Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE.

During the past two decades, recombinant human interleukin-12 (rhIL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies. Purity is a critical quality attribute (CQA) in the quality control system of rhIL-12. In our study, rhIL-12 bulks from manufacturer B showed a different pattern in non-reduced SDS-PAGE compared with size-exclusion chromatography (SEC)-HPLC. A small fragment was only detected in non-reduced SDS-PAGE but not in SEC-HPLC. The results of UPLC/MS and N-terminal sequencing confirmed that the small fragment was a 261⁻306 amino acid sequence of a p40 subunit of IL-12. The cleavage occurs between Lys260 and Arg261, a basic rich region. With the presence of 0.2% SDS, the small fragment appeared in both native PAGE and in SEC-HPLC, suggesting that it is bound to the remaining part of the IL-12 non-covalently, and is dissociated in a denatured environment. The results of a bioassay showed that the fractured rhIL-12 proteins had deficient biological activity. These findings provide an important reference for the quality control of the production process and the final products of rhIL-12.

Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag.

Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous. Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ∼98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichroism, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31±0.05 mg/mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app)=69±1 µM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics.

Tools of the trade: developing antibody-based detection capabilities for recombinant proteins.

Protein-specific antibodies serve as critical tools for detection, quantification, and characterization of recombinant proteins. Perhaps the most important and widely used antibody-based procedures for recombinant protein applications are Western immunoblotting and enzyme-linked immunosorbent assays (ELISAs). These analyses require well-characterized, sensitive, and high-affinity antibodies that specifically and selectively recognize the recombinant target protein in the native or denatured form. Although the number of commercially available antibodies is quite substantial and rapidly growing, the appropriate antibody tools for many applications currently do not exist. In this chapter, strategies to develop and characterize both polyclonal and monoclonal antibodies directed against a specific protein of interest are discussed. Experimental strategies and methods are presented for producing and selecting the best antibodies and optimizing protocols for Western analyses, ELISAs, and other applications. Once antibody and procedure optimization is completed to ensure specificity, sensitivity, accuracy, and reliability, these immune-based approaches can now serve as powerful and enabling tools in the characterization, detection and diagnostics, structure/function analysis, and quality assessment of recombinant proteins.

Efficient plant-based production of chicken interleukin-12 yields a strong immunostimulatory cytokine.

Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic in mammals. Therapeutic strategies to develop mammalian IL-12 as a vaccine adjuvant/immunomodulator for promoting cellular immunity and establishing a Th1-biased immune response further support the potential value of ChIL-12. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. We have expressed, characterized, and purified biologically active ChIL-12 in plants using a rapid Agrobacterium-mediated tobacco plant-based transient expression system. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of chicken IL-12 (ChIL-12). A histidine 6x tag was used for identity and purification of ChIL-12(His) protein. Our results demonstrated precise cleavage of the endogenous chicken p40 signal peptide in plants as well as addition of N-linked glycans. Biological activity was confirmed in vitro by interferon-gamma secretion of ChIL-12-treated chicken splenocytes. In addition, splenocytes treated with ChIL-12 expressed with or without the His tag demonstrated comparable ChIFN-gamma induction. These studies indicate that plant-based platforms for bioproduction of complex pharmaceutical proteins produce functional ChIL-12 and provide key advantages in safety, scale, and cost-effective platform for veterinary vaccine and therapeutic applications.

[Construction and screen of recombinant BCG strain expressing and secreting human interleukin 12 protein].

