基于CD7纳米抗体的新型免疫毒素对人白血病细胞在体外和体内的活性研究
发布时间：2018-09-03 09:07
【摘要】：在过去的几十年里,白血病和淋巴瘤的临床治疗取得了很大的进展。然而在T细胞白血病和淋巴瘤中,仅仅只有一小部分的T急性淋巴细胞白血病(T-ALL)或者外周T细胞淋巴瘤(PTCL)病人获得了长期无肿瘤生存。常规的细胞毒疗法(化疗或者放疗等)对病人具有副反应且疗效有限。众所周知,当白血病或者淋巴瘤患者一旦对化疗产生耐药或者出现复发,临床治疗手段将会变得十分有限,病人的生存期也随之变得很短。因此高效的、能够避免多药耐药性机制,而且对治疗人T细胞恶性肿瘤具有良好特异性和毒性的新疗法是具有十分重要的科学和临床意义。在新的治疗药物开发中,由毒素和肿瘤细胞特异性的抗体片段或者配体等融合构成的重组免疫毒素被认为对化疗耐药性的T细胞疾病是仍然有效的。运用免疫毒素的一个关键要素就是选择肿瘤细胞适合的靶点。许多研究已经表明CD7分子在大多数T细胞淋巴瘤和白血病细胞表面表达,而在一小部分正常T淋巴细胞上缺失。CD7分子作为治疗靶点的另外一个优势就是当它和抗体或者抗体衍生物结合后会迅速内化,这使得针对CD7分子的抗体非常适合作为药物运输工具。由于上述优点,多种CD7特异性的免疫毒素被制备出来并检测它们的抗白血病效应。然而,大多数的研究主要集中在植物毒素,如蓖麻毒素,皂草素及其衍生物,这些毒素由于缺乏足够的安全和疗效而未获得临床批准。另一种免疫毒素,截短形式的铜绿假单胞菌外毒素A(ETA或PE38)融合到CD7的单链抗体片段(scFv),被报道可造成约20%原代白血病细胞死亡,但是却没有进一步评估其体内抗白血病效应,这意味着T细胞白血病细胞可能对PE38不敏感或者报道的CD7单链抗体需要进一步的改进。事实上,由PE38构成的抗CD22免疫毒素在用于毛细胞白血病患者的临床试验中表现出令人印象深刻治疗效应,达到了46%的完全缓解,而且无明显的剂量限制性毒性,这表明PE38至少对一些淋巴细胞是敏感的。因此,新的抗CD7抗体或者可变区片段有可能为我们提高针对T细胞淋巴瘤和白血病免疫毒素功效提供新的选择。纳米抗体被选择成为我们要开发的新型抗CD7抗体的原因如下:纳米抗体是由casterman等人首先发现的一种单域抗体,来自骆驼重链抗体的重链可变区,具有诸多杰出的理化性质,这使它们成为了靶向递送生物活性药物的优秀候选者。研究人员已经证明纳米抗体可以与毒素以及其他功能性分子偶联,然后用这些偶联物去靶向细胞,对癌症和其他疾病进行治疗。目的:筛选cd7特异性的纳米抗体,构建新型的基于cd7纳米抗体和pe38的免疫毒素,在体外检测这些免疫毒素对t-all细胞系、t-all病人和aml病人原代细胞的效应,最后在体内评估它们抗白血病的作用。方法:用cd7阳性的jurkat细胞免疫骆驼,提取免疫后的骆驼外周血淋巴细胞,构建纳米抗体噬菌体文库,用生物淘洗的方法筛选获得抗cd7的纳米抗体。通过流式细胞分析仪检测纳米抗体vhh6、免疫毒素pg001(vhh6-pe38)和pg002(dvhh6-pe38)的特异性和亲和力。在体外通过检测蛋白质合成抑制和凋亡的方法来测定纳米抗体免疫毒素pg001和pg002对人cd7阳性的jurkat、cem和cd7阴性的rpmi8226、h460细胞系的细胞毒作用。通过annexinv和7-aad染色检测pg001和pg002对白血病患者原代细胞的细胞毒效应。pg001和pg002在体内的抗肿瘤潜力采用cem细胞异种移植肿瘤模型进行评估。结果:我们成功鉴定了具有高特异性和亲和力(~15nm)的cd7纳米抗体——vhh6。基于vhh6的单价免疫毒素(pg001)和双价免疫毒素(pg002)对cd7阳性细胞保持有高度的特异性,亲和力分别约为16nm和4nm。这两种毒素高效地以抗原依赖型的方式促进cd7阳性白血病细胞系jurkat和cem发生凋亡,但是对cd7阴性的人多发性骨髓瘤细胞系rpmi8226和人肺癌细胞系h460增殖的几乎没有影响。特别需要指出的是,免疫毒素pg002对jurkat、cem细胞的半有效浓度分别为30pm和23pm,这提示pg002治疗cd7阳性白血病具有很大的潜力。此外,pg002在单剂量为100ng/ml浓度条件下能有效介导新鲜收集的t-all和aml患者原代细胞凋亡。在异种移植肿瘤模型中,pg001和pg002能够抑制cem细胞的增殖,并可显著延长小鼠的生存期。其中,pg002相比于pg001在肿瘤模型中的治疗效果更为突出。结论:我们构建的单价(pg001)和双价(pg002)cd7纳米抗体免疫毒素以抗原特异性的方式,以及在低浓度条件下即可有效杀伤cd7阳性t-all细胞系和新鲜t-all和aml病人来源的细胞。基于免疫毒素pg001和pg002有效杀死白血病细胞,可显著延长异种移植肿瘤小鼠的生存,PG001和PG002值得进一步开展临床前和临床试验研究,从而进一步评价它们抗白血病的潜能。
[Abstract]:In the past few decades, great progress has been made in the clinical treatment of leukemia and lymphoma. However, only a small number of patients with T-cell acute lymphoblastic leukemia (T-ALL) or peripheral T-cell lymphoma (PTCL) have long-term tumor-free survival. Conventional cytotoxic therapy (chemotherapy or chemotherapy) or lymphoma has been used. It is well known that when patients with leukemia or lymphoma become resistant to chemotherapy or relapse, the means of clinical treatment will become very limited, and the patient's survival will become very short. Therefore, efficient, multi-drug resistance mechanisms can be avoided, and the treatment of human T fine. In the development of new therapeutic drugs, recombinant immunotoxins consisting of toxins and tumor cell-specific antibody fragments or ligands are considered to be still effective in the treatment of chemotherapy-resistant T-cell diseases. A key element of immunotoxins is the selection of suitable targets for tumor cells. Many studies have shown that CD7 molecules are expressed on the surface of most T-cell