2E8-Gen免疫毒素导向杀伤B系白血病细胞的研究
摘要
急性B细胞系白血病(B系ALL)是儿童血液系统最常见的恶性肿瘤，也是儿童时期死亡率很高的疾病。目前的主要治疗方法是抗肿瘤药物的联合化学治疗(化疗)，必要时加放射治疗(放疗)。尽管化疗和放疗的治疗作用勿庸置疑，但尚存在严重的缺陷，即它们在杀伤恶性细胞的同时对正常细胞也可造成不同程度的伤害。免疫毒素通常是采用具有一定导向作用的先导分子和药物耦合而成，它们对人体肿瘤具有明显的导向治疗作用，可克服当前化疗和放疗普遍存在的缺陷。因此，开展免疫毒素的肿瘤导向治疗研究具有极其重要的意义。
有效的导向治疗取决于良好的先导分子及其携带的毒素，以与白细胞表面分化抗原特异性结合的单抗作为载体，将药物特异地带到白血病细胞膜上或细胞内，以达到杀灭白血病细胞的目的，有着无可替代的优势。有些免疫毒素的动物和临床研究已取得理想的疗效。因此，开展免疫毒素的肿瘤导向治疗研究具有极其重要的意义。
目前国际上抗人CD19单抗有B4、B43、HD37、ZCH-4-2E8、SJ25C1和FMC63等几十种，其中有3株(B4、B43和HD37)被研制成免疫毒素：B43-4’，5’，7-三羟基异黄酮(B43-Gen)、B43-商陆抗病毒蛋白(B43-PAP)、HD37-去糖基蓖麻毒素A链(HD37-dgRTA)和B4-封闭性蓖麻毒素(Anti-B4-bR)；这些免疫毒素导向杀伤B系ALL的动物和临床研究已取得了较为理想的效果。国内对该靶分子的导向杀伤研究未见报道，采用抗人CD19新克隆ZCH-4-2E8作为先导分子进行导向杀伤研究国内外均未见报道。
2E8是本实验室研制的经国际鉴定为特异性识别CD19抗原的单抗，并正式纳
浙江大学硕士学位论文 中文摘要
入了国际CD命名系统。
许多毒素被用于研制兔疫毒素，如：蓖麻毒蛋白 A链kicin A chain入 商陆
抗病毒蛋白（poke忧ed antiviral protein，PAP），绿脓杆菌外毒素（exotoxin of
pseudomonas aeruglnosa，ETA），4’，5’尸－二羟基异黄酮（genistein，Gen）等。Gen
是蛋白酪氨酸激酶抑制剂，与抗体偶合后能有效的杀伤白血病细胞。Uckun等报道
B43－Gen在体外能杀死 99．999％有 CD表达的 Nalm－6细胞，对化疗失败的 B系
ALL患者仍有效。
本实验通过双功能光敏偶合剂Sulfo－SANPAH在体外进行2E8与Gen的铰联
来完成2E8．Gen免疫毒素的研制，并研究其对CD19＋白血病细胞的导向杀伤作用，
初步探讨其杀伤机制，为进一步研究第二代兔疫毒素如单链抗体免疫毒素pCFV
immunotoxinX人鼠嵌合性抗体和人源化抗体奠定基础。
材料与方法
1．ZCH－4．2E8腹水的制备、纯化和鉴定
2E8细胞经3次亚克隆化后按常规方法制备大量腹水，采用改良B。VV幻法纯
化IgM型2E8单抗。纯化后2E8样品用二硫苏糖醇0TT）还原，经十二烷基硫酸
钠一聚丙稀酚胺（SDS－PAGE）凝胶电泳鉴定其纯度。
二．2E8－Gen免疫毒素的研制
2E8与Gen偶合是通过双功能铰联剂Sulfo－SANPAH在体外进行化学铰联（按
SUlfO七ANPAH试剂说明书并参考文献X再经纯化而获得。并用色谱层析法检查游
离Gen的去除情况。
3．细胞培养及实验分组
2E8、Nalm七旧细胞白血病）、K562（红白血病）和讪卜3 门细胞白血病）
细胞株在含 20％灭活小牛血清的 RPMI 640培养液中，常规条件下培养。取处于
对数增长期、存活率达 95％以上的细胞用于实验。
实验分组如下：（）对照（NC）组：同期培养不加药；（2）2E8－Gen兔疫毒
素O）组：加不同浓度的2田乃m（）磷酸盐缓冲液o＊S，既是2田乃n第
2
浙江大学硕士学位论文 中文摘要
3次的透析外液也是其溶剂）组：加入与2m乃 相同体积的PSS：N）＊m组：
加不同浓度的Gen（500nM、40W和 100W）；（5）纯2E8组：加入与2E8．Gen中
载体单抗2E8相同浓度和相同体积的未与Gen结合的纯2E8。培养时间和起始细
胞数量按实验需要而定。测绘细胞生长曲线时细胞培养于24孔板，MTT法细胞培
养于96孔板。
4．细胞观察
用倒置相差显微镜观察培养细胞的生长状态及形态学特点，照相记录。
5．生长曲线测定
用苔盼蓝拒染法测绘各组的Nalm－6、K562和Molt3细胞培养3～5天的生长
曲线。每组均以相隔24小时为一观察点，每观察点做3个复孔。
6．MTT比色法测定
将细胞接种于96孔板中，加药后再加培养基到每孔200PL培养，实验终点后，
力入浓度为 sing 加L的 MTT溶液每孔 20pL，继续培养 4 ’J’时，经 2000rPmX20min
离心，吸去培养基，加入二甲基亚矾m）2每孔200pL以溶解沉淀物，振荡
smin后用酶标仪检测。每24 ／J’时为一观察点，每观察点做3或12个复孔。
7．双色流式细胞仪检测
荧光素标记的连接素 V／碘化丙旋unnexin V干ITC小）染色和操作步骤按
试剂盒说明书进行，l小时内用流式细胞仪检测分析。
8．统计学处理
全部数据采用“S6．12统计软件处理。数据以均值士标准差（肚m）表示，
采用学生t检验，尸
B lineage acute lymphoblastic leukemia (ALL) is the most common type of hematopoietic malignancies in children. The current primary treatment of childhood ALL is combined chemotherapy. Although the present therapies on childhood ALL are effective, severe toxicity and side effects are still the major complications due to the poor treatment selection of the chemotherapeutic drugs. Since 1975 when the technology of hybridoma and monoclonal antibody was developed, many investigators have been paying close attention to antibody targeting therapy for patients with cancer. As compared to conventional chemotherapy, antibody target therapy on leukemia cells (biotherapy) is well selective. It only kills leukemia cells expressing target molecules while leaving those without target molecules unaffected. Therefore targeting therapy can greatly reduce the opportunity of nonspecific toxicity and side effects on normal tissues.
