基于计算机辅助分子设计的抗CD20单克隆抗体的改造
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摘要
鼠源单克隆抗体在临床应用中有以下缺点:诱发人体产生人抗小鼠抗体(human anti-mounse antibody,HAMA)反应,在人体内半寿期短,不能够有效地激活人体的生物效应功能,如补体依赖的细胞毒作用(complement-dependent cytotoxicity,CDC)及抗体依赖性细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity,ADCC)。通过分子生物学技术对鼠抗体进行人源化改造在一定程度上可以克服这些缺点。本室制备的鼠源抗CD20单克隆抗体(monoclonal antibody,mAb)1-28具有潜在的应用价值。本研究在对mAb 1-28(简称1-28)功能进行验证的基础上,通过计算机辅助分子设计和分子生物学技术对其进行改造,构建了抗CD20嵌合抗体C1-28和新型小分子抗CD20抗体5S(ScFv-Fc),并对其抗原结合特性及通过CDC杀伤肿瘤细胞的功能进行了初步研究。
1 抗CD20单克隆抗体1-28的免疫学特性及功能分析
首先分析了1-28与靶细胞的结合活性。间接免疫荧光的结果显示,1-28可与人B细胞淋巴瘤Daudi细胞和Raji细胞结合,而不与人T细胞系Jurkat细胞结合,表明1-28与靶细胞的结合是特异的。流式细胞术分析结果显示,1-28与Daudi和Raji细胞结合的阳性率达99%以上。相对亲和力(relative binding affinity)测试结果显示,1-28的亲和力为美罗华亲和力的1/12左右。通过ELISA法测定出1-28的亲和常数为K=1.6×10~(10) M~(-1)。竞争实验的结果提示1-28与另外两种抗CD20抗体(美罗华、2H7)识别的是不同的抗原表位。1-28对Raji细胞和Daudi细胞都具有生长抑制效应,但是程度不同。另外,1-28可以诱导Raji细胞和Daudi细胞发生凋亡。1-28还可通过激活补体杀伤靶细胞。以Raji细胞为研究对象时,其半数杀伤浓度为1.26 nmol/L。1-28对非靶细胞Jurkat没有CDC的功能,提示这种杀伤是特异的。
The clinical application of murine monoclonal antibodies is associated with a number of drawbacks, including inducement of human anti-mouse antibody (HAMA), short circulating time in vivo, and the inability to fully harness the effectors (ADCC) and complement (CDC) repertoire of the human immune system. Genetic engineering has been used to humanize mouse mAb and can overcome the drawbacks significantly. Our group developed a mouse anti-human CD20 monoclonal antibody 1-28, which possessed potential clinical values. The immunological characteristics of mAb 1-28 were tested. With computer-aided molecular modeling design method and DNA recombination technology, chimeric anti-CD20 antibody and single-chain Fv (scFv) with human lgG1 hinge and Fc regions, designated 5S, were produced. Their binding activity and complement-dependent cytotoxicity (CDC) were analyzed. 1 The immunological characters study on mAb 1-28Specific binding activity of mAb 1-28 was verified by flow cytometry method. The relative binding affinity of mAb 1-28 was only about 1/12th of Rituxan. K value of 1-28 was 1.6×10~(10)M~(-1) measured by Scatchard analysis. The results of competitive binding assay showed that both 2H7 (mouse anti-human CD20 antibody) and Rituxan (chimeric anti-CD20 antibody) could not inhibit the binding of 1-28 to CD20 antigen expressed on the surface of Raji cells. 1-28 could alone inhibit CD20+ cells' proliferation and was sufficient to induce apoptosis of Raji cells and Daudi cells. 1-28 could also fix and activate complement to kill CD20+ cells, such as Raji, while it's ineffective to Jurkat cells.
