Human Interleukin-2 cDNA Research Articles

Matrix‐assisted refolding and purification of placenta‐derived recombinant human interleukin‐6 produced in Escherichia coli

Biological activity of human interleukin-6 (IL-6) is associated with a vast number of diseases such as rheumatoid arthritis, sepsis, and severe inflammatory diseases. In this study, human IL-6 cDNA was isolated from a cDNA library that was constructed with mRNA derived from human placental tissues. Subsequently, the complete human IL-6 cDNA was cloned and expressed in BL21DE3 cells. The recombinant human IL-6 (rhIL-6) protein was expressed in a form of an insoluble inclusion body. Inclusion bodies were solubilized under denaturing conditions and purified by immobilized metal affinity chromatography with gradual on-column refolding by the gradient elution method (from 6 to 0 M urea). The protein was purified to apparent homogeneity of about 99% with a yield of 50 mg/L. The purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion high-performance liquid chromatography, and Western blotting analysis. The bioactivity was assessed by proliferation assay of TF-1 cells in a dose-dependent manner. The present study confirms the expression of the placenta-derived IL-6 gene in a prokaryotic expression system and matrix-assisted on-column refolding and purification of rhIL-6 by immobilized metal affinity chromatography.

In vivoanti-tumour activity of recombinant MVM parvoviral vectors carrying the human interleukin-2 cDNA

The natural oncotropism and oncotoxicity of vectors derived from the autonomous parvovirus, minute virus of mice (prototype strain) [MVM(p)], combined with the immunotherapeutic properties of cytokine transgenes, make them interesting candidates for cancer gene therapy. The in vivo anti-tumour activity of a recombinant parvoviral vector, MVM-IL2, was evaluated in a syngeneic mouse melanoma model that is relatively resistant in vitro to the intrinsic cytotoxicity of wild-type MVM(p). In vitro infection of the K1735 melanoma cells prior to their injection resulted in loss of tumorigenicity in 70% of mice (7/10). Tumour-free mice were protected against a challenge with non-infected parental cells. In addition, MVM-IL2-infected tumour cells induced an anti-tumour activity on parental cells injected at a distant location. These non-infected tumour cells were injected either at the same time or 7 days before the injection of MVM-IL2-infected cells. In the latter setting, which mimics a therapeutic model for small tumours, 4/10 mice were still tumour-free after 4 months. Our results show that (i) the MVM-IL2 parvoviral vector efficiently transduces tumour cells; and (ii) the low multiplicity of infection (MOI = 1) used in our experiments was sufficient to elicit an anti-tumour effect on distant cells, which supports further studies on this vector as a new tool for cancer gene therapy.

IL-6-overexpression brings about growth impairment potentially through a GH receptor defect.

This paper is concerned with growth retardation associated with overproduction of interleukin-6 (IL-6). As a model, we used MUP/hIL-6 transgenic mice in which human IL-6 cDNA is overexpressed under the control of a MUP gene enhancer/promoter. The growth-retardation of MUP/hIL-6 transgenic mice was paralleled by reduced serum levels of IGF-I. As shown, hepatic IGF-I mRNA levels were reduced in the transgenic mice. MUP/hIL-6 transgenic mice are in a state of growth hormone (GH)-resistance, since their serum GH levels are either normal or elevated. To identify possible steps in GH signaling which might be perturbed in the transgenic mice, we examined the synthesis of GH receptor (GHR) mRNA. We noted a twofold reduction of hepatic GHR mRNA in the transgenic mice. We therefore conclude that overexpression of IL-6 brings about growth impairment in part through a GH receptor defect.

