Construction and development of a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma

Nicotinamide cofactor-dependent oxidoreductases have been widely employed during the bioproduction of varieties of useful compounds. Efficient cofactor regeneration is often required for these biotransformation reactions. Herein, we report the synthesis of an important pharmaceutical intermediate 4-hydroxy-2-butanone (4H2B) via an immobilized in situ cofactor regeneration system composed of NAD(+)-dependent glycerol dehydrogenase (GlyDH) and NAD(+)-regenerating NADH oxidase (nox). Both enzymes were immobilized on functionalized single-walled carbon nanotubes (SWCNTs) through the specific interaction between the His-tagged enzymes and the modified SWCNTs. GlyDH demonstrated ca. 100% native enzyme activity after immobilization. The GlyDH/nox ratio, pH, and amount of nicotinamide cofactor were examined to establish the optimum reaction conditions for 4H2B production. The nanoparticle-supported cofactor regeneration system become more stable and the yield of 4H2B turned out to be almost twice (37%) that of the free enzyme system after a 12-h reaction. Thus, we believe that this non-covalent specific immobilization procedure can be applied to cofactor regeneration system for bioconversions.     

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.     

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.     

Design, construction, and characterization of a large synthetic human antibody phage display library

Advances in proteomic research allow the identification of several hundred protein components in complex biological specimens. Structural information is typically lost during proteomic investigations. For this reason, the rapid isolation of monoclonal antibodies specific to proteins of interest would allow the study of structurally intact biological specimens, thus providing complementary proteomic information. Here, we describe the design, construction, characterization, and use of a large synthetic human antibody phage display library (ETH-2-Gold) containing three billion individual antibody clones. A large repertoire of antibodies with similar biochemical properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto three antibody germline segments (DP47, DPK22, and DPL16), which are frequently found in human antibodies. The ETH-2-Gold library exhibits efficient display of antibody fragments on filamentous phage, as assessed by immunoblot. Furthermore, the library is highly functional, since >90% of clones express soluble antibodies in bacteria and since good quality monoclonal antibodies have been isolated against 16 different antigens. The usefulness of the library as a tool for generating monoclonal antibodies for biomedical applications was tested using the C-domain of tenascin-C (a marker of angiogenesis) as antigen and showing that specific antibodies to this target were able to stain vascular structures in tumor sections.     

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。     

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.     

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     

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.     

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.     

Baculovirus surface display: construction and screening of a eukaryotic epitope library.

The baculovirus expression system was utilized to serve as a tool for ligand selection, demonstrating the applicability of the system to the generation and screening of eukaryotic expression libraries. The HIV-1-gp41 epitope 'ELDKWA', specific for the neutralizing human mAb 2F5, was inserted into the antigenic site B of influenza virus hemagglutinin and expressed on the surface of baculovirus infected insect cells. In order to improve the antigenicity of the epitope within the hemagglutinin, and therefore enhance the specific binding of 2F5, we inserted three additional, random amino acids adjacent to the epitope. This pool of hemagglutinin genes was directly cloned into the baculovirus Ac-omega. To identify distinct proteins displayed on the cellular surface, we developed a screening protocol to select for specific binding capacity of individual viral clones. Using fluorescence activated cell sorting (FACS) we isolated a baculovirus clone displaying the epitope with markedly increased binding capacity out of a pool of 8000 variants in only one sorting step. Binding properties of the identified ligand were examined by FACS performing a competition assay.     

Analysis of the diversity of a sheep antibody repertoire as revealed from a bacteriophage display library

We have applied bacteriophage display technology to construct and analyze the diversity of an IgG library of >1 x 108 clones from an adult sheep immunized against the hapten atrazine. We have identified eight new VH gene families (VH2-VH9) and five new Vkappa gene families (VkappaV-VkappaIX). The heavy and kappa light chain variable region gene loci were found to be far more diverse than previously thought.     

一种从抗狂犬病病毒噬菌体抗体库中筛选和验证中和抗体的方法

目的建立一种从噬菌体库抗体库中高效筛选和验证抗狂犬病病毒中和抗体的方法。方法①定性分析:从经过灭活的CVS-11筛选噬菌体抗体库中挑取单克隆于96孔板中培养,制备噬菌体抗体,取培养上清进行快速荧光灶抑制试验(rapid fluorescent focus inhibition test, RFFIT),选择可明显抑制病毒感染的克隆测序,获得具有中和活性的抗体可变区序列;②定量分析:挑选有中和活性的克隆,重新制备噬菌体抗体颗粒,纯化后进行RFFIT分析;扩增抗体可变区基因,构建真核瞬时表达质粒。瞬转HEK-293 EBNA1细胞,培养上清经Mabselect SuRe亲和纯化后测定抗体比活。结果定性分析获得的噬菌体抗体颗粒与全分子抗体体外中和活性显著相关;剔除低活性序列后,活性高于0.5 IU/mL的噬菌体抗体颗粒与其全分子抗体体外中和活性之间无显著相关;所有纯化后噬菌体抗体颗粒活性>0.5 IU/mL的序列其全分子抗体体外中和活性均>500 IU/mg。结论构建了一种从抗狂犬病病毒噬菌体库中高效筛选、验证中和抗体的方法。     

Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5′ and/or 3′ end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V_H families/segments was observed showing that ONCL did not introduce cloning biases for or against any V_H family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 × 10¹⁰ and by selecting five unique Fabs against GAPDH antigen.