A good targeting reagent depends on the directing carrier and its ligand toxin conjugated. Use of monoclonal antibody (Mab) recognizing leukocyte differentiation antigens as directing carrier has an overwhelming advantage over any other targeting molecules. Mabs can specifically bind to the antigens existing on the leukemic cell surface and carry the toxin to the surface of malignant cells resulting in killing. The preliminary application of some of the reagents in clinical practice has showed the promising results.
There are many mouse anti-human CD 19 Mabs reported in the literature, such as B4, B43, HD37, ZCH-4-2E8 (2E8), SJ25C1 and FMC63, etc. among which only B4, B43 and HD37 have been conjugated with toxins to form immunotoxins, such as B43-Genistein, B43-Pokeweed antiviral protein (B43-PAP), HD37-deglycosylated ricin A chain(HD37-dgRTA) and B4-blocked ricin (B4-bR); the effectiveness of these immunotoxins has showed promising in treatment of B precursor leukemias in both animal models and in clinical patients. According to the literature found, no CD 19 immunotoxin has been documented at home while no report at al has been published on the study using ZCH-4-2E8 as directing carrier to generate CD 19 imunotoxin both at home and abroad.
ZCH-4-2E8, a new clone of CD 19 Mab was generated in this lab and assigned into CD 19 category by the 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA6) in 1996.
Many toxins have been used to conjugate directing antibodies so far, such as ricin A chain, pokeweed antiviral protein (PAP), exotoxin of pseudomonas aeruginosa (ETA), genistein (Gen) etc. The latter is a tyrosine kinase inhibitor which can effectively kill the leukemia cells when it linked to certain directing antibodies. Uckun et al have reported that their B43-Gen can kill 99.999% of the CD 19+ Nalm-6 cells in vitro and showed promising treatment results in patients with B precursor ALL with minimal toxicity.
In this study, the new clone of CD 19, 2E8 was used to conjugate with Genistein (Gen) via a light sensitive agent Sulfo-SANPAH to generate 2E8-Gen immunotoxin. By using this immunotoxin, target killing of CD 19+ leukemia cells was carried out and the killing mechanism was studied. The results of this study have provided a better fundamentals for generating second generation of 2E8 immunotoxin such as single chain fragment of variable region immunotoxin (ScFv immunotoxin), human-mouse chimeric antibody or humanized antibody for clinical purpose.