lymphomas and leukemia cells, but are absent on a small number of normal T-lymphocytes. Another advantage of CD7 as a therapeutic target is that it is derived from antibodies or antibodies. Because of these advantages, a variety of CD7-specific immunotoxins have been prepared and tested for their anti-leukemic effects. However, most of the research has focused on phytotoxins, such as ricin, saponin and their derivatives. Another immunotoxin, a truncated form of Pseudomonas aeruginosa exotoxin A (ETA or PE38) fused with a single-chain antibody fragment (scFv) of CD7, has been reported to cause about 20% of primary leukemia cell death, but its anti-leukemia effect in vivo has not been further evaluated. This implies that T-cell leukemia cells may be insensitive to PE38 or that the reported CD7 single chain antibody needs further improvement. In fact, the anti-CD22 immunotoxin, composed of PE38, has shown impressive therapeutic effects in clinical trials of hairy cell leukemia patients, reaching a complete remission of 46% without significant dosage. Restrictive toxicity indicates that PE38 is sensitive to at least some lymphocytes. Therefore, new anti-CD7 antibodies or variable region fragments may provide new options for improving the efficacy of immunotoxins against T-cell lymphoma and leukemia. Body is a single domain antibody first discovered by Casterman et al. It comes from the heavy chain variable region of camel heavy chain antibody and has many outstanding physical and chemical properties, which makes them excellent candidates for targeted delivery of bioactive drugs. Objective: to screen cd7-specific nano-antibodies, construct novel immunotoxins based on CD7 nano-antibodies and pe38, detect the effects of these immunotoxins on T-ALL cell lines, T-ALL patients and primary cells of AML patients in vitro, and finally evaluate their anti-albumin in vivo. Methods: Camels were immunized with cd7-positive Jurkat cells. The peripheral blood lymphocytes of immunized camels were extracted and phage libraries of nano-antibodies were constructed. Anti-cd7 nano-antibodies were screened by bio-elution. The nano-antibodies vhh6, immunotoxin pg001 (vhh6-pe38) and pg002 (dvhh6-pe38) were detected by flow cytometry. Specificity and affinity. In vitro cytotoxicity of nano-antibody immunotoxins pg001 and pg002 on human cd7-positive jurkat, CEM and cd7-negative rpmi8226, H460 cell lines was determined by detecting inhibition of protein synthesis and apoptosis. The cytotoxicity of pg001 and pg002 on primary cells of leukemia patients was detected by annexin V and 7-AAD staining. The antitumor potential of pg001 and pg002 in vivo was evaluated by CEM cell xenotransplantation tumor model. Results: We successfully identified CD7 nanoantibodies with high specificity and affinity (~15nm) - vhh6. Univalent immunotoxins (pg001) and bivalent immunotoxins (pg002) based on vhh6 maintained a high level of cd7-positive cells. The specific affinity is about 16 nm and 4 nm, respectively. These two toxins efficiently promote apoptosis of cd7-positive leukemia cell lines Jurkat and CEM in an antigen-dependent manner, but have little effect on the proliferation of cd7-negative