Interleukin-2 gene therapy of chronic neuropathic pain

Previous research has revealed an antinociceptive (analgesic) effect of interleukin-2 (IL-2) in central and peripheral nervous systems. Unfortunately IL-2 is very short-lived in vivo, so it is impractical to apply IL-2 for analgesia in clinic. This study was performed to evaluate the effect of intrathecal delivery of human IL-2 gene on rat chronic neuropathic pain induced by chronic constriction injury of the sciatic nerve. Human IL-2 cDNA was cloned into pcDNA3 containing a cytomegalovirus promoter. The paw-withdrawal latency induced by radiant heat was used to measure the pain threshold. The results showed that recombinant human IL-2 had a dose-dependent antinociceptive effect, but that this only lasted for 10–25 min. The pcDNA3-IL-2 or pcDNA3-IL-2/lipofectamine complex in contrast also showed dose-dependent antinociceptive effects, but these reached a peak at day 2–3 and were maintained for up to 6 days. Liposome-mediated pcDNA3-IL-2 produced a more powerful antinociceptive effect than pcDNA3-IL-2 alone. The paw-withdrawal latencies were not affected by control treatments such as vehicle, lipofectamine, pcDNA3, or pcDNA3-lipofectamine. In the experimental groups, human IL-2 mRNA was detected by reverse transcription-polymerase chain reaction in the lumbar spinal pia mater, dorsal root ganglion, sciatic nerve, and spinal dorsal horn, but not in gastrocnemius muscle. The expressed IL-2 profile detected by western blot coincided with its mRNA profile except it was present in the spinal dorsal horn at a higher level. Furthermore, human IL-2 assayed by enzyme-linked immunosorbent assay in cerebrospinal fluid could still be detected at day 6, but lower than day 3. The antinociceptive effect of pcDNA3-IL-2 could be blocked by naloxone, showing some relationship of the antinociceptive effect produced by IL-2 gene to the opioid receptors. It is hoped that the new delivery approach of a single intrathecal injection of the IL-2 gene described here may be of some practical use as a part of a gene therapy for treating neuropathic pain.

Immunotherapy of established tumors in mice by intratumoral injection of an adenovirus vector harboring the human IL-2 cDNA: induction of CD8(+) T-cell immunity and NK activity.

Intratumoral (i.t.) injections of an adenovirus encoding the human interleukin-2 (IL-2) under the control of the RSV (Ad-pRSV-IL-2) or CMV (Ad-pCMV-IL-2) promoter were performed in established mastocytoma P815 tumors in B6D2 mice. Both early and long-term survival were found increased in mice treated with Ad-pCMV-IL-2 as compared with those obtained with Ad-pRSV-IL-2: tumor regression was observed in 30--50% of mice for the former and 5--15% for the latter. Difference in efficacy between the two vectors was directly correlated to the amount of IL-2 produced i.t. between 24 and 48 hours postinjection, which reached 10--20 ng/tumor for Ad-pCMV-IL-2 and 0.3--0.5 ng/tumor for Ad-pRSV-IL-2. In both cases, expression in the tumor was clearly detectable for a period of 7--10 days postinjection. Serum IL-2 was not detectable in mice treated with Ad-pRSV-IL-2, whereas expression peaked at a total of 1--2 ng at 24 hours but declined very rapidly in the Ad-pCMV-IL-2-treated group. Constant production of IL-2 inside the tumor was necessary for successful therapy because i.t. injections of recombinant IL-2 at levels up to 1 microg for five consecutive days did not lead to antitumoral activity. Evidence of induced systemic immunity following Ad-pCMV-IL-2 injections was obtained from rechallenge experiments in which tumor-free mice after treatment rejected a subsequent contralateral injection of a lethal dose of P815 tumor cells and from the observation that regression of nontreated tumors occurred in animals bearing bilateral tumors that were treated i.t. in a single tumor with Ad-pCMV-IL-2. P815-specific cytotoxic T lymphocytes (CTL) were found specifically in spleen cells from cured mice or rechallenged mice but not in control mice. Interestingly, limiting dilution analysis of anti-P815 CTL precursor (CTLp) frequency revealed a significant increase in mice cured of their tumor as compared to that obtained in naive mice or control mice treated or not with Ad-IL-2 but whose tumor was growing. In vivo depletion of T-cell subsets, as well as natural killer cells at the time of i.t. injections with Ad-pCMV-IL-2, demonstrated that both CD8(+) T cells and natural killer cells, but not CD4(+) T cells, were required for successful therapy. Finally, mice preimmunized with Ad-null viruses were severely compromised in their capacity to eradicate established P815 tumors after Ad-pCMV-IL-2 therapy, at least when neutralizing antibody titers reached a critical level.

