A Comparative Study of the Inhibitory Effects
Anakinra, the recombinant form of IL-1 receptor antagonist (IL-1Ra), has been approved for clinical use in the treatment of rheumatoid arthritis as the drug Kineret, but it must be administered daily by subcutaneous injection. Gene transfer may offer a more effective means of delivery. In this study, using prostaglandin E2 production as a measure of stimulation, we quantitatively compared the ability of anakinra, as well as that of IL-1Ra delivered by gene transfer, to inhibit the biologic actions of IL-1
. Human synovial fibroblast cultures were incubated with a range of doses of anakinra or HIG-82 cells genetically modified to constitutively express IL-1Ra. The cultures were then challenged with recombinant human IL-1
either simultaneously with addition of the source of IL-1Ra or 24 hours later. In a similar manner, the potencies of the two sources of IL-1Ra were compared when human synovial fibroblasts were challenged with IL-1
produced constitutively by genetically modified cells. No significant difference in inhibitory activity was observed between recombinant protein and IL-1Ra provided by the genetically modified cells, under static culture conditions, even following incubation for 4 days. However, under culture conditions that provided progressive dilution of the culture media, striking differences between these methods of protein delivery became readily apparent. Constitutive synthesis of IL-1Ra by the genetically modified cells provided sustained or increased protection from IL-1 stimulation over time, whereas the recombinant protein became progressively less effective. This was particularly evident under conditions of continuous IL-1
synthesis.
IL-1 has been implicated as a pathogenic mediator in numerous inflammatory and degenerative conditions, including rheumatoid arthritis (RA) and osteoarthritis (OA). The IL-1 receptor antagonist (IL-1Ra), a naturally occurring inhibitor of the biologic actions of IL-1, has obvious therapeutic potential in such diseases ; indeed recombinant human IL-1Ra (anakinra) has recently been approved for use in patients with RA as the drug Kineret (Amgen, Inc., Thousand Oaks, CA, USA).
Limitations of IL-1Ra as a pharmaceutical include its lack of oral availability and its short biologic half-life. This is why in clinical application Kineret must be administered by daily subcutaneous injection. Even then, it remains unlikely that a therapeutic concentration of IL-1Ra will be maintained between injections ; IL-1Ra is rapidly eliminated in the kidney, resulting in a serum half-life of 4-6 hours following intravenous injection into healthy, human volunteers. This problem is exacerbated by the pronounced spare receptor effect of IL-1. According to the literature it is necessary to maintain an IL-1Ra : IL-1 molar ratio of 10-100 or more to achieve a strong inhibitory effect.
We have proposed IL-1Ra gene transfer as a means of overcoming these problems. The advantages of IL-1Ra gene delivery include its ability to engender the continuous production of therapeutic concentrations of IL-1Ra at defined anatomic locations for extended periods of time - potentially for life. Moreover, it is theoretically possible to regulate levels of IL-1Ra gene expression in a manner commensurate with disease activity. IL-1Ra gene therapy has been evaluated in a number of different animal models of RA and OA, with extremely promising results. Indeed, a phase I human study of IL-1Ra gene therapy in RA was recently successfully completed.
During the preclinical development of IL-1Ra gene therapy, we often noticed that transfer of the IL-1Ra gene provided a far greater biologic effect than administration of the recombinant protein. An example is provided by the treatment of antigen-induced arthritis in rabbits. Lewthwaite and coworkers reported that repeated injection of recombinant human IL-1Ra had no effect in this model of RA beyond inhibition of the synovial fibrosis occurring in the chronic stage of the disease. Otani and colleagues, in contrast, observed a dramatic beneficial effect on cartilage matrix metabolism, and a moderate anti-inflammatory effect when administering IL-1Ra locally to joints via ex vivo gene transfer.
There exist several possible explanations for the improved effectiveness of IL-1Ra when delivered as a gene rather than as a recombinant protein. The most likely of these are as follows. First, gene transfer results in continuous, rather than intermittent, protein delivery, thus maintaining a constant supply of IL-1Ra at a concentration sufficient to inhibit the biologic actions of IL-1. Second, gene delivery produces a molecule that has been subjected to authentic post-translational processing. Because the recombinant molecule lacks glycosylation and has an extra amino-terminal methionine, the native molecule may have greater biologic potency than the recombinant one.
The present study was designed to compare quantitatively the relative effectiveness of these two avenues of protein delivery under controlled conditions in vitro. Cultures of primary human synovial fibroblasts (HSFs) were treated with human IL-1Ra, either administered as the recombinant protein or by co-culture with fibroblasts genetically engineered to express and secrete human IL-1Ra in a constitutive manner. Stimulation from human IL-1
was then provided by addition of recombinant IL-1
protein or by co-culture with fibroblasts genetically engineered to constitutively secrete high levels of human IL-1
. Using prostaglandin E 2 (PGE 2 ) levels in conditioned media as a readout of IL-1 stimulation in the respective cultures, protection from IL-1 stimulation by each method was evaluated under static and dynamic culture conditions, the latter of which were designed to resemble more closely the circumstance of an arthritic joint in which IL-1 is chronically produced.
The data suggest that the recombinant and transgenic molecules are similarly potent. Although the gene delivery procedure may benefit marginally from increased concentration at the cellular level, the advantage of gene transfer as a means of drug delivery arises from the sustained availability of IL-1Ra that this method permits.
