联合应用MALDI-TOFMS抗原表位确定技术与分子对接进行抗体人源化改造
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摘要
众所周知,肿瘤是最致命的疾病之一。自20世纪70年代以来,我国癌症患者的人数逐年升高,约有百分之八十的癌症患者死于肺癌、肝癌、胃癌等常见肿瘤。通常的治疗方法包括外科手术、放射治疗、化疗、生物治疗等,这些治疗方法有时单独使用,有时联合使用。
抗体治疗是生物治疗的一种,它的重要性越发明显。抗体治疗的前景是光明的,目前已批准20余种抗体药物用于治疗各种疾病,在临床上已取得预期效果。抗体药物是利用以细胞工程技术和基因工程技术为主体的抗体工程技术制备的药物,但由于其伴随的人抗鼠抗体反应(HAMA),抗体药物的应用往往受到了一定程度的限制。为了降低抗体药物的HAMA反应,基于蛋白质结构信息发展了一系列的改造抗体的方法。随着分子生物学的发展,嵌合抗体和人源化抗体的出现,成为了解决抗体应用中降低HAMA反应的有效途径,但改造的抗体往往伴随着亲和力的大幅下降。
本研究建立了一种新型的抗体人源化方法,首次联合基于质谱的表位确定技术和分子对接对抗体进行人源化改造。通过鉴定出HAb18G/CD147的抗原表位进行HAb18抗体的人源化设计,这样就在保证了抗体HAb18亲和力的基础上进行人源化改造,之后通过基因重组技术合成新的抗体片段并检测该抗体片段的生物学活性,下面将从以下三部分分别说明。
第一部分:MALDI-TOF MS确定HAb18G/CD147的抗原表位
目的:确定HAb18G/CD147的表位序列
方法:利用配套的磁架洗涤新的磁珠,洗去磁珠上的防腐剂。加入适量的HAb18抗体,在室温的条件下与磁珠结合1~2小时并轻微摇晃,使抗体与磁珠充分结合。之后用PBS(0.1% BSA,pH=7.4)洗涤磁珠4~5次,以封闭未结合抗体磁珠的活性位点。将适量的HAb18G/CD147加入到制备好的抗体磁珠混悬液中,室温下结合1~2小时,使抗原与抗体充分结合。加入胰酶溶液酶解HAb18G/CD147,用PBS洗涤掉未结合的抗原肽段,并用0.1﹪TFA(pH=2.5)洗脱HAb18G/CD147抗原表位肽并迅速冻干,用MALDI-TOF MS检测制备的样品。
结果:制备了HAb18G/CD147抗原表位肽,说明了利用免疫磁珠酶解抗原的办法是可行的,并且制备出的样品符合MALDI-TOF MS检测的标准。对制备的HAb18G/CD147抗原表位肽进行MALDI-TOF MS分析,并与PeptideCutter软件预测的胰酶酶切HAb18G/CD147各肽段分子量进行对比,得到了HAb18G/CD147抗原表位肽的序列信息。
第二部分:应用确定的表位与分子对接对抗体HAb18人源化设计
目的:确定突变的氨基酸位点
方法:根据已有的HAb18抗体模型和得到的HAb18G/CD147抗原表位序列,应用DOCKING软件建立HAb18G/CD147与HAb18的分子对接模型,并预测表位的关键氨基酸残基。在模拟出HAb18G/CD147同其抗体HAb18的对接模型后,用表面重塑的方法对HAb18可变区进行人源化设计。利用序列分析和结构分析确定鼠源抗体外露的差异残基,选定将要突变的氨基酸残基位点,以便能够合成低免疫原性又具有抗原结合活性的人源化抗体。
结果:建立了一套新的人源化抗体的方法,首次将基于MALDI-TOF MS确定抗原表位技术引入到分子对接中,建立了HAb18G/CD147和HAb18的分子对接模型,完成了HAb18可变区的人源化设计,确定突变的氨基酸位点为H42E,H90A和L43S。
第三部分:人源化抗体片段HAb18-huscFv的表达与鉴定
目的:验证该人源化方法
方法:根据设计的人源化方案,用overlapping PCR的方法合成人源化形式HAb18-huscFv的基因,克隆至表达载体pCANTAB-6His-HAb18-huscFv中,转化至E. coli中诱导表达。经Ni2+柱纯化表达的蛋白后,采用SDS-PAGE、Western blot、细胞免疫荧光等方法对表达产物进行检测、鉴定,同步构建表达其亲代鼠源形式抗体为对照。采用间接竞争ELISA法分析抗体HAb18-huscFv与HAb18G/CD147的结合活性, SPR法测定抗体HAb18-huscFv与HAb18G/CD147的亲和力,并用同步表达的鼠源形式抗体作为对照,分析两种抗体有无差异。最后采用HAMA阳性血清,对比了HAb18-huscFv和鼠源HAb18-scFv的免疫原性差异。
结果:根据已选择的突变位点,用overlapping PCR的方法合成了新的基因并克隆至表达载体,测序验证正确后转化到E. coli中诱导表达,经Ni2+柱纯化后获得了较纯的HAb18-huscFv。SDS-PAGE和Western blot检测目标蛋白分子量约为27kDa。经SPR法测定,HAb18-huscFv与HAb18-scFv相比亲和力无明显差异。用细胞免疫荧光法检测HAb18-huscFv与肝癌细胞SMMC-7721具有一定结合能力。间接竞争ELISA法结果显示:抗体HAb18-huscFv与HAb18-scFv相比具有较低的免疫原性。
结论:基于利用新方法建立的抗体人源化方案,改造后的抗体HAb18-huscFv与HAb18-scFv相比,在降低了免疫原性的同时,又保留了抗体的抗原结合能力,证明了该方法是有效可行的,为抗体人源化设计提供了新的思路。
As is known to all, cancer is the most lethal illness in the world. The number of cancer patients has been soaring in China since the 1970s. About 80 percent of cancer patients died from common cancers of the lungs, liver and stomach, according to previously released figures. The usual treatments include surgery, radiation therapy, chemotherapy and biological therapy. These treatment methods are sometimes used alone and sometimes in combination. Biological therapy is to stimulate or restore the ability of the immune system to fight infection and disease. It is thus any form of treatment that uses the body's natural abilities that constitute the immune system to fight infection and disease or to protect the body from some of the side effects of treatment.
