刘小云

论文题目：单克隆抗体导向药物分子小型化及抗肿瘤作用

作者简介：刘小云，男，1968年11月出生，1997年09月师从于中国协和医科大学甄永苏教授，于2000年07月获博士学位。

摘　要

 

    以单克隆抗体为载体的抗肿瘤导向药物既是医药生物技术领域的研究热点也是抗肿瘤药物研究的重大课题。经典的完整单抗与药物偶联物具有分子庞大、对实体瘤穿透力弱、体内分布特异性不高以及易引发人抗鼠抗体反应等诸多缺点，因此临床疗效不佳。寻找合理的途径克服以上不足是单抗导向药物发展的关键。迄今为止，主要途径包括抗体的改造、抗体作用新靶点以及新型弹头药物的发现与应用。本研究在以上诸方面进行探讨。

 

一． 抗肝癌单抗3A5 Fab'片段与平阳霉素偶联物的抗肿瘤作用

 

    单抗Fc段是引起偶联物在体内非特异性分布及人抗鼠抗体反应的主要成分，是当前抗体改造的首选目标。去除Fc段，不仅有利于降低抗体引发的不良反应。而且可降低偶联物分子量。本研究首先以大鼠抗人肝癌BEL-7402细胞单抗3A5为研究对象，以胃蛋白酶降解3A5去除Fc段，再经DTT还原获得活性Fab'片段。在此基础上以dextran T-40为中介体将 Fab'片段与抗肿瘤抗生素平阳霉素偶联，获得偶联物Fab'-PYM。酶联免疫法（ELISA）测定Fab'-PYM偶联物与靶细胞的免疫反应性、2，3，5-氯化三苯基四氮唑（TTC）法测定偶联物中平阳霉素效价、克隆形成法和MTT法测定偶联物细胞毒作用并以动物移植肿瘤模型观察偶联物的体内抗肿瘤作用。结果表明，偶联物保持了与靶细胞的免疫反应性；Fab'与平阳霉素偶联分子比为1:50; 偶联物中平阳霉素保留原效价15%；克隆形成法测定结果表明，偶联物对体外培养的靶细胞肝癌BEL-7402细胞及肠癌HT-29细胞的IC₅₀分别为0.02 mM和0.08 mM，对非靶细胞KB的IC₅₀为0.50 mM；平阳霉素对以上三种细胞的IC₅₀分别为0.51 mM、1.45 mM和0.42 mM；MTT法测定结果表明，偶联物对靶细胞小鼠结肠癌26（C26）细胞的杀伤作用明显强于游离平阳霉素，二者IC₅₀分别为1.61 mM和21.46 mM；对非靶细胞KB的作用相似，IC₅₀分别为3.45 mM和2.63 mM。表明偶联物在体外对靶细胞的细胞毒作用强于游离平阳霉素。体内抗肿瘤实验结果表明，偶联物对结肠癌26生长有显著抑制作用，其抑制率高于等剂量游离PYM。 5 mg/kg ´ 6，ip，偶联物治疗组抑瘤率为77%，游离PYM组抑瘤率为56% (p< 0.05)，动物的心、肺、肝、脾、肾、胃、小肠和股骨等病理组织检查未发现毒性变化； 5 mg/kg ´ 7，iv， Fab'-PYM、3A5-PYM和PYM抑瘤率分别为89%、73%和70%，Fab'-PYM作用明显强于3A5-PYM和PYM (p<0.01)； 10 mg/kg ´ 7，iv ，Fab'-PYM抑瘤率可达91%； 10 mg/kg ´ 4, iv，Fab’-PYM和PYM对脾内接种结肠癌26肝转移抑制率分别为91%和62%， 5 mg/kg ´ 4，iv ，Fab’-PYM对结肠癌26的肝转移抑制率为78%。以小鼠皮下接种肝癌22 (H22) 模型观察偶联物对肝癌的作用， 10 mg/kg ´ 5, iv ，Fab’-PYM和游离PYM抑瘤率分别为86%和63%； 5 mg/kg ´ 5，iv， Fab’-PYM抑瘤率为82%。

