hIL-2 Gene Research Articles

Antitumor activity of recombinant oncolytic vaccinia virus with human IL2.

The tumor microenvironment is highly immunosuppressive. The genetically modified oncolytic vaccinia virus (OVV) is a promising vector for cancer immunotherapy. The aim of the present study was to assess the antitumor effects of human interleukin-2 (hIL2)-armed OVV in vitro. The hIL2 gene was inserted into a thymidine kinase and the viral growth factor double deleted oncolytic VV (VVDD) to generate recombinant hIL2-armed OVV (rVVDD-hIL2). Viral replication capacity in A549 cells was quantified by plaque titration on CV-1 cells. Production of hIL2 in cancer cells infected by rVVDD-hIL2 was measured by enzyme-linked immunosorbent assay. Finally, 3-(4,5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay was performed to assess the antitumor effects of rVVDD-hIL2. The results showed that rVVDD-hIL2 viral particles expressed increasing levels of hIL2 in human and murine cancer cell lines with growing multiplicities of infection (MOIs). The insertion of the hIL2 gene did not impair the replication capacity of VV, and the rVVDD-hIL2 virus killed cancer cells efficaciously. The lytic effects of the recombinant oncolytic virus on tumor cells increased with the growing MOIs. In conclusion, these findings suggest that hIL2-armed VVDD effectively infects and lyses tumor cells, with high expression of hIL2.

El jarabe de Agave salmiana mejora la producción de interleucina 2 humana recombinante en Escherichia coli

The expression of heterologous proteins in Escherichia coli is strongly affected by the type of carbon source used. In this work, the expression of a synthetic codon-optimized gene of human interleukin-2 in E. coli BL21-SI, carrying plasmid pET12a-hIL2 is presented. Glucose, fructose or Agave syrup from Agave salmiana, were used as carbon sources for production of recombinant human IL-2 (rhIL-2) in 1.5-L bioreactor aerobic cultures using mineral medium. Codon optimization of the native hIL-2 gene eliminated the presence of 35 rare codons for E. coli, and improved the codon usage up to 76% compared with the native gene sequence. Cultures using 10 g/L glucose showed the lowest production of rhIL-2, and in contrast, cultures using fructose improved the production of rhIL-2 1.9-times. The utilization of fructose from Agave syrup enhanced the rhIL-2 production 3.9-times, reaching 103.42±6.61 mgIL-2/L. The specific rhIL-2 production rate (5.52±0.33 mgIL-2/gDCW·h) using Agave syrup was also the highest. These results indicate that Agave syrup stimulates the production of rhIL-2 and it is an inexpensive alternative carbon source. This research abilities the potential use the Agaves to produce alternative and valuable biotechnological products instead the alcoholic beverages.

Introducing curcumin as an electrochemical DNA hybridization indicator and its application for detection of human interleukin-2 gene

In this paper, the application of curcumin (CU) as a non-toxic electrochemical DNA hybridization indicator was described. Hybridization investigations on a pencil graphite electrode surface as a transducer using oligonucleotides containing only one base type, including poly A, poly T, poly C, and poly G as probe and as related complementary/non-complementary sequences, showed that CU has no specific interaction with each of the oligonucleotides of DNA. Furthermore, results showed good interaction between CU and the hybridized form of oligonucleotides; thus, the extent of hybridization was evaluated based on the difference between differential pulse voltammetry (DPV) signals of CU accumulated on the probe-pencil graphite electrode (PGE) and CU accumulated on the probe-target-PGE. Then, the developed biosensor was successfully applied for the detection of short sequences of human interleukin-2 (hIL-2) gene as a model. A hybridization experiment with non-complementary oligonucleotide showed that the suggested DNA sensor responds selectively to the target. At optimized conditions, two linear ranges were obtained for hIL-2 gene, first from 50 to 1000 pM and second from 0.01 to 1 μM with a detection limit of 12 pM. 7.0) containing 20 mM NaCl.

