β淀粉样蛋白人源性抗体的制备

目的:应用噬菌体展示及抗体库技术,制备并鉴定β淀粉样蛋白(Aβ)人源性抗体。方法:应用固相筛选方法,以人工合成的Aβ1-15肽为靶标分子,在大容量全合成人源性噬菌体抗体库中筛选抗Aβ人源性抗体,并进行特异性鉴定。结果:经过3轮筛选,单克隆鉴定获得噬菌体抗体F11,竞争性ELISA表明抗体对Aβ1-42的结合位点位于1～15氨基酸残基内,ELISA结果证实抗体特异性良好。结论:以Aβ1-15肽为靶标分子获得了特异性良好的人源性抗体。     

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.     

抗P-选择蛋白人源性单克隆抗体的制备

目的:获得人源性抗P-选择蛋白(selectin)特异性抗体,为相关疾病治疗奠定基础。方法:在HEK293细胞中真核分泌表达人P-选择蛋白功能性片段,以此蛋白片段作为抗原,利用本室构建的大容量全合成人源性噬菌体抗体库进行筛选,经过3轮固相筛选后,阳性克隆得到富集;将其中富集效果最好的一株单链抗体A1改造成全抗体(IgG1),重组质粒转染H293细胞后,抗体得到表达;表达后在全抗体水平上用ELISA和Western印迹分别验证了A1抗体的特异性,并通过非竞争ELISA方法初步测定这株抗体的亲和力。结果:3轮筛选得到3株特异性噬菌体抗体,其中富集效果最好的单链抗体A1改造成全抗体形式后特异性良好,抗体亲和力Kd=2×10-8 mol/L。结论:筛选得到一株特异性较好的抗P-选择蛋白人源性单克隆抗体A1,其特异性和亲和力较好,有继续开发的价值。     

Two-dimensional electrophoresis/phage panning (2D-PP): a novel technology for direct antibody selection on 2-D blots

We describe a novel method, two-dimensional electrophoresis/phage panning (2D-PP), for the generation of antibodies against proteins in crude biochemical samples, such as cellular membrane fractions. These sources have traditionally presented problems as to the development of antibodies by conventional techniques. 2D-PP involves two-dimensional resolution of proteins, blotting of the proteins onto a nitrocellulose membrane, and screening of a phage antibody library and isolation of corresponding antibodies. By 2D-PP with detergent-insoluble "lipid rafts" as a target protein complex, we obtained specific phage pools against eight antigen spots (from a total of 39 spots). These antibodies were functional in Western blotting, enzyme-linked immunosorbent assaying (ELISA), and immunoscreening of a cDNA expression library. Propagation of anti-nitrocellulose phages was the major problem in 2D-PP, but was overcome by the use of the soluble anti-nitrocellulose antibody fragment. 2D-PP constitutes a key tool for functional analysis of proteins in complex fractions.     

Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution

Phage display and two competitive panning elution conditions were used to isolate Candida-specific single chain fragment variable (scFv) antibodies. An scFv phage library constructed from splenic lymphocytes of mice immunized by idiotypic vaccination with an HM-1 killer toxin (HM-1)-neutralizing monoclonal antibody (nmAb-KT) was used for panning against Candidaalbicans membrane fraction (CaMF). Key steps were specific elution conditions to separately release the bound phages with original antigen HM-1 + HM-1 peptide 6 and CaMF. The positive phages were screened by using enzyme-linked immunosorbent assay, and after nucleotide sequencing, clone expression, and purification, clone scFv-C1 was selected for detailed characterization. The scFv-C1 showed IC₅₀ values for cell growth against various Candida species and Saccharomyces cerevisiae as 2.40 to 6.40 μM and 2.20 μM, respectively. By using surface plasmon resonance analysis, the scFv-C1 had a K_d value of 3.09 × 10⁻¹¹ M to nmAb-KT, indicating a 260-fold higher affinity than for HM-1. These results showed the generated scFv-C1 mimicking HM-1-binding affinity to nmAb-KT and in vitro antifungal activity. We believe that the effectiveness of the competitive panning elution method and antigen-specific recombinant scFv antibodies obtained in this study are excellent candidates for antimycotic drugs.     