     以上结果表明，单抗3A5 Fab'片段与PYM偶联物具有比游离平阳霉素及平阳霉素与完整抗体更强的抗肿瘤作用。

 

二． 抗IV型胶原酶单抗3D6 Fab'片段与平阳霉素偶联物的抗肿瘤作用

 

    IV型胶原酶是肿瘤细胞发生侵袭与转移的重要分子基础，也是抗癌药物新靶点。本研究首先以无花果蛋白酶酶解3D6获得F(ab')₂片段，以ELISA法测定F(ab')₂片段与IV型胶原酶的免疫反应性、克隆形成法测定F(ab')₂对KB细胞的细胞毒作用、明胶酶谱法测定F(ab')₂对IV型胶原酶活性的影响、以小鼠皮下接种Lewis癌肺转移模型观察F(ab')₂片段的体内抗肿瘤转移作用。结果表明，3D6 F(ab')₂片段对IV型胶原酶的反应性与完整抗体3D6相似并可抑制IV型胶原酶活性，其作用呈剂量效应关系；F(ab')₂对KB及BEL-7402细胞无明显细胞毒作用；体内实验结果显示，隔日iv给药，共6次，1 mg/kg、2 mg/kg和3 mg/kg剂量，F(ab')₂对Lewis癌肺转移有中度抑制作用，抑制率分别为51%、52%和54%，肺组织切片检查结果表明，F(ab')₂治疗组肺内瘤灶也明显少于对照组，但F(ab')₂对皮下瘤生长无影响；1 mg/kg剂量的单抗3D6对Lewis癌肺转移也有中度抑制作用，抑制率为58%。ip给药12次，5 mg/kg和10 mg/kg剂量，F(ab')₂片段对Lewis癌肺转移也有一定抑制作用，抑制率分别为66%和76%；动物的心、肺、肝、脾、肾、胃、小肠和股骨等病理组织检查未发现毒性变化。表明单抗3D6 F(ab')₂可能可以用于治疗肿瘤转移。

 

    为进一步探讨3D6活性片段与药物偶联物用于治疗肿瘤的可能性，本研究以b-巯基乙醇还原3D6 F(ab')₂片段获得有活性的Fab'片段并与平阳霉素偶联，获得偶联物Fab'-PYM。常规方法测定偶联物的生物学性质与体内外抗肿瘤作用。结果表明，Fab'-PYM偶联物保持了与IV型胶原酶及几种靶细胞的免疫反应性；克隆形成法测定，PYM对KB细胞和BEL-7402细胞生长均有抑制作用，IC₅₀分别为0.42 mM和0.17 mM； Fab'-PYM对两种细胞IC₅₀分别为0.02 mM和0.48 mM。 Fab'-PYM对皮下接种的H22有明显抑制作用，iv 给药4次，5 mg/kg和10 mg/kg剂量，抑瘤率分别为64%和82%，10 mg/kg剂量游离平阳霉素抑瘤率为59%；iv 给药5次，Fab'-PYM偶联物可抑制皮下接种Lewis癌的肺转移，抑制率为70%和81%；10 mg/kg游离平阳霉素的抑制率为56%。

    以上结果表明以IV型胶原酶为靶点的单抗Fab'片段与平阳霉素偶联物也可明显抑制肿瘤生长与转移。

 

三． 单抗Fab'片段与平阳霉素偶联物的分子小型化

 