Anakinra, the recombinant form of IL-1 receptor antagonist (IL-1Ra), has been approved for clinical use in the treatment of rheumatoid arthritis as the drug Kineret, but it must be administered daily by subcutaneous injection. Gene transfer may offer a more effective means of delivery. In this study, using prostaglandin E2 production as a measure of stimulation, we quantitatively compared the ability of anakinra, as well as that of IL-1Ra delivered by gene transfer, to inhibit the biologic actions of IL-1
. Human synovial fibroblast cultures were incubated with a range of doses of anakinra or HIG-82 cells genetically modified to constitutively express IL-1Ra. The cultures were then challenged with recombinant human IL-1
either simultaneously with addition of the source of IL-1Ra or 24 hours later. In a similar manner, the potencies of the two sources of IL-1Ra were compared when human synovial fibroblasts were challenged with IL-1
produced constitutively by genetically modified cells. No significant difference in inhibitory activity was observed between recombinant protein and IL-1Ra provided by the genetically modified cells, under static culture conditions, even following incubation for 4 days. However, under culture conditions that provided progressive dilution of the culture media, striking differences between these methods of protein delivery became readily apparent. Constitutive synthesis of IL-1Ra by the genetically modified cells provided sustained or increased protection from IL-1 stimulation over time, whereas the recombinant protein became progressively less effective. This was particularly evident under conditions of continuous IL-1
synthesis.
IL-1 has been implicated as a pathogenic mediator in numerous inflammatory and degenerative conditions, including rheumatoid arthritis (RA) and osteoarthritis (OA). The IL-1 receptor antagonist (IL-1Ra), a naturally occurring inhibitor of the biologic actions of IL-1, has obvious therapeutic potential in such diseases ; indeed recombinant human IL-1Ra (anakinra) has recently been approved for use in patients with RA as the drug Kineret (Amgen, Inc., Thousand Oaks, CA, USA).
Limitations of IL-1Ra as a pharmaceutical include its lack of oral availability and its short biologic half-life. This is why in clinical application Kineret must be administered by daily subcutaneous injection. Even then, it remains unlikely that a therapeutic concentration of IL-1Ra will be maintained between injections ; IL-1Ra is rapidly eliminated in the kidney, resulting in a serum half-life of 4-6 hours following intravenous injection into healthy, human volunteers. This problem is exacerbated by the pronounced spare receptor effect of IL-1. According to the literature it is necessary to maintain an IL-1Ra : IL-1 molar ratio of 10-100 or more to achieve a strong inhibitory effect.
We have proposed IL-1Ra gene transfer as a means of overcoming these problems. The advantages of IL-1Ra gene delivery include its ability to engender the continuous production of therapeutic concentrations of IL-1Ra at defined anatomic locations for extended periods of time - potentially for life. Moreover, it is theoretically possible to regulate levels of IL-1Ra gene expression in a manner commensurate with disease activity. IL-1Ra gene therapy has been evaluated in a number of different animal models of RA and OA, with extremely promising results. Indeed, a phase I human study of IL-1Ra gene therapy in RA was recently successfully completed.
During the preclinical development of IL-1Ra gene therapy, we often noticed that transfer of the IL-1Ra gene provided a far greater biologic effect than administration of the recombinant protein. An example is provided by the treatment of antigen-induced arthritis in rabbits. Lewthwaite and coworkers reported that repeated injection of recombinant human IL-1Ra had no effect in this model of RA beyond inhibition of the synovial fibrosis occurring in the chronic stage of the disease. Otani and colleagues, in contrast, observed a dramatic beneficial effect on cartilage matrix metabolism, and a moderate anti-inflammatory effect when administering IL-1Ra locally to joints via ex vivo gene transfer.
There exist several possible explanations for the improved effectiveness of IL-1Ra when delivered as a gene rather than as a recombinant protein. The most likely of these are as follows. First, gene transfer results in continuous, rather than intermittent, protein delivery, thus maintaining a constant supply of IL-1Ra at a concentration sufficient to inhibit the biologic actions of IL-1. Second, gene delivery produces a molecule that has been subjected to authentic post-translational processing. Because the recombinant molecule lacks glycosylation and has an extra amino-terminal methionine, the native molecule may have greater biologic potency than the recombinant one.
The present study was designed to compare quantitatively the relative effectiveness of these two avenues of protein delivery under controlled conditions in vitro. Cultures of primary human synovial fibroblasts (HSFs) were treated with human IL-1Ra, either administered as the recombinant protein or by co-culture with fibroblasts genetically engineered to express and secrete human IL-1Ra in a constitutive manner. Stimulation from human IL-1
was then provided by addition of recombinant IL-1
protein or by co-culture with fibroblasts genetically engineered to constitutively secrete high levels of human IL-1
. Using prostaglandin E 2 (PGE 2 ) levels in conditioned media as a readout of IL-1 stimulation in the respective cultures, protection from IL-1 stimulation by each method was evaluated under static and dynamic culture conditions, the latter of which were designed to resemble more closely the circumstance of an arthritic joint in which IL-1 is chronically produced.
The data suggest that the recombinant and transgenic molecules are similarly potent. Although the gene delivery procedure may benefit marginally from increased concentration at the cellular level, the advantage of gene transfer as a means of drug delivery arises from the sustained availability of IL-1Ra that this method permits.