Humanization of Ovarian Carcinoma Anti2idiotype Single2chain

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 18 (4) : ,,,, (, ),, PCR, 6B11 ScFv, 6B11V L 2V H IgG1 CH3 (V L 2V H 2CH3), E coli BL21 (DE3) IPTG SDS2PAGE, 50 kd, 100 kd ELISA West2 ern, COC16629 IgG1, scfv,, IgG1,, Q754,R Humanization of Ovarian Carcinoma Anti2idiotype Single2chain CUI Heng 3 ( V L 2V H 2CH3) Antibody, CHANG Xiao2hong, FENGJie, LIU Bei, CAO Shan2jin ( Gynecologic Oncology Center, People s Hospital, Peking University, Beijing , China) Abstract In order to reduce the human anti2mouse antibody response and obtain optimal antigenicity, anti2 idiotype single2chain which mimicking ovarian cancer antigen has been humanized Using overlap PCR and DNA recombinant technique, the sequence of 6B11 ScFv was changed by heavy chain and light chain reversal Prokaryotic expression vector was produced by genetic fusion of 6B11V L 2V H to human IgG1 hinge and CH3 re2 gion Transformed E coli BL21 (DE3) was propagated and induced by IPTG SDS2PAGE showed that a pro2 tein band with molecular weight of 50 kd appeared as the expected size after transformation Molecular weight of 100 kd might be examined by electrophoresis in non2denaturing systems The fusion protein was analyzed with ELISA, inhibition ELISA tests and Western blotting, respectively The humanized anti2idiotype minibody showed the capacity of bivalent binding to ovarian cancer monoclonal antibody coc16629 and goat anti2human immunoglobulin IgG1 It is a useful reagent for clinical uses Key words humanization, anti2idiotypic mimibody, human IgG1, ovarian neoplasm, recombinant fusion gene : , : (No ) 863 (No ) 3 Tel : (010) , Fax : (010) , E2mail : net,,1956 2,, Received :November 1,2001 ;Accepted :December 28,2001 Supported by the National Natural Science Foundation of China (No ) and National High Technology and Development Program ( No ) Patent number application of China (No ) 3 Corresponding author Tel : (010) , Fax : (010) , E2mail : net

2 4 : GC CA GTC TC CAAA GC TC C T GA TC TA CA TA [1 ], 5 20 % 40 %, F 4 : 5 2 CA G GC GGA GGT GGC TC T GGC GGT G, 2 3, GC G GA T C GCA G GT CA A A C T GCA G GA GT C,, T G GC C C T G G GA TA T T GCA GC C C T C C C A G 2 3, F 5 : 5 2 G GA TA T T GCA GC C C TC C CA GA C C C T CA GT C T GA C T T GT TC T T TC TC T GGGC T T T CA C T GCA CA C T TA T GGTA TA GGA GTA2 3,, F 6 : 5 2 A CA C T TA T GGTA TA GGA GTA GGC T,, G GA T T C GT CA GC C T T CA G G GA A G G GT C T [2 ] G GA GT G GC T G GCA CA C T T T T G GT G GA A T, 2 3, R 1 : 3 2 C GGGA TC C T GA GGA GA C GGT GA C C,, GT G GT GC C T T G GC C C CA GA CA T C GA A GT 6B11ScFv A C CA C GC TA T2 5 OC16629 R 2 : 3 2 A CA T C GAA GTA C CA C GC TA T T T TC COC16629 [3,4 ] 6B11 ScFv GTA GTA A TA A G G GC TA T T C GA GCA CA GT, 6B11V L 2V H, IgG1 A GTA T GT G GCA GTA T C T G2 5 R 3 : 3 2 GTA GTA T GT GGCA GTA TC T GCA GT CH3 (V L 2V H 2CH3), GT C CA CA C T G GC GA T GT T GA G GA A TA C C (6B11V L V H hc), T G GT T GT T G GA G GC GT C C T T G GA GA CA G COC R 4 : 3 2 GGA GGC GTC C T T GGA GA CA GT GA G 1 C C G GC T C T TCA GGGC T GT GT TA TA GTA C T TA T TA T TA T TC CA C CAAA T GT GT GC CA R 5 :3 2 GC CA GA GC CA C C TC C GC C T GAA C C pet30a ( + ) 6B11V H V L hc GC C T C CA C C C C GT T T TA T T TC CAA C T T T G, 6B11 ScFv(V H 2V L ) [5 ] ; T C C C C CC TCC GAA C GT GTA C GGAAAA T2 5 puc18 ; JM109 R 6 :3 2 TC C GAA C GT GTA C GGAAAA T GT GT DH5 BL21 (DE3) A C T C T GA GA GCA GA A A TA A A C T C C CA GA 1 2 T CA TCA GCC TCCA C TC T GC T GA TC TT GA2 5 Nco, BamH, Sac, Smal, T4 DNA Promega 1 3 PCR ( 6B11 ScFv ) A C C T GTC T GGGA CCCCA GAAAA TC GGT bp F 1 :5 2 C GGAA T TC C CA T GGGA GA CA TC GA GC T CA C TCA GTC TC CA C TC TC C C T GC C T G T CA GT C T T GGA GA TCAA GC C TC CA TC TC T T GCA GA2 3 F 2 : 5 2 A A GC C T C CA T C T C T T GCA GA T C TA GT CA GA A C C T T GTA CA CA GTA A T G GA A A CA CC TA TTTA CA TT GGTA CC T GCA GAA G2 3 F 3 :5 2 TA CA T T GGRA C C T GCA GAA GC CA G GT T T C CAA C C GA T T T TC T GGGGTC C CA2 3 R 7 : 3 2 C T C CA C T C T GC T GA T C T T GA GT GT GA A A TC T GTC C C T GA TC CA C T GC CA C T GA 1 4 COC16629 HRP2 COC16629, 6B11 6B11, IgG IgG 1 5 PCR 6B11 V L V H (A) F3 + R7 Frag 2 (1) Solution