    分子小型化是单抗导向药物发展的趋势，具有分子小型化特点的单抗常规化疗药物偶联物有良好的应用前景。本研究在抗IV型胶原酶单抗3D6 Fab'片段与平阳霉素偶联物基础上进一步探索其小型化改建的可能性。以不同分子量的三种偶联剂葡聚糖T-40 （DT-40，MW 40，000）、葡聚糖T-10 （DT-10，MW 10，000）和环糊精 （CD， MW 1135）为中介体（linker），分别将Fab'片段与平阳霉素偶联，比较了偶联分子比、平阳霉素生物效价变化及细胞毒作用。结果表明，以DT-40、DT-10和CD为中介体，偶联分子比（Fab':Linker:PYM） 分别为1:1:50、1:1:20和1:2.4:7.3，平均分子量分别为170 kDa、95 kDa和68 kDa，偶联物中平阳霉素最小抑菌浓度（MIC）分别为5.00 mM、3.44 mM和4.03 mM；对靶细胞KB的IC₅₀分别为0.01 mM、 0.05 mM和0.02 mM，对非靶细胞BEL-7402的IC₅₀分别为0.48 mM、0.51 mM和0.51 mM。表明环糊精可能是平阳霉素与抗体偶联物的理想中介体。进一步在体内观察Fab'-CD-PYM的抗肿瘤作用，小鼠皮下接种H22，接种后第7天开始治疗，隔日静脉注射给药一次，共4次，10 mg/kg平阳霉素对肿瘤生长无明显作用，但Fab'-CD-PYM仍可明显抑制H22的生长，停药一周（实验第21天），5 mg/kg 和10mg/kg治疗组，抑瘤率分别为73% 和 81%。

    以上结果表明，Fab'-PYM偶联物分子经裁减后不仅具有分子结构小型化的特点，而且仍有高度抗肿瘤作用，值得深入研究。

 

四． 力达霉素用于构建高效、小型化单抗导向药物

 

    小型、高效弹头药物的发现与应用是进一步降低偶联物分子量，提高偶联物对实体瘤穿透力的又一有效手段。多肽类抗生素力达霉素（Lidamycin，LDM，又称C1027）具有细胞毒作用强，体内抗肿瘤活性高且分子量较低（MW约12 kDa）的特点，是导向药物理想弹头。本实验以力达霉素为弹头药物，构建了力达霉素与单抗3A5 及3D6 Fab'片段偶联物，并研究其抗肿瘤作用。

 

     单抗3A5 Fab'-LDM偶联物与靶细胞有较强免疫反应性；偶联物分子量为65 kDa，与目前报道的分子量最小并有明显体内抗肿瘤作用的单链抗体免疫毒素（67 kDa）相当；克隆形成法测定，Fab'-LDM对靶细胞BEL-7402的作用与游离力达霉素相当，IC₅₀分别为2.25×10^-17 M和2.64×10^-16 M；Fab'-LDM和力达霉素对HT-29的IC₅₀分别为1.57×10^-15 M和2.65×10^-15 M；对非靶细胞KB的IC₅₀分别为2.49×10^-15 M和2.27×10^-15 M。MTT法测定偶联物和力达霉素对C26细胞的IC₅₀分别为3.17×10^-10 M和1.76×10^-9 M；对KB细胞的IC₅₀分别为8.57×10^-10 M和1.14×10^-9 M；表明偶联物较好地保留了力达霉素的细胞毒作用。偶联物对BALB/c小鼠皮下接种结肠癌26有明显生长抑制作用，静脉注射给药三次， 偶联物可明显抑制C26皮下瘤的生长，动物生存时间延长。

