The availability of highly purified recombinant cytokines has facilitated advances in immunological research and the development of cytokine-based immunotherapies. Several prokaryotic and eukaryotic systems have been used for the production of recombinant cytokines. Alternative approaches to cytokine production using retroviral, adenoviral, and direct DNA gene transfer also have met with success. Genetic manipulation of autologous or allogeneic cells transduced to secrete cytokines also was highly effective in several murine models (19
). No genetically engineered biotinylated cytokines are commercially available at present.
The PinPoint vector system allows the production of unique, monobiotinylated proteins which can be affinity purified using SoftLink soft-release avidin resin. In addition, the biotinylation tag can be easily removed enzymatically to produce nonbiotinylated product, should this be required. Therefore, we attempted to use this system to generate a monobiotinylated human IL-2. One potential caveat was the presence of an endogenously biotinylated protein normally present in E. coli, which may coisolate with rbhIL-2. While we did not observe significant levels of this protein in our rbhIL-2 preparation, potential solutions to this problem include dialysis of the rbhIL-2 preparation by using a size exclusion filter through which the endogenous protein can pass but rbhIL-2 cannot. Alternatively, molecular genetic techniques could be used to tag the endogenously biotinylated protein and facilitate its removal from rbhIL-2 preparations. Another caveat, which we did observe, was reduced activity of rbhIL-2 compared to nonbiotinylated rhIL-2. One possible explanation for this observation could be steric interference, or structural alterations, due to the presence of the biotinylation stub.
Having produced a genetically engineered rbhIL-2, we determined whether it could be used to monitor T-cell activation. Traditionally, T-cell activation has been monitored by [3
H]TdR incorporation, a process that is closely related to underlying changes in the T-cell number. Other methods to monitor in vitro T-lymphocyte activation include detection of T-cell cytokines such as IL-2 and gamma interferon by ELISA (16
), measurement of CD8 T-cell activation by granzyme B enzyme-linked immunospot assay (17
), use of the IL-2-dependent cell line CTLL-2, MTT reduction (22
), and detection of the lymphoid activation marker CD69 by flow cytometry (20
). Additional assays include light absorbency to measure lymphocyte proliferation (6
) and a combination of intracellular cytokine expression, cytokine secretion, and cytokine receptor expression (1
). However, most of these assays require substantial time and the use of radioactive isotopes and/or are expensive to run.
We provide an alternative method to monitor T-cell activation by using FITC-SA or HRP-SA conjugated to rbhIL-2. This assay can be completed in 1 day and does not require the use of radioactive isotopes. This method is comparable in sensitivity to the traditional [3
H]TdR incorporation assay when stimulating cells with PHA and is up to 10-fold more sensitive when analyzing TT-stimulated cells by chemiluminescence techniques (Fig. ). In addition, the time required to complete the assay is one-quarter to one-sixth of that required to measure [3
H]TdR incorporation. It remains to be determined whether the use of a biotinylated anti-CD25 (IL-2Rα) MAb would be an equally effective tool for monitoring T-cell activation by the described strategy. However, rbhIL-2 would be expected to bind only IL-2R capable of binding IL-2 itself (3
). IL2R binding by rbhIL-2 is indicated by the fact that purified rbhIL-2 stimulates the proliferation of an IL-2-dependent T-cell line whereas similarly purified material from bacteria transfected with a biotinylation vector lacking the IL-2 insert does not (Fig. ). In addition, rbhIL-2 binds primarily to T cells (Table ), the binding correlates with CD25 expression (Fig. ), and it increases with the level of T-cell activation, as measured by [3
H]TdR incorporation (Fig. ). In addition, bhIL-2 would not be expected to bind to Fc receptors, a potential complication when using intact anti-CD25 MAb. Furthermore, therapeutic use of anti-CD25 MAb would probably be problematical, since host Ab would be generated to the variable regions and any mouse components that are present. This is important since rbhIL-2 has the potential for application in vaccines and immunotherapeutics (8
). For example, our laboratory has recently developed a targeting strategy which has the ability to target biotinylated proteins to APC (23
). We are currently exploring its use in similarly targeting cytokines to APC in order to influence the degree and direction of the immune response. Furthermore, by changing the specificity of the targeting component, it may also be possible to target biotinylated cytokines to tumors, a strategy previously shown to be effective for the elimination of cancer cells (8
In summary, an rbhIL-2 has been generated which has potential application not only in immunodiagnostics but in clinical immunotherapeutics as well.