Recombinant adenoviral vectors have adjuvant activity and stimulate T cell responses against tumor cells.

The host-immune response against adenoviruses forms a major obstacle for their use as gene therapy vectors for treatment of genetic defects. None the less, they are the preferred vectors for in vivo gene transfer in experimental gene therapy protocols for cancer. In this article we demonstrate the antitumor efficacy of adenovirus-mediated transfer of human interleukin-2 cDNA in the rat-CC531 model for hepatic metastases of colorectal cancer: intratumoral administration of 10 plaque-forming units of the hlL-2-expressing adenoviral vector, AdCAIL-2, resulted in a cessation of tumor growth in 80% of the injected tumors. In control groups receiving AdCnull, a vector with the same viral backbone, but lacking transgene expression, none of the tumors responded. However, intratumoral treatment with this vector significantly enhanced tumor regression induced by systemic IL-2 protein treatment, which was used as a positive control. In addition we show, by performing delayed-type of hypersensitivity assays, that AdCnull when injected intratumorally enhances recognition of tumor antigens by T lymphocytes to the same extent as intratumoral treatment with the IL-2-expressing vector. The replication-deficient adenoviruses appear to have a therapeutic advantage in cytokine-mediated immunotherapy: even adenovirus vectors that do not express a transgene, show adjuvant activity and stimulate an antitumor immune response.

In vivo introduction of the interleukin 6 gene into human keratinocytes: induction of epidermal proliferation by the fully spliced form of interleukin 6, but not by the alternatively spliced form.

The achievement of keratinocyte gene therapy in clinical practice requires fundamental experiments using human keratinocytes or skin. We have recently demonstrated that the in vivo introduction of the interleukin 6 (IL-6) gene into rat keratinocytes induces epidermal proliferation and lymphocyte infiltration into the skin. In this study, we first amplified the human IL-6 cDNA from oligo-dT-primed keratinocyte cDNA and then detected the fully spliced (FS) form and the alternatively spliced (AS) form of IL-6 cDNA. Sequence analysis showed that the AS form, which was composed of the IL-6 coding region with all of exon II deleted except for the first guanine, was identical to that reported to be present in lymphocytes. We constructed the expression vectors phIL6 of the FS form and phIL6S of the AS form. We transplanted human skin onto nude rats and introduced phIL6 and phIL6S into the human keratinocytes using the naked DNA method. Keratinocytes prepared 24 h after introduction from the areas treated with them were examined by reverse transcriptase (RT)-PCR and enzyme linked immunosorbent assay (ELISA). RT-PCR showed that the amounts of FS IL-6 mRNA and AS IL-6 mRNA were similar, whereas the ELISA showed that the amount of FS IL-6 peptide was four times that of the AS IL-6 peptide. Histological examination 48 h after introduction showed that the FS form had induced epidermal proliferation, whereas the AS form had not. The epidermal thickening without lymphocyte infiltration induce by the FS form indicates that keratinocyte proliferation is caused by a direct effect of overexpressed IL-6, and not by a secondary effect of infiltrating lymphocytes. This is the first report of the introduction of a human gene into human keratinocytes to produce a biologically active transgenic gene product in human skin using the naked DNA method.

Receptor-mediated interleukin-2 gene transfer into human hepatoma cells.