Human single-chain Fv antibodies against Burkholderia mallei and Burkholderia pseudomallei

Much effort has been devoted to the development of mouse monoclonal antibodies that react specifically with Burkholderia mallei and Burkholderia pseudomallei for diagnostic and/or therapeutic purposes. Our present study focused on the screening of a phage-displayed nonimmune human single-chain Fv (scFv) antibody library against heat-killed B. mallei and B. pseudomallei for the generation of human scFv antibodies specific to the two pathogenic species of bacteria. Using two different panning procedures, we obtained seven different scFv phage antibodies that interacted with the heat-killed whole bacterial cells of B. mallei and B. pseudomallei. Our results demonstrate that panning of a human scFv antibody library against heat-killed whole bacterial cells may provide a valuable strategy for developing human monoclonal antibodies against the highly pathogenic bacteria.     

REAL-Select: Full-Length Antibody Display and Library Screening by Surface Capture on Yeast Cells

We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1∶1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.     

Therapeutic antibodies: Discovery and development using the ProteOn XPR36 biosensor interaction array system

Therapeutic monoclonal antibodies are becoming a significant and rapidly growing class of therapeutic pharmaceuticals. Their discovery and development requires fast and high-throughput methodologies for screening and selecting appropriate candidate antibodies having high affinity for the target as well as high specificity and low cross-reactivity. This study demonstrates the use of the ProteOn XPR36 protein interaction array system and its novel approach, termed One-Shot Kinetics, for the rapid screening and selection of high-affinity antibodies. This approach allows multiple quantitative protein binding analyses in parallel, providing association, dissociation, and affinity constants for several antibodies or supernatants simultaneously in one experiment. We show that the ProteOn XPR36 system is a valuable tool for use across multiple stages of the therapeutic antibody discovery and development process, enabling efficient and rapid screening after panning, affinity maturation, assay validation, and clone selection.     

Identification and repair of positive binding antibodies containing randomly generated amber codons from synthetic phage display libraries

Phage display technology allows for the rapid isolation and characterization of monoclonal antibodies that have vast potential for therapeutic and diagnostic applications. However, the panning process, which utilizes a host strain that suppresses termination by the amber codon, has an inherent bias toward clones containing randomly generated amber stop codons, complicating identification of positive binding antibodies when the antibody genes are finally expressed in a nonsupressor host. Here, we perform biopanning against a Histone 2A peptide using streptavidin- or anti-biotin-coated beads. After four rounds, a dominant clone is characterized but contains a spurious amber stop codon. A protocol is given that readily corrects the amber codon, allowing for soluble antibody production once the phagemid is transformed into a nonsuppressor bacterial strain. This work also highlights the ability to isolate antibodies against a protein antigen by using only a small peptide (15 amino acids) representing a portion of the antigen.     

Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

A prerequisite for the enrichment of antibodies screened from phage display libraries is their stable expression on a phage during multiple selection rounds. Thus, if stringent panning procedures are employed, selection is simultaneously driven by antigen affinity, stability and solubility. To take advantage of robust pre-selected scaffolds of such molecules, we grafted single-chain Fv (scFv) antibodies, previously isolated from a human phage display library after multiple rounds of in vitro panning on tumor cells, with the specificity of the clinically established murine monoclonal anti-CD22 antibody RFB4. We show that a panel of grafted scFvs retained the specificity of the murine monoclonal antibody, bound to the target antigen with high affinity (6.4-9.6 nM), and exhibited exceptional biophysical stability with retention of 89-93% of the initial binding activity after 6 days of incubation in human serum at 37 degrees C. Selection of stable human scaffolds with high sequence identity to both the human germline and the rodent frameworks required only a small number of murine residues to be retained within the human frameworks in order to maintain the structural integrity of the antigen binding site. We expect this approach may be applicable for the rapid generation of highly stable humanized antibodies with low immunogenic potential.     

De novo identification of cell-type specific antibody-antigen pairs by phage display subtraction. Isolation of a human single chain antibody fragment against human keratin 14.