2 Expression and characterization of Chimeric Anti-CD20 AntibodyThe light- and heavy-chain genes were amplified from hybridoma cell line 1-28 secreting anti-human CD20 mAb by RT-PCR method, and then cloned to T vector, sequenced. MAb 1-28 protein was separated by reduced SDS-PAGE, and the light- and heavy-chain bands were excised from preparative gel, digested by trypsin, and subjected to peptide mass fingerprinting. The light- and heavy-chain DNA sequences were verified by their protein sequences. To acquire a positive control in the experiments, V genes of 2H7 were synthesized by overlapping PCR method. Both V genes of 1-28 and 2H7 were cloned into chimeric antibody expression vector (pCMV-VH & pCMV-VL), generating chimeric anti-human CD20 mAb expression vectors. Plasmids were transfected into 293T cells with Lipofectamine? 2000. High level expression of the two anti-human CD20 chimeric mAbs were obtained and their m.w. in agreement with that of human IgG. The binding activity of C2H7 was well reserved, but C1-28 lost its binding activity to Daudi and Raji cells completely. In complement-dependent cytotoxicity assay, C2H7 could fix and activate both human complement and rabbit complement to kill target cells specifically.3 Design of a new kind of anti-CD20 antibodyThe spatial structure of mAb 1-28 variable region was modeled theoretically with homology method carried on http://www.expasy.ch. The stability of mAb 1-28 Fv was analyzed according to inter-molecular hydrogen-bond forming theory and reaction free energy theory. The result showed that the stability of the complex Fv was weak due to the structural non-match and electrostatic non-complementarity. The weak stability of the complex Fv induced the low bioactivity. The residues characters of N and C terminal of antibody 1-28 were studied based on distance geometry and computer graphics technology. The single chain Fv antibody (abbr. scFv) was constructed and the mode was VH-Linker-VL. The result showed that the stability of the antibody 1-28 was increased while the active domain conformation was not changed due to the
1 抗CD20单克隆抗体1-28的免疫学特性及功能分析
首先分析了1-28与靶细胞的结合活性。间接免疫荧光的结果显示,1-28可与人B细胞淋巴瘤Daudi细胞和Raji细胞结合,而不与人T细胞系Jurkat细胞结合,表明1-28与靶细胞的结合是特异的。流式细胞术分析结果显示,1-28与Daudi和Raji细胞结合的阳性率达99%以上。相对亲和力(relative binding affinity)测试结果显示,1-28的亲和力为美罗华亲和力的1/12左右。通过ELISA法测定出1-28的亲和常数为K=1.6×10~(10) M~(-1)。竞争实验的结果提示1-28与另外两种抗CD20抗体(美罗华、2H7)识别的是不同的抗原表位。1-28对Raji细胞和Daudi细胞都具有生长抑制效应,但是程度不同。另外,1-28可以诱导Raji细胞和Daudi细胞发生凋亡。1-28还可通过激活补体杀伤靶细胞。以Raji细胞为研究对象时,其半数杀伤浓度为1.26 nmol/L。1-28对非靶细胞Jurkat没有CDC的功能,提示这种杀伤是特异的。
The clinical application of murine monoclonal antibodies is associated with a number of drawbacks, including inducement of human anti-mouse antibody (HAMA), short circulating time in vivo, and the inability to fully harness the effectors (ADCC) and complement (CDC) repertoire of the human immune system. Genetic engineering has been used to humanize mouse mAb and can overcome the drawbacks significantly. Our group developed a mouse anti-human CD20 monoclonal antibody 1-28, which possessed potential clinical values. The immunological characteristics of mAb 1-28 were tested. With computer-aided molecular modeling design method and DNA recombination technology, chimeric anti-CD20 antibody and single-chain Fv (scFv) with human lgG1 hinge and Fc regions, designated 5S, were produced. Their binding activity and complement-dependent cytotoxicity (CDC) were analyzed. 1 The immunological characters study on mAb 1-28Specific binding activity of mAb 1-28 was verified by flow cytometry method. The relative binding affinity of mAb 1-28 was only about 1/12th of Rituxan. K value of 1-28 was 1.6×10~(10)M~(-1) measured by Scatchard analysis. The results of competitive binding assay showed that both 2H7 (mouse anti-human CD20 antibody) and Rituxan (chimeric anti-CD20 antibody) could not inhibit the binding of 1-28 to CD20 antigen expressed on the surface of Raji cells. 1-28 could alone inhibit CD20+ cells' proliferation and was sufficient to induce apoptosis of Raji cells and Daudi cells. 1-28 could also fix and activate complement to kill CD20+ cells, such as Raji, while it's ineffective to Jurkat cells.
