Construction And Expression Of Covalently Linked Bivalent Single-chain Antibodies Against Human Colorectal Carcinoma

Colorectal carcinoma is one of the common malignant tumors which have the higher incidence, which occupies the third place in our country and the death rate of that are increasing continuously in recent years. Therefore, efficient diagnosis and therapeutic approaches are important for colorectal carcinoma research. Although in recent years some progress has been made in respect to application of monoclonal antibodies for the therapy and diagnosis of colorectal carcinoma , most mAbs are of murine origin, they usually have antigenic activity to human , and their large molecules also lead to poor diffusion and long persistence in circulation, which severely limits the efficacy of clinical utilization. Genetic engineered antibody fragments like Fab and scFv which have small molecular weight ( Mr 25 ~30kDa) can improve diffusion and circulation clearance , while they are low avidity because of their monovalent, and their small sizes lead to too fast clearance in circulation. Recently bivalent antibody fragments which are constructed using monovalent antibody fragment like scFv and Fab display optimized effects in tumor imaging and therapy. Bivalent can promise higher avidity than monovalent, and the medial molecular size can guarantee the diffusion and moderate clearance in circulation.ND - 1 is a murine monoclonal antibody against tumor - associated antigen LEA, which mainly expressed in human colorectal carcinoma, It was developed by Professor Song Jindan in 1983 by immunizing Balb/c mice with CCL — 187 human colorectal carcinoma cell line. The histological determination of nearly thousand pathologic samples showed that ND - 1 can bind specially to well dif-ferentiated and moderately differentiated colorectal carcinoma tissues and its specificity is superior to IgG against CEA which has been commercialized in the US. Single - chain Fv of ND - 1 (ND - 1 scFv) against human colorectal carcinoma was constructed by fusing gene of variable fragment of heavy chain and light chain and expressed in E. coli. It showed that ND - lscFv displayed fast imaging and clearance , but for the other part, monovalent and so small size lead to low avidity and low accumulation in tumor tissues, thus its function is limited in tumor therapy.Therefore , we constructed the bivalent ND - 1 single - chain antibody [ ND -lsc(Fv)2 -5 and ND - lsc(Fv)2 - 15]by genetic recombination of the gene of ND -lscFv. It was contructed by different linker [G4S and (G4S)3] cova-lently linked the two scFv antibody fragments. The binding sites and molecular weight have been doubled at the same time , so as to gain high activity and overcome shortage of too fast clearance rate of scFv. The construction of ND - lsc (Fv)2can provide evidence for the next nude mice bearing experiments in vivo and clinical diagnosis and therapy of colorectal carcinoma.Materials and MethodsFrom the expression vector pET -28a( + )ND - lscFv, the gene of ND -1 scFv was amplified and added a linker sequence ( encoding G4 S) to the upstream of the gene of ND -1 scFv and restrict sites Sal I and Hind HI by primer PI and P3. The PCR product was ligated into clone vector of pMD18 - T to form pMD18 -T - scFv —5. Positive clone containing pMD18 - T - scFv —5 was i-dentified by DNA sequencing. Using pMD18 - T - scFv - 5 as the template DNA, the gene of scFv - 5 was amplified and added a linker sequence ( encoding ( G4S)2)to the upstream of the gene of scFv -5 and restrict sites Sal I and Hind M by primer P2 and P3. The PCR product was ligated into clone vector of pMD18 -T to form pMD18 -T -scFv -15. Positive clone containing pMD18 -T - scFv - 15 was identified by DNA sequencing. pMD18 - T - scFv - 5 and pMD18 -T - scFv - 15 were double digested respectively with Sail and Hind HI, the product of scFv - linker was recovered and linked into pET -28a( + ) ND -lscFv which is prepared by double digestion with Sail and HindlH. The novel vector pET -28a( + ) ND - lsc( Fv)2 was transformed into E. coli BL21. Condition for optimal expression was adjusted, then ND - 1 sc ( Fv) 2 protein was expressed in the optimal condition. Target protein was purified by Ni - NTA resin affinity chromatography, and then it was refolded. Its molecular weight and purity were analyzed by SDS - PAGE. Immunoflourescence Assay was conducted to determinate the specificity of ND - lsc( Fv)2to CCL - 187 by comparing to Hela cell line. The immunity among ND - lmAb, ND - lsc( Fv)2and ND - lscFv was detected by ELISA.ResultsUsing the expression vector pET -28a( + )ND - lscFv as template DNA, PCR produced an about 750bp fragment by using primer PI and P3 which was compatible to designed sequence of scfv - 5 and proofed by the DNA sequencing. Using pMD18 -T - scFv -5 as template DNA, PCR produced an about 750bp fragment by using primer P2 and P3 which was compatible to designed sequence of scfv - 15 and proofed by the DNA sequencing. The 750 bp fragment was recovered by double digestion ,and which was ligated into pET -28a( + ) ND - lscFv to form pET -28a( + ) ND - lsc( Fv)2. After transformed into E. coli BL21, it was induced by lmM IPTG at 37X1 for 3 hours. Inclusion body was harvested and purified by Ni — NTA resin affinity chromatography. SDS — PAGE showed that the purity of purified ND - 1 sc ( Fv ) 2 was about 90% or 86% , its molecular weight is about 55kDa , according to its predicted Mr value. Immunoflourescence Assay showed that ND - 1 sc ( Fv) 2 had specific binding activity to CCL-187 cell line. ELISA revealed that ND - lsc(Fv)2 had significantly higher activity than ND - lscFv( P <0. 01) ,and the activity of ND - lsc (Fv)2 -15 was closer to ND -1 than ND-lsc(Fv)2 -5.ConclusionWe had constructed successfully ND -1 sc ( Fv) 2 gene against human color-ectal carcinoma [ ND -lsc(Fv)2 -5 and ND -lsc(Fv)2-15] . Their activity were both obviously higher than ND -1 scFv and had reached desired result, ND - lsc(Fv)2 - 15 was closer to ND -1 than ND -lsc(Fv)2-5 . They may become potentially useful in clinical diagnosis and therapy as a carrier for human colorectal carcinoma.