Targeted Biologic Activity Study Of Recombinant Immunotoxin CD25scFv-α-luffin-KDEL In Vitro

BackgroundCutaneous T-cell lymphoma(CTCL) is a malignant lymphoma characterized by a clonal accumulation of CD4+ helper T lymphocytes in the skin. CTCL accounts for about 2/3 of total cutaneous lymphomas. Studies show that phenotype of CTCL is mainly Th2 or regular T cells, and 50%~60% of CTCL expresses IL-2R. Up to now, there is no effective treatment to the advanced CTCL. With gradually deepening study on biological characteristics and antitumor immunologic mechanism of CTCL, immunotoxin therapy for targeting tumors might become a good choice.Immunotoxin is a fusion protein comprised of toxin molecule and targeting vector which has specific targeted features. The fusion protein can be recombined and expressed in Escherichia coli by genetic engineering technology. The fusion proteins has some characters, such as, good stability, excellent tissue permeability, low immunogenicity and specific tumor targeted activity. All of these enable immunotoxins an optional therapy to target and kill tumor cells. DAB389IL-2(denileukin diftitox, Ontak), a recombinant fusion protein of diphtheria toxin and IL-2, has been approved by the FDA for the treatment of relapsed or refractory CTCL with CD25+ expression, however, its serious side effects on liver, vasculature and hematopoietic system limit its use. The possible reasons are as follows: 1. The specificity is not sufficient. IL-2 is the targeting vector of DAB389IL-2, but IL-2 lies usually in the surface of many cytokines and normal cells(such as T cells and NK cells), and advanced CTCL expresses mainly IL-2Rα(CD25) which has low affinity with IL-2; 2. The majority of people had been vaccinated or had touched diphtheria bacilli vaccine in childhood, thus, diphtheria toxin antibody preexist in their bodies.Ribosome inactivating proteins(RIPs), mostly from plants, can depurinate from eukaryotic ribosomes and prokaryotic ribosomes, leading to the interruption of protein translation. RIPs are divided into two groups according to their different construction, type 1 RIPs and type 2 RIPs. The type 1 RIPs can use to prepare immunotoxins. Luffin is one of the classic toxins of type I RIP and it is the most toxic in luffin family. It has been reported that luffin had been used for targeted killing of melanoma cells when coupled with monoclonal anti-melanochrome antibody in 1900’s.Single-chain antibody(ScFv) is a small molecule antibody comprised of the variable region of heavy chain(VH) and the variable region of light chain (VL) by linker. ScFv has been used extensively for targeting vector of immunotoxin by genetic engineering technology. CD25 single-chain antibody (CD25scFv) can specificly combine with tumor cells expressing CD25. It has been used as targeting vector to construct the recombinant immunotoxin for the treatment of Hodgkin lymphoma in experiments. Based on the theories above, we useα-luffin-KDEL originated from Luffa cylindrica as the toxin molecule and CD25scFv as targeting vector to construct recombinant CD25scFv-α-luffin-KDEL, and to explore its targeting activity to tumor cells expressing CD25. We look forward to further study on its effect on the treatment of CTCL expressing CD25.ObjectiveWe useα-luffin-KDEL from Luffa cylindrica as the toxin molecule and CD25scFv as targeting vector to complete the construction, expression and purification of CD25scFv-α-luffin-KDEL. In order to explore its targeting biologic activity, we tested the effect of CD25scFv-α-luffin-KDEL on cell proliferation, apoptosis and distribution of cell cycle of Hut-78 (originated from cutaneous T-cell lymphoma and expressing high affinity CD25) , Jurkat cells(expressing low affinity CD25) and Raji cells(originated from Burkitt lymphoma and almost no expression of CD25).Methods1.The cDNA sequence data of the signal sequence deleted region ofα-luffin available in the GenBank(GenBank: X62371.1) were used to design gene-specific primers contained NcoI and XhoI sites for PCR amplification of the cDNA encoding the matureα-luffin andα-luffin-KDEL. Then, the PCR products were cloned into the plasmid pET28a(+), and verified by DNA sequence analysis, respectively. The verified recombinant vector was further transformed into Escherichia coli (DE3)pLysS strain to express the recombinant proteins after IPTG induction. The expressed recombinant proteins were assayed by SDS-PAGE and were purified by Ni-NTA agarose affinity, then were examined by western blotting. The cytotoxicity of the purified recombinantα-luffin andα-luffin-KDEL on JEG-3, HepG2 and MCF-7 cells were tested by the Cell Counting Kit-8 assay. The cells were treated with various concentrations of recombinant proteins and at different time periods. The data were processed