Construction Of Eukaryotic Vectors For The Expression Of Full-length Humanized Antibody And Surface Reshaping Antibody RCAb1

In this experiment, a series of eukaryotic expression vectors were constructed andscreened for the expression of full-length humanized antibody using the well-establishedantigen-antibodysystemHAb18G/CD147-HAb18;Then, thevariableregiongenesVHandVL of mAb CAb1 against human colorectal cancer were cloned, and their reliability andaccuracy were validated through reconstitution of a human-mouse chimeric Fab of CAb-1;Furthermore, the mutated variable regions of the surface reshaping antibody rCAb1 weredesigned and obtained; Finally, the recombinant rCAb1 were expressed and purified inmammaliancellsbytheconstructedeukaryoticexpressionvector.Experimentalstudyonthecontentsabovecanbedividedintothreeparts:Part I: Constructing and screening high efficient eukaryotic vectors with our ownintellectualpropertyfortheexpressionoffull-lengthhumanantibodyObjective:ToobtaineukaryoticexpressionvectorsforantibodyexpressionMethods: According to standard recombinant DNA operations, the followingeukaryotic vectors were constructed: 1Weakened screening system using"crippled"DHFR(pIRES1-18L; pIRES2-18H); 2 Site integration system (pDHL-18FRT); 3 Binary vectorsystem (18H-pDHA , 18L-pCI); 4 Reversed binary vector system (18H-PCI, 18L-p105); 5Multicistron expression vector (18H-L-pCI-FRT); 6 Weakened multicistron expressionvector(18H-L-pCII);and 7TraditionalpAH/pAGsystem(pAH4604-18, pAG4622-18).Allthe vectors contain the variable region genes of mAb HAb18 and the constant regions ofhuman IgG1γ1 andκchain. After transfected them into COS-7 cells respectively, wedetected the chimeric antibody cHAb18 in the culture supernatant by dot blot analysis. Atthe same time, we measured the expression levels of each system by sandwich ELISA.Subsequently, some of the constructs were transfected into different phenotypes of CHOcellsbyelectroporation andpositive cloneswere selected with limited dilution method.The antibody genes in continuous daughter cells were assessed by RT-PCR. For the scale-upculture, the products in the supernatant were purified. SDS-PAGE and Western blot wereemployed for the detection of antibody expression; And finally immunofluorescencestaining and flow cytometry were also used for the specificity analysis of the expressionproducts.Results: All the vectors containing antibody genes were constructed. PCR or enzymerestriction analysis both showed the corresponding segments as expected size, whichsuggested that all the constructs were successfully obtained. After transfected into COS-7cells, dot-blot hybridization showed that almost all transfectants could detect the expressedhuman IgG compared with those of non-transfectants, except for vectors 18 H-L-pCII anddual-vector pAH/pAG. However, when using the sandwich ELISA coated with theextracellular domain of the HAb18G/CD147, the results showed that all transfectants coulddetect the expressed chimeric antibody cHAb18, and the expression level reached to ahigher level at 48 h and the maximum expression level at 120 h after transfection. Theproductivity of transfectants with plasmids pIRES1-18L/pIRES2-18H was about 1.4milligram per liter. Further, we used pIRES1-18L/pIRES2-18H,pDHL-18FRT and18H-L-pCII to transfect different phenotypes of CHO cells. The Sandwich ELISA showedthat all transfectants could detect the expressed chimeric antibody cHAb18 compared withthose of non-transfectants. Among all the transfectants with plasmidspIRES1-18L/pIRES2-18H, the productivity varies from 0.3 to 16 milligram per liter; andtransfectantswith plasmids pDHL-18FRTwith pOG44 could reachto 10 milligramperliter;However,thetransfectantswithplasmid18H-L-pCIIwasratherlowerevenunderscreeningpressure and productivity was less than 1.5 milligram per liter. RT-PCR could detectantibody gene expression to the stable transfectants 1F6 with 30 continuous passages. Inlarge scale culture, we obtained 7.5 mg of expression products from about 500 ml culturesupernatant through affinity chromatographycolumn. SDS-PAGE andWestern blot showedthe purified products were approximately 150 kDa under nonreducing