     3D6 Fab'-LDM分子量也为65 kDa，对BEL-7402和KB细胞IC₅₀分别为1.04×10^-13 M 和1.99×10^-15 M；力达霉素对两种细胞IC₅₀分别为2.0×10^-15 M和2.21×10^-15 M；MTT法测定Fab’-LDM及力达霉素对转移性肺癌PG细胞的杀伤作用，IC₅₀分别为4.2×10^-10 M和3.7×10^-10 M； 对血管内皮细胞IC₅₀分别为5.7×10^-11 M 和7.2×10^-11 M。体内实验结果表明 Fab'-LDM有明显抗肿瘤作用，0.05 mg/kg、0.1 mg/kg和 0.2 mg/kg剂量，iv给药3次，Fab'-LDM对皮下接种的肝癌22抑制率分别为91%、89%和86%，而0.1 mg/kg力达霉素抑瘤率为77%，低于等剂量Fab'-LDM偶联物 (p<0.01)；0.05 mg/kg、0.1 mg/kg和 0.2 mg/kg，iv给药4次，Fab'-LDM也可明显抑制H22生长并延长荷瘤动物存活时间；Fab'-LDM偶联物对皮下接种的Lewis癌肺转移也有明显抑制作用，0.05 mg/kg和 0.1 mg/kg剂量，iv给药2次， Fab'-LDM对Lewis癌肺转移的抑制率分别为91%和94%，高于等剂量游离力达霉素(p<0.01)。

    以上结果表明，力达霉素与不同单抗Fab'片段偶联物具有分子量小，抗瘤活性高的特点，是单抗导向药物的发展方向。

   

    综上所述，本研究的结果表明：1. 单抗Fab'片段可与抗肿瘤药物构成高效单抗导向药物。2. 具有分子小型化特点的单抗偶联物具有比游离药物及完整抗体药物偶联物更强的抗肿瘤作用。

 

 

ABSTRACT

 

    Monoclonal antibody (mAb)-targeted cancer therapy is one of the key projects in the field of cancer therapy and medicinal biotechnology. On account of the large molecular size, poor penetration into solid tumors, relatively non-specific distribution in vivo, and human anti-mouse antibody response (HAMA), the conventional antibody-drug conjugates failed to show satisfactory antitumor effects in humans. Finding of novel molecular targets, reconstruction of mAb and discovery of novel "effector" agents are critical for coming over the deficiency of conventional mAb conjugates. The studies focus on these fields.

 

1. Antitumor effects of pingyangmycin conjugated with Fab' fragment of mAb 3A5 directed against hepatoma

    Fc fragment of mAb is one of the critical factors related to non-specific distribution of mAb conjugate and HAMA reaction; therefore, it is beneficial to remove Fc fragment. In the study, 3A5, an IgG1 rat mAb directed against human hepatoma BEL-7402 cells, was digested with pepsin and then reduced by dithioerytol (DTT) to produce its Fab' fragment. The Fab' fragment was then conjugated to pingyangmycin (PYM), an antitumor antibiotic, through dextran T-40 to produce Fab'-PYM conjugate. Fab'-PYM conjugate retained 15% activity of PYM and showed immunoreactivity with antigen-related cancer cells including BEL-7402, hepatoma 22 (H22), colon carcinoma HT-29 and colon carcinoma 26 (C26) cells. Determined by clonogenic assay, the Fab'-PYM conjugate showed obvious cytotoxicity to BEL-7402 and HT-29 cells with IC₅₀ values of 0.02 mM and 0.08 mM, respectively, whereas the IC₅₀ values of PYM to these cells were 0.51 mM and 1.15 mM, respectively. As for KB cells, the antigen irrelevant cells, IC₅₀ values of Fab'-PYM and PYM were 0.51 mM and 0.42 mM, respectively. Determined by MTT assay with C26 and KB cells, IC₅₀ values of Fab'-PYM were 1.61 mM and 3.45 mM, respectively, while IC₅₀ values of PYM were 21.46 mM and 2.63 mM, respectively. By ip route of 6 injections at dose of 5 mg/kg, Fab'-PYM conjugate and PYM suppressed the growth of C26 in BALB/c mice by 77% and 56% (P<0.05), respectively. By iv route of 7 injections at dose of 5 mg/kg and 10 mg/kg, Fab'-PYM also markedly suppressed the growth of C26 in mice, the inhibitory effects were much higher than equivalent 3A5-PYM conjugate and free PYM (p<0.01). Given 5 injections, iv, at dose of 5 mg/kg and 10 mg/kg, Fab'-PYM inhibited the growth of H22 by 82% and 86%, respectively, whereas 5 mg/kg PYM suppressed H22 by 62%, which was lower than that of equivalent Fab'-PYM (p<0.05). Given by ip injections at dose of 5 mg/kg and 10 mg/kg, Fab'-PYM conjugate also showed remarkable suppression on the liver metastases of C26 inoculated into spleen by 78% and 91%, whereas 10 mg/kg PYM inhibited the metastasase by 62% (p<0.01). These results indicate that 3A5 Fab'-PYM conjugate is more effective against tumor than free PYM.

