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Intracytoplasmic and nuclear staining of melanoma cells and fibroblasts with A10-5 Fab. Paraformaldehyde-fixed Mel-B melanoma cells (A) and FF2207 fibroblasts (B) were incubated on glass coverslips with biotinylated A10-5 Fab, followed by staining with FITC-labeled streptavidin. Cells were analyzed by confocal microscopy. Control 43 Fab did not stain the cells (not shown).
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The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma-associated Ags in patients. In this study, we isolated a recombinant phage-Fab clone (A10-5) from a phage-Fab library derived from the B cells o...
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A phagemid system was developed for the monovalent display of combinatorial antibody Fab libraries on the surface of filamentous phage M13. Fab fragments were fused to the carboxyl-terminal domain of the gene III protein. Phage displaying Fab fragments on their surface, or Phabs, were enriched by 10(3)- to 10(5)-fold on antigen-coated surfaces over...
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... Therefore, the phage display method appears to be an important complement to other robust target discovery tools, including genomic and proteomic approaches. Several research groups have used such a subtractive antibody phage display strategy for the identification of target-specific antigens expressed on human tumor cells, including melanoma (Cai and Garen, 1995; Li et al., 2001; Noronha et al., 1998), colorectal carcinoma (Topping et al., 2000), and lung adenocarcinoma (Ridgway et al., 1999) cell lines. Although subsequent identification of cognate antigens remains a significant bottleneck, these studies resulted in the discovery of novel cancer-specific cell surface targets. ...
In the post-genomic era, proteomics together with genomic tools have led to powerful new strategies in basic and clinical
research. These combined “omics” technologies are being integrated into the drug target discovery process. Unlike the genome,
the proteome is a highly dynamic entity that requires techniques capable of analyzing on selected populations of proteins
in specific biological conditions that reflect the proteins’ functional characteristics. Antibodies have become one of the
most important reagents for the analysis of selected populations of proteins, and the application of phage-display antibody
libraries to high-throughput antibody generation against large numbers of various antigens provides a tool for proteome-wide
protein expression analysis. In this review, we will discuss the utility of phage-display antibodies in proteomics applications,
specifically for the discovery of novel disease markers and therapeutic targets.
KeywordsProteomics–Phage display–Antibody–Mass spectrometry–Herapeutics
... Linkage of murine scFv molecules through a hinge region with human constant domains has been initially described by Hu and colleagues (Hu et al., 1996). Pioneering work demonstrated the proof of priniciple that such molecules retain functional properties (Hu et al., 1996; Shan et al., 1999; Hombach et al., 2000; Li et al., 2001). In the present study, based on the computer-guided homology modeling and molecular docking method, the 3D complex structure of Fv fragment from IgM-type murine antibody 1–28 was analyzed theoretically. ...
A novel murine IgM-type anti-human CD20 monoclonal antibody (mAb) 1-28 was prepared in our Lab, which can induce apoptosis and inhibit proliferation of Daudi and Raji cells. However, the efficacy of 1-28mAb in human cancer therapy is likely to be limited by human anti-mouse antibody responses. A chimeric antibody, C1-28, containing 1-28mAb variable region genes fused to human constant region genes (gamma 1, kappa) was constructed. However, C1-28 lost the antigen-binding activity. Here, using sequence similarity and known 3D structure of antibody variable regions as template, the spatial conformations of 1-28 variable regions (i.e. V(H) and V(L)) were analyzed with computer-guided homology modeling methods. According to the surface electrostatic distribution and interaction free energy analysis, the relationship between structure and stability of 1-28 variable regions was studied theoretically and a new chimeric anti-CD20 antibody scFv-Ig named 5S was designed. Expression level of 5S in the culture supernatant was determined to be around 50mug/mL using sandwich ELISA method with chimeric antibody Rituxan as reference. 5S retained its murine counterpart's binding activity by fluorescence-activated cell-sorting analysis. Furthermore, it could kill CD20 positive Daudi and Raji cells by complement-dependent cytotoxicity. For binding affinity often decreased even lost when IgM antibody was constructed into chimeric IgG1 form, our success give a hint about how to construct a IgG1-type chimeric antibody from IgM-type murine antibody to preserve its binding activity.