OBJECTIVE: To screen and construct the recombinant Bacillus Calmette-Guerin (rBCG) strain expressing and secreting the human interleukin 12 (rBCG-12). METHODS: IL-12 complete gene including p40 and p35 subunits was amplified by PCR from a plasmid pORF-h IL-12 and cloned into E. coli-Mycobacteria shuttle vector pMV361. The recombinant vector was named as rpMV-IL-12. rpMV-IL-12 was confirmed by DNA sequencing, and then rpMV-IL-12 was used, by electroporation way, to transform the BCG for getting the recombinant rBCG-12 strain. The positive rBCG-12 clone was screened and identified by kanamycin resistance and IL-12 target gene PCR amplification. The IL-12 protein expression of rBCG-12 was induced with heat shock reaction. Then rBCG-12 culture supernatant and bacterial precipitation were collected respectively and analyzed with SDS-PAGE electrophoresis for their protein components. RESULTS: The recombinant shuttle plasmid rpMV-IL-12 was constructed successfully. The sequence of amplified IL-12 gene was consistent with GenBank report. By SDS-PAGE electrophoresis, it was confirmed that the IL-12 protein could express and secrete into the supernatant of rBCG-12 strain culture. CONCLUSION: The recombinant BCG strain expressing human IL-12 protein, rBCG-12 strain, is constructed and screened successfully. It is the foundation for further research on rBCG-12 immune function.

Identification and molecular cloning of functional chicken IL-12.

By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains. These molecules are the first discovered nonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively. The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, while expression was not detected in stimulated primary chicken embryo fibroblast cells. Following transient expression of both molecules in COS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammalian counterparts. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomeric IL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-gamma synthesis and proliferative activity of freshly exposed chicken splenocytes. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates, despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolutionary dichotomy of birds and mammals >300 million years ago. In this article, we describe the first nonmammalian IL-12 molecule and show that this chicken IL-12 molecule is bioactive.

Production and in vivo testing of a recombinant bovine IL-12 as an adjuvant for Salmonella typhimurium vaccination in calves.

A recombinant bovine interleukin-12 (boIL-12) that contains a histidine hexamer, rboIL-12His, was produced, purified and administered to calves. We first tried the purification of heterodimer IL-12 from a mixture of p40 homodimer, p40 monomer, and p40-p35 heterodimer with a p35 subunit tagged with a histidine hexamar at its C-terminal (p35His). A recombinant baculovirus expressing p35His was generated and used for superinfection with a recombinant baculovirus expressing p40 subunit. The expressed subunits, p40 and p35His, were assembled into a 70kDa heterodimer in insect cells, released into culture medium, and then purified using a nickel chelate column. The purified rboIL-12His was bioactive for induction of IFN-gamma in bovine peripheral blood mononuclear cells (PBMCs) in vitro. The purified rboIL-12His was then administered to calves with inactivated Salmonella Typhimurium (ST). When sera were assayed by ELISA, specific anti-ST IgG1 antibodies were detected in all ST immunized calves, but, specific anti-ST IgG2 antibodies were detected only in calves administered ST along with rboIL-12His, indicating a possible switch to a Th1 response. Administration of commercially available Salmonella vaccine did not elicit IgG2 antibodies in calves. These results suggest that co-administration of IL-12 with inactivated ST cells could induce a Th1-type response in calves.

Molecular cloning of gerbil interleukin 12 and its expression as a bioactive single-chain protein.

Complementary DNAs coding for gerbil interleukin 12 (IL-12) p40/p35 subunits were cloned by a combination of cross species reverse transcriptase-polymerase chain reaction (RT-PCR) and 3' rapid amplification of cDNA ends (RACE) techniques. IL-12 p40/p35 had 79% nucleotide identity and 81% amino acid homology to mouse IL-12 p40/p35. The p40/p35 subunits were expressed as a single polypeptide separated by a short hinge sequence that allowed for proper folding and assembly. COS-7 cells transfected with DNA encoding the single-chain gerbil IL-12 (pSCjIL12) secreted high levels of the protein which stimulated proliferation of ConA-activated gerbil spleen lymphoblasts in a dose-dependent manner.

Exposure to ultraviolet radiation causes dendritic cells/macrophages to secrete immune-suppressive IL-12p40 homodimers.