Antibody therapeutics is of growing importance as medical treatments in different types of biological therapies. It continue to be one of the bright spots in pharmaceutical drug development, with 20 monoclonal antibodies approved for the treatment of various diseases, and many more in clinical trials with promising results. Antibody drugs developed based on genetic engineering, cell engineering technology and antibody engineering technologies for the preparation of the main drug. Widespread use of the antibody may be limited, however, by the immune response typically elicited by murine mAbs. Such an immune response may cause allergic reactions and reduce the effectiveness of re-treatment by immune complex formation. In order to reduce immunogenicity, different strategies have been developed based on protein structural information. With the biology development, people can humanize mouse antibody with DNA recombination and antibody library which developed antibody techniques from chimeric and reshaped to human antibody, and now variety of monoclonal antibody derivatives. However, these methods are often accompanied by loss of antibody affinity.
This work described a novel antibody humanization method by epitope mapping based on MALDI-TOF MS of its antibody bound epitope peptides combined with antibody-antigen molecular docking by computer modeling. Studies were undertaken employing a mAb of clinical utility, HAb18, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We fabricated a humanized version of HAb18-scFv, HAb18-huscFv, and detected its biological activity.
Part I: identification of HAb18G/CD147 epitope by MALDI-TOF MS analysis
Objective: to ascertain the sequence of HAb18G/CD147 epitope
Method: first, the Dynabeads should be washed before use to remove preservatives. The washing procedure was facilitated by using a magnet. After calculating the required amount of Dynabeads and HAb18, we incubated the beads and HAb18 at room temperature for 1~2 hours with gentle rotation of the tube. Then, we separated the beads coated with HAb18 using a magnet and washed the beads for 4~5 times in Phosphate buffered saline (0.1% BSA, pH=7.4) by a magnet. When the purified monoclonal antibody HAb18 was bound to the magnetic beads, HAb18G/CD147 was added to the affinity matrix and proteolytic digestion performed by trypsin. The released supernatant, non-epitope, peptides and the protease were removed by washing with PBS buffer. For unequivocal identification of epitope peptides, the antibody-bound peptide fragment mixture was eluted by 0.1﹪TFA(pH=2.5). After freeze-drying, the combined sample solution was ready for MALDI-TOF MS analysis.
Results: HAb18G/CD147 epitope peptide was obtained by limited proteolysis of antigens bound to an immobilized monoclonal antibody. The epitope sequence was identified by MALDI-TOF MS analysis.
Part II: humanization design of HAb18 by antibody-antigen molecular docking combined with epitope mapping by mass spectrometric.
Objective: determine the residues that need to be mutated to their human counterpart.
Method: the sequence of HAb18G/CD147 epitope was determined by using the procedure of epitope excision in combination with MALDI-TOF MS analysis. After that, HAb18 structure modeling was constructed by homology modeling. Then, the docking of HAb18G/CD147 and HAb18 was performed by InsightII. Furthermore, we identified the mutated amino acid residues for humanization based on analyzing of antibody spatial structure and interaction among residues by means of computer assisted molecule design.
Results: we proposed a method of verifying the epitope and the key residues based on analyzing the complex model of HAb18G/CD147 extracellular portion and its mAb based on computer assisted molecule docking. Besides, we identified the mutated amino acid residues for humanization and determined the candidate mutation sites were that H42E, H90A and L43S were replaced by H42G, H90T and L43P.
Part III: expression and characterization of HAb18-huscFv
Objective: confirms the feasibility of the method of humanization.
Method: the humanized gene sequences of HAb18-huscFv were synthesized by overlapping PCR technique after computer aided CDR grafted humanization design of the variable region of HAb18. The genetic fragment of HAb18-huscFv was cloned to construct pCANTAB-6His-HAb18-huscFv. The recombinant plasmids containing HAb18-huscFv were identified by restriction enzyme analysis and PCR method. After that, the expression vector pCANTAB-6His-HAb18-huscFv was expressed in E. coli. Expression proteins were purified by affinity chromatography and detected by SDS-PAGE and Western blot. Indirect competitive ELISA was performed to analyze HAb18-huscFv binding activity to HAb18G/CD147 and its immunogenicity compared with HAb18-scFv. Cell immunofluorescence was used to compare binding activity of HAb18-huscFv and HAb18-scFv to antigen HAb18G/CD147 of liver cancer cells SMMC-7721. SPR biochemical analysis was used to detect association constant (Kon) and dissociation constant (Koff) of HAb18-huscFv and HAb18-scFv.