 

2. Antitumor effects of pingyangmycin conjugated with Fab' fragment of mAb directed against type IV collagenase

    Type IV collagenase is a critical proteolytic enzyme involoving in tumor invasion and metastasis. Inhibition of type IV collagenase activity results in anti-metastatic activity. In the experiment, 3D6, a mouse mAb directed against type IV collagenase, was digested with ficin to produce its F(ab')₂ fragment. F(ab')₂ fragment markedly inhibited the gelatinase activity in a dose dependent manner, ranging from 12.5 mM to100 mM. However, no obvious cytotoxicity to KB and BEL-7402 cells was observed, the IC₅₀ values were higher than 200 mM. Administered by iv injection at dose of 1 mg/kg, 2 mg/kg ,and 3 mg/kg, F(ab)'₂ fragment suppressed the pulmonary metastases of Lewis lung carcinoma by 51%, 52% and 54%, respectively, whereas the intact antibody 3D6 at 1 mg/kg suppressed the metastases by 58 %. At ip dose of 5 mg/kg and 10 mg/kg, F(ab')₂ suppressed the metastases by 66% and 76%, respectively. However, no inhibitory effects on the growth of subcutaneous tumor were observed. The results indicate that F(ab')₂ fragment of 3D6 is active in suppression gelatinase and tumor metastasis.

    To explore the possibility for mAb 3D6 and its conjugates to hse in tumor therapy, F(ab')₂ fragment of 3D6 was further reduced by b-mercaptoethanol to produce Fab' fragment and then conjugated with PYM. The IC₅₀ values of mAb 3D6 Fab'-PYM conjugate to KB and BEL-7402 cells were 0.02 mM and 0.48 mM, more effective than that of free PYM. Given 4 injections, iv,  Fab'-PYM at dose of 5 mg/kg and 10 mg/kg inhibited the growth of H22 by 64% and 82%，respectively; whereas free PYM at dose of 10 mg/kg inhibited the growth of H22 by 59% (p<0.05). In addition, Fab'-PYM at dose of 5 mg/kg and 10 mg/kg, iv, also suppressed the metastases of Lewis lung carcinoma by 68% and 81%, respectively, more effective than that of equivalent PYM (p<0.01). These results indicate that Fab'-PYM conjugate highly inhibits tumor growth and metastasis and demonstrates the potential for therapeutic application.

 

3. Molecular downsizing of Fab'-PYM conjugate

    Molecular downsizing of the mAb conjugates is beneficial for its better penetration into solid tumors. To further reduce the size of 3D6 Fab'-PYM conjugate, three linkers including dextran T-40 (DT40), dextran T-10 (DT10) and β-cyclodextrin (CD) were applied to construct three different conjugates, Fab'-DT40-PYM, Fab'-DT10-PYM and Fab'-CD-PYM. The conjugation ratio (Fab':linker:PYM) were 1:1:50, 1:1:20 and 1:2.4:7.3; and the molecular weights were 170 kDa, 95 kDa and 70 kDa, respectively. Determined by TTC assay, MIC values were 5.00 mM, 3.44 mM and 4.03 mM；and determined by clonogenic assay, IC₅₀ values to KB cells were 0.01 mM, 0.05 mM and 0.02 mM, respectively; whereas IC₅₀ values to BEL-7402 were 0.48 mM、0.51 mM and 0.51 mM, respectively. These results indicate that CD is a better linker for Fab' and PYM. Antitumor effect of Fab'-CD-PYM conjugate was further evaluated in mice bearing H22. Treatment started on day 7 after tumor inoculation, no inhibitory effect on the growth of H22 in mice treated with PYM at dose of 10 mg/kg was observed, whereas Fab'-CD-PYM conjugate is still able to remarkably suppress the growth of H22. Given by iv injections at dose of 5 mg/kg and 10 mg/kg for a total of 4 doses, Fab'-CD-PYM conjugate suppressed the growth of H22 by 73% and 81%, respectively; whereas PYM at dose of 10 mg/kg suppressed the growth of H22 by 14% (p<0.01). These results indicate that the CD-linked Fab'-PYM conjugate with much smaller molecular size is much more active than free PYM. 