UV-induced immune suppression is a risk factor for sunlight-induced skin cancer. Exposure to UV radiation has been shown to suppress the rejection of highly antigenic UV-induced skin cancers, suppresses delayed and contact hypersensitivity, and depress the ability of dendritic cells to present Ag to T cells. One consequence of UV exposure is altered activation of T cell subsets. APCs from UV-irradiated mice fail to present Ag to Th1 T cells; however, Ag presentation to Th2 T cells is normal. While this has been known for some time, the mechanism behind the preferential suppression of Th1 cell activation has yet to be explained. We tested the hypothesis that this selective impairment of APC function results from altered cytokine production. We found that dendritic cells/macrophages (DC/Mphi) from UV-irradiated mice failed to secrete biologically active IL-12 following in vitro stimulation with LPS. Instead, DC/Mphi isolated from the lymphoid organs of UV-irradiated mice secreted IL-12p40 homodimer, a natural antagonist of biologically active IL-12. Furthermore, when culture supernatants from UV-derived DC/Mphi were added to IL-12-activated T cells, IFN-gamma secretion was totally suppressed, indicating that the IL-12p40 homodimer found in the supernatant fluid was biologically active. We suggest that by suppressing DC/Mphi IL-12p70 secretion while promoting IL-12p40 homodimer secretion, UV exposure preferentially suppress the activation of Th1 cells, thereby suppressing Th-1 cell-driven inflammatory immune reactions.

IL-10 is induced in the reperfused myocardium and may modulate the reaction to injury.

Reperfusion of the ischemic myocardium is associated with a dramatic inflammatory response leading to TNF-alpha release, IL-6 induction, and subsequent neutrophil-mediated cytotoxic injury. Because inflammation is also an important factor in cardiac repair, we hypothesized the presence of components of the inflammatory reaction with a possible role in suppressing acute injury. Thus, we investigated the role of IL-10, an anti-inflammatory cytokine capable of modulating extracellular matrix biosynthesis, following an experimental canine myocardial infarction. Using our canine model of myocardial ischemia and reperfusion, we demonstrated significant up-regulation of IL-10 mRNA and protein in the ischemic and reperfused myocardium. IL-10 expression was first detected at 5 h and peaked following 96-120 h of reperfusion. In contrast, IL-4 and IL-13, also associated with suppression of acute inflammation and macrophage deactivation, were not expressed. In the ischemic canine heart, CD5-positive lymphocytes were the predominant source of IL-10 in the myocardial infarct. In the absence of reperfusion, no significant induction of IL-10 mRNA was noted. In addition, IL-12, a Th1-related cytokine associated with macrophage activation, was not detected in the ischemic myocardium. In vitro experiments demonstrated late postischemic cardiac-lymph-induced tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression in isolated canine mononuclear cells. This effect was inhibited when the incubation contained a neutralizing Ab to IL-10. Our findings suggest that lymphocytes infiltrating the ischemic and reperfused myocardium express IL-10 and may have a significant role in healing by modulating mononuclear cell phenotype and inducing TIMP-1 expression.

IL-12p70 production by Leishmania major-harboring human dendritic cells is a CD40/CD40 ligand-dependent process.

Leishmaniasis, a vector-borne parasitic disease, is transmitted during a sandfly blood meal as the parasite is delivered into the dermis. The parasite displays a unique immune evasion mechanism: prevention of IL-12 production within its host cell, the macrophage (i.e., where it differentiates and multiplies). Given the close proximity of skin dendritic cells (DC) to the site of parasite delivery, their critical role in initiating immune responses and the self-healing nature of Leishmania major (Lm) infection, we examined the interaction between myeloid-derived human DC and Lm metacyclic promastigotes (infectious-stage parasites) to model the early "natural" events of infection. We found that DC can take up Lm and, after this internalization, undergo changes in surface phenotype suggesting "maturation". Despite the intracellular location of the parasite and resultant up-regulation of costimulatory and class II molecules, there was no detectable cytokine release by these Lm-harboring DC. However, using intracellular staining and flow cytometry to analyze cytokine production at the single-cell level, we found that Lm-harboring DC, but not monocytes, produce large amounts of IL-12p70 in a CD40 ligand (CD40L)-dependent manner. Finally, DC generated from mononuclear cells from patients with cutaneous leishmaniasis (Lm), once loaded with live metacyclic promastigotes, were found to reactivate autologous primed T lymphocytes and induce a CD40L-dependent IFN-gamma response. Our results link the required CD40/CD40L interactions for healing with DC-derived IL-12p70 production and provide a mechanism to explain the genesis of a protective T cell-mediated response in the face of local immune evasion within the macrophage at the site of Leishmania delivery.