Receptor-mediated gene delivery is an attractive method for gene transfer in vitro and shows promise for in vivo gene therapy applications. In the current study, we have selected the cytokine interleukin-2 (IL-2) gene to explore the feasibility of receptor-mediated gene transfer into human hepatocellular carcinoma HepG2 cells, using Epstein-Barr virus (EBV)-based vectors. We have developed a targeted DNA delivery system for the treatment of liver cancer by gene therapy. This system utilizes the hepatocyte-specific asialoglycoprotein receptor, which is uniquely expressed on liver cell membranes but not present on other cell types. Galactosylated histone, a ligand to the asialoglycoprotein receptors, was synthesized, and a new EBV-based expression vector bearing the human IL-2 cDNA was constructed and conjugated to the ligand through ionic interactions. The ligand/IL-2 DNA complex was able to bind specifically to cell-surface receptors on the target cell and, when incubated with HepG2 cells, resulted in elevated levels of IL-2 gene expression. These results indicate that therapeutic genes like IL-2 in ligand/DNA complex can be transferred into hepatoma cells via the hepatocyte receptor. This study constitutes an encouraging first step in the assessment of receptor-mediated gene transfer as a technique for gene therapy in liver cancer.

Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy.

NK-92 is a highly cytotoxic natural killer (NK) tumor cell line that possesses properties that make it an excellent candidate for adoptive cellular immunotherapy. However, the cytotoxicity of NK cells is dependent on cytokines such as interleukin 2 (IL-2). Although NK-92 cells maintain cytotoxicity for a time after withdrawal of IL-2, clinical use will probably require prolonged treatment with fully activated cells to eliminate disease effectively. The ability to support cytotoxic cells with exogenously administered IL-2 is limited by associated toxicity. Therefore, we describe the transfection of the IL-2-dependent NK-92 cell line with human IL-2 (hIL-2) cDNA by particle-mediated gene transfer to create two IL-2-independent variants, NK-92MI and NK-92 CI, and describe their characterization and comparison with parental cells. Both variants were shown to contain, express, and synthesize the hIL-2 cDNA. IL-2 synthesis was higher in NK-92MI cells compared with NK-92CI cells, with no expression in parental cells. Functionally, the cytotoxicity of all three cell lines was similar and coincubation with IL-2-independent variants did not affect hematopoietic progenitor cells. NK-92MI and NK-92CI cells were more radiosensitive than NK-92 cells, with proliferation inhibited at lower radiation doses and increased morality and decreased cytotoxicity compared with parental cells. Data presented here show that we have created by particle-mediated gene transfer two IL-2-independent variants of NK-92 that are identical to parental cells in virtually all respects, including high cytotoxic activity. The nonviral transfection of these cells makes them suitable for clinical applications. These IL-2-independent cells should allow prolonged treatment with fully active natural killer cells without the need for exogenous IL-2 support.

Specific targeting of cytokine-secreting cells: a bispecific diabody recognizing human interleukin-6 and CD3 induces T cell-mediated killing.

Cytokines have been implicated in the pathophysiology of many diseases. Although there have been many attempts to neutralize the activity of cytokines in vivo and in vitro, no strategies have been developed to specifically eliminate cells that overexpress cytokines. Considering the fact that cytokines in part remain cell associated on secretion, we have constructed a bispecific diabody consisting of a nonneutralizing scFv antibody recognizing human interleukin-6 (IL-6) and an scFv corresponding to the monoclonal antibody (mAb) OKT3, which recognizes and activates the human T cell receptor. Here we show that the diabody recognized both human IL-6 and human CD3. In the presence of human T cells, the diabody induced killing of human hepatoma cells that had been transfected with a human IL-6 cDNA. The extent of killing was dependent on the ratio of effector/target cells and increased with increasing concentrations of the diabody. Untransfected control cells or human hepatoma cells that secrete the IL-6-related cytokine leukemia inhibitory factor (LIF) remained unaffected. We conclude that diabodies recognizing cytokines can be used to specifically target cytokine-secreting cells.