Results: on the basis of humanization design, we fabricated a humanized version of HAb18scFv that H42E, H90A and L43S were replaced by H42G, H90T and L43P. After PCR and sequencing, the expression plasmid pCANTAB-6His-HAb18-huscFv was expressed in E. coli. The expressed products were briefly purified and analyzed by SDS-PAGE, western blot, cell immunofluorescence, indirect competitive ELISA and SPR biochemical analysis. These results suggest HAb18-huscFv had similar biological functions compared with HAb18-scFv, but a lower immunogenicity. Conclusion: here, we describe a novel antibody humanization method by epitope mapping based on MALDI-TOF MS analysis combined with antibody-antigen molecular docking. By this means, the functional conformation of the antibody-combining site was preserved for the retention of ligand-binding property, which requires maintenance of the CDRs and their interaction with each other and with the rest of the structure of the antibody-combining site.
抗体治疗是生物治疗的一种,它的重要性越发明显。抗体治疗的前景是光明的,目前已批准20余种抗体药物用于治疗各种疾病,在临床上已取得预期效果。抗体药物是利用以细胞工程技术和基因工程技术为主体的抗体工程技术制备的药物,但由于其伴随的人抗鼠抗体反应(HAMA),抗体药物的应用往往受到了一定程度的限制。为了降低抗体药物的HAMA反应,基于蛋白质结构信息发展了一系列的改造抗体的方法。随着分子生物学的发展,嵌合抗体和人源化抗体的出现,成为了解决抗体应用中降低HAMA反应的有效途径,但改造的抗体往往伴随着亲和力的大幅下降。
本研究建立了一种新型的抗体人源化方法,首次联合基于质谱的表位确定技术和分子对接对抗体进行人源化改造。通过鉴定出HAb18G/CD147的抗原表位进行HAb18抗体的人源化设计,这样就在保证了抗体HAb18亲和力的基础上进行人源化改造,之后通过基因重组技术合成新的抗体片段并检测该抗体片段的生物学活性,下面将从以下三部分分别说明。
第一部分:MALDI-TOF MS确定HAb18G/CD147的抗原表位
目的:确定HAb18G/CD147的表位序列
方法:利用配套的磁架洗涤新的磁珠,洗去磁珠上的防腐剂。加入适量的HAb18抗体,在室温的条件下与磁珠结合1~2小时并轻微摇晃,使抗体与磁珠充分结合。之后用PBS(0.1% BSA,pH=7.4)洗涤磁珠4~5次,以封闭未结合抗体磁珠的活性位点。将适量的HAb18G/CD147加入到制备好的抗体磁珠混悬液中,室温下结合1~2小时,使抗原与抗体充分结合。加入胰酶溶液酶解HAb18G/CD147,用PBS洗涤掉未结合的抗原肽段,并用0.1﹪TFA(pH=2.5)洗脱HAb18G/CD147抗原表位肽并迅速冻干,用MALDI-TOF MS检测制备的样品。
结果:制备了HAb18G/CD147抗原表位肽,说明了利用免疫磁珠酶解抗原的办法是可行的,并且制备出的样品符合MALDI-TOF MS检测的标准。对制备的HAb18G/CD147抗原表位肽进行MALDI-TOF MS分析,并与PeptideCutter软件预测的胰酶酶切HAb18G/CD147各肽段分子量进行对比,得到了HAb18G/CD147抗原表位肽的序列信息。
第二部分:应用确定的表位与分子对接对抗体HAb18人源化设计
目的:确定突变的氨基酸位点
方法:根据已有的HAb18抗体模型和得到的HAb18G/CD147抗原表位序列,应用DOCKING软件建立HAb18G/CD147与HAb18的分子对接模型,并预测表位的关键氨基酸残基。在模拟出HAb18G/CD147同其抗体HAb18的对接模型后,用表面重塑的方法对HAb18可变区进行人源化设计。利用序列分析和结构分析确定鼠源抗体外露的差异残基,选定将要突变的氨基酸残基位点,以便能够合成低免疫原性又具有抗原结合活性的人源化抗体。
结果:建立了一套新的人源化抗体的方法,首次将基于MALDI-TOF MS确定抗原表位技术引入到分子对接中,建立了HAb18G/CD147和HAb18的分子对接模型,完成了HAb18可变区的人源化设计,确定突变的氨基酸位点为H42E,H90A和L43S。
第三部分:人源化抗体片段HAb18-huscFv的表达与鉴定
目的:验证该人源化方法
方法:根据设计的人源化方案,用overlapping PCR的方法合成人源化形式HAb18-huscFv的基因,克隆至表达载体pCANTAB-6His-HAb18-huscFv中,转化至E. coli中诱导表达。经Ni2+柱纯化表达的蛋白后,采用SDS-PAGE、Western blot、细胞免疫荧光等方法对表达产物进行检测、鉴定,同步构建表达其亲代鼠源形式抗体为对照。采用间接竞争ELISA法分析抗体HAb18-huscFv与HAb18G/CD147的结合活性, SPR法测定抗体HAb18-huscFv与HAb18G/CD147的亲和力,并用同步表达的鼠源形式抗体作为对照,分析两种抗体有无差异。最后采用HAMA阳性血清,对比了HAb18-huscFv和鼠源HAb18-scFv的免疫原性差异。
结果:根据已选择的突变位点,用overlapping PCR的方法合成了新的基因并克隆至表达载体,测序验证正确后转化到E. coli中诱导表达,经Ni2+柱纯化后获得了较纯的HAb18-huscFv。SDS-PAGE和Western blot检测目标蛋白分子量约为27kDa。经SPR法测定,HAb18-huscFv与HAb18-scFv相比亲和力无明显差异。用细胞免疫荧光法检测HAb18-huscFv与肝癌细胞SMMC-7721具有一定结合能力。间接竞争ELISA法结果显示:抗体HAb18-huscFv与HAb18-scFv相比具有较低的免疫原性。
结论:基于利用新方法建立的抗体人源化方案,改造后的抗体HAb18-huscFv与HAb18-scFv相比,在降低了免疫原性的同时,又保留了抗体的抗原结合能力,证明了该方法是有效可行的,为抗体人源化设计提供了新的思路。
As is known to all, cancer is the most lethal illness in the world. The number of cancer patients has been soaring in China since the 1970s. About 80 percent of cancer patients died from common cancers of the lungs, liver and stomach, according to previously released figures. The usual treatments include surgery, radiation therapy, chemotherapy and biological therapy. These treatment methods are sometimes used alone and sometimes in combination. Biological therapy is to stimulate or restore the ability of the immune system to fight infection and disease. It is thus any form of treatment that uses the body's natural abilities that constitute the immune system to fight infection and disease or to protect the body from some of the side effects of treatment.