Material and Methods
1. Generation and purification of the 2E8 antibody
Before generation of large quantity of 2E8 antibody, three subcloning were performed to avoid the possible mutation of the hybridoma cells in which some none-secreting cells might exist. A large quantity of 2E8 ascites was made using the method previously described in the literature. Purification of the antibody was carried out using the Bouvet method with modification and followed by chromatography. The purity of antibody was checked with SDS-PAGE.
2. Preparation of 2E8-Gen immunotoxin
2E8-Gen was prepared to conjugate
有效的导向治疗取决于良好的先导分子及其携带的毒素，以与白细胞表面分化抗原特异性结合的单抗作为载体，将药物特异地带到白血病细胞膜上或细胞内，以达到杀灭白血病细胞的目的，有着无可替代的优势。有些免疫毒素的动物和临床研究已取得理想的疗效。因此，开展免疫毒素的肿瘤导向治疗研究具有极其重要的意义。
目前国际上抗人CD19单抗有B4、B43、HD37、ZCH-4-2E8、SJ25C1和FMC63等几十种，其中有3株(B4、B43和HD37)被研制成免疫毒素：B43-4’，5’，7-三羟基异黄酮(B43-Gen)、B43-商陆抗病毒蛋白(B43-PAP)、HD37-去糖基蓖麻毒素A链(HD37-dgRTA)和B4-封闭性蓖麻毒素(Anti-B4-bR)；这些免疫毒素导向杀伤B系ALL的动物和临床研究已取得了较为理想的效果。国内对该靶分子的导向杀伤研究未见报道，采用抗人CD19新克隆ZCH-4-2E8作为先导分子进行导向杀伤研究国内外均未见报道。
2E8是本实验室研制的经国际鉴定为特异性识别CD19抗原的单抗，并正式纳
浙江大学硕士学位论文 中文摘要
入了国际CD命名系统。
许多毒素被用于研制兔疫毒素，如：蓖麻毒蛋白 A链kicin A chain入 商陆
抗病毒蛋白（poke忧ed antiviral protein，PAP），绿脓杆菌外毒素（exotoxin of
pseudomonas aeruglnosa，ETA），4’，5’尸－二羟基异黄酮（genistein，Gen）等。Gen
是蛋白酪氨酸激酶抑制剂，与抗体偶合后能有效的杀伤白血病细胞。Uckun等报道
B43－Gen在体外能杀死 99．999％有 CD表达的 Nalm－6细胞，对化疗失败的 B系
ALL患者仍有效。
本实验通过双功能光敏偶合剂Sulfo－SANPAH在体外进行2E8与Gen的铰联
来完成2E8．Gen免疫毒素的研制，并研究其对CD19＋白血病细胞的导向杀伤作用，
初步探讨其杀伤机制，为进一步研究第二代兔疫毒素如单链抗体免疫毒素pCFV
immunotoxinX人鼠嵌合性抗体和人源化抗体奠定基础。
材料与方法
1．ZCH－4．2E8腹水的制备、纯化和鉴定
2E8细胞经3次亚克隆化后按常规方法制备大量腹水，采用改良B。VV幻法纯
化IgM型2E8单抗。纯化后2E8样品用二硫苏糖醇0TT）还原，经十二烷基硫酸
钠一聚丙稀酚胺（SDS－PAGE）凝胶电泳鉴定其纯度。
二．2E8－Gen免疫毒素的研制
2E8与Gen偶合是通过双功能铰联剂Sulfo－SANPAH在体外进行化学铰联（按
SUlfO七ANPAH试剂说明书并参考文献X再经纯化而获得。并用色谱层析法检查游
离Gen的去除情况。
3．细胞培养及实验分组
2E8、Nalm七旧细胞白血病）、K562（红白血病）和讪卜3 门细胞白血病）
细胞株在含 20％灭活小牛血清的 RPMI 640培养液中，常规条件下培养。取处于
对数增长期、存活率达 95％以上的细胞用于实验。
实验分组如下：（）对照（NC）组：同期培养不加药；（2）2E8－Gen兔疫毒
素O）组：加不同浓度的2田乃m（）磷酸盐缓冲液o＊S，既是2田乃n第
2
浙江大学硕士学位论文 中文摘要
3次的透析外液也是其溶剂）组：加入与2m乃 相同体积的PSS：N）＊m组：
加不同浓度的Gen（500nM、40W和 100W）；（5）纯2E8组：加入与2E8．Gen中
载体单抗2E8相同浓度和相同体积的未与Gen结合的纯2E8。培养时间和起始细
胞数量按实验需要而定。测绘细胞生长曲线时细胞培养于24孔板，MTT法细胞培
养于96孔板。
4．细胞观察
用倒置相差显微镜观察培养细胞的生长状态及形态学特点，照相记录。
5．生长曲线测定
用苔盼蓝拒染法测绘各组的Nalm－6、K562和Molt3细胞培养3～5天的生长
曲线。每组均以相隔24小时为一观察点，每观察点做3个复孔。
6．MTT比色法测定
将细胞接种于96孔板中，加药后再加培养基到每孔200PL培养，实验终点后，
力入浓度为 sing 加L的 MTT溶液每孔 20pL，继续培养 4 ’J’时，经 2000rPmX20min
离心，吸去培养基，加入二甲基亚矾m）2每孔200pL以溶解沉淀物，振荡
smin后用酶标仪检测。每24 ／J’时为一观察点，每观察点做3或12个复孔。
7．双色流式细胞仪检测
荧光素标记的连接素 V／碘化丙旋unnexin V干ITC小）染色和操作步骤按
试剂盒说明书进行，l小时内用流式细胞仪检测分析。
8．统计学处理
全部数据采用“S6．12统计软件处理。数据以均值士标准差（肚m）表示，
采用学生t检验，尸
B lineage acute lymphoblastic leukemia (ALL) is the most common type of hematopoietic malignancies in children. The current primary treatment of childhood ALL is combined chemotherapy. Although the present therapies on childhood ALL are effective, severe toxicity and side effects are still the major complications due to the poor treatment selection of the chemotherapeutic drugs. Since 1975 when the technology of hybridoma and monoclonal antibody was developed, many investigators have been paying close attention to antibody targeting therapy for patients with cancer. As compared to conventional chemotherapy, antibody target therapy on leukemia cells (biotherapy) is well selective. It only kills leukemia cells expressing target molecules while leaving those without target molecules unaffected. Therefore targeting therapy can greatly reduce the opportunity of nonspecific toxicity and side effects on normal tissues.
