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.     

Microselection – affinity selecting antibodies against a single rare cell in a heterogeneous population

Rare cells not normally present in the peripheral bloodstream, such as circulating tumour cells, have potential applications for development of non‐invasive methods for diagnostics or follow up. Obtaining these cells however require some means of discrimination, achievable by cell type specific antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV‐irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies per single cell selection, including three highly K562 cell type specific.     

Selection of cell‐type specific antibodies on tissue‐sections using phage display

With the advent of modern technologies enabling single cell analysis, it has become clear that small sub‐populations of cells or even single cells can drive the phenotypic appearance of tissue, both diseased and normal. Nucleic acid based technologies allowing single cell analysis has been faster to mature, while technologies aimed at analysing the proteome at a single cell level is still lacking behind, especially technologies which allow single cell analysis in tissue. Introducing methods, that allows such analysis, will pave the way for discovering new biomarkers with more clinical relevance, as these may be unique for microenvironments only present in tissue and will avoid artifacts introduced by in vitro studies. Here, we introduce a technology enabling biomarker identification on small sub‐populations of cells within a tissue section. Phage antibody libraries are applied to the tissue sections, followed by washing to remove non‐bound phage particles. To eliminate phage antibodies binding to antigens ubiquitously expressed and retrieve phage antibodies binding specifically to antigens expressed by the sub‐population of cells, the area of interest is protected by a ‘shadow stick’. The phage antibodies on the remaining areas on the slide are exposed to UV light, which introduces cross‐links in the phage genome, thus rendering them non‐replicable. In this work we applied the technology, guided by CD31 expressing endothelial cells, to isolate recombinant antibodies specifically binding biomarkers expressed either by the cell or in the microenvironment surrounding the endothelial cell.     

An anti-leukemic single chain Fv antibody selected from a synthetic human phage antibody library

The display of human antibody repertoire on the cell surface of the filamentous bacteriophage has offered a novel strategy for selecting antibodies to a diverse range of purified targets. However, the selection of antibodies with biological functions has not yet been fully investigated. To select phage antibodies with therapeutic potential, a synthetic human single chain Fv (scFv) phage antibody library was panned on whole premyelocytic leukemia cell line (HL60). Phages binding to common receptors and undesirable phages were subtracted by incubating the library with human glioma cells. High affinity binding phages to HL60 cells were enriched by fluorescence-activated cell sorting. After the 6th round of selection, 50% of the selected phage antibodies showed significant binding to HL60 cells, whereas none of the analyzed phage antibodies bound to human pre-B cells (Nalm-6). In addition to binding, one scFv antibody inhibited HL60 cell proliferation by 90% compared to irrelevant scFv antibodies. Taken together the data demonstrate that specific scFv antibodies with biological functions can be isolated by using whole cells as affinity matrix.     

Using phage display to select antibodies recognizing post-translational modifications independently of sequence context

Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Antibody reactivity was lost by antigen treatment with sulfatase or preincubation with soluble tyrosine sulfate, indicating its specificity. The isolation of this antibody signals the potential of phage antibody libraries in the derivation of reagents specific for post-translational modifications, although the extensive screening required indicates that such antibodies are extremely rare.     

Identification of CD4 and transferrin receptor antibodies by CXCR4 antibody-guided Pathfinder selection.

To generate human antibodies against CXCR4, a seven-transmembrane chemokine receptor and a principal coreceptor for HIV-1, several rounds of Pathfinder and Step-back selection from a large phage display antibody library were performed on Jurkat cells. A mAb against CXCR4 or biotinyated phage antibodies were used as guide molecules. Over 100 pan-Jurkat-cell-positive antibodies were characterized, but none were CXCR4 specific. However, several antibodies against CD4 and the transferrin receptor were identified. Our results indicate that, although Pathfinder and Step-back selection can be used to select phage antibodies on whole cells, the successful selection of certain targets is still complex and limited. The reason is probably, in part, due to the inaccessibility of the targeted extracellular structures and the range of the horseradish peroxidase-labeled guide molecule. Refinements of these techniques are required to improve target specificity and selectivity.     