RT-PCR amplification of various canine cytokines and so-called house-keeping genes in a species-specific macrophage cell line (DH82) and canine peripheral blood leukocytes.

Total ribonucleic acid (RNA) isolated from a continuous canine macrophage cell line (DH82) was used in reverse transcription polymerase chain reactions (RT-PCR) for the detection of transcripts of interleukin (IL)-8, -12, and tumour necrosis factor-alpha (TNF). Three different methods of RNA isolation (standard guanidinium-thiocyanate method with and without application of RNA matrix, and boiling) were used and compared in regard to RT-PCR results. The most suitable method was used to establish RT-PCR amplification of mRNA transcripts of IL-2, -10, and interferon-gamma (IFN) in RNA isolated from canine peripheral blood leukocytes. Integrity of RNA isolates was ensured by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or beta-actin, IL-8, -12, and TNF were amplified from RNA isolated by various methods. Use of guanidinium-thiocyanate with and without RNA matrix gave the most consistent results. Boiling as a mean of RNA isolation was quick and easy, but the RT-PCR results were extremely variable and multiple smaller bands were observed in the agarose gel in some preparations. IL-2, -10 and IFN transcripts were amplified from RNA isolated with guanidinium-thiocyanate from leukocytes stimulated with concanavalin A. DNase-treatment of RNA isolates was necessary to assure the destruction of genomic DNA and to avoid amplification of genomic sequences. This was especially a problem when using primers for GAPDH, beta-actin, IL-12, and TNF. Lack of DNase-treatment may lead to false positive results. This may be especially a problem when amplification of so-called house-keeping genes is used as internal control for RNA integrity. These findings demonstrated that isolation of total RNA with guanidinium-thiocyanate followed by DNase-treatment gave reliable and consistent results for detection of cytokine transcripts by RT-PCR in a canine macrophage cell line and canine peripheral blood leukocytes.

Human eosinophils produce biologically active IL-12: implications for control of T cell responses.

The present study assessed the capacity of eosinophils (EOS) to synthesize the cytokine IL-12. Blood-derived, highly purified human EOS from six atopic patients and two nonatopic individuals were treated in culture with IL-4, IL-5, granulocyte-macrophage CSF, IFN-gamma, TNF-alpha, IL-1alpha, RANTES, and complement 5a, respectively. The expression of both IL-12 protein and mRNAs for the p35 and p40 IL-12 subunits was strongly induced in all donors by the Th2-like cytokines IL-4 and granulocyte-macrophage CSF and was also moderately induced by TNF-alpha and IL-1alpha. IL-5 treatment resulted in IL-12 synthesis in four atopic donors and one nonatopic donor, whereas IFN-gamma induced IL-12 synthesis in only two atopic donors. In contrast, RANTES exclusively induced mRNA for the p40 subunit without detectable protein release, and complement 5a had no effect on IL-12 mRNA or protein expression. EOS-derived IL-12 was biologically active, because supernatants derived from IL-4-treated EOS superinduced the Con A-induced expression of IFN-gamma by a human Th1-like T cell line. This activity was neutralized by anti-IL-12 Abs. In conclusion, EOS secrete biologically active IL-12 after treatment with selected cytokines, which mainly represent the Th2-like type. Consequently, EOS may promote a switch from Th2-like to Th1-like immune responses in atopic and parasitic diseases.

Molecular cloning and mRNA expression of porcine interleukin-12.

