, PBS group shorter than other treatment groups, which was significant difference. However, the difference between each treatment groups was not significant.11. CIK combined Ad CN205-IL12 group, the CIK combined Ad CN205-GFP group and the group of single Ad CN205-GFP group showed large tumor tissue necrosis, while PBS group, CIK group and group Ad CN205-IL2 showed less necrosis area; Infiltration of humanized CD3 positive cell in CIK combined Ad CN205-IL12 group significantly higher than other groups. Suggesting that IL-12 can enhance the infiltration of CIK cells and promote the proliferation or survival of CIK cells in tumor;12. GFP or IL-12 was only expressed in tumor tissue, and there was not expression of GFP or IL-12 in liver tissue. CIK combined with oncolytic virus treatment did not affect the specificity expression of exogenous gene in the tissue. The results of immunohistochemistry showed that the virus can be detected only in tumor cells, and the presence of viral Hexon was undetectable in normal liver, suggesting that CIK combined with viral treatment does not affect the specificity of viral replication.ConclusionCIK cells combined IL-12 expressing double target oncolytic virus Ad CN205-IL12 showed synergistic anti-tumor effect. Oncolysis by virus and high concentration of IL-12 expression in tumor site can increased the infiltration of CIK cells in tumor tissue and enhance the anti-tumor effect of CIK cells.The second part: the mechanism of CIK cells delivering Ad5/11 chimeric virus targeted tumor therapyResearch purposesFor the mass growth or metastasis characteristics of hepatocellular carcinoma, we are going to explore the feasibility of carrying the Ad5/11 chimeric virus by CIK cells to achieve systemic administration, and the related mechanisms of viral transfer and delivery were also studied.Research content and methods1. The expression of CAR, αvβ5, αvβ3, CD46 molecule on CIK cell surface were detected flow cytometry; The virus infection ability to CIK of Ad5 virus was detemind by GFP-positive rate after infected by Ad5-CMV-GFP virus;2. The virus infection ability to CIK of Ad5-CMV-GFP-11 F chimeric virus was determined by GFP-positive rate after infected by Ad5-CMV-GFP-11 F virus; The virus binding ability to CIK of Ad5-CMV-GFP-11 F chimeric virus detected by Texsa Red labeled virus observed through a confocal laser scanning microscopy;3. To study the correlation between and CD46 cells and Ad5/11 chimeric virus during virus infection, Texas Red labeled virus, FITC labeled CD46 molecule were observed by confocal laser scanning microscopy;4. Through an in vitro model to simulate in vivo situation in which CIK would be sheared by blood stream during transport. CIK cells infected with the virus, after washing the CIK cells with 10 times the volume of PBS, CIK co-cultured with tumor cells, transfer situation of virus was observed by confocal laser scanning microscopy;5. Using CIK cells as vehicles to carry adenovirus, after virus loading to CIK cells, the infection ability, proliferation, gene expression activity and the antitumor ability were detected by co-culturing with hepatoma Huh-7 cell and PLC/RPF/5 cell line.6. Observate the transfer process of the virus from CIK cells migrate to tumor cells by confocal laser scanning microscopy.(CIK cell membrane was labeled using Di O, virus was labeled by Texas red).7. To research and observate the relative of virus transfer processes and cell skeleton movement, we use cytochalasin D to inhibit the cytoskeletal movement.Results1. CIK cell do not express the recptors of CAR(αvβ5, αvβ3, etc.) which is necessary for the type 5 adenovirus infection. On the other hand, CIK cells express the complement regulatory molecule CD46 receptor which associated with type 11 adenovirus infection; Adenovirus type 5 can not infect and adsorb CIK cells.2. Ad5 / 11 chimeric adenovirus can infect both epithelial originated tumor cell and CIK cells; Ad5 / 11 chimeric adenovirus can be adsorbed on the surface of CIK cells; The infection efficiency of CIK cells in 100 MOI can reach 45.2 ± 3.4% while Ad5 virus can not infect CIK cells;3. Ad5 / 11 chimeric adenovirus can cause polarization at the cell surface, but only limited virus can enter the into the CIK cells;4. When with Ad5/11 chimeric adenovirus loaded CIK cells co-cultured with tumor cells, the virus can be transferred from the surface of CIK cells to tumor cells;5. CIK can carry Ad5 / 11 chimeric adenovirus and transfer to the tumor cells, and keep oncolytic virus killing activity for Hu H7 cells. At the time point of 72 h CIK cells carrying Ad CN205-GFP-11 F group the tumor cell survival significantly lower than the single-CIK group(p = 0.0008), and CIK carrying Ad CN205-GFP group(p = 0.0168); For PLC / RPF / 5 cells, CIK cells carrying Ad CN205-GFP-11 F group tumor survival rate was significantly lower than that CIK group(p <0.0001), and CIK carry Ad CN205-GFP group(p = 0.005);6. The transfer of Ad5/11 chimeric adenovirus from CIK cell surface to the tumor cells depends on the interaction between CIK cells and the tumor cells;7. Inhibition of cytoskeletal movement by cytochalasin D can arrest viral transfer process;ConclusionAd5/11 chimeric adenoviruses can infection and adsorption to CIK cells. After loading to CIK cells, Ad5/11 chimeric adenovirus can transfer to tumor cells by cell membrane interaction between CIK cells and tumor. The transferred virus maintain the activity of infection and proliferation.