Selection of scFv phages on intact cells under low pH conditions leads to a significant loss of insert-free phages

Display of functional antibody fragments on the surface of filamentous bacteriophages allows fast selection of specific phage antibodies against a variety of target antigens. However, enrichment of single chain variable fragment (scFv)-displaying phages is often hampered by the abundance of bacteriophages lacking antibody fragments. Moderate adhesive binding activities and production advantages of these "empty" phages results in their subsequent enrichment during selection on target cells. To date, very limited effort has been made to develop strategies removing nonspecific binding phages during the selection processes. To efficiently reduce insert-free phages when panning on intact cells, we increased the washing stringency by lowering the pH of the buffer with citric acid. Under standard washing procedures (pH 7.4), only approximately 73% of recovered phages were insert-free after three rounds of selection. Using stringent washing procedures (pH 5.0), approximately 12% of recovered phages contained no scFv. Using this protocol, we have cloned an antibody fragment from a mouse/human hybridoma cell line directed against the disialoganglioside GD2. This study confirms that selection of phage antibodies on cells is efficiently enhanced by assays augmenting the stringency to remove nonspecific binding phages.     

A novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p75NTR

The neurotrophin receptor p75^NTR is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75^NTR antibody or phage scFv library pre-panned against p75^NTR are internalized by neurons expressing p75^NTR; (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75^NTR antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75^NTR expression is upregulated in motor neurons in response to injury and in disease, the p75^NTR antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.     

Selection of tumor-specific internalizing human antibodies from phage libraries

Antibody internalization into the cell is required for many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes or viral DNA into cells. To generate directly tumor-specific internalizing antibodies, a non-immune single chain Fv (scFv) phage antibody library was selected on the breast tumor cell line SKBR3. Internalized phage were recovered from within the cell and used for the next round of selection. After three rounds of selection, 40 % of clones analyzed bound SKBR3 and other tumor cells but did not bind normal human cells. Of the internalizing scFv identified, two (F5 and C1) were identified as binding to ErbB2, and one (H7) to the transferrin receptor. Both F5 and H7 scFv were efficiently endocytosed into SKBR3 cells, both as phage antibodies and as native monomeric scFv. Both antibodies were able to induce additional functional effects besides triggering endocytosis: F5 scFv induces downstream signaling through the ErbB2 receptor and H7 prevents transferrin binding to the transferrin receptor and inhibits cell growth. The results demonstrate the feasibility of selecting internalizing receptor-specific antibodies directly from phage libraries by panning on cells. Such antibodies can be used to target a variety of molecules into the cell to achieve a therapeutic effect. Furthermore, in some instances endocytosis serves as a surrogate marker for other therapeutic biologic effects, such as growth inhibition. Thus, a subset of selected antibodies will have a direct therapeutic effect.     

Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules

Phage-display of antibody libraries is a powerful tool to select antibodies for specific epitopes. We describe a strategy for selecting highly specific scFv-clones that discriminate between various conformational states of cell surface receptors. This approach adapts the M13 pIII phage-display technology toward a cell suspension-based strategy, which allows panning against complex, multimeric, fully functional cell membrane epitopes without alteration of structure due to purification or immobilization. As the functional properties are preserved, phage can be specifically depleted or selected for neo-epitopes exposed after physiological alterations of the targeted molecules. This subtractive strategy allows highly specific selection for single-chain antibodies directed against functionally regulated epitopes on the cell surface molecules that can be tailored for diagnostic and therapeutic applications. Using this protocol, activation-specific single-chain antibodies can be obtained within 4-6 weeks.     

Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection

Much work has been done to develop tumor-targeting antibodies by selecting a phage antibody library on cancer cell lines. However, when tumor cells are removed from their natural environment, they may undergo genetic and epigenetic changes yielding different surface antigens than those seen in actual cases of cancer. We developed a strategy that allows selection of phage antibodies against tumor cells in situ on both fresh frozen and paraffin-embedded tissues using laser capture microdissection. By restricting antibody selection to binders of internalizing epitopes, we generated a panel of phage antibodies that target clinically represented prostate cancer antigens. We identified ALCAM/MEMD/CD166, a newly discovered prostate cancer marker, as the target for one of the selected antibodies, demonstrating the effectiveness of our approach. We further conjugated two single chain Fv fragments to liposomes and demonstrated that these nanotargeting devices were efficiently delivered to the interior of prostate cancer cells. The ability to deliver payload intracellularly and to recognize tumor cells in situ makes these antibodies attractive candidates for the development of targeted cancer therapeutics.     