... [33][34][35] The power of the panning methodology was exemplified by retrieval of an antibody pool with near perfect selectivity (>99%) for target cells, with high functionality (86% apoptosis inducing), acceptable antibody diversity (9.7% unique clones) and high affinity (subnanomolar to nanomolar). These data compare favourably both to the isolation of antibodies from similarly designed antibody libraries, 5,[13][14][15] as well as to other types of libraries designed to promote isolation of antibodies with specificity for internalizing receptors. 7 A further prominent feature of the current screening methodology is its rapid nature. ...
Novel panning and screening methodology was devised to isolate high affinity human recombinant scFv antibody fragments with functionally associated properties in B lymphoma cells. The approach was used to generate a panel of apoptosis-inducing antibodies specific for antigens differentially expressed in B lymphoma vs. T leukaemia cells. The selections resulted in an antibody pool with near perfect selectivity (>99%) for the B lymphoma target cells. Randomly picked clones (72) revealed 7 unique antibody genotypes. Six of these rapidly induced apoptosis in target cells. Following the conversion to full IgGs, the antibodies were shown to be specific for HLA-DR/DP, the B-cell receptor mu chain and for CD54/ICAM-1. The latter receptor was not previously associated with apoptotic properties in B-cell lymphomas. Anti-ICAM-1 IgG induced apoptosis in a broad range of B lymphoma cell lines and were shown by immunohistochemistry to bind strongly to B lymphoma tissue obtained from 5 different B lymphoma patients. The recombinant IgG antibodies had affinities in the subnanomolar (0.3 nM) to nanomolar (3 nM) range. The described technology is generally applicable for the rapid isolation of high affinity human antibodies with specificity for differentially expressed cell surface receptors with intrinsic negative or positive signalling properties from naïve phage libraries.
... To overcome this problem, scaled-up methods using at least 10-fold more phage and cell material should be developed. Other methods that might be useful in the identification of phage-isolated proteins include the screening of target cell cDNA libraries against phage display-selected peptides (2,23) or peptide mass spectrometry (3). Such methods might become valuable tools for the identification of novel cell specific recep-tors via their ligands, peptides expressed by phage. ...
Cell-binding ligands for RG2 rat glioma were identified in our recent study from a library of peptides that are displayed as fusion molecules on phage particles. Here, one of the phage clones was used to affinity purify those cell membrane components to which the displayed peptides bind. This phage clone, displaying the ELRGDSLP peptide, was shown to recognize glioma cells specifically in comparison to control phage-expressing peptides of either similar or irrelevant sequences. Blocking experiments with synthetic RGDS peptide demonstrated that the phage-glioma cell recognition occurs via the RGD motif known to be present in many integrin-binding proteins. To form an affinity matrix that would bind to glioma cell membrane molecules, ELRGDSLP phage particles were cross-linked using dextran polymer. Whole cell lysate from RG2 rat glioma cells was passed through the matrix, resulting in the isolation of cell membrane components having strong affinity to the peptides on phage and molecules associated with those components. One of the isolated proteins was found to be CD44s, a cell surface adhesion molecule involved in glioma cell invasion and migration, which likely formed a complex with an RGD-binding integrin. Cell membrane proteins isolated with this innovative approach could be used for the design of cell-specific anticancer treatments.
... Although phage libraries have been used previously to identify tumor-specific antigens, the number of specific mAbs isolated has typically been small (20,23,34,35). Previously used antibody libraries were constructed in phagemid vectors that on average have only a single scFv molecule displayed on each phagemid particle. ...
