Phage antibody library technology in tumor therapy: a review / 生物工程学报

Tumor is a serious threat to human health. At present, surgical resection, chemoradiotherapy, targeted therapy and immunotherapy are the main therapeutic strategies. Monoclonal antibody has gradually become an indispensable drug type in the clinical treatment of cancer due to its high efficiency and low toxicity. Phage antibody library technology (PALT) is a novel monoclonal antibody preparation technique. The recombinant immunoglobulin variable region of heavy chain (VH)/variable region of light chain (VL) gene is integrated into the phage vector, and the antibody is expressed on the phage surface in the form of fusion protein to obtain a diverse antibody library. Through the process of adsorption-elution-amplification, the antibody library can be screened to obtain the antibody molecule with specific binding antigen as well as its gene sequence. PALT has the advantages of short antibody production cycle, strong plasticity of antibody structure, large antibody yield, high diversity and direct production of humanized antibodies. It has been used in screening tumor markers and preparation of antibody drugs for breast cancer, gastric cancer, lung cancer and liver cancer. This article reviews the recent progress and the application of PALT in tumor therapy.

Selection and identification of human monoantibody TRAb Fab fragment / 天津医药

Objective To select and express a human thyrotrophin receptor antibody (TRAb) Fab fragment from phage antibody library constructed with phage display technology. Methods With immobilized antigen, the reconstructed humanized TRAb Fab library was enriched by five rounds panning (adsorption-elution-amplification). The TSAb Fab and TBAb Fab fragment were selected by coated fusion proteins of hTSHRn and hTSHRc. The positive clones were identified and selected by Phage-ELISA. TRAb positive clones were identified by PCR and double restriction enzyme digestion. The soluble TRAb (TSAb, TBAb) Fab fragments were expressed. TRAb (TSAb, TBAb) Fab fragments were identified by Western blotting assay. The DNA fragment was sequenced from the positive clones. Results Following five rounds of biopanning, TRAb (TSAb,TBAb) Fab phage antibody was screened. The enrichment effect reached to 77 times and 94 times. The soluble TRAb (TSAb,TBAb) Fab antibodies were expressed from positive clones and identified by phage ELISA. Western blotting analysis showed that the phage displaying Fab had significant binding activity with antigens. These sequence analysis showed that all of the heavy chain Fd gene and light chain gene were derived from human immunoglobulin variable region. The light chain variable region of the monoclonal 48 was homologous to the immunoglobulin light chain Vλ homology of 94.4%, and the heavy chain variable region of the monoclonal 48 was homologous to the immunoglobulin heavy Fd chain VH4 homology of 88.9%. The light chain variable region of the monoclonal 56 was homologous to the immunoglobulin light chain Vλ homology of 95.6%, and the heavy chain variable region of the monoclonal 56 was homologous to the immunoglobulin heavy Fd chain VH3 homology of 84.6%. Conclusion We have successfully selected TRAb (TSAb, TBAb) Fab fragment from a human phage display immune antibody library.

Screening and primary identification of anti-CCR7 scFv from large phage library / 中国药理学通报

Aim To screen and identify anti-CCR7 single chain fragments variable(scFv)from lager phage library and to detect the scFv efficiency.Methods The insert ratio of ScFv antibodies library was identified by PCR.The products digested by Sfi I/Not I double enzyme.Panning against breast cancer cell and CCR7 were performed four and three rounds respectively.Positive clones were transformed to E.coli HB2151, and their dissolvability was assayed.The soluble scFv was purified by affinity chromatography, and its relative molecular mass was determined by Western blot.The ELISA assay was used to identify the immunocompetence of the antibody.Immunocytochemical staining and radioimmunoimaging were employed to determine the affinities of scFv binding with CCR7 in cell line and in nude mice.Results The insert ratio of ScFv gene was 90%(18/20), enzyme digest reaction showed the aim products on 1% agarose gel.ScFvs were obviously enriched after 7 round panning.Western blot result showed soluble scFv's molecular mass was about 34 KD.ELISA analysis showed dissolved antibody had high immunocompetence to MDA-MB-435 s cells.Immunocytochemical staining and radioimmunoimaging indicated that ScFvs were bound efficiently to MDA-MB-435 s cells which expressed CCR7.Conclusions ScFvs against CCR7 are successfully acquired by screening the phage antibody library.The soluble ScFvs have high affinity and specifical binding to human breast cancer cells.