Production of the Human Anti-Metastatic Melonoma Interleukin-2 in Maize Vegetative Biomass for Clinical Use

The E.coli produced recombinant human interleukin-2 (rhIL-2) has been approved by the Food and Drug Administration for the immunotherapeutic treatment of the end-stage metastatic melanoma and renal cell cancer. However, the E.coli produced rhIL-2 is very expensive (~$11,400 USD per treatment). In the present study, we explored the feasibility of producing rhIL-2 in transgenic Zea mays (maize) vegetative biomass instead of its production in E. coli because (1) maize vegetative biomass is abundant, (2) maize can cheaply produce recombinant proteins while obtaining its energy from freely available sun via photosynthesis, has an easy scale-up via reproduction system, can be easily grown by farmers in the field with minimum level of training, and has conserved protein folding machinery including glycosylation similar to that of human. The human hIL-2 gene was codon optimized to maximize its expression in plants. A plasmid construct containing the rhIL-2 regulated by a rubisco green-specific promoter, an endoplasmic reticulum-specific signal peptide, 6-histodin tag was developed and nos terminator The construct was transferred into the maize genome via the gene gun bombardment, and fertile plants developed. Molecular analysis confirmed that the human IL-2 had integrated, transcribed and translated in up to the 4th (T3) generation maize plants. When the same gene construct was transferred into the tobacco genome and the rhIL-2 was purified and its biological activity compared with the FDA approved commercially available E coli- produced version against the marine splenic CD4+ the mice cells, the plant-produced version was as effective as the commercially available E. coli-produced version. Research is needed to test the rhIL-2 producing maize in the field, and to perform preclinical and clinical trial for its potential commercial release.

Specific Immune Response Induced by PML-Rarα-hIL-2 Vaccine in BALB/C Mice

Specific active immunotherapy with DNA vaccines offers the hope for a natural nontoxic alternative to eradicate minimal residual disease in cancer patients. However, little data are regarding the DNA vaccines in leukemia. Our previous study has successfully developed a vector containing PML-RARα segment fused to human Interleukin- 2 (hIL-2) gene. In the present study, we analyzed the specific immune response by immunization with the recombinant plasmid in BALB/c mice. The recombinant plasmids were injected into BALB/C mice with a total of 200 μg DNA in normal saline at 14-day intervals for three cycles. The plasmid containing the same sequence of PML-RARα segment or hIL-2 gene alone and blank plasmid served as controls. Two weeks after the final DNA boost, the transcription and expression of PML-RARα and hIL-2 in injection site were detected by RT-PCR and immunohistological methods; the specific cytotoxicity of spleen cells from the vaccinated BALB/C mice was detected by LDH release assay; interferon-γ secretion was measured by ELISA; antibodies against human PML-RARα were detected by indirect immunofluorescene analysis (flow cytometry); and T-cell receptor rearrangement excision circles (TRECs) in DNA from mice thymic cells were detected by real-time quantitative RT-PCR (TaqMan), thereby to evaluate the content of naïve T cells. After immunization, the PML-RARα/hIL-2 mRNA and protein could be identified in the injection site of PML-RARα- hIL-2 vaccinated mice. Time dependent antibody production and an increase in INF–γ could be detected in vaccinated mice serum (p<0.05). Specific cytotoxicity on NB4 cells was significantly higher than that from control groups (p=0.012). TRECs level form PML-RARα-hIL-2 vaccine group was significantly higher than that from PML-RARα, hIL-2 vaccine or control groups (p<0.05). In conclusions, the reconstructed PML-RARα plasmids might display the common feature of DNA vaccine, which can induce specific humoral and cellular immune responses in BALB/c mice in vivo and can induce the increase of naïve T cells in mice thymus, it might relate to stronger cellular immune response, it is to our knowledge, the first description on the change of naïve T cells in response to vaccine.