Antibody therapeutics is of growing importance as medical treatments in different types of biological therapies. It continue to be one of the bright spots in pharmaceutical drug development, with 20 monoclonal antibodies approved for the treatment of various diseases, and many more in clinical trials with promising results. Antibody drugs developed based on genetic engineering, cell engineering technology and antibody engineering technologies for the preparation of the main drug. Widespread use of the antibody may be limited, however, by the immune response typically elicited by murine mAbs. Such an immune response may cause allergic reactions and reduce the effectiveness of re-treatment by immune complex formation. In order to reduce immunogenicity, different strategies have been developed based on protein structural information. With the biology development, people can humanize mouse antibody with DNA recombination and antibody library which developed antibody techniques from chimeric and reshaped to human antibody, and now variety of monoclonal antibody derivatives. However, these methods are often accompanied by loss of antibody affinity.
This work described a novel antibody humanization method by epitope mapping based on MALDI-TOF MS of its antibody bound epitope peptides combined with antibody-antigen molecular docking by computer modeling. Studies were undertaken employing a mAb of clinical utility, HAb18, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We fabricated a humanized version of HAb18-scFv, HAb18-huscFv, and detected its biological activity.
Part I: identification of HAb18G/CD147 epitope by MALDI-TOF MS analysis
Objective: to ascertain the sequence of HAb18G/CD147 epitope
Method: first, the Dynabeads should be washed before use to remove preservatives. The washing procedure was facilitated by using a magnet. After calculating the required amount of Dynabeads and HAb18, we incubated the beads and HAb18 at room temperature for 1~2 hours with gentle rotation of the tube. Then, we separated the beads coated with HAb18 using a magnet and washed the beads for 4~5 times in Phosphate buffered saline (0.1% BSA, pH=7.4) by a magnet. When the purified monoclonal antibody HAb18 was bound to the magnetic beads, HAb18G/CD147 was added to the affinity matrix and proteolytic digestion performed by trypsin. The released supernatant, non-epitope, peptides and the protease were removed by washing with PBS buffer. For unequivocal identification of epitope peptides, the antibody-bound peptide fragment mixture was eluted by 0.1﹪TFA(pH=2.5). After freeze-drying, the combined sample solution was ready for MALDI-TOF MS analysis.
Results: HAb18G/CD147 epitope peptide was obtained by limited proteolysis of antigens bound to an immobilized monoclonal antibody. The epitope sequence was identified by MALDI-TOF MS analysis.
Part II: humanization design of HAb18 by antibody-antigen molecular docking combined with epitope mapping by mass spectrometric.
Objective: determine the residues that need to be mutated to their human counterpart.
Method: the sequence of HAb18G/CD147 epitope was determined by using the procedure of epitope excision in combination with MALDI-TOF MS analysis. After that, HAb18 structure modeling was constructed by homology modeling. Then, the docking of HAb18G/CD147 and HAb18 was performed by InsightII. Furthermore, we identified the mutated amino acid residues for humanization based on analyzing of antibody spatial structure and interaction among residues by means of computer assisted molecule design.
Results: we proposed a method of verifying the epitope and the key residues based on analyzing the complex model of HAb18G/CD147 extracellular portion and its mAb based on computer assisted molecule docking. Besides, we identified the mutated amino acid residues for humanization and determined the candidate mutation sites were that H42E, H90A and L43S were replaced by H42G, H90T and L43P.
Part III: expression and characterization of HAb18-huscFv
Objective: confirms the feasibility of the method of humanization.
Method: the humanized gene sequences of HAb18-huscFv were synthesized by overlapping PCR technique after computer aided CDR grafted humanization design of the variable region of HAb18. The genetic fragment of HAb18-huscFv was cloned to construct pCANTAB-6His-HAb18-huscFv. The recombinant plasmids containing HAb18-huscFv were identified by restriction enzyme analysis and PCR method. After that, the expression vector pCANTAB-6His-HAb18-huscFv was expressed in E. coli. Expression proteins were purified by affinity chromatography and detected by SDS-PAGE and Western blot. Indirect competitive ELISA was performed to analyze HAb18-huscFv binding activity to HAb18G/CD147 and its immunogenicity compared with HAb18-scFv. Cell immunofluorescence was used to compare binding activity of HAb18-huscFv and HAb18-scFv to antigen HAb18G/CD147 of liver cancer cells SMMC-7721. SPR biochemical analysis was used to detect association constant (Kon) and dissociation constant (Koff) of HAb18-huscFv and HAb18-scFv.