2 Expression and characterization of Chimeric Anti-CD20 AntibodyThe light- and heavy-chain genes were amplified from hybridoma cell line 1-28 secreting anti-human CD20 mAb by RT-PCR method, and then cloned to T vector, sequenced. MAb 1-28 protein was separated by reduced SDS-PAGE, and the light- and heavy-chain bands were excised from preparative gel, digested by trypsin, and subjected to peptide mass fingerprinting. The light- and heavy-chain DNA sequences were verified by their protein sequences. To acquire a positive control in the experiments, V genes of 2H7 were synthesized by overlapping PCR method. Both V genes of 1-28 and 2H7 were cloned into chimeric antibody expression vector (pCMV-VH & pCMV-VL), generating chimeric anti-human CD20 mAb expression vectors. Plasmids were transfected into 293T cells with Lipofectamine? 2000. High level expression of the two anti-human CD20 chimeric mAbs were obtained and their m.w. in agreement with that of human IgG. The binding activity of C2H7 was well reserved, but C1-28 lost its binding activity to Daudi and Raji cells completely. In complement-dependent cytotoxicity assay, C2H7 could fix and activate both human complement and rabbit complement to kill target cells specifically.3 Design of a new kind of anti-CD20 antibodyThe spatial structure of mAb 1-28 variable region was modeled theoretically with homology method carried on http://www.expasy.ch. The stability of mAb 1-28 Fv was analyzed according to inter-molecular hydrogen-bond forming theory and reaction free energy theory. The result showed that the stability of the complex Fv was weak due to the structural non-match and electrostatic non-complementarity. The weak stability of the complex Fv induced the low bioactivity. The residues characters of N and C terminal of antibody 1-28 were studied based on distance geometry and computer graphics technology. The single chain Fv antibody (abbr. scFv) was constructed and the mode was VH-Linker-VL. The result showed that the stability of the antibody 1-28 was increased while the active domain conformation was not changed due to the
引文
1. Fink-Bennett DM, Thomas K. 90Y-ibritumomab tiuxetan in the treatment of relapsed or refractory B-cell non-Hodgkin's lymphoma.J Nucl Med Technol. 2003 Jun;31(2):61-8; quiz 69-70.
2. Kaminski MS, Estes J, Zasadny KR, el al. Ridioimmunotherapy with iodine 1311 tositumomab for relapsed or refractory B-cell non-Hodgkin lymphoma: updated results and long-term follow up of the University of Michigan experience. Blood. 2000, 96(15): 1259-1266.
3. Breedveld FC. Therapeutic monoclonal antibodies. Lancet. 2000, 335-740.
4. Gopal AK, Press OW. Clinical applications of anti-CD20 antibodies. J Lab Clin Med. 1999 Nov; 134(5): 445-450.
5. Tedder TF, Mclntyre G, Schlossman SF. Heterogeneity in the B1 (CD20) cell surface molecule expressed by human B-lymphocytes. Mol Immunol. 1988 Dec; 25(12): 1321-1330.
6. Deans JP, Li H, Polyak MJ. CD20-mediated apoptosis: signalling through lipid rafts. Immunology. 2002 Oct; 107(2): 176-182.
