基于CD7納米抗體的新型免疫毒素對人白血病細胞在體外和體內的活性研究
發(fā)布時間：2018-09-03 09:07
【摘要】：在過去的幾十年里,白血病和淋巴瘤的臨床治療取得了很大的進展。然而在T細胞白血病和淋巴瘤中,僅僅只有一小部分的T急性淋巴細胞白血病(T-ALL)或者外周T細胞淋巴瘤(PTCL)病人獲得了長期無腫瘤生存。常規(guī)的細胞毒療法(化療或者放療等)對病人具有副反應且療效有限。眾所周知,當白血病或者淋巴瘤患者一旦對化療產(chǎn)生耐藥或者出現(xiàn)復發(fā),臨床治療手段將會變得十分有限,病人的生存期也隨之變得很短。因此高效的、能夠避免多藥耐藥性機制,而且對治療人T細胞惡性腫瘤具有良好特異性和毒性的新療法是具有十分重要的科學和臨床意義。在新的治療藥物開發(fā)中,由毒素和腫瘤細胞特異性的抗體片段或者配體等融合構成的重組免疫毒素被認為對化療耐藥性的T細胞疾病是仍然有效的。運用免疫毒素的一個關鍵要素就是選擇腫瘤細胞適合的靶點。許多研究已經(jīng)表明CD7分子在大多數(shù)T細胞淋巴瘤和白血病細胞表面表達,而在一小部分正常T淋巴細胞上缺失。CD7分子作為治療靶點的另外一個優(yōu)勢就是當它和抗體或者抗體衍生物結合后會迅速內化,這使得針對CD7分子的抗體非常適合作為藥物運輸工具。由于上述優(yōu)點,多種CD7特異性的免疫毒素被制備出來并檢測它們的抗白血病效應。然而,大多數(shù)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,這些毒素由于缺乏足夠的安全和療效而未獲得臨床批準。另一種免疫毒素,截短形式的銅綠假單胞菌外毒素A(ETA或PE38)融合到CD7的單鏈抗體片段(scFv),被報道可造成約20%原代白血病細胞死亡,但是卻沒有進一步評估其體內抗白血病效應,這意味著T細胞白血病細胞可能對PE38不敏感或者報道的CD7單鏈抗體需要進一步的改進。事實上,由PE38構成的抗CD22免疫毒素在用于毛細胞白血病患者的臨床試驗中表現(xiàn)出令人印象深刻治療效應,達到了46%的完全緩解,而且無明顯的劑量限制性毒性,這表明PE38至少對一些淋巴細胞是敏感的。因此,新的抗CD7抗體或者可變區(qū)片段有可能為我們提高針對T細胞淋巴瘤和白血病免疫毒素功效提供新的選擇。納米抗體被選擇成為我們要開發(fā)的新型抗CD7抗體的原因如下:納米抗體是由casterman等人首先發(fā)現(xiàn)的一種單域抗體,來自駱駝重鏈抗體的重鏈可變區(qū),具有諸多杰出的理化性質,這使它們成為了靶向遞送生物活性藥物的優(yōu)秀候選者。研究人員已經(jīng)證明納米抗體可以與毒素以及其他功能性分子偶聯(lián),然后用這些偶聯(lián)物去靶向細胞,對癌癥和其他疾病進行治療。目的:篩選cd7特異性的納米抗體,構建新型的基于cd7納米抗體和pe38的免疫毒素,在體外檢測這些免疫毒素對t-all細胞系、t-all病人和aml病人原代細胞的效應,最后在體內評估它們抗白血病的作用。方法:用cd7陽性的jurkat細胞免疫駱駝,提取免疫后的駱駝外周血淋巴細胞,構建納米抗體噬菌體文庫,用生物淘洗的方法篩選獲得抗cd7的納米抗體。通過流式細胞分析儀檢測納米抗體vhh6、免疫毒素pg001(vhh6-pe38)和pg002(dvhh6-pe38)的特異性和親和力。在體外通過檢測蛋白質合成抑制和凋亡的方法來測定納米抗體免疫毒素pg001和pg002對人cd7陽性的jurkat、cem和cd7陰性的rpmi8226、h460細胞系的細胞毒作用。通過annexinv和7-aad染色檢測pg001和pg002對白血病患者原代細胞的細胞毒效應。pg001和pg002在體內的抗腫瘤潛力采用cem細胞異種移植腫瘤模型進行評估。結果:我們成功鑒定了具有高特異性和親和力(~15nm)的cd7納米抗體——vhh6�；趘hh6的單價免疫毒素(pg001)和雙價免疫毒素(pg002)對cd7陽性細胞保持有高度的特異性,親和力分別約為16nm和4nm。這兩種毒素高效地以抗原依賴型的方式促進cd7陽性白血病細胞系jurkat和cem發(fā)生凋亡,但是對cd7陰性的人多發(fā)性骨髓瘤細胞系rpmi8226和人肺癌細胞系h460增殖的幾乎沒有影響。特別需要指出的是,免疫毒素pg002對jurkat、cem細胞的半有效濃度分別為30pm和23pm,這提示pg002治療cd7陽性白血病具有很大的潛力。此外,pg002在單劑量為100ng/ml濃度條件下能有效介導新鮮收集的t-all和aml患者原代細胞凋亡。在異種移植腫瘤模型中,pg001和pg002能夠抑制cem細胞的增殖,并可顯著延長小鼠的生存期。其中,pg002相比于pg001在腫瘤模型中的治療效果更為突出。結論:我們構建的單價(pg001)和雙價(pg002)cd7納米抗體免疫毒素以抗原特異性的方式,以及在低濃度條件下即可有效殺傷cd7陽性t-all細胞系和新鮮t-all和aml病人來源的細胞�；诿庖叨舅豴g001和pg002有效殺死白血病細胞,可顯著延長異種移植腫瘤小鼠的生存,PG001和PG002值得進一步開展臨床前和臨床試驗研究,從而進一步評價它們抗白血病的潛能。
[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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R733.7
，
本文編號：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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R733.7
，
本文編號：2219526
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