Construction Of SM5-1-PE38KDEL Fusion Toxin (mutants) And Experimental Therapy On Xenograft Human Hepatocellular Carcinoma

Primary liver cancer is one of the most common cancers in China, which is an aggressive malignant disease with poor prognosis. It is urgent to find targeted therapy for liver cancer. Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, especially advances in immunoconjugate technology have revitalized the "magic bullet" concept of immunotherapeutics for the treatment of cancer. More importantly, antibody fusion proteins such as immunotoxins which were derived by coupling bacterial or plant toxins to scFv antibody fragments using recombinant DNA technology have greatly increased the potential for cancer therapeutic opportunities. Numbers of such reagents are in clinical trials.Pseudomonas exotoxin A (PE) is a three-domain protein in which domain Ia(1-252 )is involved in recognition of receptors on eukaryotic target cells, domain â…¡ (253-364) promotes translocation of PE into the cytosol, and domain â…¢ (405-613) enzymatically ADP-ribosylates elongation factor 2. It is genetically altered so that it can not bind to the toxin receptor present on most normal cells. PE38KDEL is such a truncated form of PE in which domain Ia (residues 1 -252) and a portion of domain Ib (residues 365-380) have been deleted, furthermore, C-terminal residues 609-613 (REDLK) of PE was replaced by "KDEL". Once recombinant immunotoxins(ITs) bind to receptors on cancer cells, the toxin enters and kills the cells.p230 is highly expressed on the surface of tumor cells, and is rarely expressed on the normal tissues, so it is a ideal target for tumor immunotherapy. SM5-1 is a murine monoclonal antibody that specifically binds to p230 with high affinity, and it has potent therapy effect for tumors.Vascular leak syndrome (VLS) is a major and often dose-limiting side effect of immunotoxins and cytokines. Earlier studies identified a three-amino acid motif that is shared by toxins, ribosome-inactivating proteins, and interleukin-2, all of which cause this problem. It is postulated that this syndrome is initiated by damage to vascular endothelial cells. To PE, three motifs, 348-350(GDL), 423-425(GDV),605-607(GDL) are identified to induce VLS.Therefore p230 is an excellent target for immunotoxin therapy. The murine Mab SM5-1 possesses high specificity for hepatocellular carcinoma and minimally reactive with a variety of normal tissues. Successful development of tumor-targeted therapeutic agents is dependent, in part, on the site-specific delivery of therapeutic agents. One such molecule is truncated Pseudomonas exotoxin A(PE38KDEL), a 38 kDa ribosome-inactivating toxin, which contain three three-amino acid motifs related VLS.The present study describes our investagation of a panel of recombinant immunotoxins consist of a single-chain analogue of the antibody SM5-1 genetically fused to a PE38KDEL and mutated PE38KDEL in amino acid flanking X(D)Y sequence. Our purpose is to construct high effective, low toxicity immunotoxin for cancer therapy.Production of SM5-1-PE38KDEL immunotoxins in E.coli. The plasmid vector pET-32a containing the fusion gene was transformed into E.coli(BL21) ,and the target protein was induced by the addition of IPTG. The proteins were purified using by His-Ni++ metal affinity chromatography and gel filtration.The in vitro activity of immunotoxin prepared with mutant rPE38KDELs. We have generated 9 immunotoxins SM5-1-PE38KDEL with mutation in amino acid flanking X(D)Y sequence of truncated Pseudomonas Exotoxin A (PE38KDEL) in the three-dimensional structure. These immunotoxins have been evaluated for activity by cell-free prorein inhibition system and MTS assay. When tested in the reticulocyte assay, the SM5-l-PE38KDEL.wt and SM5-1-PE38KDEL mutl had similar activities, although the SM-PE38KDEL.wt was slightly more active. Three mutants SM-PE38KDEL.mut3, SM-PE38KDEL mut5 and SM-PE38KDEL.mut7 retained this activity, two-to five-fold less active in the reticulocyte assay; other four mutants were 40- to 200-fold less active in the reticulocyte assay. This suggests that these mutant amino acids may be particularly critical for the activity of the immunotoxin. In light of these results, we selected the SM-PE38KDEL.mutl, mut3, mut5 and mut7 for specific cytotoxicity against two antigen-positive(ch-hep-l and ch-hep-3)and anantigen-negative(BEL7404) cell line. In contrast to these four mutants, mutl had only one fold less active than wild-type, three other mutants were five- to 36-fold less active as immunotoxins in the hepatocellular carcinoma cell lines. In light of these results, we selected these four mutants for in vivo PVL testing.The ability of immunotoxins containing PE38KDEL to induce PVL in vivo. ICR mice were i.v. administered with a single dose of wild-type immunotoxin (lmg-4.0mg/kg) and mutants (lmg-12mg/kg).To wild-type, necropsy was performed 24hr following immunotoxin administration. The appearance of large amounts of clear fluid in the thoracic cavity(hydrothorax) was found with the dose of lmg/kg. Acute toxicity was apparent in mice at 2-4mg/kg. Mice treated with 4mg/kg all died within 24hr,and a half mice treated with 2mg/kg died within 48hr. To mutants, at 3mg/kg, there was no fluid accumulation and below 8mg/kg no fluid was detected. As comparable to wild-type at lmg/kg, mutl and mut7 did not induce PVL.The therapeutic activity of SM5-1-PE38KDEL versus mutl in nude hepatocellular carcinoma mice. Because the immunotoxin SM5-l-PE38KDEL.mutl had good activity in vitro and did not induce PVL in vivo, it was assessed in vivo its therapeutic activity in nude mice compared to SM5-l-PE38KDEL.wt using equitoxic cytotoxic doses. The dose of 0.5mg/kg and 1.2mg/kg was used in SM5-l-PE38KDEL.wt and SM5-l-PE38KDEL.mutl respectively. At 5 weeks, 75 % mice in control groups grew solid tumor, but two groups of SM5-l-PE38KDEL.wt and mutl not, yet, and the mice of SM-5-lPE38KDEL.wt group all lost 5-10% of body weight, therefore at equitoxic IC50, SM5-l-PE38KDEL.mutl was more safe than wild-type. On the based of this result, We assessed their therapeutic activity in nude mice which had grown tumors. Once tumors were measurable (~30-50mm2), animals were treated (i.v. via tail vein) with saline, SM5-l(sFv), CD25-PE38KDEL), PE38KDEL, SM-5-l-PE38KDELand SM5-l-PE38KDEL.mutl for three consecutive days. After four weeks, at equitoxic IC50, the average tumor sizes of the mice of SM-5-l-PE38KDEL(0.5mg/kg) and SM5-l-PE38KDEL.mutl)(1.2mg/kg) were retained 30-50mm3; At the half of lethal doses, the tumors of the mice of SM5-l-PE38KDEL.mutl ( 5mg/kg) gradually decreased and disappeared at 40 days.