7. Polyak MJ, Deans JP. Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure. Blood, 2002, 99(9): 3256-3262,
8. Chang KL, Arber DA, Weiss LM. CD20: A Review. Applied Immunohistochem. 1996. 4: 1
9. Stashenko P, Nadler LM, Hardy R, et al. Characterization of a human B lymphocyte-specific antigen. J Immunol. 1980 Oct; 125(4): 1678-85.
10. Kosmas C, Stamatopoulos K, Stavroyianni N, et al. Anti-CD20-based therapy of B cell lymphoma: state of art. Leukemia. 2002, 16: 2004-2015.
11. Shan D, Ledbetter JA, Press OW. Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood. 1998 Mar 1; 91 (5): 1644-1652.
12. Brown DA, London E. Structure and function of shingolipid- and cholesterol-rich membrane rafts. J Biol chem. 2000. 275: 17221-17224.
13. Resh MD. Myristylation and palmitylation of Src family members: the fats of the matter. Cell. 1994. 76: 411-413.
14. Mayer J, Navratil M, Vasova J, Vorlicek J. Non-cytostatic treatment of malignancies. Anti-CD20 monoclonal antibody (Rituximab, Mabthera) in the treatment of non-Hodgkin's lymphoma. VnitrLek. 2001 Sep; 47 Suppl 1: 57-62.
15. Shan D, Ledbetter JA, Press OW. Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. Cancer Immunol Immunother. 2000. Mar; 48 (12): 673-683.
16.王玉刚,沈倍奋.CD20抗原及治疗性抗CD20抗体.中国肿瘤生物治疗杂志.2005.12(1):76-79.
17. Stein R, Qu Z, Chen S, et al. Characterization of a new humanized anti-CD20 monoclonal antibody, IMMU-106, and Its use in combination with the humanized anti-CD22 antibody, epratuzumab, for the therapy of non-Hodgkin's lymphoma. Clin Cancer Res, 2004, 10(8): 2868-2878.
18. Teeling JL, French RR, Cragg MS, et al. Characterization of new human CD20 monoclonal antibodies with potent cytolyticactivity against non-Hodgkin lymphomas. Blood. 2004 Sep 15; 104(6): 1793-1800.
19.李爱玲,孙瑛勋,洪海燕等.利用基因重组抗原研制人CD20单克隆抗体及其功能的研究.中华微生物学和免疫学杂志.2002,22(4):385-388.
20.李爱玲,黎燕,孙瑛勋等.抗人CD20单克隆抗体诱导Daudi细胞凋亡的功能研究.免疫学杂志.2002,18(4):292-295.
21.李爱玲.抗CD20和CD34单抗的研制及其功能研究.第二军医大学博士论文.2002.
22. Horning SJ. Treatment approaches to the low-grade lymphomas. Blood. 1994 Feb 15; 83(4): 881-884.
23. Ghetie MA, Bright H, Vitetta ES. Homodimers but not monomers of Rituxan (chimeric anti-CD20) induce apoptosis in human B-lymphoma cells and synergize with a chemotherapeutic agent and an immunotoxin. Blood. 2001 Mar 1; 97(5): 1392-1398.
24. Bator JM, Reading CL. Measurement of antibody affinity for cell surface antigens using an enzyme-linked immunosorbent assay. J Immuno/Methods, 1989, 125(1-2). 167—176.
25. Michel RB, Mattes MJ. Intracellular accumulation of the anti-CD20 antibody 1F5 in B-lymphoma cells. Clin Cancer Res. 2002 Aug;8(8):2701-2713.
26. Reff ME, Career K, Chambers KS, Chinn PC, Leonard JE, Raab R, Newman RA, Hanna N, Anderson DR. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood. 1994 Jan 15; 83(2): 435-445.
27. Elvin AK, Tai TW, Harold MP, et al. Sequences of Proteins of Immunological Interest,. U. S.: Public Health Service Natinal Institutes of Health, 1991, Fifth Edition.
