Four-way ligation for construction of a mammalian cell-based full-length antibody display library

A unique four-way ligation strategy was developed for rapid construction of a full-length antibody library. A mammalian expression vector was constructed that contained dual mammalian expression cassettes and sequences recognized by the unique restriction enzymes BsmBI, BstXI, and SfiI. Both full-length light-chain and variable domain of heavy-chain genes were inserted into the vector in one step by four-way ligation, and full-length bivalent antibodies were displayed on mammalian cell surfaces. Using this strategy, only 2 weeks were required to successfully construct high-quality, full-length human antibody libraries.     

Human anti-EGFL7 recombinant full-length antibodies selected from a mammalian cell-based antibody display library

Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.     

Large-scale sequencing analysis of the full-length cDNA library of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the human cancers clearly linked to viral infections. Although the major risk factors for HCC development have been elucidated, the hepatocellular carcinogenesis pathway resulting in malignant transformation of liver cells remains to be clarified. Recently, some results of microarray and comparative genomic hybridization analysis have been provided as comprehensive studies of genomic instability in HCC, including mutation, deletion and DNA copy losses. In this work, the full-length cDNA library has been constructed and sequenced, and the sequencing results have been further clustered and analyzed. The results show that 1,342 genes have been found, and about 300 of these genes may be important in e.g. cell proliferation, DNA repair and apoptosis. After further analysis of DNA sequences, the deletion genotypes of at least 24 genes have been found. However, the functional changes of these deletion mutants and their significance in hepatocellular carcinogenesis remain to be clarified. This research may be one of the best to obtain the candidate genes for hepatocellular carcinogenesis.     

Construction and characterization of a pseudo-immune human antibody library using yeast surface display

Lymphocytes from eight individuals out of 60 healthy donors, whose plasmas showed relatively higher antibody titer for a target antigen of death receptor 5 (DR5), were selected for the source of antibody genes to construct so called an anti-DR5 pseudo-immune human single-chain fragment variable (scFv) library on the yeast cell surface (approximately 2x10(6) diversity). Compared with a large nonimmune human scFv library (approximately 1x10(9) diversity), the repertoire of the pseudo-immune scFv library was significantly biased toward the target antigen, which facilitated rapid enrichments of the target-specific high affinity scFvs during selections by fluorescence activated cell sortings. Isolated scFvs, HW5 and HW6, from the pseudo-immune library showed much higher specificity and affinity for the targeted antigen than those from the nonimmune library. Our results suggest that a pseudo-immune antibody library is very efficient to isolate target-specific high affinity antibody from a relatively small sized library.     

The development of a novel vector for construction of a full-length cDNA library

A new original vector pEM-(dT)₄₀(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The vector pGEM-(dT)₄₀f(+) is initially transformed into single stranded and then into a linear form and its (dT)₄₀ tail at the 3′-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector circularization and synthesis of the second strand cDNA. This approach has the following advantages: (1) it significantly simplifies cDNA library construction, which includes three steps; (2) full-length cDNA library construction is achieved by adding a (dC)_n homopolymer tail to the 5′end; (3) preparation of a clone library requires a few milligrams of total RNA; (4) it is possible to obtain cDNA clones up to 10 kbp; (5) it does not require PCR reaction (which can induce artifact mutations in cDNA sequences); (6) this approach does not employ restrictase treatment and chimeric cDNA products are not formed.     

Screening and identification of a specific peptide binding to hepatocellular carcinoma cells from a phage display peptide library

To screen and identify the novel probe markers binding hepatocellular carcinoma specifically and sensitively, a phage‐displayed 12‐mer peptide library was used to make biopanning with the modified protocols on HepG2 cells. After four rounds of panning, the consensus sequences were obtained, and the PC28, a phage clone with most specific and sensitive binding to HepG2 cells, was identified as the best positive clone. The peptide probe HCSP4 (sequence SLDSTHTHAPWP) was synthesized based on the sequencing result of PC28. The specificity and sensitivity of HCSP4 were primarily analyzed using immunofluorescence, flow cytometry, and other methods. The results show that HCSP4 can bind to hepatocellular carcinoma cells with satisfactory specificity and sensitivity. It may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

噬菌体抗体库的优化

噬菌体抗体组合文库技术作为噬菌体展示和抗体组合文库两种技术的集成,由于它具有库容量大、特异性高、和敏感性强的优点而被誉为抗体技术的第三次革命。但是由于一些技术上的原因,使得它无法得到广泛的应用,本文就其优化进行综述。     

Construction of a large phage display antibody library by in vitro package and in vivo recombination

Capacity and diversity are extremely important to the quality of various phage display libraries. In this work, λ phage-based in vitro package was applied to construct a filamentous phage display antibody library so as to enlarge its capacity and introduce more sequence diversity in the final library. In vivo recombination via Cre recombinase/lox sites was also exploited to create V_H/V_L combination diversity based on multivalent package of λ phage packaging extracts on phagemid DNA concatemers. The library constructed with 10 μg concatenated phagemid DNA and ten vials of λ phage packaging extracts was calculated to contain 1.40×10¹⁰ independent clones. Higher capacity can be easily achieved when more materials are consumed. This strategy is somewhat more efficient than prior methods.     