用抗细胞表面分子单链抗体筛选和鉴别其相互作用肽

展示于噬茵体表面的肽库适于用抗体筛选其特异性结合表达肽。scFv-C193是一个抗KGla细胞表面分子的单链抗体,其抗原仍不清楚,为了筛选并鉴定其识别分子,用8cFv-C193筛选了1个展示于T7噬茵体表面的人胎肝cDNA片段库。经4轮生物吸附后分离单个阳性噬斑,并对其表达产物做电泳分析及斑点杂交。结果鉴别出1个scFv-C193结合肽,scFv-C193+克隆噬菌体DNA插入片段的PCR扩增和序列分析结果表明它是1个43bpDNA片段表达产物。BLAST分析EST人类基因数据库,发现它97％相同于CD34₊造血干/祖细胞mRNA的1-43bp,提示scFv-C193识别片段存在于人类造血干／祖细胞基因中,单克隆单链抗体scFv-C193可能用于研究这些人类基因的性质。     

Production and characterization of a humanized single-chain antibody against human integrin alphav beta3 protein

Anti-angiogenesis therapy is an emerging strategy for cancer treatment. This therapy has many advantages over existing treatments, such as fewer side effects, fewer resistance problems, and a broader tumor type spectrum. Integrin αvβ3 is a heterodimeric transmembrane glycoprotein that has been demonstrated to play a key role in tumor angiogenesis and metastasis. We have used a phage antibody display to humanize a mouse monoclonal antibody (mAb E10) against human integrin αvβ3 with a predetermined CDR3 gene. Three human phage antibodies were developed. Analysis of the humanized phage antibodies by phage ELISA revealed that the antibodies retained high antigen-binding activity and detected the same epitope as the parent mAb E10. A humanized single chain Fv (scFv) antibody was expressed in Escherichia coli in a soluble form. Analysis of the purified scFv indicated that it has the same specificity and affinity as the original mAb. Cell viability assays and xenograft model results suggested that the humanized scFv possesses anti-tumor growth activity in vitro and in vivo. This successful production of a humanized scFv with the ability to inhibit αvβ3-mediated cancer cell growth may provide a novel candidate for integrin αvβ3-targeted therapy.     

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.     

Targeted gene delivery to mammalian cells by filamentous bacteriophage.

We report that prokaryotic viruses can be re-engineered to infect eukaryotic cells resulting in expression of a reporter gene inserted into the bacteriophage genome. Phage capable of binding mammalian cells expressing the growth factor receptor ErbB2 and undergoing receptor-mediated endocytosis were isolated by selection of a phage antibody library on breast tumor cells and recovery of infectious phage from within the cell. As determined by immunofluorescence, F5 phage were efficiently endocytosed into 100 % of ErbB2 expressing SKBR3 cells. To achieve reporter gene expression, F5 phage were engineered to package the green fluorescent protein (GFP) reporter gene driven by the CMV promoter. These phage when applied to cells underwent ErbB2-mediated endocytosis leading to GFP expression. GFP expression occurred only in cells overexpressing ErbB2, was dose-dependent reaching, 4 % of cells after 60 hours and was detected with phage titers as low as 2.0 x 10(7) cfu/ml (500 phage/cell). The results demonstrate that bacterial viruses displaying the appropriate antibody can bind to mammalian receptors and utilize the endocytic pathway to infect eukaryotic cells, resulting in expression of a reporter gene inserted into the viral genome. This represents a novel method to discover targeting molecules capable of delivering a gene intracellularly into the correct trafficking pathway for gene expression by directly screening phage antibodies. This should significantly facilitate the identification of appropriate targets and targeting molecules for gene therapy or other applications where delivery into the cytosol is required. This approach can be adapted to directly select, rather than screen, phage antibodies for targeted gene expression. The results also demonstrate the potential of phage antibodies as an in vitro or in vivo targeted gene delivery vehicle.     

人转铁蛋白受体及其特异性抗体的制备

目的:从胎盘中提取转铁蛋白受体并获得抗转铁蛋白受体的抗体。方法:人新鲜胎盘组织被破碎后,用去污剂TritonX-100裂解细胞膜,释放膜蛋白。利用膜蛋白中的转铁蛋白受体能与铁-转铁蛋白复合物特异性结合的特性对其进行亲和纯化。对纯化得到的目的蛋白,经脱盐后进行ELISA及肽质量图谱分析,证明为所需的转铁蛋白受体后,以其包被免疫管,从全合成人源噬菌体抗体库中筛选抗体。结果:从人源噬菌体抗体库中筛选到5个能够与转铁蛋白受体特异性结合的噬菌体单链抗体。结论:以人源转铁蛋白受体为抗体,可从全人源噬菌体抗体库中筛选到其特异性的抗体。     