by SPSS 13.0 software package. The difference of cytotoxicity of the two proteins was compared with the analysis of variance.2.The CD25scFv gene contained BglII and NcoI sites was amplified by PCR from pET32a-anti-CD25-PE38KDELmutant plasmid, then was inserted into the plasmid pET32a(+), and verified by DNA sequence analysis. After the recombinant plasmid pET32a-CD25scFv and pET28a-α-luffin-KDEL were digested with NcoI and XhoI, the linearized pET32a-CD25scFv and the luffin-KDEL fragment were ligated to construct the recombinant vector pET32a-CD25scFv-α-luffin-KDEL. After the recombinant plasmid was confirmed by restriction endonuclease digestion and sequencing, the recombinant pET32a-CD25scFv-α-luffin-KDEL was further transformed into Escherichia coli BL21DE3 strain to express the recombinant proteins after IPTG induction. The expressed recombinant proteins were assayed by SDS-PAGE, and purified by Ni-NTA agarose affinity and refolded. The gained recombinant proteins were examined by western blotting. The expression of CD25 on the surface of Hut-78 cells, Jurkat cells and Raji cells were detected by flow cytometry, respectively. After the Hut-78 cells, Jurkat cells and Raji cells were treated with the recombinant CD25scFv-α-luffin-KDEL, cell proliferation rates were measured by CCK-8; and apoptosis was determined by Annexin V/PI double staining; and distribution of cell cycle was analyzed by PI staining.Results1. (1) The cDNA of the mature peptideα-luffin was successfully amplified. There is only one nucleotide mutation when compared with the sequence in GeneBank, which doesn’t affect the coding of amino acid sequence. it was found that matureα-luffin shared 96% amino acid similarity with luffin-a. (2) pET28a-α-luffin and pET28a-α-luffin-KDEL were successfully constructed. The recombinant matureα-luffin andα-luffin-KDEL (molecular weight 28kDa), were successfully expressed in a partly soluble form in Escherichia coli BL21DE3plysS after optimization of expression conditions. After Ni-NTA agarose affinity purification and western blotting verification, the interest proteins were obtained. (3) The results of CCK-8 viability assays showed that the two recombinant proteins inhibited the growth of JEG-3 cells, HepG2 cells and MCF-7 cells in dose- and time-dependent manners. The inhibition rates existed obvious difference. The cytotoxicity ofα-luffin to JEG-3 was significantly stronger, as compared with MCF-7 cells. In a word,α-luffin-KDEL has stronger cytotoxicity to the three kinds of tumor cells thanα-luffin.2. (1) The recombinant plasmid, pET32a-CD25scFv-α-luffin-KDEL was constructed successfully. The fusion protein(molecular weight about 80kDa) was expressed in the inclusion bodies form in Escherichia coli BL21DE3. After purification, denaturation, renaturation and western blotting verification, the interest protein was obtained. (2) 87.5% of the Hut-78 cells, 26.9% of the Jurkat cells and only 0.2% of the Raji cells incubated with the anti-CD25-FITC antibody exhibited positive staining by performing flow cytometry(FCM). (3) The results of CCK-8 viability assays showed that the recombinant CD25scFv-α-luffin-KDEL inhibited the growth of Hut-78 cells, Jurkat cells and Raji cells in dose- and time-dependent manners. The inhibition of the CD25scFv-α-luffin-KDEL to Hut-78 cells was stronger than to Jurkat cells and Raji cells. When compared the cytotoxicity of the recombinant protein to Jurkat cells and to Raji cells, there was not significant difference. Inadditionly , the cytotoxicity of CD25scFv-α-luffin-KDEL is stronger thanα-luffin-KDEL to Hut-78 cells and Jurkat cells. When compared the cytotoxicity ofα-luffin-KDEL to the three types of cells, there was not significant difference. The result of apoptosis and cell cycle analysis showed that CD25scFv-α-luffin-KDEL promoted the apoptosis of Hut-78 cells and Jurkat cells. At the same time, CD25scFv-α-luffin-KDEL made Hut-78 cells and Jurkat cells in S phase decreased and those in G0/G1 phase increased. The cells were arrested in G0/G1 phase and G2/M phase.Conclusions: (1) The mature peptideα-luffin andα-luffin-KDEL were cloned from the fresh immature seeds of Luffa cylindrica. The expressed recombinant proteins in Escherichia coli possess antitumor activities in vitro and the activities are possibly varied with the cell species. Moreover, the recombinantα-luffin-KDEL possesses better antitumor activies than the recombinantα-luffin. (2) The recombinant CD25scFv-α-luffin-KDEL, generated by usingα-luffin-KDEL from Luffa cylindrica as the toxin molecule and CD25scFv as targeting vector obviously, possess the antitumor potency of targeting the CD25-positive tumor cells in vitro, the mechanism of which may be related with its pro-apoptotic effect and inhibition of DNA synthesis in S phase.