A good targeting reagent depends on the directing carrier and its ligand toxin conjugated. Use of monoclonal antibody (Mab) recognizing leukocyte differentiation antigens as directing carrier has an overwhelming advantage over any other targeting molecules. Mabs can specifically bind to the antigens existing on the leukemic cell surface and carry the toxin to the surface of malignant cells resulting in killing. The preliminary application of some of the reagents in clinical practice has showed the promising results.
There are many mouse anti-human CD 19 Mabs reported in the literature, such as B4, B43, HD37, ZCH-4-2E8 (2E8), SJ25C1 and FMC63, etc. among which only B4, B43 and HD37 have been conjugated with toxins to form immunotoxins, such as B43-Genistein, B43-Pokeweed antiviral protein (B43-PAP), HD37-deglycosylated ricin A chain(HD37-dgRTA) and B4-blocked ricin (B4-bR); the effectiveness of these immunotoxins has showed promising in treatment of B precursor leukemias in both animal models and in clinical patients. According to the literature found, no CD 19 immunotoxin has been documented at home while no report at al has been published on the study using ZCH-4-2E8 as directing carrier to generate CD 19 imunotoxin both at home and abroad.
ZCH-4-2E8, a new clone of CD 19 Mab was generated in this lab and assigned into CD 19 category by the 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA6) in 1996.
Many toxins have been used to conjugate directing antibodies so far, such as ricin A chain, pokeweed antiviral protein (PAP), exotoxin of pseudomonas aeruginosa (ETA), genistein (Gen) etc. The latter is a tyrosine kinase inhibitor which can effectively kill the leukemia cells when it linked to certain directing antibodies. Uckun et al have reported that their B43-Gen can kill 99.999% of the CD 19+ Nalm-6 cells in vitro and showed promising treatment results in patients with B precursor ALL with minimal toxicity.
In this study, the new clone of CD 19, 2E8 was used to conjugate with Genistein (Gen) via a light sensitive agent Sulfo-SANPAH to generate 2E8-Gen immunotoxin. By using this immunotoxin, target killing of CD 19+ leukemia cells was carried out and the killing mechanism was studied. The results of this study have provided a better fundamentals for generating second generation of 2E8 immunotoxin such as single chain fragment of variable region immunotoxin (ScFv immunotoxin), human-mouse chimeric antibody or humanized antibody for clinical purpose.
Material and Methods
1. Generation and purification of the 2E8 antibody
Before generation of large quantity of 2E8 antibody, three subcloning were performed to avoid the possible mutation of the hybridoma cells in which some none-secreting cells might exist. A large quantity of 2E8 ascites was made using the method previously described in the literature. Purification of the antibody was carried out using the Bouvet method with modification and followed by chromatography. The purity of antibody was checked with SDS-PAGE.