Results: on the basis of humanization design, we fabricated a humanized version of HAb18scFv that H42E, H90A and L43S were replaced by H42G, H90T and L43P. After PCR and sequencing, the expression plasmid pCANTAB-6His-HAb18-huscFv was expressed in E. coli. The expressed products were briefly purified and analyzed by SDS-PAGE, western blot, cell immunofluorescence, indirect competitive ELISA and SPR biochemical analysis. These results suggest HAb18-huscFv had similar biological functions compared with HAb18-scFv, but a lower immunogenicity. Conclusion: here, we describe a novel antibody humanization method by epitope mapping based on MALDI-TOF MS analysis combined with antibody-antigen molecular docking. By this means, the functional conformation of the antibody-combining site was preserved for the retention of ligand-binding property, which requires maintenance of the CDRs and their interaction with each other and with the rest of the structure of the antibody-combining site.
引文
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10. Newman, R., et al., "Primatization" of Recombinant Antibodies for Immunotherapy of Human Diseases: A Macaque/Human Chimeric Antibody Against Human CD4. Nature Biotechnology, 1992. 10(11): p. 1455-1460.
11. Queen, C., et al., A humanized antibody that binds to the interleukin 2receptor. Proceedings of the National Academy of Sciences of the United States of America, 1989. 86(24): p. 10029.
12. Green, L., et al., Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nature genetics, 1994. 7(1): p. 13-21.
13. S derlind, E., et al., Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nature Biotechnology, 2000. 18(8): p. 852-856.
14. Jones, P., et al., Replacing the complementarity-determining regions in a human antibody with those from a mouse. 1986.
15. Tsurushita, N., P. Hinton, and S. Kumar, Design of humanized antibodies: from anti-Tac to Zenapax, Methods 36 (2005). Article View Record in Scopus| Cited By in Scopus (19): p. 69-83.
16. Iwahashi, M., et al., CDR substitutions of a humanized monoclonal antibody (CC49): contributions of individual CDRs to antigen binding and immunogenicity. Molecular Immunology, 1999. 36(15-16): p. 1079-1091.
17. Kashmiri, S., et al., Development of a minimally immunogenic variant of humanized anti-carcinoma monoclonal antibody CC49. Critical reviews in oncology/hematology, 2001. 38(1): p. 3-16.
18. Leung, S., et al., Construction and characterization of a humanized, internalizing, B-cell (CD22)-specific, leukemia/lymphoma antibody, LL21. Molecular Immunology, 1995. 32(17-18): p. 1413-1427.
19. Yang, W., et al., CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. Journal of molecular biology, 1995. 254(3): p. 392-403.
20. Morea, V., A. Lesk, and A. Tramontano, Antibody modeling: implications for engineering and design. Methods, 2000. 20(3): p. 267-279.
21. Hwang, K., et al., Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods, 2005. 36(1): p. 35-42.
22. Ewert, S., A. Honegger, and A. Plukthun, Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering. Methods, 2004. 34(2): p. 184-199.
23. Jespers, L., et al., Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen. Nature Biotechnology, 1994. 12(9): p. 899-903.
24. Rader, C., D. Cheresh, and C. Barbas, A phage display approach for rapid antibody humanization: designed combinatorial V gene libraries. Proceedings of the National Academy of Sciences of the United States of America, 1998. 95(15): p. 8910.
25. Roguska, M., et al., Humanization of murine monoclonal antibodies through variable domain resurfacing. Proceedings of the National Academy of Sciences of the United States of America, 1994. 91(3): p. 969.
26. Roguska, M. and J. Pedersen, A comparison of two murine monoclonal antibodies humanized by CDR-grafting and variable domain resurfacing. Protein Engineering Design and Selection, 1996. 9(10): p. 895.
27. Weinblatt, M., et al., Adalimumab, a fully human anti-tumor necrosis factorαmonoclonal antibody, for the treatment of rheumatoid arthritis in patients taking concomitant methotrexate: the ARMADA trial. Arthritis &Rheumatism, 2003. 48(1): p. 35-45.
28. discontinues Vectibix, A., treatment in PACCE trial evaluating Vectibix(tm) as part of triple combination regimen. Pressemelding (Amgen) 22.3. 2007.
29. Vaughan, T., J. Osbourn, and P. Tempest, Human antibodies by design. Nature Biotechnology, 1998. 16(6): p. 535-539.
30. Smith, G., Filamentous fusion phage: novel expression vectors that display cloned antigens on the surface of the virion. Science, 1985. 228(4705): p. 1315-1317.
31. Fukazawa, T., B. Walter, and L. Owen-Schaub, Adenoviral Bid overexpression induces caspase-dependent cleavage of truncated Bid and p53-independent apoptosis in human non-small cell lung cancers. Journal of Biological Chemistry, 2003. 278(28): p. 25428.
32. Watanabe, T., et al., Adenoviral gene transfer in the peripheral nervous system. Journal of Orthopaedic Science, 2006. 11(1): p. 64-69.