Adenovirus-mediated expression of melanoma antigen gp75 as immunotherapy for metastatic melanoma.

Melanocyte differentiation antigens, such as the brown locus protein gp75, are potential biological targets for immunotherapy. We investigated whether expression of the murine gp75 cDNA mediated by an adenovirus (Ad) vector could induce melanoma rejection using this model self antigen that usually induces tolerance, and whether Ad vector-directed production of interleukin-2 (IL2) might augment this response. To evaluate this approach, Ad vectors were constructed containing the murine gp75 cDNA (Ad.gp75) and the human IL2 cDNA (Ad.IL2). Efficacy was evaluated in C57BI/6 mice challenged i.v. with 10(5) B16 cells, using the number of lung metastases as the efficacy parameter. Naive control mice developed 175 +/- 12 metastases by day 14. Controls receiving intranasal Ad.IL2 1 day after B16 cell injection, intraperitoneal (i.p.) mitomycin-C-treated B16 cells +/- i.p. Ad.IL2 before B16 cell challenge and Ad.beta gal-treated mice had similar numbers of metastases as controls (P > 0.1). In marked contrast, preimmunization with intradermal Ad.gp75 provided dramatic reduction in the number of lung metastases (52 +/- 7, 29% of control). Addition of regional (intranasal delivery to the lung) Ad.IL2 to intradermal Ad.gp75 preimmunization 1 day following tumor challenge provided further protection (18 +/- 6, 10% of control). Depletion of CD4+ and CD8+ T-cell subsets effectively blocked the protective effect seen following immunization. Adoptive transfer of macrophage-depleted splenocytes from Ad.gp75-immunized mice similarly afforded significant protection against B16 tumor cell challenge. Further, serum obtained 21 days following Ad.gp75 immunization showed no detectable anti-gp75 antibody by immunoprecipitation. These results suggest that immunization with Ad.gp75 induces cellular immune responses that are capable of rejecting B16 melanoma in a host that is usually tolerant to gp75 antigen.

Construction and characterization of retroviral vectors for interleukin-2 gene therapy.

Several investigators have employed interleukin-2 (IL-2) gene transfer to enhance the immunogenicity of tumor cell vaccines. We describe in this report the construction and characterization of retroviral vectors for IL-2 gene therapy. Human IL-2 cDNA with a chimeric rat preproinsulin/IL-2 DNA leader sequence was subcloned into the pLXSN (long terminal repeat promoter) and pLNCX (cytomegalovirus [CMV] promoter) vectors to generate the plasmids pLXSN-iIL2 and pLNCX-iIL2, respectively. Human IL-2 cDNA with a chimeric human tissue factor/IL-2 DNA leader sequence was utilized to construct the vector pLXSN-tIL2. The levels of IL-2 secreted by transduced tumor cells and fibroblasts were evaluated by enzyme-linked immunosorbent assay (ELISA) of culture supernatants and compared with those of normal peripheral blood mononuclear cells (PBMC) activated in vitro with calcium ionophore and phorbol 12-myristate 13-acetate. The highest levels of IL-2 secreted by transduced tumor cells (760 units/10(6) cells/24 h), adult fibroblasts (625 units/10(6) cells/24 h), and embryonic fibroblasts (3,975 units/10(6) cells/24 h) were 150- to 1,000-fold higher than than secreted by the activated PBMC (4 units/10(6) cells/24 h). Similar levels of IL-2 were expressed by human fibroblasts transduced with pLXSN vectors employing the preproinsulin (pLXSN-iIL2) or tissue factor (pLXSN-tIL2) leader sequences (range in IL-2 units/10(6) cells/24 h pLXSN-iIL2 = 375-625 vs. pLXSN-tIL2 = 90-440). Because IL-2-transduced cells for clinical applications are generally irradiated to prevent cellular proliferation, we evaluated the effects of radiation on IL-2 production. Radiation doses between 1,500 and 10,000 cGy resulted in gradual decreases in IL-2 secretion by transduced cells. The range of the decrease in IL-2 secretion was 7-11% by day 7, 0-29% by day 14, and 25-50% by day 35. For clinical applications, stable production of the vector in high concentrations is an important consideration. The retroviral vector pLXSN-tIL2 produced the highest viral titer and was chosen for further characterization. Southern blot analysis of SacI-digested genomic DNA from the LXSN-tIL2 producer cell line and SacI-digested pLXSN-tIL2 plasmid DNA revealed the expected 3.2-kbp fragment, suggesting the absence of transgene rearrangement and the suitability of this vector as a candidate for clinical applications.