Engineering Human Tumor-specific Cytotoxic T Cells to Function in a Hypoxic Environment

Hypoxia occurs in many tumors and reduces the effectiveness of radio- and chemotherapy. Hypoxia also impedes immune responses to tumors, reducing T lymphocyte production of cytokines such as interleukin-2 (IL-2) and interferon gamma, as well as the survival and proliferation of these cells. We constructed a lentiviral vector encoding a bidirectional hypoxia-inducible responsive element (HRE) derived from human vascular endothelial growth factor, which drives the hIL-2 gene and a marker gene. We used a model of human B cell lymphoma to show that tumor-specific T cells modified with this vector upregulate hIL-2 expression when oxygen tension is low in vitro and in vivo. The consequence of this effect is to increase T-cell survival and proliferation whilst sustaining effector function, even in O(2) concentrations as low as 1%. The phenotype of the transduced cells is unchanged, as is their ability to migrate to tumor. HRE-IL-2-modified cytotoxic T lymphocytes (CTLs) produce faster and more complete tumor regression than parental CTLs and increase overall survival. Hypoxia-resistant T cells may thus be of value in the treatment of human tumors in which areas of hypoxia may otherwise account for resistance to this therapeutic strategy.

Development of a PML-RARα-IL-2 Recombinant Plasmid DNA for APL.

Specific active immunotherapy with DNA vaccines is one of the best approaches to eradicate minimal residual disease in cancer patients, when appropriate tumor-specific antigens are identified for this disease. However, little data are regarding the DNA vaccines in acute leukemia, recent reports has shown that a human PML-RARα breakpoint-drived DNA vaccine can prevent APL in mice model, but the host immune response were weakly, due to the weak immunogenicity. In order to improve the effect of DNA vaccine for acute promyelocytic leukemia (APL) therapy, we develop a vector coexpressing PML-RARα gene fused to human Interleukin- 2 (hIL-2) gene. PML-RARα fusion gene segment and the full-lenght hIL-2 gene were amplified from NB4 cells or Jurkat cells. Both PCR products were cloned into PIRES plasmid respectively to construct a recombinant plasmid PML-RARα-IRES-hIL-2. The recombinant plasmids were then transfected into K562 or A549 cells respectively. The expression of the PML-RARα/hIL-2 mRNA and protein in transfected cells were identified by RT-PCR, dot blotting, ELISA and Western-Blot respectively. By in vivo assays, BALB/c mice were vaccinated at 6–8 week of age with a total of 200 μg DNA in normal saline, injected into two sites in the quadriceps muscles on day 0, 7 and 21. The plasmid containing the same sequence of PML-RARα gene and blank plasmid served as controls. Two weeks after the final DNA boost, both PML-RARα/hIL-2 mRNA and protein, serum INF-γ and anti-NB4 cells specific cytotoxicity of splenocytes following 7 days of stimulation in vitro with freeze thawing NB4 cells and recombinant human IL-2 were assessed by ELISA and LDH assays. The results showed that the sequence of the fragments inserted in multi-clone site (MCS) A and MCS B of PIRES plasmid were absolutely correct by double restriction enzyme cutting analysis (Sal I/Not I) and sequence analysis, the PML-RARα/hIL-2 mRNA and protein could be identified in transfected K562 or A549 cells and in mice quadriceps muscles. The level of serum INF-γ and cytotoxicity of splenocytes against NB4 cells from immunized mice was significant increased than that from control groups. Our results indicated that the recombinant plasmid expressing PML-RARα and hIL-2 was successfully constructed, which can induce more effective immune response and anti-APL cells effect in animal models. The data suggest that effective vaccination approaches should be possible against APL. It could be farther used in the research as PML-RARα DNA vaccine for APL.