condition, and itbecame two newbandsaftertreatmentwith reduction reagent, with the molecularweightof50 kDa and 25 kDa, respectively. And the interested protein could bind with goatanti-human IgG, this indicated that the molecular was human IgG. Immunofluorescence showedthepurifiedIgGcouldbindwithcellsbearingHAb18G/CD147specifically.Conclusion: Through designing and reconstruction, a series of eukaryotic expressionvector for full-length human antibody expression were constructed. Among these vectors,weakened screening system (pIRES1-18L; pIRES2-18H) and site integration system(pDHL-18FRT) showed a high efficiency expression. And they could be further used forother antibody expression. What's more, a high expression levels and stable cell linesexpressed cHAb18 was obtained, and the expression products maintained good specificityand affinity with that of parental antibody. Finally, recombinant IgG product cHAb18 waspurified, and this further corroborated the correctness of the constructed vectors and theirfitnessfortheexpressionoffull-lengthhumanIgG.Part II: Cloning and identification of the light and heavy chain gene of mAb CAb1againsthumancolorectalcancerObjective: To obtain the variable region genes and mouse-human chimeric Fd orchimeric light chain genes of mAb CAb1, then prepare the corresponding small moleculeantibodycFabMethods: First ofall, we extracted totalRNAfromhybridoma cellCAb-1. The Fd andlight chain genes were amplified by RT-PCR using the synthesized primers. The amplifiedproducts were inserted into T vector to obtain pMD18-T/Fd and pMD18-T/L for analysisandsequencing.ThechimericcFdorchimericlightchainwasobtainedbyPCRfromvectorpComb3C/cFab, in which the amplified VH and VL from pMD18-T/Fd and pMD18-T/Lwith the corresponding primers B4, B4 for and A8, A8, were ligated with human CH1 andCL in pComb3C, respectively. cFd and cL of CAb-1 were then amplified by usingpComb3C/cFd-cLastemplateand joined with pET32a(+)through Nde I/Sal I(cFd)orNdeI/Xho I (cL) digestion, respectively. The resultant constructs pET-CAbH and pET-CAbLwere transformed into E.coli. BL21-DE3 and checked for the presence of the genes bycolony PCR and restriction digestion. Then cFd and cL were expressed and purified,respectively. After cFd and cL proteins were mixed at the equal molar concentration, weused a modified stepwise dialysis in vitro refolding system to obtain cFab. SDS-PAGE andWestern blotwere employed for the detection of the refolding products; After purified withProtein G column, we further used indirect ELISA, immunofluorescence staining and flow cytometry to detect the binding activity of the products. Finally, competition ELISA wasusedtodeterminewhethertheobtainedcFabcouldcompetewiththemurineCAb1.Results: About 62.5μg total RNA was obtained from 1×107 hybridoma cells, andclearly 5S, 18S and 28S of bands can be observed by RNAelectrophoresis. The amplifiedVH and VLgenes of CAb-1 have 351 bp (117 amino acids) and 336 bp (112 amino acids),respectively.AndtheCH1ofCAb-1 belongs to mouse IgG1, and the CL belongs toκchain. By optimizing their free energy of mRNAsecondary structures, two non-fusion expressionvector pET-CAbH and pET-CAbL were successfully constructed. The cFd and cL can beexpressed efficiently in E.coli with expression 29.2% and 23.6% of total bacteria proteinsafter6 h inductionat30°C, respectively. Byreconstitution in vitro, SDS-PAGEshowed thatcFd and cLwere refolded into cFab with 70.2%ofprotein recoveryrate at100μg/ml initialtotal proteins. Immunofluorescence and FACS results showed that the refolded cFab couldbind with colon cancer cells SW480 and Hce-8693, but not normal cells. Finally,competitive ELISAshowed the purified cFab can compete with murine CAb-1 F(ab')2, andviceversa.Conclusions: The variable region genes of CAb1 were cloned successfully usingdesigned primers of mouse IgG1 antibody. The cFd and cL of CAb1 were expressed andprepared through optimization of the free energy of the mRNA secondary structure. Weobtained functional refolded cFab successfully in vitro, and the products could bind withcolon cancer cells specifically. Refolded cFab could compete with murine CAb-1, whichindicated both of them