Sequence of interleukin-2 isolated from human placental poly A+ RNA: possible role in maintenance of fetal allograft.

There are several cell types within the placenta that produce cytokines which can contribute to the regulatory mechanisms that ensure normal pregnancy. The immunological milieu at the maternofetal interface is considered to be crucial for survival of the fetus. Interleukin-2 (IL-2) is expressed by the syncytiotrophoblast, the cell layer between the mother and the fetus. IL-2 appears to be a key factor in maintenance of pregnancy. Therefore, it was important to determine the sequence of human placental interleukin-2. Direct sequencing of human placental IL-2 cDNA was determined for the coding region. Subclone sequencing was carried out for the 5'- and 3'-untranslated regions (5'-UTR and 3'-UTR). The 5'-UTR for human placental IL-2 cDNA is 294 bp, which is 247 nucleotides longer than that reported for cDNA IL-2 derived from T cells. The sequence of the coding region is identical to that reported for T cell IL-2, while sequence analysis of the polymerase chain reaction (PCR) product showed that the cDNA from the 3' end was the same as that reported for cDNA from T cells. Human placental IL-2 cDNA is 1,028 base pairs (excluding the poly A tail), which is 247 bp longer at the 5' end than that reported for IL-2 T cell cDNA. Therefore, the extended 5'-UTR of the placental IL-2 cDNA may be a consequence of alternative promoter utilization in the placenta.

Molecular cloning and expression of DNA encoding ovine interleukin 2

We have generated DNA encoding the mature form of ovine interleukin 2 (IL-2) by polymerase chain reaction (PCR) using primers complementary to sequences at the 5' and 3' ends of human, murine and bovine IL-2 cDNA. The predicted PCR product of 400 bp was ligated into the yeast Ty-P1 galactose-inducible expression vector pOGS40 which was used to transform yeast spheroplasts. The fusion protein, with a Factor Xa proteolytic cleavage site between ovine IL-2 and the P1 fusion partner, was expressed from galactose-induced transformed yeast. P1:IL-2 fusion protein, which self-assembles into virus-like particles (VLPs) due to the interaction of the P1 protein, was purified from lysates of mechanically disrupted yeast by centrifugation on a discontinuous sucrose gradient. Fusion protein was detected in Western blot analysis with polyclonal antisera raised to recombinant bovine IL-2. Soluble recombinant ovine IL-2 was released from the P1 fusion protein by cleavage with Factor Xa enzyme. After purification recombinant ovine IL-2 was functionally active as shown by its ability to support the proliferation of Con A-activated T cells and was capable of generating maedi visna virus-specific cytotoxic T cells from primed precursor cells. The availability of recombinant ovine IL-2 will greatly help the analysis of the specificity of pathogen-specific cells in the sheep.

