抗Ⅳ型胶原酶单抗scFv片段与力达霉素构建的基因工程强化融合蛋白及其抗肿瘤作用的研究
摘要
应用单克隆抗体(单抗)及其工程抗体衍生物对于肿瘤，尤其是转移性肿瘤进行治疗是一个巨大和快速发展的研究领域。单克隆抗体针对特定的单一抗原表位，具有高度的特异性，这是抗体药物发挥治疗作用的重要基础。抗体药物的重要特点之一是可以定向制造(tailored making)，根据需要制备具有不同治疗作用的抗体药物。即随着对某些靶分子生物活性的探讨与确定，针对特定的靶分子，定向制备针对特定靶分子的抗体药物。抗肿瘤抗体药物的研究表明，其特异性主要表现为特异性结合，选择性杀伤，体内靶向性分布以及具有更强的疗效。而且，特定的化疗药物与不同抗体偶联可以作为一种技术平台(technical platform)制备系列化的抗体药物。因而，利用化学偶联方法制备的免疫偶联物或运用基因工程技术构建的基于单链抗体scFv的融合蛋白是针对特定的靶分子定向制备的高效小型的肿瘤靶向性药物，这类导向药物的开发研制符合目前单抗导向药物的小型化、高效化、低免疫原性的研究趋势，显示出巨大的应用潜力。本研究在抗体工程药物的制备和活性分析方面进行探讨。
一 单抗3G11的生物学活性分析及其偶联物3G11-LDM的实验治疗
Ⅳ型胶原酶能够破坏基质的代谢平衡，促进肿瘤细胞穿过基底膜和细胞外基质所构成的组织学屏障，进而侵袭周围组织并向远端转移；它在人类前列腺癌、结直肠癌、乳腺癌、鳞癌、恶性星形细胞瘤、转移瘤等细胞株以及肿瘤新生血管内皮细胞中均呈高表达，而且与肿瘤患者的预后密切相关。抑制Ⅳ型胶原酶的活性可以抑制肿瘤细胞的侵袭转移和肿瘤血管生成。本研究采用链霉卵白素—生物素—酶联复合物(SABC)染色方法，对取自北京友谊医院2001-2002年间病理科手术切除的80例肿瘤组织标本进行免疫组织化学(IHC)染色，检测单抗3G11对人体不同肿瘤的选择性识
With the invention of the hybridoma technology that allows production of monoclonal antibody (mAb), a new era of biological therapy for invasive and metastasis cancer has been initiated. A variety of mAbs, with high specificity and affinity for their target antigens, have been utilized in the recent decades for delivery of anticancer agents such as drugs, enzymes, radionuclides, or toxins in cancer therapy. The specificity of mAb to unique epitope is the key to antitumor activity of antibody-based therapeutics. Because of the specificity of mAbs directed to mutant or overexpressed oncogenes, and other tumor-associated antigens, antibody-based drugs have been achieved by tailored making according to various needs. The results of the research on mAbs have shown that their specificities mainly presents high special affinity, selective cytotoxicity to tumor cells in vitro, and targeting distribution in tumors and more effective therapy. Moreover, the antibody-based therapy for cancer has been in favor of offering promise as a technical platform and obtaining a variety of high potent agents for clinical application. Therefore, utilizing chemical method for immunoconjugates or DNA recombinant technology for scFv-fusion protein, miniature and high powerful agents specially targeting to tumors have been made for various needs. Since the promising potent of antibody-based therapeutics has been revealed, this studies focused on characterizations of novel molecular targets, construction and biological analysis of antibody-based drugs.
1. The characterization of mAb 3G11 and the antitumor effects of immunoconjugate 3G11-LDM
Type IV collagenases, also termed gelatinases, including MMP-2 of 72 kDa and MMP-9 of 92 kDa play an extremely important role during tumor growth and progression and display as an attractive target for monoclonal antibody-directed therapy. Type IV collagenases degrade type IV collagen which is the major component of basement membrane and other collagens, and destroy the integrality of basement membrane and extracellular matrix, which is in favor of the invasion and metastasis of tumor cells. In addition, the activities of type IV collagenases are higher in endothelial cells stimulated by angiogenesis factors than normal cells. Inhibition of the activities of type IV collagenase may inhibit tumor invasion and metastasis, and angiogenesis. The
一 单抗3G11的生物学活性分析及其偶联物3G11-LDM的实验治疗
Ⅳ型胶原酶能够破坏基质的代谢平衡，促进肿瘤细胞穿过基底膜和细胞外基质所构成的组织学屏障，进而侵袭周围组织并向远端转移；它在人类前列腺癌、结直肠癌、乳腺癌、鳞癌、恶性星形细胞瘤、转移瘤等细胞株以及肿瘤新生血管内皮细胞中均呈高表达，而且与肿瘤患者的预后密切相关。抑制Ⅳ型胶原酶的活性可以抑制肿瘤细胞的侵袭转移和肿瘤血管生成。本研究采用链霉卵白素—生物素—酶联复合物(SABC)染色方法，对取自北京友谊医院2001-2002年间病理科手术切除的80例肿瘤组织标本进行免疫组织化学(IHC)染色，检测单抗3G11对人体不同肿瘤的选择性识
With the invention of the hybridoma technology that allows production of monoclonal antibody (mAb), a new era of biological therapy for invasive and metastasis cancer has been initiated. A variety of mAbs, with high specificity and affinity for their target antigens, have been utilized in the recent decades for delivery of anticancer agents such as drugs, enzymes, radionuclides, or toxins in cancer therapy. The specificity of mAb to unique epitope is the key to antitumor activity of antibody-based therapeutics. Because of the specificity of mAbs directed to mutant or overexpressed oncogenes, and other tumor-associated antigens, antibody-based drugs have been achieved by tailored making according to various needs. The results of the research on mAbs have shown that their specificities mainly presents high special affinity, selective cytotoxicity to tumor cells in vitro, and targeting distribution in tumors and more effective therapy. Moreover, the antibody-based therapy for cancer has been in favor of offering promise as a technical platform and obtaining a variety of high potent agents for clinical application. Therefore, utilizing chemical method for immunoconjugates or DNA recombinant technology for scFv-fusion protein, miniature and high powerful agents specially targeting to tumors have been made for various needs. Since the promising potent of antibody-based therapeutics has been revealed, this studies focused on characterizations of novel molecular targets, construction and biological analysis of antibody-based drugs.
1. The characterization of mAb 3G11 and the antitumor effects of immunoconjugate 3G11-LDM
Type IV collagenases, also termed gelatinases, including MMP-2 of 72 kDa and MMP-9 of 92 kDa play an extremely important role during tumor growth and progression and display as an attractive target for monoclonal antibody-directed therapy. Type IV collagenases degrade type IV collagen which is the major component of basement membrane and other collagens, and destroy the integrality of basement membrane and extracellular matrix, which is in favor of the invasion and metastasis of tumor cells. In addition, the activities of type IV collagenases are higher in endothelial cells stimulated by angiogenesis factors than normal cells. Inhibition of the activities of type IV collagenase may inhibit tumor invasion and metastasis, and angiogenesis. The
引文
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