高效、低毒免疫毒素SM5-1-PE38KDEL（突变体）的构建及其抗肿瘤作用机理的研究
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摘要
原发性肝癌是我国最常见的恶性肿瘤之一,术后复发率较高,更主要的是手术无法切除肉眼看不见的肿瘤组织,而放、化疗对造血和免疫系统均有严重的抑制作用,甚至导致机体免疫力进一步下降,使肿瘤广泛转移。肿瘤免疫治疗是继手术、放疗、化疗之后的第四种治疗模式,使肿瘤的彻底根治有了新的希望。
免疫毒素是由具有导向能力的分子和具有细胞毒性的分子组合而成的具有特异性杀伤能力的杂合分子。这种以特异性单抗为“载体”、以毒素为“弹头”组成的杂合分子又称为“生物导弹”,但是在相当长的一段时间内内,免疫毒素治疗效果并未达到令人满意的程度。这其中主要有两个原因,一是毒素所致的严重副反应;二是缺乏高度特异性的肿瘤单克隆抗体。因而,寻找肿瘤特异性抗体和改善毒素的毒副作用,并进而探讨对肝癌有效的治疗已成为目前临床的迫切需求。
绿脓杆菌外毒素(PE),是66kD的单链蛋白,氨基末端构成Ⅰa区,为细胞结合结构区域。第253-364氨基酸组成Ⅱ区,负责毒素的转位。Ⅲ区,405-613,具有ADP核糖基化活性。PE的细胞毒机制是:在受体或抗体受体介导下内吞,在胞内蛋白水解酶的作用下Ⅰ、Ⅱ区被去除,Ⅲ区转移入内质网,再转移至胞质,在那罩PE的DⅢ能使肽链延长因子2的ADP核糖基化,不能与核蛋白体相互作用,肽链不能延长,从而阻碍蛋白质的合成,导致真核细胞的死亡。尽管免疫毒素采用了PE衍生物的形式,大大减少了非特异结合,但仍存在着对正常细胞的剂量依赖的非特异或特异的损害,特别是对血管内皮细胞有明显的损害,造成血管渗漏综合症(Vascular Leak Syndrome,VLS):血管通透性增加,液体渗出,大量蛋白质进入组织间隙,水肿,从而体重增加,低蛋白血症,并造成组织缺氧,多器官衰竭等,严重限制了临床应用。PE分子有三个区域与血管渗漏综合症有关:348-350、430-432及605-607。根据PE分子的晶体结构,这三个区域均位于PE分子的表面,与血管内皮细胞接触,引起渗漏综合症。因此,我们设想如果能对此三区域及其周边相邻的氨基酸进行突变,使PE失去与血管内皮细胞接触作用同时保留其细胞毒活性,那就能大大减少血管渗漏综合症的发生,使免疫毒素的临床应用成为可能。
SM5-1是本研究所自主筛选克隆的小鼠抗人单克隆抗体,其抗原p230特异
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 Ⅱ (253-364) promotes translocation of PE into the cytosol, and domain Ⅲ (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 an
antigen-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.
免疫毒素是由具有导向能力的分子和具有细胞毒性的分子组合而成的具有特异性杀伤能力的杂合分子。这种以特异性单抗为“载体”、以毒素为“弹头”组成的杂合分子又称为“生物导弹”,但是在相当长的一段时间内内,免疫毒素治疗效果并未达到令人满意的程度。这其中主要有两个原因,一是毒素所致的严重副反应;二是缺乏高度特异性的肿瘤单克隆抗体。因而,寻找肿瘤特异性抗体和改善毒素的毒副作用,并进而探讨对肝癌有效的治疗已成为目前临床的迫切需求。
绿脓杆菌外毒素(PE),是66kD的单链蛋白,氨基末端构成Ⅰa区,为细胞结合结构区域。第253-364氨基酸组成Ⅱ区,负责毒素的转位。Ⅲ区,405-613,具有ADP核糖基化活性。PE的细胞毒机制是:在受体或抗体受体介导下内吞,在胞内蛋白水解酶的作用下Ⅰ、Ⅱ区被去除,Ⅲ区转移入内质网,再转移至胞质,在那罩PE的DⅢ能使肽链延长因子2的ADP核糖基化,不能与核蛋白体相互作用,肽链不能延长,从而阻碍蛋白质的合成,导致真核细胞的死亡。尽管免疫毒素采用了PE衍生物的形式,大大减少了非特异结合,但仍存在着对正常细胞的剂量依赖的非特异或特异的损害,特别是对血管内皮细胞有明显的损害,造成血管渗漏综合症(Vascular Leak Syndrome,VLS):血管通透性增加,液体渗出,大量蛋白质进入组织间隙,水肿,从而体重增加,低蛋白血症,并造成组织缺氧,多器官衰竭等,严重限制了临床应用。PE分子有三个区域与血管渗漏综合症有关:348-350、430-432及605-607。根据PE分子的晶体结构,这三个区域均位于PE分子的表面,与血管内皮细胞接触,引起渗漏综合症。因此,我们设想如果能对此三区域及其周边相邻的氨基酸进行突变,使PE失去与血管内皮细胞接触作用同时保留其细胞毒活性,那就能大大减少血管渗漏综合症的发生,使免疫毒素的临床应用成为可能。
SM5-1是本研究所自主筛选克隆的小鼠抗人单克隆抗体,其抗原p230特异
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 Ⅱ (253-364) promotes translocation of PE into the cytosol, and domain Ⅲ (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 an
antigen-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.
引文
1. King DJ, Byron OD, Mountain A, et al. Expression, purification and characterization of B72 Fv fragments.Biochem J, 1993,290(Pt 3):723-728.
2. Huston JS, Levinson D, Mudget-Hunter M, et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produce in E. coli.Proc Natl Acad Sci USA, 1988,85:5879-5884.
3. Colcher D, Pavlikova G, Beresford G, et al. Single-chain antibodies in pancreatic cancer. Ann N Y Acid Sci, 1999, 880: 263-280.
4. Zu Putlitz J, Skerra A, Wands JR. Intracellular expression of a cloned antibody fragment interferes with hepatitis B virus surface antigen secretion. Biochem Biophys Res Commun, 1999, 24(255): 785-791.
5. Mansfield E, Amlot P, Pastan I, et al. Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD22-bearing cells and
2. Huston JS, Levinson D, Mudget-Hunter M, et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produce in E. coli.Proc Natl Acad Sci USA, 1988,85:5879-5884.
3. Colcher D, Pavlikova G, Beresford G, et al. Single-chain antibodies in pancreatic cancer. Ann N Y Acid Sci, 1999, 880: 263-280.
4. Zu Putlitz J, Skerra A, Wands JR. Intracellular expression of a cloned antibody fragment interferes with hepatitis B virus surface antigen secretion. Biochem Biophys Res Commun, 1999, 24(255): 785-791.
5. Mansfield E, Amlot P, Pastan I, et al. Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD22-bearing cells and