Effects of huTNF-α and hIL-2 gene transfection on the expression of MDR1 and LRP genes in lung cancer cell lines

To study the effects of huTNF-α and hIL-2 gene transfection on the expression of MDR1 and LRP genes in lung cancer cell lines. huTNF-α and hIL-2 gene plasmids were constructed and transfected into A549, GLC-82, H446 and H460 cells with lipofectinmin. Positive clones were screened out by G418. The expressions of MDR1 and LRP genes were detected at mRNA level by reverse transcription polymerase chain reaction (RT-PCR) in the non transfected cells and the cloned cells. MDR1 gene was positive in A549, GLC-82, H446 and H460 cell lines, LRP gene was positive in A549, GLC-82 and H460 cell lines; The transfected cell lines expressed both huTNF-α and hIL-2 gene, and the A549, H446 and H460 cell lines transfected with hIL-2 gene had no MDR1 expression at mRNA level compared with the non transfected ones. MDR1 and LRP genes are expressed in lung cancer cell lines, which indicates the presence of intrinsic drug resistance before any form of therapy. MDR1 gene is not expressed in hIL-2 transfected cell lines, which demonstrates that hIL-2 gene modulates the MDR1 gene expression at mRNA level, and may reverse the multidrug resistance of lung cancer.

Construction of IL-2 gene-modified human hepatocyte and its cultivation with microcarrier.

To construct interleukin-2 gene-modified human hepatocyte line (L-02/IL-2) and investigate the changes of the function of liver cells and IL-2 secretion in culture with microcarrier,laying the foundation for further experimentation on hepatocyte transplantation. hIL-2 gene was transduced into L-02 hepatocytes by recombinant retroviral vector pLNCIL-2, and the changes of morphology and clonogenicity rate of the transduced cells were observed, the secretion levels of hIL-2 in cultural supernatant were detected by ELISA and NeoR gene was amplified by PCR. The growth of L-02/IL-2, the special biochemistry items and the levels of IL-2 were detected after cultivation with microcarrier. The clonogenicity rate of the L-02/IL-2 cells was lower than that of L-02/Neo cells and L-02 cells. The levels of hIL-2 could reach 32,000 pg/10(6) cells per day and kept secreting for more than ten weeks. NeoR gene segment was respectively obtained by PCR from both L-02/IL-2 and L-02/Neo cell's genomic DNA. At the 6(th) day in culture with microcarrier, the matrix-induced liver cell aggregates were formed, the number of alive L-02/IL-2 cell were 16.8+/-0.53 x 10(6)/flask and the levels of ALB and UREA were 52.54+/-1.28 mg/L and 5.29+/-0.17 mmol/L, respectively. These data had not significantly changed as compared with those of L-02 cells (P>0.05); However, the levels of IL-2 in IL-2/L-02 cells remarkably exceeded that in L-02 cells in the whole culture process (P<0.001). The IL-2 gene-modified hepatocyte line has been successfully constructed. The L-02/IL-2 cellular aggregates cultured with microcarrier have a high capacity of IL-2 production as well as protein synthesis and amino acid metabolism.

Conversion of the genetic background of transgenic mice by in vitro fertilization.

We attempted to convert the genetic background of transgenic (Tg) mice in a short period of time by applying in vitro fertilization techniques. Tg mice were obtained by injecting human interleukin-2 (hIL-2) gene into the fertilized eggs of the C57BL/6N strain. These Tg mice were back-crossed 8 times to the inbred C3H/HeN strain using the hIL-2 gene as the genetic selection marker. In order to shorten the length of the back-crossing time, in vitro fertilization was performed with eggs collected from immature Tg females and spermatozoa from mature C3H/HeN males, and successfully fertilized eggs (2-cell stage) were transferred to pseudopregnant recipients to obtain the offspring of next generation. When no offspring were obtained through the procedures using immature Tg females, in vitro fertilization was performed with mature Tg males and mature C3H/HeN females to continue successive back-crosses. With this method, it was possible to perform 8 successive back-crosses in 18 months.