The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte-specific antigens from a phage display antibody repertoire by using phage display subtraction. Phage display is a method of displaying foreign molecules on the surface of filamentous bacteriophage particles. It allows the interaction between two cognate molecules to be analysed through affinity selections. Recently, large repertoires of phage displayed human antibody fragments have been constructed. From such repertoires, antibodies can be obtained in vitro without the need for immunization or the hybridoma technology. A novel subtractive strategy for selecting antibodies from phage libraries was applied. Phage antibodies were selected against immobilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand. A competing cell lysate was used to reduce retrieval of phage antibodies with specificities to commonly non-differentially expressed antigens. A monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identified as demonstrated by ELISA assays and immunoblotting analysis. The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotting based on 2D PAGE and a corresponding 2D PAGE protein database. In accordance with the expected tissue localization of K14, the identified scFv stained the basal layer of human epidermis by indirect immunofluorescence analysis. Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody-antigen pairs that characterize a specific cell type.     

基于细胞的抗单增李斯特菌单域重链抗体的筛选及鉴定

【目的】获得针对单增李斯特菌的特异性单域重链抗体,并对筛选过程中特异性克隆的富集规律进行分析,为筛选具有种属特异性的噬菌体展示抗体提供参考。【方法】采用固相筛选技术,以热灭活的单增李斯特菌菌体为抗原,通过四轮常规筛选和一轮消减筛选,从驼源天然噬菌体展示文库中筛选针对单增李斯特菌的单域重链抗体。采用Phage-ELISA法,对后四轮筛选洗脱物中随机挑选的噬菌体进行鉴定,阳性克隆进行基因测序及结合特异性分析。通过多序列比对分析将获得的基因序列进行分组和统计。【结果】成功筛选到2株单增李斯特菌特异性的单域重链抗体。【结论】在优化的筛选条件下,基于全细胞的筛选方法能够获得特异性识别单增李斯特菌的单域重链抗体,消减筛选对于去除非特异性克隆是有效的和必要的。     

Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.     

抗EGFRvIII噬菌体抗体库的构建和筛选

目的：应用噬菌体展示技术筛选针对表皮生长因子受体突变体Ш (epidermal growth factor receptor variant type Ⅲ, EGFRvIII)的单链抗体 (single chain Fv, scFv)。方法：利用原核表达纯化的人EGFRvIIIex蛋白和高表达EGFRvIIIex的小鼠成纤维细胞系NIH3T3免疫小鼠,扩增VH和VL片段并拼装成scFv 基因,连接至噬菌粒pCANTAB 5E,电击转化Hpd3cells,构建噬菌体单链抗体库,并进行3轮富集筛选。在第4轮筛选时,采用了降低抗原浓度的方法。然后将筛选得到的阳性克隆测序分析,转化E.coli HB2151,IPTG 诱导可溶性scFv 的表达。结果：构建了库容为7.9×107 的噬菌体单链抗体库。经过第4轮低浓度抗原筛选,得到了较高亲和力的克隆。取单个阳性克隆测序分析结果表明,该抗EGFRvIII scFv 基因序列长807 bp,编码268个氨基酸。IPTG诱导后表达的可溶性scFv 可分别与纯化的EGFRvIIIex抗原以及细胞表面的EGFRvIIIex结合。结论：利用噬菌体抗体库筛选得到了高亲和力的抗EGFRvIII scFv,为开发针对EGFRvIII的抗体药物提供了靶向载体分子。     

Generation of human monoclonal antibodies against <Emphasis Type="Italic">Propionibacterium acnes</Emphasis> by applying the phage display method to human peripheral blood mononuclear cells immunized in vitro

Propionibacterium acnes is a gram-positive, non-spore-forming, rod-shaped bacterium that is often detected in normal human skin flora. P. acnes has been associated with many diseases. In this study, we attempted to generate anti-P. acnes human monoclonal antibodies. A phage antibody library was first generated from human peripheral blood mononuclear cells immunized in vitro with P. acnes using the phage display method, and P. acnes-specific phage antibodies were obtained using solid phase panning. Antigen-specific variable region genes were then amplified and recombined into vectors expressing human IgG antibodies. The results indicated that the recombinant human IgG antibodies exhibited P. acnes-specific binding. This study demonstrates that the combined use of an in vitro immunization protocol and the phage display method enables the generation of human monoclonal antibodies against pathogenic bacteria and toxic antigens.     