identifying the same epitope; Finally, The functional cFab alsoindicatedthatthevariableregiongenesofCAb-1arecorrectandreliable,andthestrategyisalsoeffectiveincheckingtheobtainedvariablegenes.Part III: The expression and purification of the resurfacing antibody rCAb1 againsthumancolorectalcancerObjective:To obtain resurfacing antibodyrCAb1againsthuman colorectalcancerwiththe screened vectors, and then preliminary verify the binding activity of the expressedantibodyMethods: First, the variable region genes of CAb1 were analyzed and blastP withsequences in non-redundant human immunoglobulin VL and VH sequences of Genbank, local antibody structure databases was established. All the chosen sequences were dividedinto four categories, which were selected from two species (Mus musculus and Homosapiens)sinceeachcontaininglightandheavychaintypes.Weselected200sequencesformeach category for analysis patterns of surface exposed residues that most closely matchedthe patterns found on VL and VH of CAb1 according to the scores of similarity. After wegot the differential and abnormal residues of the variable region sequences, thethree-dimensional of Fv structure of CAb1 was modeled and the intermolecular hydrogenbonding was calculated. According to the results, the candidate sites for mutation weredetermined. After that, the variable region sequences were obtained by overlapping PCR.Then, theywere inserted into Tvector respectively to get cloning vector pMD18-T/VH andpMD18-T/VL for sequencing. After that, the VH and VL were cloned into the weakenedscreeningsystemtogetexpressionvectorpIRES1-CAbLandpIRES2-CAbH.Afteranalysisby PCR and restriction enzyme digestion, they were transfected into COS-7 cells; theculture supernatantwasdetected bysandwich ELISA. Forthe scale-up culture,the productsin the supernatant were purified. SDS-PAGE and Western blot were employed for thedetection of antibody expression; And also, immunofluorescence staining and flowcytometry were used for the specificity analysis of the expression products to colon cancercells. Finally,competitionELISAwasemployedforthebindingactivitywithmurine CAb1,and the relative affinity was calculated in accordance with the antibody concentration ratiowheninhibitionratewas50%.Results: The different CDR and SDR of CAb1 sequences was marked according toKabat, Abm, Chothia and Contact rules, after alignment and screened antibody sequences,the surface exposed positions in a set of heavy and light chain variable region frameworkwere determined and the mostcloselyidentical to the set ofMusmusculusorhomo sapienssurface residues wasalso labeled.We finallydetermined the initial candidate mutation siteswere T018S, N086S and Q018P according to the intermolecular hydrogen bondinginteractions and amino acids surface accessibility (A residue was defined as beingaccessible when its relative accessibility was greater than 30%). Using overlapping PCR,the mutated variable regions of VH and VLwere obtained and cloned into pMD18-T. AftertheconstructiontheexpressionvectorpIRES1-CAbLandpIRES2-CAbH,SandwichELISA results showed the transfectants with the two plasmids expressed human IgG. By selectingwith MTX, the expression product was finally purified. SDS-PAGE and Western blotshowed that the protein molecular weight of about 150 kDa, after reduction for the twobands, the molecular weight of about 52 kDa and 27 kDa. The products could bind withcolon cancer cells specifically. Competitive ELISA showed the purified rCAb1 couldcompete with murine CAb1, the relative affinity was about 55% of murine parentalantibody.Conclusion: The candidate mutation sites of the CAb1 for surface remodeling wereobtained by analysis the variable region sequences of CAb1 against human colorectalcancer; the mutation variable region genes of CAb1 were obtained successfully usingover-lapping PCR. Dual-vector pIRES1-CAbL and pIRES2-CAbH were constructed, andthey couldbe used for the expression of resurfacing antibody rCAb1. The prepared rCAb1could bind with colon cancer cells specifically. Compared with that of the parental murineantibody CAb1, the expressed rCAb1 maintains desirable binding activity and specifity tocoloncancercells.