Development of a novel mammalian cell surface antibody display platform

Antibody display systems have been successfully applied to screen, select and characterize antibody fragments. These systems typically use prokaryotic organisms such as phage and bacteria or lower eukaryotic organisms, such as yeast. These organisms possess either no or different post-translational modification functions from mammalian cells and prefer to display small antibody fragments instead of full-length IgGs. We report here a novel mammalian cell-based antibody display platform that displays full-length functional antibodies on the surface of mammalian cells. Through recombinase-mediated DNA integration, each host cell contains one copy of the gene of interest in the genome. Utilizing a hot-spot integration site, the expression levels of the gene of interest are high and comparable between clones, ensuring a high signal to noise ratio. Coupled with fluorescence-activated cell sorting (FACS) technology, our platform is high throughput and can distinguish antibodies with very high antigen binding affinities directly on the cell surface. Single-round FACS can enrich high affinity antibodies by more than 500 fold. Antibodies with significantly improved neutralizing activity have been identified from a randomly mutagenized library, demonstrating the power of this platform in screening and selecting antibody therapeutics.     

Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma

Background

Hep27 monoclonal (Hep27 Mab) is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102). We attempted to produce a single-chain fragment (scFv), a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques.

Results

The sequences encoding the variable regions of heavy (V_H) and light (V_L) chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser)₃ and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa). Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab).

Conclusion

This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy.     

Development of a novel mammalian cell surface antibody display platform

Antibody display systems have been successfully applied to screen, select and characterize antibody fragments. These systems typically use prokaryotic organisms such as phage and bacteria or lower eukaryotic organisms, such as yeast. These organisms possess either no or different post-translational modification functions from mammalian cells and prefer to display small antibody fragments instead of full-length IgGs. We report here a novel mammalian cell-based antibody display platform that displays full-length functional antibodies on the surface of mammalian cells. Through recombinase-mediated DNA integration, each host cell contains one copy of the gene of interest in the genome. Utilizing a hot-spot integration site, the expression levels of the gene of interest are high and comparable between clones, ensuring a high signal to noise ratio. Coupled with fluorescence-activated cell sorting (FACS) technology, our platform is high throughput and can distinguish antibodies with very high antigen binding affinities directly on the cell surface. Single-round FACS can enrich high affinity antibodies by more than 500-fold. Antibodies with significantly improved neutralizing activity have been identified from a randomly mutagenized library, demonstrating the power of this platform in screening and selecting antibody therapeutics.Key words: antibody display, mammalian display, antibody library, vector, antibody screen, affinity maturation     

Construction, screening and identification of a phage display antibody library against the Eimeria acervulina merozoite

A single-chain antibody library against Eimeria acervulina merozoites was constructed by phage display approach. Antibody-displaying phage was selected in four panning rounds against cryopreserved E. acervulina merozoites. Five clones were randomly selected from the fourth panning round, and their nucleotide sequences were aligned and compared to mouse germ-line sequences. Soluble antibody was produced in a non-suppressor Escherichia coli strain, purified by protein A affinity chromatography, and characterized by Western-blotting. Immunofluorescence assay showed localization of the produced recombinant antibody fragment on the surface E. acervulina merozoites. These resultant antibody fragments showed high specificity and binding capacity for soluble antigens and intact fixed merozoites which seems promising as diagnostic, therapeutic and/or vaccine tools against coccidiosis.     

Specific glycosidase activity isolated from a random phage display antibody library

Carbohydrates serve as key receptor sites in various cellular events such as viral attachment, tumor formation, and tissue inflammation. A potential route to control these events is to manipulate targeted carbohydrate structures in vivo using specifically designed glycohydrolases. Here we show that a stereospecific catalytic activity designed toward a particular sugar and linkage can be readily isolated from a phage display antibody library derived from a nonimmunized host. The activity was isolated using a transition-state analogue mimicking an alpha-glucosidasic linkage as antigen and showed a 20-fold specificity for that sugar and linkage. The DNA sequence, however, contains a large deletion in the antibody gene, which also changes the downstream reading frame, resulting in a translated sequence containing only 57 amino acids that has a predominantly hydrophobic amino terminal and a strongly hydrophilic carboxy terminal. The isolated catalytic activity has a strong pH dependence, attributable to one or more of the numerous potentially charged groups in the carboxyl terminal. While the protein readily forms more stable multimers, the 7.3-kD monomer represents by far the smallest glycosidase enzyme reported to date and can provide substantial new information toward understanding and modifying glycosidase activity.     

Screening and identification of a targeting peptide to hepatocarcinoma from a phage display peptide library

Ligands specific to cell surface receptors have been heavily investigated in cancer research. Phage display technology is a powerful tool in this field and may impact clinical issues including functional diagnosis and targeted drug delivery. In this study, a hepatocellular carcinoma cell line (HepG2) and a normal hepatocyte line (L-02) were used to carry out subtractive screening in vitro with a phage display-7 peptide library. After four rounds of panning, there was an obvious enrichment for the phages specifically binding to the HepG2 cells, and the output/input ratio of phages increased about 976-fold (from 0.3x10(-7) to 292.8x10(-7)). A group of peptides capable of binding specifically to the hepatoma cells were obtained, and the affinity of these peptides to the targeting cells and tissues was studied. Through a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and immunofluorescence, the S1 phage and synthetic peptide HCBP1 (sequence FQHPSFI) were shown to bind to the tumor cell surfaces of two hepatoma cell lines and biopsy specimens, but not to normal hepatocytes, other different cancer cells, or nontumor liver tissues. In conclusion, the peptide HCBP1 may be a potential candidate for targeted drug delivery in therapy of hepatoma cancer.     

Automation of phage display for high-throughput antibody development

Antibodies are important tools for the detection of diagnostic markers and are the most well-known examples of specific molecular interactions. We have developed automated technology that enables the selection of antibodies and other interacting molecules from large recombinant libraries. The physical link between phenotype and genotype in phage display allows selective isolation and amplification of a particular phage encoding a desired antibody fragment. Successive rounds of phage selection, amplification and screening are performed at high throughput, using a pin-based magnetic particle processor. The integration of this with existing DNA and protein array technology enables industrial screening of complex libraries and opens up a new level of functional genomic analysis.