Interleukin (IL)-12 is a heterodimeric cytokine consisting of 35 and 40 kDa subunits, produced primarily by phagocytic cells in response to bacteria or bacterial products. IL-12 is important in the regulation of both innate and antigen-specific immunity through its stimulatory effects on NK cells and cytotoxic lymphocytes. Reverse transcriptase-polymerase chain reaction with primers derived from human sequence was used to clone the p35 and p40 subunits of porcine IL-12. Predicted amino acid sequences for both subunits are approximately 85% homologous to their human cognates but contain a 3aa addition and a 4aa deletion in p35 and p40 subunits, respectively. The high degree of similarity indicates the proteins may be cross reactive, an important consideration in pig-human xenotransplantation. Both subunits of pIL-12 are constitutively expressed in a variety of porcine tissues. Highest levels of the p40 subunit were found in lymphoid tissues including inguinal and mesenteric lymph nodes, Peyer's patches, spleen and thymus. The p35 subunit was also detected in these tissues. Levels of mRNA encoding the p40 subunit, but not the p35 subunit, were rapidly increased in alveolar macrophages stimulated with lipopolysaccharide or killed Staphylococcus aureus. Thus, the heterodimeric subunits appear to be differentially regulated at the transcriptional level. Since p40 also self-associates to form inactive homodimers, differential expression may be a mechanism for regulating IL-12 activity.

Cloning of a partial cDNA for rat interleukin-12 (IL-12) and analysis of IL-12 expression in vivo.

Experimental models of autoimmunity in the rat may feature selective activation of either the Th1 or Th2 subset of helper T cells. Interleukin-12 (IL-12) is a key cytokine in the development of Th1 responses. In order to study IL-12 in the rat we used polymerase chain reaction (PCR) primers based on murine IL-12 to amplify a partial cDNA from rat tissue. The product was cloned and sequenced: it shows 94% nucleotide identity with the murine gene and 94% identity of predicted amino acid sequence. Primers based on the rat IL-12 sequence were used to analyse IL-12 expression in vivo using semi-quantitative PCR. We studied RNA from lymphoid tissues of two rat strains which differ in their response to mercuric chloride (HgCl2): Brown Norway (BN) rats develop autoimmunity with a predominant Th2 response; Lewis rats are resistant. Interleukin-12 expression was higher in Lewis than BN, and higher in spleen than lymph node. After HgCl2, IL-12 expression increased in BN towards the time when the autoimmune response autoregulates. Variation in baseline levels of IL-12 expression may account for the Th2 predisposition of BN rats compared to Lewis rats; IL-12 may play a role in the autoregulation of the Th2 response induced by HgCl2.

Molecular cloning and characterization of murine IL-12 genes.

IL-12 is a heterodimeric cytokine composed of two covalently linked chains, p40 and p35. p40 expression appears to be restricted to monocytes/macrophages and B cells and is highly regulated, while p35 is more ubiquitously and constitutively expressed. To investigate the mechanism involved in the regulation of IL-12 expression, we molecularly cloned and characterized the murine p40 and p35 genes. The p40 gene spans over 14 kb, consists of eight exons and seven introns, and was shown to be localized on chromosome 11A5-B2 by fluorescence in situ hybridization. A single major transcription initiation site was detected by primer extension analysis, and a TATA box was found at approximately 30 bp upstream from the transcription initiation site. The 5' flanking region preceding the transcription initiation site induced the enhanced expression of a promoterless reporter gene after LPS stimulation when transfected into a macrophage-like cell line. In contrast, the p35 gene spans over 8 kb and consists of seven exons and six introns on chromosome 6C, and multiple transcription initiation sites were detected. The 5' flanking region lacks canonical TATA and CAAT boxes at the appropriate position, but, instead, contains GC-rich sequences and constitutively mediated promoter activity when placed upstream of a promoterless reporter gene and transfected into a B cell lymphoma cell line. Thus, the characteristics of promoter regions of p40 and p35 genes are quite different, and this would account for the different regulations of p40 and p35 expression.