human multiple myeloma cell lines RPMI 8226 and human lung cancer cell line h460. The semi-effective concentrations of pg002 on Jurkat and CEM cells were 30pm and 23pm respectively, suggesting that pg002 has great potential in the treatment of cd7-positive leukemia. In addition, pg002 can effectively mediate the primary apoptosis of freshly collected T-ALL and AML cells at a single dose of 100ng/ml. In xenograft tumor models, pg001 and pg002 can inhibit the apoptosis. Pg001 and pg002 CD7 nano-antibodies can effectively kill cd7-positive T-ALL fine particles in antigen-specific manner and at low concentrations. Cell lines and fresh T-all and AML patient-derived cells. Based on the effective killing of leukemia cells by immunotoxins pg001 and pg002, the survival of xenograft tumor mice can be significantly prolonged. PG001 and PG002 are worthy of further preclinical and clinical studies to further evaluate their anti-leukemia potential.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.7
，
本文编号：2219526
[Abstract]:In the past few decades, great progress has been made in the clinical treatment of leukemia and lymphoma. However, only a small number of patients with T-cell acute lymphoblastic leukemia (T-ALL) or peripheral T-cell lymphoma (PTCL) have long-term tumor-free survival. Conventional cytotoxic therapy (chemotherapy or chemotherapy) or lymphoma has been used. It is well known that when patients with leukemia or lymphoma become resistant to chemotherapy or relapse, the means of clinical treatment will become very limited, and the patient's survival will become very short. Therefore, efficient, multi-drug resistance mechanisms can be avoided, and the treatment of human T fine. In the development of new therapeutic drugs, recombinant immunotoxins consisting of toxins and tumor cell-specific antibody fragments or ligands are considered to be still effective in the treatment of chemotherapy-resistant T-cell diseases. A key element of immunotoxins is the selection of suitable targets for tumor cells. Many studies have shown that CD7 molecules are expressed on the surface of most T-cell lymphomas and leukemia cells, but are absent on a small number of normal T-lymphocytes. Another advantage of CD7 as a therapeutic target is that it is derived from antibodies or antibodies. Because of these advantages, a variety of CD7-specific immunotoxins have been prepared and tested for their anti-leukemic effects. However, most of the research has focused on phytotoxins, such as ricin, saponin and their derivatives. Another immunotoxin, a truncated form of Pseudomonas aeruginosa exotoxin A (ETA or PE38) fused with a single-chain antibody fragment (scFv) of CD7, has been reported to cause about 20% of primary leukemia cell death, but its anti-leukemia effect in vivo has not been further evaluated. This implies that T-cell leukemia cells may be insensitive to PE38 or that the reported CD7 single chain antibody needs further improvement. In fact, the anti-CD22 immunotoxin, composed of PE38, has shown impressive therapeutic effects in clinical trials of hairy cell leukemia patients, reaching a complete remission of 46% without significant dosage. Restrictive