The significance and potential application of this approach for the treatment of tumours is also addressed. Interleukin-2 receptor alpha (IL-2Rα; generously provided by Dr Jim Miller, University of Rochester) in pcEVX-3 was PCR amplified using primers (Table 1) to add the KpnI and BamHI restriction sites, remove the hydrophobic transmembrane region and, for some constructs, addition of a 6 × Histidine tag (6 × His). This product was cloned into pBluescript (pBluescript IL-2Rα). The (GGGGS)x linker of various repeat lengths was either synthesized (GENEART Inc., Toronto, ON, Canada) or was made by annealing
primers from complimentary oligonucleotides (Table 1) and then cloned into pBluescript using the EcoRI and KpnI restriction sites. The (GGGGS)x linker was excised and cloned into the pBluescript IL-2Rα plasmid. Τhe linker and IL-2Rα BMN673 were excised using the EcoRI and BamHI sites and directionally cloned into the pBluescript IL-2/PSAcs plasmid containing murine IL-2 and the PSA cleavage sequence (HSSKLQ) resulting in the pBluescript IL-2/PSAcs/linker/IL-2Rα plasmid. This plasmid was then verified by sequencing and subsequently cloned into pcDNA3.1 (Invitrogen, Carlsbad,
CA) using the XhoI HIF-1 pathway and BamHI restriction sites to obtain flanking restriction enzyme sites so that it could be shuttled into pVL1392 for expression in the BD BaculoGold™ transfer vector system (BD Biosciences, San Jose, CA) using the XbaI and BamHI sites. To change the cleavage sequence (cs) from HSSKLQ (PSAcs) to SGESPAYYTA (MMPcs) the pBluescript plasmid containing the mouse IL-2
and the PSAcs portion of the fusion cAMP protein was linearized using NotI and PCR was performed using the IL-2 forward primer and the MMPcs reverse primer (Table 1). This PCR product was then digested with SalI and EcoRI restriction endonucleases and cloned into pBluescript to create the pBluescript IL-2/MMPcs plasmid. The pVL1392 vector containing the mouse IL-2/PSAcs/(GGGGS)4/IL-2Rα + 6 × His fusion protein was digested with EcoRI and BamHI and the fragment containing the (GGGGS)4 linker and IL-2Rα was isolated and cloned into the pBluescript IL-2/MMPcs plasmid using the EcoRI and BamHI sites. The fragment encoding the entire fusion protein was then shuttled into pcDNA3.1 using the XhoI and BamHI sites and subsequently shuttled into pVL1392 using XbaI and BamHI for expression. A human phage display library constructed from peripheral blood lymphocytes was used to screen for phage expressing single-chain fragments of antibodies capable of binding to human IL-2 on their surface (phscFvs). The library was generated in the pAP-III6 vector,22,23 a monovalent display vector, by PCR amplification of VL and VH immunoglobulin domains from peripheral blood lymphocyte cDNA prepared from approximately 100 donors.