28. Spira G, Yuan R, Paizi M, et al. Simultaneous expression of Kand λ light chains in a murine IgG3 anti-Cryptococcus neoformans hybridoma cell line. Hybridoma. 1994, 13(6): 531-535.
29. Krishnan IS, Hansen HJ, Gold DV, et al. Co-secretion of two distinct kappa light chains by the mu-9 hybridoma. Hybridoma. 1999, 18(4): 325-333.
30. Cabilly S, and Riggs AD. Immunoglobulin transcripts and molecular history of a hybridoma that produced antibody to carcinoembryonic antigen. Gene. 1985, 40: 157-161.
31. Loset GA, Roux KH, Zhu P, et al. Differential segmental flexibility and reach dictate the antigen binding mode of chimeric IgD and IgM: implications for the function of the B cell receptor. J Immunol. 2004 Mar 1; 172(5): 2925-2934.
32. Tobita T, Oda M, Azuma T. Segmental flexibility and avidity of IgM in the interaction of polyvalent antigens. Mol Immunol. 2004 Jan; 40(11): 803-811.
33. Blundell TL, Sibanda BL, Sternberg MJE, Thornton JM. Knowledge-based prediction of protein structures and the design of novel molecules. Nature, 1987, 326, 347～352.
34. Blundell T, Carney D, Gardner S, et al.. Knowledge-based protein modeling and design. Eur J Biochem, 1988, 172, 513～520.
35. Sail A, Overington JP, Johnson MS, et al.. From comparisons of protein sequences and structures to protein modeling and design. Trends Biochem Sci, 1990, 15, 235～240.
36. Sanchez R, Sail A. Advances in comparative protein structure modeling. Curt Opin Struct Biol, 1997, 7, 206～214.
37. Sanchez R, Pieper U, Melo F, et al.. Protein structure modeling for structural genomics. Nat Struct Biol, 2000, 7 (suppl.), 986～990.
38. Wu AM, Tan GJ, Sherman MA, et al. Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange. Protein Eng. 2001 Dec; 14(12): 1025-1033.
39. Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990 Feb 20; 211 (4): 943-958.
40. Morea V, Arthur ML, Anna T. Antibody modeling: implications for engineering and design. Methods, 2000, 20, 267～279.
41. Glockshuber R, Malia M, Pfitzinger I, et al. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry. 1990 Feb 13; 29(6): 1362-1367.
42. Shan D, Press OW, Tsu TT, Hayden MS, Ledbetter JA. Characterization of scFv-lg constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths. J ImmunoL 1999 Jun 1; 162(11): 6589-6595.
43. Krott A A, Lah M, Oddie G W, et al. Single chain Fv fragments of anti-neuraminidase antibody NC10 containing five and ten residue linkers form dimmers and with zero residue linker a tdmer. Protein Eng, 1997, 10: 423～428
44. Todorovska A, Roovers R C, Dolezal O, et al. Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J Immunol Methods, 2001, 248(1-2): 47～66.
45. Maloney DG, Gdllo-Lopez AJ, White CA, et al. IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma. Blood, 1997, 90(6): 2188-2195.
46. Ghobrial I, Witzig T. Radioimmunotherapy: a new treatment modality for B-cell non-Hodgkin's lymphoma. Oncology (Huntingt). 2004 May; 18(5): 623-30; discussion 633-4, 637-8, 640.
47.谢志刚.基因工程抗HER2/neu×抗CD16双特异性抗体的构建、表达及其功能的初步研究.军事医学科学院博士论文.2004.
48. Hale G, Clark M, Waldmann H. Therapeutic poptential of rat monoclonal antibodies: isotype specificity of antibody-dependent cell-mediated cototoxicity with human lymphocytes. J Immunol. 1985. 134: 3056-3061.
49. Cragg MS, Morgan SM, Chan HT, et al. Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts. Blood. 2003 Feb 1; 101 (3): 1045-1052.