Construction of human naive phage antibody library and primary screening of the gab antibodies against gp96 / 中国综合临床

Objective To construct a naive human Fab fragment phage display library,from which the anti-gp96 antibodies may be panned by the gp96 purified from the tissue of urothelial carcinoma in the urinary bladder and provide a basis to new therapy for the malignant tumors.Methods Peripheral blood lymphocytes were isolated from 800 ml of blood,which was obtained from four healthy blood donors.The heavy chain Fd and light chain cDNA synthesized from the total RNA of lympbocytes were amplified by PCR with variable regions 5' and 3' primers of heavy and light chain, and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E.coli XL1-Blue by electroperation.The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody Fabs.The phagemids abstracted from amplified E.coli were cut with endonucleases such as Sac Ⅰ,Xba Ⅰ,Xho Ⅰ and Spe Ⅰ, and both the phage antibody Fabs and phage-raids abstracted from amplified E.coil were amplified by PCR to monitor the insertion of the genes of light chain or heavy chain Fd fragment.The gp96 purified from the urothelial carcinoma tissue of the bladder by affinity chromatog-raphy on eoncanavalin-A sephnrose and DEAE-sephnrose ion exchange chromatography were utilized as antigens to process three rounds of panning to the original Fab antibody library.Results The quantity of total RNA and cDNA were qualified.By combination of light chain and heavy chain genes, an antibody library containing 6.6×106 clones was obtained, and both the cutting of enzymes and PCR showed that there were the genes of light chain or heavy chain Fd fragment in the phagemids.The gp96 protein was obtained from urothelial carcinoma tissue in the urinary bladder.After having been panned by gp96, the original antibody library gained enrichment by 68 times.Conclusion Utilizing the technology of phage surface display, specific antibody can be gained from the human naive Fab phage display library,which can be used for immunological therapy for tumors.

Selection and preliminary identification of human Fab fragement antibody against amylin from phage antibody library / 中国免疫学杂志

Objective:To obtain antibodies against amylin from a 'naive' human Fab fragment antibody phage diasplay library and to analyze the specificity of antigen binding activity.Methods:Panning and screening Fab antibody from the antibody library,the positive clones with well reactivity to amylin were selected after five times selection of 'adsorption-elution-enrichment'.Then the plasmid DNA which was extracted from the clones,was digested with Spe Ⅰ and Nhe Ⅰ to delete gⅢ (about 660 bp).The digested 47 000 bp DNA which was purified after separation of bands from agarose gel was ligated with T4-DNA ligase.The constructed expressing phagemids were transformed to the BL21(DE3)pLysS,soluble Fab was expressed in it by the induction of IPTG and its characteristics and specificity were determined by ELISA and Western blot.Results:Soluble Fab antibodies were expressed in E.coli.According with molecular weight of IgG Fab,protein band of about 47 kD was shown by SDS-PAGE.Western blot using the goat anti human IgG-HRP showed their binding activities.ELISA showed their specificity with amylin antigens and they did not react with bovine serum albumin.Conclusion:The high level expression and identification of the soluble human anti- amylin Fab fragment antibodies has been obtained successfully,which lays a solid foundation for further researching about the biological and pathological activities of amylin.

Construction of gene library of human-originated single-chain variable fragment antibody associated with lung adenocarcinoma / 重庆医科大学学报

Objective:To construct human single-chain variable fragment (scFv) antibodies gene library associated with lung adenocarcinoma. Methods:Total RNA was extracted from the lymphatic tissue near the lung adenocarcinoma.VH and VL fragments were amplified with RT-PCR,the purified VH and VL fragments were used to produce scFv fragment by splicing-overlap-extension PCR. The scFv gene was cloned in pCANTAB-5E Phasmid,then transferred to Ecoli TG1.The insertion ratio of antibody gene was identified with RT-PCR,the positive enzyme-cutting products were identified with 1.5% agarose gel electrophoresis. Results:Clear 28 S and 18 S strips were found in total RNA;The size of VH is about 370 bp,VL is about 350 bp.The size of scFv is about 750 bp.After transformation into Ecoli TG1,2.2?107 CFU/?g ampicillin resistant bacteria colonies grow after overnight culture,and the positive insert ratio was 83.3%. Conclusion:Human single-chain variable fragment(scFv) antibodies gene associated with lung adenocarcinoma was constructed successfully. It could provide bases to further screen the antibody library construction.