492 18 min F6 + R4 Frag 2(1) Solution 94 5 min, 55 5 min, 72 5 OC16629 ( Ag), HRP2COC16629, (B) PCR (1) PCR F2 + R6 + Frag 2 (1) Solution Frag 2 (2) Solution Solution F5 + R3 + Frag 2(1) Solution

3 min F6 + R4 Frag 2(1) Solution 94 5 min, 55 5 min, 72 5 OC16629 ( Ag), HRP2COC16629, (B) PCR (1) PCR F2 + R6 + Frag 2 (1) Solution Frag 2 (2) Solution Solution F5 + R3 + Frag 2(1) Solution Frag 2(2) (2) PCR F2 + R5 + Frag 2 (2) Solution Frag 2 (3) Solution F4 + R2 + Frag 2 (2) Solution Frag 2 (3) Solution (3) PCR F1 + R1 + Frag 2 (3) Solution + Frag 2(3) Solution PCR Fragments s,55 30 s,72, 30 s ; 30 PCR 1 %, Noc BamH puc18, JM109, B V L 2V H IgG1 CH3 pet30a ( + ) 6B11V H V L hc Noc PCR 1 2 DNA BamH, puc182 ( ) No 2 6B11V L 2V H Noc BamH ( ) ; T4 DNA Sma, BL21 (DE3), Sma, bp Sac IPTG 37 6B11 ScFv, EcoR, pet306b11v H V L hc, bp, (Fig 2) pet30a6b11 V L V H hc SDS2PAGE, Bio2Rad 37 4 h,sds2page, COC16629 IgG, DAB mgπml PAGE [6 ], Sigma 1110 (Fig 3) ELISA : 6B11V L V H h C 6B11 ( Ab2 ) 6B11ScFv A 490 ELISA 6B11, HRP2COC min, 96,37 1 h, 490 nm,, HRP2COC16629 : = (12A 490 ΠA 490 ) 100 % PCR PCR 1 % (Fig 1) 750 bp 1000 bp, 6B11 Fig 1 1 % Agarose gel eletrophoresis of PCR poducts 1,2,3,4,5,6 :plasmids have target fragment ; M:DNA molecular weight markers pet30a pet30a ( + ) 6B11V L V H hc, 50 kd, 3 molπl,8 molπl, 90 %

4 : 493 COC16629 (Fig 4) 2 5 PEG2 1 mgπml, PAGE 100 kd, (Fig 5) Fig 2 1 % Agarose gel eletrophoresis of cloned vector digested with EcoR, Nco, BamH, Sac, Sma, respectively 1 :Molecular weight markers

plasmid digested with BamH ΠEcoR (387) ; 7 :Cloned plasmid digested with Sma (1 358 bp) ; 8 :Cloned plasmid digested with EcoR ΠSac (1 136 bp) ; 9 :Molecular weight markers (<X174 DNAΠHae ) ; Fig 4

systems showed molecular weight M Protein marker 1,2,3 Renatured fusion protein (1 mgπml) Fig 3 SDS2PAGE analysis of expression and purity of 6B11V L V H hc fusion proteins 1 Renatured fusion protein