toxicity indicates that PE38 is sensitive to at least some lymphocytes. Therefore, new anti-CD7 antibodies or variable region fragments may provide new options for improving the efficacy of immunotoxins against T-cell lymphoma and leukemia. Body is a single domain antibody first discovered by Casterman et al. It comes from the heavy chain variable region of camel heavy chain antibody and has many outstanding physical and chemical properties, which makes them excellent candidates for targeted delivery of bioactive drugs. Objective: to screen cd7-specific nano-antibodies, construct novel immunotoxins based on CD7 nano-antibodies and pe38, detect the effects of these immunotoxins on T-ALL cell lines, T-ALL patients and primary cells of AML patients in vitro, and finally evaluate their anti-albumin in vivo. Methods: Camels were immunized with cd7-positive Jurkat cells. The peripheral blood lymphocytes of immunized camels were extracted and phage libraries of nano-antibodies were constructed. Anti-cd7 nano-antibodies were screened by bio-elution. The nano-antibodies vhh6, immunotoxin pg001 (vhh6-pe38) and pg002 (dvhh6-pe38) were detected by flow cytometry. Specificity and affinity. In vitro cytotoxicity of nano-antibody immunotoxins pg001 and pg002 on human cd7-positive jurkat, CEM and cd7-negative rpmi8226, H460 cell lines was determined by detecting inhibition of protein synthesis and apoptosis. The cytotoxicity of pg001 and pg002 on primary cells of leukemia patients was detected by annexin V and 7-AAD staining. The antitumor potential of pg001 and pg002 in vivo was evaluated by CEM cell xenotransplantation tumor model. Results: We successfully identified CD7 nanoantibodies with high specificity and affinity (~15nm) - vhh6. Univalent immunotoxins (pg001) and bivalent immunotoxins (pg002) based on vhh6 maintained a high level of cd7-positive cells. The specific affinity is about 16 nm and 4 nm, respectively. These two toxins efficiently promote apoptosis of cd7-positive leukemia cell lines Jurkat and CEM in an antigen-dependent manner, but have little effect on the proliferation of cd7-negative human multiple myeloma cell lines RPMI 8226 and human lung cancer cell line h460. The semi-effective concentrations of pg002 on Jurkat and CEM cells were 30pm and 23pm respectively, suggesting that pg002 has great potential in the treatment of cd7-positive leukemia. In addition, pg002 can effectively mediate the primary apoptosis of freshly collected T-ALL and AML cells at a single dose of 100ng/ml. In xenograft tumor models, pg001 and pg002 can inhibit the apoptosis. Pg001 and pg002 CD7 nano-antibodies can effectively kill cd7-positive T-ALL fine particles in antigen-specific manner and at low concentrations. Cell lines and fresh T-all and AML patient-derived cells. Based on the effective killing of leukemia cells by immunotoxins pg001 and pg002, the survival of xenograft tumor mice can be significantly prolonged. PG001 and PG002 are worthy of further preclinical and clinical studies to further evaluate their anti-leukemia potential.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.7
，
本文编号：2219526
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