Development of progressive kidney damage and myeloma kidney in interleukin-6 transgenic mice

Interleukin-6 (IL-6) is a pleiotropic cytokine that has been postulated as playing a role in the pathogenesis of multiple myeloma, chronic autoimmune diseases, and alcoholic liver cirrhosis. We generated transgenic mice carrying a fusion between the mouse metallothionein-I (MT-I) gene promoter and the human IL-6 cDNA. MT-I/IL-6 transgenics express IL-6 constitutively in the liver and secrete the cytokine in the blood. They show initially activation of acute-phase response genes and accumulation of alpha 2- and beta-globulins in the plasma, which is followed by polyclonal hypergammaglobulinemia. MT-I/IL-6 transgenics die between 12 to 20 weeks of age. Histologic examination of transgenic animals at different ages and after necropsy showed, as expected from previous studies of IL-6 disregulation in vivo, an increase in the number of megakaryocytes in the spleen and bone marrow and, at later stages, IgG plasmacytosis in the spleen, lymph nodes, and thymus. However, no plasma cell infiltration was detected in other organs. The distinguishing feature of MT-I/IL-6 transgenics is the development of a progressive kidney pathology, in which the initial membranous glomerulonephritis is followed by focal glomerulosclerosis and finally by extensive tubular damage that reproduces the damage observed in patients at terminal stages of multiple myeloma (myeloma kidney). The pathogenetic role of IL-6 overproduction and of the resulting serum protein overload in the kidney damage is discussed.

Human lung tumor cell secretion of interleukin-2 for protection against tumor engraftment.

Lung cancer continues to claim large numbers of human lives each year despite advances made in conventional therapies. The use of biologic response modifiers to modulate the immune system against human tumors is an alternate form of immunotherapy. Interleukin-2 (IL-2), or T-cell growth factor, is an important modulator of activated T cells. We show here that tumor cells transduced with human IL-2 cDNA provide protective immunity against engraftment of IL-2-secreting, as well as parental non-IL-2-secreting, tumor cells in vivo. In an attempt to increase the antigen-induced proliferation and cytotoxicity T cells within the vicinity of tumor antigen, we have transduced human lung tumor cell lines (generated from whole tumor specimens obtained fresh from the operating room) with a vector containing the IL-2 gene. Cell lines secreting 0.5-20 Cetus units/ml of IL-2 were generated. Control cell lines were similarly established using the same retroviral vector containing the gene for adenosine deaminase (ADA). The growth of tumor xenografts of the vector-modified cell lines was observed in severe combined immunodeficient (scid) mice. Using C.B-17 scid mice, we have observed that the local secretion of IL-2 by these human lung tumor cell lines will prevent engraftment of that tumor into scid mice. The parental tumor as well as the tumor containing the ADA gene grow aggressively in the scid mouse. Growth arrest also correlated strongly with the amount of IL-2 secreted by the tumor cells. The local secretion of IL-2 by the transduced cell line will abrogate the tumorigenicity of the parental cell line as well as an allogeneic tumor. The inhibition of growth occurs only when the tumors are placed in close proximity to each other. After gamma irradiation, transduced tumor cells will continue to secrete IL-2. These results indicate that (a) human lung tumor cell lines can be transduced with IL-2-containing retroviral vectors; (b) local and sustained release of IL-2 will induce an antitumor response by the host against the IL-2-secreting as well as the control tumor cells; (c) secretion of IL-2 continues after the cells are irradiated. This study suggests that cytokine-secreting human lung tumors may be used in vaccination protocols for cancer patients.

Generation of interleukin-2-secreting melanoma cell populations from resected metastatic tumors.

This study aimed to determine the feasibility of producing patient-specific, interleukin-2 (IL-2)-secreting tumor cell vaccines for the treatment of metastatic melanoma. Primary tumor cell cultures were established from 26/33 resected metastatic melanoma samples. Recombinant retroviral gene transfer and expression in these cultures was optimized using an amphotropic, defective retrovirus carrying the LacZ gene. All cell cultures were infectable; those that proliferated more rapidly were infected at a higher frequency. Addition of fibroblast growth factor to the culture medium increased the rate of cell proliferation and the efficiency of infection. A single infection with an identical retrovirus carrying a human IL-2 cDNA resulted in the generation of unselected cell populations secreting up to 300 units IL-2/10(6) cells.48 hr. Multiple infections increased the level of IL-2 secretion to 5,000 units/10(6) cells.48 hr. The recombinant viral genome could be detected at approximately single copy in the multiply infected cells; no helper virus was detected. IL-2 secretion from infected cultures was maintained following cryopreservation and x-irradiation. These data demonstrate that heterogeneous tumor cell populations secreting IL-2 can be generated from individual patients to be used as autologous, irradiated cell vaccines.