Preparation and Identification of Anti-lactoferrin Single Chain Fv Antibody / 医学研究杂志

Objective To prepare a specific anti-lactoferrin single chain variable fragment(ScFv).Methods Anti-lactoferrin clones were screened from a 'naive' phage antibody library against the immobilized lactoferrin antigen,then the clones were transformed to the E.coli HB2151 to give soluble expression of antibody fragments.The culture supernatant containing ScFv was purified by immobilized metal affinity chromatography,and then determined with SDS-PAGE and ELISA.Results The results demonstrated that ScFvs were specific;they did not react with transferrin,lysozyme and bovine serum albumin in ELISA.The SDS-PAGE showed that the ScFvs had high purity through affinity chromatography and the molecular weight of them was about 32 kD.Conclusions The successful generation of the ScFvs against lactoferrin provides a basis for further study and clinical applications for dry eye and other ocular diseases.

Screening and identification of human anti-HIF-1? scFv of lung adenocarcinoma / 第三军医大学学报

Objective To screen human anti-hypoxia-inducible factor (HIF)-1? scFv of lung adenocarcinoma from large phage antibody library and identify the positive clones. Methods Panning of large phage antibody library against lung adenocarcinoma cell line A549 and HIF-1? was conducted respectively to select specific antibodies. E. coli HB2151 was infected to induce the expression of soluble scFv. The binding activity and specificity were tested by ELISA and immunocytochemical assay. The expression and relative molecular weight of the soluble scFv was detected by SDS-PAGE and Western blot analysis. Results After panning,the target scFv was enriched,and ELISA results showed that positive reactions to HIF-1? were detected in 5 of 10 random clones with a positive ratio of 50%. Immunocytochemical analysis showed the specific affinity of the antibodies to A549 cells. The soluble human anti-HIF-1? scFv fragments of lung adenocarcinoma were expressed in E. coli HB2151 and then confirmed by SDS-PAGE. The result of Western blotting showed that the relative molecular weight of the soluble scFv was about 30?103. The binding activity and specificity were confirmed by ELISA. Conclusion Human anti-HIF-1? scFv of lung adenocarcinoma is successfully obtained with large phage antibody library technique.

Application of two bacteria colony-PCR methods in the screening of phage antibody library / 医学研究生学报

Objective:To value the application of two bacteria colony-PCR methods in the screening of phage antibody library. Methods:Five positive monoclonal bacterium were respectively suspended in either deionized water or 0.1% Triton X-100, and then boiled to be used as template in PCR. . The DNAs products of PCR were extracted and digested by two enzymes, and then determined by electrophoresis. Results:The inserted genes were detected in all the 5 clones after PCR and enzyme digestion .Conclusion:Bacteria colony-PCR can be used in screening positive recombinant colonies. The bacteria colony-PCR method with bacteria colonies suspended in deionized water is valuable in large scale positive recombinant bacterium screening.

Construction of and screening for phage antibody library of human lung adenocarcinoma / 第三军医大学学报

Objective To construct human single-chain antibody gene library associated with lung adenocarcinoma, and screen lung adenocarcinoma cell A549-specific antibody. Methods The lymphatic tissue near lung adenocarcinoma was used to construct human single-chain antibody gene library. The specific antibody to lung adenocarcinoma cell A549 was screened from the antibody library. Positive clone bacteria were transformed to E. coli HB2151, and their dissolvability was observed. The soluble scFv was purified by affinity chromatography and its relative molecular mass was determined by SDS-PAGE and Western blotting. The specificity of scFv to human lung adenocarcinoma cells was identified with ELISA. Results The phage antibody library of 4.6?107 was constructed successfully. Single-chain antibody of human lung adenocarcinoma was enriched. The ratio of yield was increased gradually, 181 times higher after 5 rounds of panning than after the first round of panning. SDS-PAGE and Western blotting results showed soluble scFv' molecular mass was about 30 000. ELISA showed dissolved antibody had high specificity to bind A549 cells, not MDA-MB-435. Conclusion The single-chain antibody gene library of human lung adenocarcinoma was constructed successfully, and specific antibodies to lung adenocarcinoma were screened, providing the basis for single-chain antibody radionuclide imaging and treatment.

Screening and identification of phage antibodies against human lipopolysaccharide binding protein / 第三军医大学学报

Objective To obtain and identify monoclonal antibody against human lipopolysaccharide binding protein (LBP). Methods Three clones of antibody with better activity against human LBP were isolated after human antibody library screening by human LBP and 5 rounds of enrichment. After plasmid extracting, geneⅢ cutting, self-recircling and electric transforming, soluble Fab was expressed in E.coli. XL1-blue by the induction of IPTG and its characteristics were determined. Results The positive antibody was concentrated to 8.16?104 folds and the highest activity of the three clones was 106 . Conclusion Phage antibody against human LBP has been obtained successfully, which lays a solid foundation for researching its function.