4 4 : 493 COC16629 (Fig 4) 2 5 PEG2 1 mgπml, PAGE 100 kd, (Fig 5) Fig 2 1 % Agarose gel eletrophoresis of cloned vector digested with EcoR, Nco, BamH, Sac, Sma, respectively 1 :Molecular weight markers ( DNAΠEcoR + Hind ) ; 2 :Cloned plasmid ; 3 :Cloned plasmid digested with EcoR ; 4 :Cloned plasmid digested with Nco ΠEcoR (1 167) ; 5 :Cloned plasmid digested with Nco ΠBamH (780) ; 6 :Cloned plasmid digested with BamH ΠEcoR (387) ; 7 :Cloned plasmid digested with Sma (1 358 bp) ; 8 :Cloned plasmid digested with EcoR ΠSac (1 136 bp) ; 9 :Molecular weight markers (<X174 DNAΠHae ) ; Fig 4 Western blot analysis of pet306b11v L V H hc production M Protein marker 1 Detected with COC Detected with goat2anti2human IgG monoclone antibody Fig 5 Electrophoresis in non2denaturing systems showed molecular weight M Protein marker 1,2,3 Renatured fusion protein (1 mgπml) Fig 3 SDS2PAGE analysis of expression and purity of 6B11V L V H hc fusion proteins 1 Renatured fusion protein ; 2 Inclusion body ; 3 Negative control ; 4,5 pet306b11v L V H hc, 37, 4 hours after induced with 014 mmol L - 1 IPTG; 6 Sonicated supernatant ; 7 pet30a ( + )ΠBL21 (DE3),37, 4 hours after induced with 0 4 mmoll - 1 IPTG; 8 pet30a ( + )ΠBL21 (DE3) ; M Protein marker 2 6, 6B11 COC16629, OC16629 (Ag) 6B11 (Ab2) 6B11ScFv,6B11V L V H hc ( Fig 6) 6B11V L V H hc (OC16629) (COC16629), (Fig 7) 6616 % 6B11V L 2V H 6B , Pessi [7 ] (minibody) 61,

494 18 Fig 6 Ab2 antibody activity assay of 6B11V L V H hc fusion protein by direct ELISA 1 Negative control ; 2 OC16629 (Ag) ; 3 6B11(Ab2) ; 4 6B11ScFv ; 5 6B11V L V H hc 100 KD 2Π3,, [9 ],,,,,, (

5 Fig 6 Ab2 antibody activity assay of 6B11V L V H hc fusion protein by direct ELISA 1 Negative control ; 2 OC16629 (Ag) ; 3 6B11(Ab2) ; 4 6B11ScFv ; 5 6B11V L V H hc 100 KD 2Π3,, [9 ],,,,,, ( References) Fig 7 Inhibition of binding of COC16629 to ovarian serous adeno2 carcinoma antigen by 6B11V L V H hc 1996 Hu Shi2 zhen [8 ] CEA (V L 2V H 2CH3), Ig F(ab ) 2 ScFv diabody, di2 abody, Ig Gynecol),2002,3(1) :44 47,, 6B11ScFv (hinge) IgG1 CH3 (V H 2V L 2CH3), (anti2id minibody) (6B11V H V L hc) [5 ], 6B11ScFv, hinge2ch3, 6B11V H V L hc, DNA 6B11V L V H hc, 6B11 6B11 IgG1 CH3, (dimer), 1 Daly M,Obrams G I Epidemiology and risk assessment for ovarian can2 cer Semin Oncol,1998,25 : Schott M E, Milenic D E, Yokota T, Whitlow M, Wood J F, Fordyce WA, Cheng R C, Schlom J Differential metabolic patterns of iodinated versus radiometal chelated anticarcinoma single2chain Fv molecules Cancer Res, 1992,52 : ,,,,,,,,, 6B11 ( Zhang Xiang2yun, Qian He2 nian, Qiu Bin2sheng, Cui Heng, Feng Jie, Huang Hua2liang,Fu Tian2 yun, Li Wen2jin, Wang Jian2liu, Zhou Ping, Li Xiao2ping Cloning and sequence analyzing of 6B11 anti2idiotypic antibody ScFv genes J Beijing Med Univ),1996,28 :5 8 4 Qian H N, Lu W Y Anti2idiotypic monoclonal antibodies against anti2 ovarian monoclonal antibody COC16629 : generatioon and application Chin Med J, 1994,107 : ,,,, (Cui Heng,Chang Xiao2hong, Feng Jie,Liu Bei,Cao Shan2jin The constnuction and expression of hu2 manized ovarian carcinoma anti2idiotype antibody Chin J Clin Obstetr 6, : ( Wang Jia2zheng, Fan Ming, Protein Techniques Handbook Beijing : Science Press),2000 : Pessi A, Bianchi E, Crameri A, Venturini S, Tramontano A, Sollazzo M A designed metal2binding protein with a novel fold Nature, 1993, 326 : Hu S, Shively L, Raubitschek A, Sherman M, Williams L E, Wong J Y, Shively J E, Wu A M Minibody : A novel engineered anti2 carcino2 embryonic antigen antibody fragnent ( single2chain Fv2CH3) which ex2 hibits rapid, high level targeting of xenografts Cancer Res, 1996, 56 : Yazaki P J, Wu A M, Tsai S W, Williams L E, Ikler D N, Wong J Y, Shively J E, Raubitschek A A Tumor targeting of radiometal labeled anti2cea recombinant T84 66 diabody and t84166minibody : compari2 son to radioiodinated fragments Bioconjug Chem,2001, 12 (2) :

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