2. Kaminski MS, Estes J, Zasadny KR, el al. Ridioimmunotherapy with iodine 1311 tositumomab for relapsed or refractory B-cell non-Hodgkin lymphoma: updated results and long-term follow up of the University of Michigan experience. Blood. 2000, 96(15): 1259-1266.
3. Breedveld FC. Therapeutic monoclonal antibodies. Lancet. 2000, 335-740.
4. Gopal AK, Press OW. Clinical applications of anti-CD20 antibodies. J Lab Clin Med. 1999 Nov; 134(5): 445-450.
5. Tedder TF, Mclntyre G, Schlossman SF. Heterogeneity in the B1 (CD20) cell surface molecule expressed by human B-lymphocytes. Mol Immunol. 1988 Dec; 25(12): 1321-1330.
6. Deans JP, Li H, Polyak MJ. CD20-mediated apoptosis: signalling through lipid rafts. Immunology. 2002 Oct; 107(2): 176-182.
7. Polyak MJ, Deans JP. Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure. Blood, 2002, 99(9): 3256-3262,
8. Chang KL, Arber DA, Weiss LM. CD20: A Review. Applied Immunohistochem. 1996. 4: 1
9. Stashenko P, Nadler LM, Hardy R, et al. Characterization of a human B lymphocyte-specific antigen. J Immunol. 1980 Oct; 125(4): 1678-85.
10. Kosmas C, Stamatopoulos K, Stavroyianni N, et al. Anti-CD20-based therapy of B cell lymphoma: state of art. Leukemia. 2002, 16: 2004-2015.
11. Shan D, Ledbetter JA, Press OW. Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood. 1998 Mar 1; 91 (5): 1644-1652.
12. Brown DA, London E. Structure and function of shingolipid- and cholesterol-rich membrane rafts. J Biol chem. 2000. 275: 17221-17224.
13. Resh MD. Myristylation and palmitylation of Src family members: the fats of the matter. Cell. 1994. 76: 411-413.
14. Mayer J, Navratil M, Vasova J, Vorlicek J. Non-cytostatic treatment of malignancies. Anti-CD20 monoclonal antibody (Rituximab, Mabthera) in the treatment of non-Hodgkin's lymphoma. VnitrLek. 2001 Sep; 47 Suppl 1: 57-62.
15. Shan D, Ledbetter JA, Press OW. Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. Cancer Immunol Immunother. 2000. Mar; 48 (12): 673-683.
16.王玉刚,沈倍奋.CD20抗原及治疗性抗CD20抗体.中国肿瘤生物治疗杂志.2005.12(1):76-79.
17. Stein R, Qu Z, Chen S, et al. Characterization of a new humanized anti-CD20 monoclonal antibody, IMMU-106, and Its use in combination with the humanized anti-CD22 antibody, epratuzumab, for the therapy of non-Hodgkin's lymphoma. Clin Cancer Res, 2004, 10(8): 2868-2878.
18. Teeling JL, French RR, Cragg MS, et al. Characterization of new human CD20 monoclonal antibodies with potent cytolyticactivity against non-Hodgkin lymphomas. Blood. 2004 Sep 15; 104(6): 1793-1800.
19.李爱玲,孙瑛勋,洪海燕等.利用基因重组抗原研制人CD20单克隆抗体及其功能的研究.中华微生物学和免疫学杂志.2002,22(4):385-388.
20.李爱玲,黎燕,孙瑛勋等.抗人CD20单克隆抗体诱导Daudi细胞凋亡的功能研究.免疫学杂志.2002,18(4):292-295.
21.李爱玲.抗CD20和CD34单抗的研制及其功能研究.第二军医大学博士论文.2002.
22. Horning SJ. Treatment approaches to the low-grade lymphomas. Blood. 1994 Feb 15; 83(4): 881-884.
23. Ghetie MA, Bright H, Vitetta ES. Homodimers but not monomers of Rituxan (chimeric anti-CD20) induce apoptosis in human B-lymphoma cells and synergize with a chemotherapeutic agent and an immunotoxin. Blood. 2001 Mar 1; 97(5): 1392-1398.