33. Schaffitzel, C., et al., Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. Journal of immunological methods, 1999. 231(1-2): p. 119-135.
34. Tomizuka, K., et al., Double trans-chromosomic mice: Maintenance of two individual human chromosome fragments containing Ig heavy andκloci and expression of fully human antibodies. Proceedings of the National Academy of Sciences, 2000. 97(2): p. 722.
35. Cohen, C., et al., Direct phenotypic analysis of human MHC class I antigen presentation: visualization, quantitation, and in situ detection of human viral epitopes using peptide-specific, MHC-restricted human recombinant antibodies. The Journal of Immunology, 2003. 170(8): p.4349.
36. Wilkinson, L. and J. Edwards, Binding of antibodies raised against tumour necrosis factor alpha (TNF-α) to blood vessels and macrophages in inflamed synovial tissue. Rheumatology international, 1991. 11(1): p. 19-25.
37. Tanaka, K., et al., Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom, 1988. 2(8): p. 151-153.
38. Zhao, Y. and B. Chait, Protein epitope mapping by mass spectrometry. Analytical chemistry, 1994. 66(21): p. 3723-3726.
39. Van de Water, J., et al., Detection of molecular determinants and epitope mapping using MALDI-TOF mass spectrometry. Clinical immunology and immunopathology, 1997. 85(3): p. 229.
40. Kumar, A., et al., Buildup rates of the nuclear Overhauser effect measured by two-dimensional proton magnetic resonance spectroscopy: implications for studies of protein conformation. Journal of the American Chemical Society, 1981. 103(13): p. 3654-3658.
41. Deisenhofer, J., et al., X-ray structure analysis of a membrane protein complex:: Electron density map at 3 resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis. Journal of molecular biology, 1984. 180(2): p. 385-398.
42. Engh, R. and R. Huber, Accurate bond and angle parameters for X-ray protein structure refinement. Acta Crystallographica Section A: Foundations of Crystallography, 1991. 47(4): p. 392-400.
43. Yu, X., et al., The glycosylation characteristic of hepatoma-associatedantigen HAb18G/CD147 in human hepatoma cells. The international journal of biochemistry & cell biology, 2006. 38(11): p. 1939-1945.
44. Ellis, S., K. Nabeshima, and C. Biswas, Monoclonal antibody preparation and purification of a tumor cell collagenasestimulatory factor. Cancer research, 1989. 49(12): p. 3385.
45. Guo, H., et al., Characterization of the gene for human EMMPRIN, a tumor cell surface inducer of matrix metalloproteinases. Gene, 1998. 220(1-2): p. 99-108.
46. Biswas, C., et al., The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily. Cancer research, 1995. 55(2): p. 434.
47. Papoutsi, M., et al., Induction of the blood-brain barrier marker neurothelin/HT7 in endothelial cells by a variety of tumors in chick embryos. Histochemistry and Cell Biology, 2000. 113(2): p. 105-113.
48. Chen, Z., et al., Targeting radioimmunotherapy of hepatocellular carcinoma with iodine (131I) metuximab injection: clinical phase I/II trials. International Journal of Radiation Oncology Biology Physics, 2006. 65(2): p. 435-444.
49. Lesk, A., Introduction to bioinformatics. 2002: Oxford University Press Oxford.
50. MUEGGE, I. and I. ENYEDY, Docking and scoring. Computational Medicinal Chemistry and Drug Discovery: p. 405-436.
51. Browne, W., et al., A possible three-dimensional structure of bovine [alpha]-lactalbumin based on that of hen's egg-white lysozyme. Journal of molecular biology, 1969. 42(1): p. 65-70.
52. Fiser, A., Comparative protein structure modelling. From ProteinStructure to Function with Bioinformatics, 2008: p. 57-90.
53. Kabat, E. and T. Wu, Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. The Journal of Immunology, 1991. 147(5): p. 1709.
54. Whitelegg, N. and A. Rees, WAM: an improved algorithm for modelling antibodies on the WEB. Protein Engineering Design and Selection, 2000. 13(12): p. 819.
55. Al-Lazikani, B., A. Lesk, and C. Chothia, Standard conformations for the canonical structures of immunoglobulins1. Journal of molecular biology, 1997. 273(4): p. 927-948.
56. Yu, X.L., et al., Crystal structure of HAb18G/CD147: implications for immunoglobulin superfamily homophilic adhesion. J Biol Chem, 2008. 283(26): p. 18056-65.
57. Hubbard, S. and J. Thornton, NACCESS, Computer Program, Department of Biochemistry and Molecular Biology. University College London, 1993.
58. Mariuzza, R. and R. PoIjak, The basics of binding: mechanisms of antigen recognition and mimicry by antibodies. Current opinion in immunology, 1993. 5(1): p. 50-55.
59. Slavin-Chiorini, D., et al., A CDR-grafted (humanized) domain-deleted antitumor antibody. Cancer biotherapy & radiopharmaceuticals, 1997. 12(5): p. 305-316.
60.杨滨,杨向民,陈志南,基于表面重塑方法的抗体HAb18可变区人源化设计.科学技术与工程, 2007(14): p. 3378-3382.
61. Sharon, J., Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold. Proceedings of the National Academy of Sciences of the United States of America, 1990. 87(12): p. 4814.
62.徐筱杰,陈丽蓉,化学及生物体系中的分子识别.化学进展, 1996(03): p. 15-27.