Mammalian cell surface display for monoclonal antibody-based FACS selection of viral envelope proteins

The elicitation of broadly and efficiently neutralizing antibodies in humans by active immunization is still a major obstacle in the development of vaccines against pathogens such as the human immunodeficiency virus (HIV), influenza virus, hepatitis C virus or cytomegalovirus. Here, we describe a mammalian cell surface display and monoclonal antibody (mAb)-mediated panning technology that allows affinity-based selection of envelope (Env) variants from libraries. To this end, we established an experimental setup featuring: 1) single and site specific integration of Env to link genotype and phenotype, 2) inducible Env expression to avoid cytotoxicity effects, 3) translational coupling of Env and enhanced green fluorescent protein expression to normalize for Env protein levels, and 4) display on HEK cells to ensure native folding and mammalian glycosylation. For proof of concept, we applied our method to a chimeric HIV-1 Env model library comprising variants with differential binding affinities to the V3-loop-directed mAbs 447–52D and HGN194. Fluorescence-activated cell sorting selectively enriched a high affinity variant up to 56- and 55-fold for 447–52D and HGN194, respectively, after only a single round of panning. Similarly, the low affinity variants for each antibody could be selectively enriched up to 237-fold. The binding profiles of membrane-bound gp145 and soluble gp140 chimeras showed identical affinity ranking, suggesting that the technology can guide the identification of Env variants with optimized antigenic properties for subsequent use as vaccine candidates. Finally, our mAb-based cellular display and selection strategy may also prove useful for the development of prophylactic vaccines against pathogens other than HIV.     

Development of recombinant single-chain variable fragment against hepatitis A virus and its use in quantification of hepatitis A antigen

Phage display technology has been utilized for identification of specific binding molecules to an antigenic target thereby enabling the rapid generation and selection of high affinity, fully human antibodies directed towards disease target appropriate for antibody therapy. In the present study, single chain Fv antibody fragment (scFv) to hepatitis A virus (HAV) was selected from phage displayed antibody library constructed from peripheral blood lymphocytes (PBLs) of a vaccinated donor. The variable heavy (V(H)) and light chains (V(L)) were amplified using cDNA as template, assembled into scFv using splicing by overlap extension PCR (SOE PCR) and cloned into phagemid vector as a fusion for display of scFv on bacteriophage. The phage displaying antibody fragments were subjected to three rounds of panning with HAV antigen on solid phase. High affinity antibodies reactive to hepatitis A virus were identified by phage ELISA and cloned into a bacterial expression vector pET20b. The scFv was purified by immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The binding activity and specificity of the scFv was established by its non-reactivity towards other human viral antigens as determined by ELISA and immunoblot analysis. The scFv was further used in the development of an in-house IC-ELISA format in combination with a commercially available mouse monoclonal antibody for the quantification of hepatitis A virus antigen in human vaccine preparations. The adjusted r2 values obtained by subjecting the values obtained by quantification of the NIBSC standards using the commercial and the in-house ELISA kits by regression analysis were 0.99 and 0.95. 39 vaccine samples were subjected to quantification using both the kits. Regressional statistical analysis through the origin of the samples indicated International Unit (IU) values of 0.0416x and 0.0419x, respectively for the commercial and in-house kit respectively.     

The Molecular Engineering of an Anti-Idiotypic Antibody for Pharmacokinetic Analysis of a Fully Human Anti-Infective

Anti-idiotype monoclonal antibodies represent a class of reagents that are potentially optimal for analyzing the pharmacokinetics of fully human, anti-infective antibodies that have been developed as therapeutic candidates. This is particularly important where direct pathogen binding assays are complicated by requirements for biosafety level III or IV for pathogen handling. In this study, we describe the development of a recombinant, anti-idiotype monoclonal antibody termed E1 for the detection of a fully human, serotype-specific, therapeutic antibody candidate for the BSLIII pathogen Dengue virus termed 14c10 hG1. E1 was generated by naïve human Fab phage library panning technology and subsequently engineered as a monoclonal antibody. We show that E1 is highly specific for the fully-folded form of 14c10 hG1 and can be employed for the detection of this antibody in healthy human subjects’ serum by enzyme linked immunosorbent assay. In addition, we show that E1 is capable of blocking the binding of 14c10 hG1 to dengue virus serotype 1. Finally, we show that E1 can detect 14c10 hG1 in mouse serum after the administration of the therapeutic antibody in vivo. E1 represents an important new form of ancillary reagent that can be utilized in the clinical development of a therapeutic human antibody candidate.     

Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format.

Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.     

Shotgun phage display — Selection for bacterial receptins or other exported proteins

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins. Published: May 1, 2003