Construction And Identification Of A Novel IP10-scFv Immunocytokine

Immunocytokines are recombinant fusion proteins consisting of tumor-specific antibody and cytokine, which combine the unique targeting ability of antibody with the multifunctional activities of cytokine. The antibody-cytokine fusion proteins retaining both antibody- and cytokine-associated functions achieve high cytokine concentration in the tumor microenvironment sufficient to elicit a significant antitumor activity without accompanying systemic toxicity.At first, the mouse chemokine CXCL10 (interferon-y inducible protein 10, IP-10) gene was amplified from RAW 264.7 cells for immunocytokine IP10-scFv construction. The PCR product was cloned into a pGEM-T vector and sequenced to eliminate the mutant.A single-chain Fv antibody fragment (scFv) specific for acidic isoferritin (AIF) was constructed with the Recombinant Phage Antibody System (RPAS). The variable regions of heavy chain (Vh) and light chain (Vl) from hybridoma 4c9 cells, which secret monoclonal antibody (mAb) against AIF, were connected with a flexible linker encoding (Gly4Ser)3 polypeptide, and the constructs of VH-linker-VL were then cloned into a phagemid pCANTAB 5 E. The recombinant phagemids pCAN4c9 were introduced into Escherichia coli (E. coli) TG1 cells, and rescued with a helper phage M13KO7. AIF-positive recombinant phages were selected from the phage-displayed scFv library by biopanning with AIF. The scFv gene from GP5 clone was sequenced and submitted to GenBank/EMBL nucleotide sequence database under accession number of AY 13 4749. AIF4c9 scFv has 786 nucleotides (nt) encoding 261 amino acids (aa), which containing 118 aa of Vh, 109 aa of Vl. The results of alignments with sequences of GenBank/EMBL demonstrate that AIF4c9 Vh gene is a member of Vh III gene family IA subgroup and displays a sequence identity of 93% with 3F1H10 gene, while the Vl gene belongs to Vk19 gene family V subgroup with a sequence identity of 98% to 101.4.1 gene. It suggests that AIF4c9 Vh gene probably contributes more than Vl to the combining site to AIF.AIF4c9 scFv gene was subcloned into pET28a vector under the control of T7 promoter, and overexpressed in E. coli BL21(DE3). The results of sodium doecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting demonstrated that the recombinant protein predominantly expressed in the cytoplasm as inclusion bodies with no activity. For efficient recovery of AIF4c9 scFv with bioactivity, an on-column refolding procedure based on Ni-chelating chromatography was developed, in which the denatured and reduced protein was isolated through binding to the column via His-tags, and refolded with an linear decreased urea gradient refolding buffer. It was found that introducing of the redox conditions at 3 mol/L urea concentration by adding oxidized/reduced glutathione (GSSG/GSH) into the refolding buffer led to a significant increased yield of the functional scFv. With the optimized on-column refolding procedure,the active AIF4c9 scFv was recovered efficiently from inclusion bodies (up to 15 mg/ml) with a refolding yield of approximate 75% confirmed by spectrophotometer. The refolded scFv retains the specific binding activity to AIF with an affinity constant (KDscfv) of 7.29x10-8 mol/L, which is about 20-fold less than that of mAb4c9 (KDmAb=4.16x10-9 mol/L). The overall yield of AIF4c9 scFv with bioactivity in E. coli flask culture was about 62 mg/L.A novel immunocytokine of IP10-scFv was constructed by linking mouse CXCL10 gene to the Vh region of AIF4c9 scFv with a flexible linker encoding (Gly4Ser)3 polypeptide, the sequence of which appears in GenBank/EMBL database under accession number of AY251299. The CXCL10 and AIF4c9 scFv genes were sequentially subcloned into an expression vector pcDNA3 under the control of cytomegalovinis promoter (Pcmv) resulted in an expression plasmid of pc3IP104c9. NSO myeloma cells transfected with the linearized plasmid were selected in 400 ug/ml G418 followed by screening for antibody secretion in ELISA, and the AIF-positive cells were expanded and cloned for ascite...