63.邢金良等,抗人HAb18G分子单链抗体基因的构建及在大肠杆菌中的分泌表达.中国免疫学杂志, 2003(07): p. 479-482.
2. Hwang, W. and J. Foote, Immunogenicity of engineered antibodies. Methods, 2005. 36(1): p. 3-10.
3. Izumi, Y., et al., Tumour biology: herceptin acts as an anti-angiogenic cocktail. Nature, 2002. 416(6878): p. 279-280.
4. Hudson, P. and C. Souriau, Engineered antibodies. Nature medicine, 2003. 9(1): p. 129-134.
5. Guisti, R., et al., FDA drug approval summary: panitumumab (Vectibix, TM). Oncologist, 2007. 12: p. 577-83.
6. Rosse, W., New insights into paroxysmal nocturnal hemoglobinuria. Current opinion in hematology, 2001. 8(2): p. 61.
7. K hler, G. and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity. 1975.
8. Bach, J., L. Chatenoud, and G. Fracchia, Safety and efficacy of therapeutic monoclonal antibodies in clinical therapy. Immunology today, 1993. 14(9): p. 421-422.
9. Dickman, S., CANCER THERAPY: Antibodies Stage a Comeback in Cancer Treatment. Science, 1998. 280(5367): p. 1196.
10. Newman, R., et al., "Primatization" of Recombinant Antibodies for Immunotherapy of Human Diseases: A Macaque/Human Chimeric Antibody Against Human CD4. Nature Biotechnology, 1992. 10(11): p. 1455-1460.
11. Queen, C., et al., A humanized antibody that binds to the interleukin 2receptor. Proceedings of the National Academy of Sciences of the United States of America, 1989. 86(24): p. 10029.
12. Green, L., et al., Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nature genetics, 1994. 7(1): p. 13-21.
13. S derlind, E., et al., Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nature Biotechnology, 2000. 18(8): p. 852-856.
14. Jones, P., et al., Replacing the complementarity-determining regions in a human antibody with those from a mouse. 1986.
15. Tsurushita, N., P. Hinton, and S. Kumar, Design of humanized antibodies: from anti-Tac to Zenapax, Methods 36 (2005). Article View Record in Scopus| Cited By in Scopus (19): p. 69-83.
16. Iwahashi, M., et al., CDR substitutions of a humanized monoclonal antibody (CC49): contributions of individual CDRs to antigen binding and immunogenicity. Molecular Immunology, 1999. 36(15-16): p. 1079-1091.
17. Kashmiri, S., et al., Development of a minimally immunogenic variant of humanized anti-carcinoma monoclonal antibody CC49. Critical reviews in oncology/hematology, 2001. 38(1): p. 3-16.
18. Leung, S., et al., Construction and characterization of a humanized, internalizing, B-cell (CD22)-specific, leukemia/lymphoma antibody, LL21. Molecular Immunology, 1995. 32(17-18): p. 1413-1427.
19. Yang, W., et al., CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. Journal of molecular biology, 1995. 254(3): p. 392-403.
20. Morea, V., A. Lesk, and A. Tramontano, Antibody modeling: implications for engineering and design. Methods, 2000. 20(3): p. 267-279.
21. Hwang, K., et al., Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods, 2005. 36(1): p. 35-42.
22. Ewert, S., A. Honegger, and A. Plukthun, Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering. Methods, 2004. 34(2): p. 184-199.
23. Jespers, L., et al., Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen. Nature Biotechnology, 1994. 12(9): p. 899-903.
24. Rader, C., D. Cheresh, and C. Barbas, A phage display approach for rapid antibody humanization: designed combinatorial V gene libraries. Proceedings of the National Academy of Sciences of the United States of America, 1998. 95(15): p. 8910.
25. Roguska, M., et al., Humanization of murine monoclonal antibodies through variable domain resurfacing. Proceedings of the National Academy of Sciences of the United States of America, 1994. 91(3): p. 969.
26. Roguska, M. and J. Pedersen, A comparison of two murine monoclonal antibodies humanized by CDR-grafting and variable domain resurfacing. Protein Engineering Design and Selection, 1996. 9(10): p. 895.
27. Weinblatt, M., et al., Adalimumab, a fully human anti-tumor necrosis factorαmonoclonal antibody, for the treatment of rheumatoid arthritis in patients taking concomitant methotrexate: the ARMADA trial. Arthritis &Rheumatism, 2003. 48(1): p. 35-45.
28. discontinues Vectibix, A., treatment in PACCE trial evaluating Vectibix(tm) as part of triple combination regimen. Pressemelding (Amgen) 22.3. 2007.
29. Vaughan, T., J. Osbourn, and P. Tempest, Human antibodies by design. Nature Biotechnology, 1998. 16(6): p. 535-539.
30. Smith, G., Filamentous fusion phage: novel expression vectors that display cloned antigens on the surface of the virion. Science, 1985. 228(4705): p. 1315-1317.
31. Fukazawa, T., B. Walter, and L. Owen-Schaub, Adenoviral Bid overexpression induces caspase-dependent cleavage of truncated Bid and p53-independent apoptosis in human non-small cell lung cancers. Journal of Biological Chemistry, 2003. 278(28): p. 25428.
32. Watanabe, T., et al., Adenoviral gene transfer in the peripheral nervous system. Journal of Orthopaedic Science, 2006. 11(1): p. 64-69.
33. Schaffitzel, C., et al., Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. Journal of immunological methods, 1999. 231(1-2): p. 119-135.