The production of strains highly expressing human interleukin-2 cDNA.

The plasmid pTLIL-2, which only directed a low-level expression of human interleukin 2(IL-2) cDNA in E. coli, was reconstructed: a series of deletions were made in 3' non-coding region of human IL-2 cDNA, and 7 recombinant plasmids with different deletions were selected, on the other hand, a Tac promoter sequence from pDR540 was inserted to upstream of IL-2 cDNA in pTLIL-2 so that pTLIL-2DT, which contains double Tac promoters, was constructed. And then, E. coli JM103 was transformed with the above 8 recombinant plasmids respectively. The expression efficiency of IL-2 cDNA in E. coli transformed with different plasmids was evaluated by means of SDS-PAGE and measuring 3H-TdR incorporation of IL-2-dependent activated T lymphocytes in the presence of bacterial extracts. Three engineering strains with high efficiency of IL-2 expression were selected, and all of these strains could produce recombinant IL-2(rIL-2) 4 times more than E. coil JM103/pTLIL-2.

Cells transfected with human interleukin 6 cDNA acquire binding sites for the hepatitis B virus envelope protein.

Earlier studies revealed that human interleukin 6 (IL-6) contains recognition sites for the hepatitis B virus (HBV) envelope (env) protein, and that IL-6 and anti-IL-6 antibodies, respectively, inhibited the interaction of cells expressing a receptor for HBV with the preS(21-47) segment of the HBV env protein, encompassing the complementary attachment site for IL-6. This suggested that IL-6 mediates HBV-cell interactions. We report that: (a) Chinese hamster ovary cells transfected with human IL-6 cDNA and Spodoptera frugiperda ovarian insect cells infected with recombinant baculovirus carrying human IL-6 cDNA expressed receptors for the preS(21-47) region of the HBV env protein, indicating that expression of IL-6 on the surface of cells is sufficient to endow them with receptors for HBV. (b) Among peptides covering the entire sequence of human IL-6 and the corresponding antipeptide antibodies, the peptide IL-6[35-66] and anti-IL-6[35-66] most effectively inhibited the interaction between human hepatoma HepG2 cells and the preS(21-47) ligand, suggesting that this region of the human IL-6 sequence encompasses a binding site for the HBV env protein. (c) Studies with replacement set peptides from the preS(21-47) sequence indicated that residues 21-25, 28, 31, 33-35, 39, and 43-45 can be replaced by alanine (serine) residues, while all the other residues are essential for maintaining the cell receptor/IL-6 binding activity. Further delineation of complementary sites on IL-6 and on the HBV env protein may contribute to the design of compounds inhibiting HBV replication.

Generation of plasmacytomas with the chromosomal translocation t(12;15) in interleukin 6 transgenic mice.

The mechanisms through which pristane or mineral oil can induce plasmacytomas in BALB/c or NZB mice are not fully understood, but involvement of interleukin 6 (IL-6), a growth factor for plasmacytomas and myelomas, has been strongly suggested. To clarify the role of IL-6 in plasmacytomagenesis, a human IL-6 cDNA was introduced into mouse germ lines under the transcriptional control of the murine major histocompatibility complex class I (H-2Ld) promoter. IL-6 transgenic mice of C57BL/6 origin developed a massive plasmacytosis but not plasmacytomas. However, introduction of BALB/c genetic background into IL-6 transgenic mice could generate monoclonal transplantable plasmacytomas with the chromosomal translocation t(12;15). These results provide firm evidence of the critical role of IL-6 in the plasmacytoma development.