Screening of human anti-ricin ScFv from large phage library / 中国免疫学杂志

Objective:To clone human anti-ricin antibodies from large phage antibody library.Methods:Panning for a large phage library against ricin toxin was conducted to select specific antibodies against ricin. The binding activities and specificities were tested by ELISA method. Soluble ScFvs were prepared through infecting E coli. HB2151 with the selected phage antibodies and induction with IPTG. Results:Forty positive clones were obtained after 5 rounds of panning, and 12 clones had specific binding ability to ricin toxin. DNA fingerprinting showed 7 different band patterns indicating 7 different positive clones. DNA sequencing showed that variable regions of these ScFvs belonged to different subgroups.Conclusion:Human anti-ricin antibodies were successfully obtained from large phage antibody library.

Construction and primary identification of a naive Fab fragment phage library / 中国免疫学杂志

Objective:To construct a human Fab fragment phage display library and provide a platform for human antibody preparation.Methods:Peripheral blood lymphocytes were collected from healthy donor.The heavy chain Fd fragment and light chain of human immunoglobulin's genes were amplified by RT-PCR,and then cloned into phagemid pComb3XSS to generate human phage antibody library.Cutting with endonucleases such as SacⅠ,XbaⅠ,XhoⅠand SpeⅠto identify the insertion of the light chain or heavy chain Fd genes.IL-2 and digoxin as the antigen was used to scan the phage antibody library.Results:A phage antibody library of Fab had 8.4?107 members and it's recombinant rate was 70%.Through DNA sequencing of one positive clone,it was showed that its heavy chain belonged to IgG subvariety and its light chain to ? family.Conclusion:The success of constructing a nave human phage antibody library proves the useful of phage display system in human antibody preparation,and it can be used to select,purify and express of amylin Fab antibody.

Construction of human recombinant antinuclear antibody Fab library on the surface of phage / 解放军医学杂志

Objective To construct a phage antibody Fab library of human antinuclear antibody. Methods Total RNA was extracted from peripheral blood lymphocytes of 4 patients suffering from autoallergic disease, with antinuclear IgG antibody titers in the serum higher than 1∶10 000 as estimated by indirect immunofluorescence technique. Taking oligo-dT as the primers, human immunoglobulin light chains ?/? genes as well as heavy chains Fd genes were reversely transcribed to cDNA by PCR. The ?/? light chains were initially cloned into pComb3Hss vector to construct a human recombinant light chain library, and the heavy chain genes were subsequently inserted into the corresponding sites of ?/?-pComb3Hss plasmid to generate a recombinant ?/?-Fd-pComb3Hss plasmid. The plasmid was then transformed into E. coli XL1-Blue, which was subsequently infected by the helper phage M13KO7. A random recombinant antibody library was expressed on the surface of filamentous phage. Results Human immunoglobulin ?/? light chain and heavy chain Fd genes (660bp) were amplified successfully. The light chain library with the capacity size of 2?104, and the human recombinant Fab antibody library with the capacity size of 4?104 were also successfully obtained. Finally, the phage antibody library of the Fab fragment of human antinuclear antibody, containing 2?109 CFU/ml phage, was constructed. Conclusion A phage antibody library of Fab fragment of human antinuclear antibody is constructed successfully. This research lays a foundation for the preparation for phage library of Fab fragment of human antinuclear antibody, and also paves the way for the advanced assessment for its effect on the immuno-targeting therapy of tumors.

The construction and bio-panning of mouse ScFv phage-displayed antibody library against pemphigus vulgaris antigen / 中国免疫学杂志

Objective:To construct mouse ScFv against pemphigus vulgaris antigen (PVA) through phage displayed antibody technology Methods:Total RNA was isolated from the spleen cells of mice immunized with recombinant pemphigus vulgaris antigen (rPVA) after six weeks, and then reverse transcripted into cDNA Primers specific for mouse V H and V L were synthesized to amply the V H and V L by ploymerase chain reaction (PCR) V L gene was modified and amplified with linker primer (which contain 3′ region of V H, linker, and 5′ region of V L), the resultant gene called V L′ Purified V L′ and V H were subjected to SOE (splicing by overlap extension) ligation, and resultant ScFv gene was digested with SfiI and NotI, cloned to the identically digested pCantab 5E Library of TG1 transfected with recombinant phagemid were derived with diversity of 1 5?10 6 Results:The pemphigus vulgaris phage antibody library was constructed, and one positive TG1 clone was obtained, which can highly expresses phage displayed antibody Conclusion:Specific genetic antibody against PVA can be made from phage displayed antibody library

Cloning of human ScFv against digoxin from a semi-synthetic phage antibody library / 中国免疫学杂志