2. Preparation of 2E8-Gen immunotoxin
2E8-Gen was prepared to conjugate
引文
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[23]Sharkey RM, Behr TM, Mattes MJ, et al. Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22. Cancer Immunol Immunother, 1997, 44(3): 179-88
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[28]James JS, Dubs G. FDA approves new kind of lymphoma treatment. Food and Drug Administration. AIDS Treat News, 1997, 284:2-3
[29]Dillman RO. Infusion reactions associated with the therapeutic use of monoclonal antibodies in the treatment of malignancy. Cancer Metastasis Rev, 1999, 18(4): 465-71
[30]Uckun FM, Bellomy K, O'Neill K, et al. Toxicity, biological activity, and pharmacokinetics of TXU (anti-CD7)-pokeweed antiviral protein in chimpanzees and adult patients infected with human immunodeficiency virus. J Pharmacol Exp Ther, 1999, 291(3): 1301-7
[31]Francisco JA, Siegall CB. Single-chain immunotoxins targeted to CD40 for the treatment of human B-lineage hematologic malignancies. Leuk Lymphoma, 1998, 30(3-4): 237-45
[32]Schnell R, Vitetta E, Schindler J, et al. Clinical trials with an anti-CD25 ricin A-chain experimental and immunotoxin (RFT5-SMPT-dgA) in Hodgkin's lymphoma. Leuk Lymphoma, 1998, 30:525-37
[33]Miller JL. FDA approves antibody-directed cytotoxic agent for acute myeloid leukemia. Am J Health Syst Pharm, 2000, 57(13): 1202-4
[34]Grillo-Lopez AJ, Dallaire BK, McClure A, et al. Monoclonal antibodies: a new era in the treatment of non-Hodgkin's lymphoma. Curr Pharm Biotechnol, 2001, 2(4): 301-11
[35]Messinger Y, Yanishevski Y, Avramis VI, et al. Treatment of human B-cell precursor leukemia in SCID mice using a combination of the investigational biotherapeutic agent B43-PAP with cytosine arabinoside. Clin Cancer Res, 1996, 2(9): 1533-42
[36]Ek O, Gaynon P, Zeren T, et al. Treatment of human B-cell precursor leukemia in SCID mice by using a combination of the anti-CD19 immunotoxin B43-PAP with the standard chemotherapeutic drugs vincristine, methylprednisolone, and L-asparaginase. Leuk Lymphoma. 1998, 31: 143-9
[37]Ek O, Reaman GH, Crankshaw DL, et al. Combined therapeutic efficacy of the
thymidylate synthase inhibitor ZD1694 (Tomudex) and the immunotoxin B43(anti-CD19)-PAP in a SCID mouse model of human B-lineage acute lymphoblastic leukemia. Leuk Lymphoma, 1998, 28: 509-14
[38]Uckun FM, Messinger Y, Chen CL, et al. Treatment of therapy-refractory B-lineage acute lymphoblastic leukemia with an apoptosis-inducing CD19-directed tyrosine kinase inhibitor. Clin Cancer Res, 1999, 5(12): 3906-13
[39]Grossbard ML, Freedman AS, Ritz J, et al. Serotherapy of B-cell neoplasms with anti-B4-blocked ricin: a phase Ⅰ trial of daily bolus infusion. Blood, 1992, 79(3): 576-85
[40]Grossbard ML, Multani PS, Freedman AS, et al. A Phase Ⅱ study of adjuvant therapy with anti-B4-blocked ricin after autologous bone marrow transplantation for patients with relapsed B-cell non-Hodgkin's lymphoma. Clin Cancer Res, 1999, 5(9): 2392-8
[41]Longo DL, Duffey PL, Gribben JG, et al. Combination chemotherapy followed by an immunotoxin (anti-B4-blocked ricin) in patients with indolent lymphoma: results of a phase Ⅱ study. Cancer J, 2000, 6(3): 146-50
[42]Herrera L, Farah RA, Pellegrini VA, et al. Immunotoxins against CD19 and CD22 are effective in killing precursor-B acute lymphoblastic leukemia cells in vitro. Leukemia, 2000, 14(5): 853-8
[43]Le-Gall F, Kipriyanov SM, Moldenhauer G, Little M. Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. FEBS Lett. 1999, 453(1-2): 164-8
[44]Chiron MF. Recombinant immunotoxins and chimeric toxins for targeted therapy in oncology. Bull Cancer, 1997, 84(12): 1135-40
[45]Mansfield E, Amlot P, Pastan I, FitzGerald DJ. Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD22-bearing cells and tumors. Blood. 1997, 90(5): 2020-6