 

4. Construction of highly effective mAb conjugates with downsized-molecules by using lidamycin

    The discovery of novel "effector" agents with highly potent antitumor activity is critical for the preparation of highly effective mAb conjugates. Because of its extremely potent cytotoxicity against cancer cells and its marked antitumor efficacy in vivo, lidamycin (LDM, also called C1027) is a satisfactory "effector" agent for mAb conjugate. In the study, LDM was linked to Fab' fragment of both mAb 3A5 and mAb 3D6 to construct Fab'-LDM conjugates and the antitumor effects of the conjugates were studied.

    The molecular weight of 3A5 Fab'-LDM was approximately 65 kDa. Determined by clonogenic assay with BEL-7402 and HT-29 cells, the IC₅₀ values of Fab'-LDM were 2.25×10^-17 M and 1.57×10^-15 M, whereas IC₅₀ values of LDM were 2.64×10^-16 M and 2.65×10^-15 M, respectively. As for KB cells, IC₅₀ values of Fab'-LDM and LDM were 2.49×10^-15 M and 2.27×10^-15 M, respectively. Determined by MTT assay with colon carcinoma 26 (C26) cells and KB cells, IC₅₀ values of Fab'-LDM were 3.17×10^-10 M and 8.57×10^-10 M; while IC₅₀ values of LDM were 1.76×10^-9  M and 1.14×10^-9 M, respectively. At dose of 0.05 mg/kg, 0.1 mg/kg and 0.2 mg/kg, iv, Fab'-LDM extremely suppressed the growth of C26 in mice and increased the survival of the mice bearing tumor. These results indicate that Fab'-LDM conjugate with much smaller molecular size shows highly antitumor efficacy.

    In the study, LDM was also linked to Fab' fragment of mAb 3D6. The molecular weight of Fab'-LDM conjugate is 65 kDa. Fab'-LDM displayed inhibitory effects on gelatinase activity. Fab'-LDM showed high cytotoxicity to KB and PG cells, the antigen- related cancer cells, with IC₅₀ values of 1.99×10^-15 M (clonogenic assay) and 4.2×10^-10 M (MTT assay), respectively, which was very similar to that of free LDM. Howerver, the cytotoxicity of Fab'-LDM to BEL-7402 cells, the antigen irrelevant cells, was approximately 52-fold lower than that of LDM. Given by iv at 0.05 mg/kg, 0.1 mg/kg and 0.2 mg/kg, ×3, Fab'-LDM suppressed the growth of H22 in mice by 86%, 89% and 91%, respectively, more effective than that of equivalent LDM (p<0.01). Fab'-LDM also remarkably increased the survival time of mice bearing H22 at a total of 4 doses. Moreover, Fab'-LDM of 0.05 mg/kg and 0.1 mg/kg suppressed metastases of Lewis lung carcinoma by 91% and 94%, respectively.

    These results indicate that highly active immunoconjugates can be prepared by linking Fab' fragment to antitumor drugs and the downsized conjugate molecule is more effective than free drug and the intact mAb conjugate.