34. Tomizuka, K., et al., Double trans-chromosomic mice: Maintenance of two individual human chromosome fragments containing Ig heavy andκloci and expression of fully human antibodies. Proceedings of the National Academy of Sciences, 2000. 97(2): p. 722.
35. Cohen, C., et al., Direct phenotypic analysis of human MHC class I antigen presentation: visualization, quantitation, and in situ detection of human viral epitopes using peptide-specific, MHC-restricted human recombinant antibodies. The Journal of Immunology, 2003. 170(8): p.4349.
36. Wilkinson, L. and J. Edwards, Binding of antibodies raised against tumour necrosis factor alpha (TNF-α) to blood vessels and macrophages in inflamed synovial tissue. Rheumatology international, 1991. 11(1): p. 19-25.
37. Tanaka, K., et al., Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom, 1988. 2(8): p. 151-153.
38. Zhao, Y. and B. Chait, Protein epitope mapping by mass spectrometry. Analytical chemistry, 1994. 66(21): p. 3723-3726.
39. Van de Water, J., et al., Detection of molecular determinants and epitope mapping using MALDI-TOF mass spectrometry. Clinical immunology and immunopathology, 1997. 85(3): p. 229.
40. Kumar, A., et al., Buildup rates of the nuclear Overhauser effect measured by two-dimensional proton magnetic resonance spectroscopy: implications for studies of protein conformation. Journal of the American Chemical Society, 1981. 103(13): p. 3654-3658.
41. Deisenhofer, J., et al., X-ray structure analysis of a membrane protein complex:: Electron density map at 3 resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis. Journal of molecular biology, 1984. 180(2): p. 385-398.
42. Engh, R. and R. Huber, Accurate bond and angle parameters for X-ray protein structure refinement. Acta Crystallographica Section A: Foundations of Crystallography, 1991. 47(4): p. 392-400.
43. Yu, X., et al., The glycosylation characteristic of hepatoma-associatedantigen HAb18G/CD147 in human hepatoma cells. The international journal of biochemistry & cell biology, 2006. 38(11): p. 1939-1945.
44. Ellis, S., K. Nabeshima, and C. Biswas, Monoclonal antibody preparation and purification of a tumor cell collagenasestimulatory factor. Cancer research, 1989. 49(12): p. 3385.
45. Guo, H., et al., Characterization of the gene for human EMMPRIN, a tumor cell surface inducer of matrix metalloproteinases. Gene, 1998. 220(1-2): p. 99-108.
46. Biswas, C., et al., The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily. Cancer research, 1995. 55(2): p. 434.
47. Papoutsi, M., et al., Induction of the blood-brain barrier marker neurothelin/HT7 in endothelial cells by a variety of tumors in chick embryos. Histochemistry and Cell Biology, 2000. 113(2): p. 105-113.
48. Chen, Z., et al., Targeting radioimmunotherapy of hepatocellular carcinoma with iodine (131I) metuximab injection: clinical phase I/II trials. International Journal of Radiation Oncology Biology Physics, 2006. 65(2): p. 435-444.
49. Lesk, A., Introduction to bioinformatics. 2002: Oxford University Press Oxford.
50. MUEGGE, I. and I. ENYEDY, Docking and scoring. Computational Medicinal Chemistry and Drug Discovery: p. 405-436.
51. Browne, W., et al., A possible three-dimensional structure of bovine [alpha]-lactalbumin based on that of hen's egg-white lysozyme. Journal of molecular biology, 1969. 42(1): p. 65-70.
52. Fiser, A., Comparative protein structure modelling. From ProteinStructure to Function with Bioinformatics, 2008: p. 57-90.
53. Kabat, E. and T. Wu, Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. The Journal of Immunology, 1991. 147(5): p. 1709.
54. Whitelegg, N. and A. Rees, WAM: an improved algorithm for modelling antibodies on the WEB. Protein Engineering Design and Selection, 2000. 13(12): p. 819.
55. Al-Lazikani, B., A. Lesk, and C. Chothia, Standard conformations for the canonical structures of immunoglobulins1. Journal of molecular biology, 1997. 273(4): p. 927-948.
56. Yu, X.L., et al., Crystal structure of HAb18G/CD147: implications for immunoglobulin superfamily homophilic adhesion. J Biol Chem, 2008. 283(26): p. 18056-65.
57. Hubbard, S. and J. Thornton, NACCESS, Computer Program, Department of Biochemistry and Molecular Biology. University College London, 1993.
58. Mariuzza, R. and R. PoIjak, The basics of binding: mechanisms of antigen recognition and mimicry by antibodies. Current opinion in immunology, 1993. 5(1): p. 50-55.
59. Slavin-Chiorini, D., et al., A CDR-grafted (humanized) domain-deleted antitumor antibody. Cancer biotherapy & radiopharmaceuticals, 1997. 12(5): p. 305-316.
60.杨滨,杨向民,陈志南,基于表面重塑方法的抗体HAb18可变区人源化设计.科学技术与工程, 2007(14): p. 3378-3382.
61. Sharon, J., Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold. Proceedings of the National Academy of Sciences of the United States of America, 1990. 87(12): p. 4814.
62.徐筱杰,陈丽蓉,化学及生物体系中的分子识别.化学进展, 1996(03): p. 15-27.
63.邢金良等,抗人HAb18G分子单链抗体基因的构建及在大肠杆菌中的分泌表达.中国免疫学杂志, 2003(07): p. 479-482.