Enrichment of a single clone from a high diversity library of phage-displayed antibodies by panning with Anopheles gambiae (Diptera: Culicidae) midgut homogenate

A high diversity library of recombinant human antibodies was selected on complex antigen mixtures from midguts of female Anopheles gambiae Giles. The library of phage-displayed single chain variable region fragment constructs, derived from beta-lymphocyte mRNA of na?ve human donors, was repeatedly selected and reamplified on the insoluble fraction of midgut homogenates. Five rounds of panning yielded only one midgut-specific clone, which predominated the resulting antibody panel. In A. gambiae, the epitope was found throughout the tissues of females but was absent from the midgut of males. The cognate antigen proved to be detergent soluble but very sensitive to denaturation and could not be isolated or identified by Western blot of native electrophoresis gels or by immunoprecipitation. Nevertheless, immunohistology revealed that this sex-specific epitope is associated with the lumenal side of the midgut. Severe bottlenecking may limit the utility of phage display selection from na?ve libraries for generating diverse panels of antibodies against complex mixtures of antigens from insect tissues. These results suggest that the selection of sufficiently diverse antibody panels, from which mosquitocidal or malaria transmission-blocking antibodies can be isolated, may require improved selection methods or specifically enriched pre-immunized libraries.     

Exploiting recombination in single bacteria to make large phage antibody libraries

The creation of large phage antibody libraries has become an important goal in selecting antibodies against any antigen. Here we describe a method for making libraries so large that the complete diversity cannot be accessed using traditional phage technology. This involves the creation of a primary phage scFv library in a phagemid vector containing two nonhomologous lox sites. Contrary to the current dogma, we found that infecting Cre recombinase-expressing bacteria by such a primary library at a high multiplicity of infection results in the entry of many different phagemid into the cell. Exchange of Vh and Vl genes between such phagemids creates many new V h/Vl combinations, all of which are functional. On the basis of the observed recombination, the library is calculated to have a diversity of 3x1011. A library created using this method was validated by the selection of high affinity antibodies against a large number of different protein antigens.     

Phage displayed antibodies to heat stable alkaline phosphatase: framework region as a determinant of specificity

Human antibodies against specific targets of tumor cells are the most desirable molecules for possible immunotherapy. They could be developed by using the combinatorial antibody library displayed on a phage. We selected four human antibody fragments (scFv) binding to the oncoplacental antigen Heat Stable Alkaline Phosphatase (HSAP, the placental isozyme of alkaline phosphatase) from a synthetic human antibody library. Characterization of these scFvs showed they bound HSAP with moderate affinity but did not have isozyme specificity, as determined by binding to cell lines exhibiting differential expression of isozymes of alkaline phosphatase. The V(H) sequences of two of these scFvs were similar and although both bound to HSAP only one was cross-reactive with albumin. The sequences revealed a difference in the framework region (FR1) of these antibodies, indicating a role for this region in the determination of specificity. This is also significant considering that the heavy chains generated the diversity of the synthetic library used in this study, and only a single light chain showing binding to BSA was used for the entire library.     

噬菌体抗体库的构建及抗乳腺癌细胞单链抗体的筛选

构建抗人乳腺癌细胞MCF 7的噬菌体单链抗体库 ,从中筛选MCF 7细胞特异性单链抗体。用MCF-7细胞免疫BALB C小鼠 ,取脾脏 ,提取总RNA ,用RT-PCR技术扩增小鼠抗体重链 (V_H)和轻链 (V_L)可变区基因 ,经重叠PCR(SOE-PCR) ,在体外将V_H和V_{L连接成单链抗体 (scFv)基因 ,并克隆到噬菌粒载体pCANTAB5E中 ,电转化至大肠杆菌TG1,经辅助噬菌体超感染 ,构建噬菌体单链抗体库。从该抗体库中筛选特异性识别MCF-7细胞的噬菌体单链抗体 ,将表面展示单链抗体的单克隆噬菌体转化大肠杆菌TOP10进行可溶性表达。成功地构建了库容为12×10⁶ 的抗MCF-7乳腺癌细胞的单链抗体库 ,初步筛选到了与MCF 7细胞特异性结合的scFv,Westernblot检测表明 ,在大肠杆菌TOP10中实现了单链抗体可溶性表达}