24. Bator JM, Reading CL. Measurement of antibody affinity for cell surface antigens using an enzyme-linked immunosorbent assay. J Immuno/Methods, 1989, 125(1-2). 167—176.
25. Michel RB, Mattes MJ. Intracellular accumulation of the anti-CD20 antibody 1F5 in B-lymphoma cells. Clin Cancer Res. 2002 Aug;8(8):2701-2713.
26. Reff ME, Career K, Chambers KS, Chinn PC, Leonard JE, Raab R, Newman RA, Hanna N, Anderson DR. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood. 1994 Jan 15; 83(2): 435-445.
27. Elvin AK, Tai TW, Harold MP, et al. Sequences of Proteins of Immunological Interest,. U. S.: Public Health Service Natinal Institutes of Health, 1991, Fifth Edition.
28. Spira G, Yuan R, Paizi M, et al. Simultaneous expression of Kand λ light chains in a murine IgG3 anti-Cryptococcus neoformans hybridoma cell line. Hybridoma. 1994, 13(6): 531-535.
29. Krishnan IS, Hansen HJ, Gold DV, et al. Co-secretion of two distinct kappa light chains by the mu-9 hybridoma. Hybridoma. 1999, 18(4): 325-333.
30. Cabilly S, and Riggs AD. Immunoglobulin transcripts and molecular history of a hybridoma that produced antibody to carcinoembryonic antigen. Gene. 1985, 40: 157-161.
31. Loset GA, Roux KH, Zhu P, et al. Differential segmental flexibility and reach dictate the antigen binding mode of chimeric IgD and IgM: implications for the function of the B cell receptor. J Immunol. 2004 Mar 1; 172(5): 2925-2934.
32. Tobita T, Oda M, Azuma T. Segmental flexibility and avidity of IgM in the interaction of polyvalent antigens. Mol Immunol. 2004 Jan; 40(11): 803-811.
33. Blundell TL, Sibanda BL, Sternberg MJE, Thornton JM. Knowledge-based prediction of protein structures and the design of novel molecules. Nature, 1987, 326, 347～352.
34. Blundell T, Carney D, Gardner S, et al.. Knowledge-based protein modeling and design. Eur J Biochem, 1988, 172, 513～520.
35. Sail A, Overington JP, Johnson MS, et al.. From comparisons of protein sequences and structures to protein modeling and design. Trends Biochem Sci, 1990, 15, 235～240.
36. Sanchez R, Sail A. Advances in comparative protein structure modeling. Curt Opin Struct Biol, 1997, 7, 206～214.
37. Sanchez R, Pieper U, Melo F, et al.. Protein structure modeling for structural genomics. Nat Struct Biol, 2000, 7 (suppl.), 986～990.
38. Wu AM, Tan GJ, Sherman MA, et al. Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange. Protein Eng. 2001 Dec; 14(12): 1025-1033.
39. Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990 Feb 20; 211 (4): 943-958.
40. Morea V, Arthur ML, Anna T. Antibody modeling: implications for engineering and design. Methods, 2000, 20, 267～279.
41. Glockshuber R, Malia M, Pfitzinger I, et al. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry. 1990 Feb 13; 29(6): 1362-1367.
42. Shan D, Press OW, Tsu TT, Hayden MS, Ledbetter JA. Characterization of scFv-lg constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths. J ImmunoL 1999 Jun 1; 162(11): 6589-6595.
43. Krott A A, Lah M, Oddie G W, et al. Single chain Fv fragments of anti-neuraminidase antibody NC10 containing five and ten residue linkers form dimmers and with zero residue linker a tdmer. Protein Eng, 1997, 10: 423～428
44. Todorovska A, Roovers R C, Dolezal O, et al. Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J Immunol Methods, 2001, 248(1-2): 47～66.
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