Objective:To clone human anti digoxin ScFv from a human semi synthetic phage antibody library.Methods:①Digoxin BSA conjugate(Dig BSA) was preparaed by a modified periodate oxidation method.②A semi synthetic phage antibody library was panned against immobilized Dig BSA.Collected clones were analysed by ELISA,inhibition ELISA and DNA Sequencing.Results:①During the four rounds panning against Dig BSA,the enrichment of the eluted phage particles was observed;②Analysis of the eluted clones identified one clone that could bind Dig as well as other digitalis.③Sequencing analysis showed that the variable genes of the positive clone belonged to VH5 and V?1 subgroup respectively.Conclusion:Human Dig specific ScFv that could bind Dig and other digitalis has been cloned from a semi synthetic phage antibody library which may provide a potential reagent for the diagnosis and therapy of Dig toxication.

Study of anti-lipid A antibodies of bacterial endotoxin from phage antibody library / 中华传染病杂志

Objective To study the treatment of sepsis caused by G - bacteria, anti-lipid A antibodies of bacterial endotoxin were screened from phage antibody library. Methods The mRNA was extracted from human B-lymphocytes against lipid A of bacterial endotoxin, reversely transcripted and amplified by polymerase chain reaction using general primers scanning Fd and light chain of IgG. The amplified fragments were inserted into pCOMB3 vector and electrotransfected competent E.coli XL 1-blue cells. Furthermore, the recombinant phage was lysed by coculture with helper VCSM13. Results Fab displayed on the surface as fusion protein with the N terminal of coat protein Ⅲ, and 4.8?10 6 clone library was established. Antibodies against lipid A of bacterial endotoxin were screened. Specific antibodies against lipid A of bacterial endotoxin were enriched by 100 times after three rounds of panning with lipid A.Conclusions Three clones exhibited specific binding to lipid A is identified by direct and competitive ELISA methods. The succcess of isolating anti-lipid A proves the usefulness of phage display system in human McAb preparation. The result shows that we have got the recombinant phage antibody.

The Screening of Human Anti-Hepatoma Fd Fragments Guided with the Chemeric Light Chain of HAb18 / 中国肿瘤生物治疗杂志

Objective: To select anti-HAb18G (hepatoma associated antigen) human Fd fragments with guided selection of L chain of chimeric Fab-HAb18. Methods: The human Fd repertories genes were amplified by RT-PCR from PBMC of hepatoma patients, and cloned into the vector pComb3X with chimeric L chain to construct the human-mouse hybrid Fab phage library. HAblSGE, extracellular region of HAblSG, was used as antigen to screen. The positive clones was obtained by ELISA and FCM with p Ⅲ-fusion Fab antibody. The DNA sequences were analyzed. Results: A human-mouse Fab antibody library were constructed with 2?107 PFU. After 6 rounds panning, 7 positive clones were obtained with ELISA and FCM. And sequences of 2 clones with better affinity were same. The CHI belonged to the IgG2 class as the parent Fd, and the variable region belonged to VH3 family. Conclusions: Through the construction of the HuMFab phage antibody library and chemeric L chain-guided selection, we get the available human Fd fragments for subsequent research.

The Selection of Thrombin-specific scAbs by Alternative Panning Strategy / 微生物学通报

Specific scAbs could be obtained through biopanning from phage antibody libraries by use of antigens as target molecules. scAbs specific to thrombin were separated from mouse antibody library by the panning strategy of alternating liquid-solid phase in this paper. Thrombin was biotinylated by photobiotin at first, then avidin-coated magnetic beads were utilized to isolate specific scAbs. The eluted phages were amplified and subject to the second round panning in microtiter plate to remove the unspecific reombinant phages. 4 specific scAbs were separated from 23 phage clones after four rounds of alternative panning.

Construction of Fab fragment phage antibody library and selection of clones expressing anti-human ?_1-AR antibody / 中国免疫学杂志

Objective:To construct a human Fab phage antibody library and to obtain some recombinant clones which can express Fab fragment antibody against ? 1-adrenergic receptor.Methods:Fd heavy chain gene and ??? light chains gene of IgG obtained by RT-PCR from peripheral lymphocytes of DCM patients whose anti ? 1-AR antibodies are present were cloned into pComb3 vector and the human Fab phage antibody library was constructed.The library was panned by phage display technology with ? 1-ARECⅡ as antigen.Results:A human Fab phage antibody library with 1.4?10 6 capability was constructed successfully,a positive clone against human ? 1-AR was screened from the phage antibody library.Conclusion:The recombinant clones which express Fab antibody against ? 1-AR can be obtained by phage display technology.