The identification of tumor-specific cell surface antigens is a critical step toward the development of targeted therapeutics for cancer. The epitope space at the tumor cell surface is highly complex, composed of proteins, carbohydrates, and other membrane-associated determinants including post-translational modification products, which are difficult to probe by approaches based on gene expression. This epitope space can be efficiently mapped by complementary monoclonal antibodies. By selecting human antibody gene diversity libraries directly on the surface of prostate cancer cells, we have taken a functional approach to identifying fully human, tumor-specific monoclonal antibodies without prior knowledge of their target antigens. Selection conditions have been optimized to favor tumor-specific antibody binding and internalization. To date, we have discovered >90 monoclonal antibodies that specifically bind and enter prostate cancer cells, with little or no binding to control cells. These antibodies are able to efficiently deliver intracellular payloads when attached to nanoparticles such as liposomes. In addition, a subset of the antibodies displayed intrinsic antiproliferative activity. These tumor-specific internalizing antibodies are likely to be useful for targeted therapeutics either alone or in combination with effector molecules. The antigens they bind constitute a tumor-specific internalizing epitope space that is likely to play a significant role in cancer cell homeostasis. Targeting components of this epitope space may facilitate development of immunotherapeutic and small molecule-based strategies as well as the use of other therapeutic agents that rely upon delivery to the interior of the tumor cell.
... Many different selection methods have been described, including biopanning on immobilized antigen coated onto solid supports such as ELISA plates, immunotubes or magnetic beads (solid-phase panning) (Clackson et al., 1991;Griffiths et al., 1994;Duenas et al., 1996;Marks et al., 1991;Sawyer et al., 1997), selection in solution using biotinylated antigen (solution-phase pannin g) (Hawkins et al., 1992), panning on fixed prokaryotic cells (Bradbury et al., 1993) or on mammalian cells (Cai and Garen, 1995) including cultured cells (Li et al., 2001) and primary cells (Ditzel et al., 2000;Williams et al., 2002) , subtractive selection using sorting procedures, enrichment on tissue sections or pieces of tissue (de Kruif et al., 1995;Van Ewijk et al., 1997) and, in principle, selections using living animals (Trepel et al., 2002). Solid-phase panning and solution phase panning are commonly used if purified antigen is available. ...
Neutralizing antibodies play a major role in host defense against viral infections. Passive administration of antibodies specific for HIV-1 can protect monkeys from infections mediated by the HIV-1 envelope glycoprotein (Env) in a concentration dependent manner (Shibata et al., 1999; Baba et al., 2000; Ruprecht et al., 2001; Xu et al., 2002; Veazey et al., 2003; Burton, 2002; Ferrantelli and Ruprecht, 2002; Mascola et al., 1999; Mascola et al., 2000; Mascola, 2002; Parren et al., 2001). In some of these experiments human monoclonal antibodies (hmAbs) were used that exhibit potent and broad HTV neutralizing activity in vitro (Burton, 1997; Burton, 2002; Ferrantelli and Ruprecht, 2002). Recent clinical trials found that two of these broadly HIV neutralizing hmAbs (nhmAbs), 2F5 and 2G12, could produce a modest decrease in viral load without side effects in humans (Armbruster et al., 2002; Stiegler et al., 2002). However, the potency of 2F5 and 2G12 used in combination in this clinical trial was not sufficient to reduce the HIV-1 plasma RNA levels to the low levels observed after treatment with HAART (Stiegler et al., 2002). Increases in the potency of the currently available broadly HTV nhmAbs and the development of new neutralizing hmAbs might be helpful here although problems associated with neutralization escape are likely to be severe in any attempts to use antibodies therapeutically. Importantly, finding immunogens that are able to elicit broadly HIV nhmAbs could be facilitated by the exploration of the interaction of these antibodies with the Env-an approach known as “retrovaccinology” (Burton, 2002). However, only a few broadly cross-reactive HTV nhmAbs have been identified to date and efforts to use mimetics of their epitopes or portions of the epitopes as immunogens are ongoing but of limited success so far (Zwick et al., 2001a). The identification of new broadly cross-reactive HIV nhmAbs and their conserved epitopes is therefore of obvious importance for the development of effective HIV vaccines.
... Alternative screening strategies such as plaque lift assays [39] permit the exhaustive screening of small phage-expression libraries (diversity 10 6 ), and consequently may offer advantages for the discovery of human Abs to novel targets. Human Abs to melanoma antigens have been identified by screening phage-expression libraries constructed from B lymphocytes obtained from a patient who had been vaccinated with autologous melanoma cells [4, 5] or from a patient in remission following immunotherapy [22, 26] . It is likely that using phage-expressed Ab libraries enriched with melanoma-reactive Abs arising from the in vivo humoral response facilitated the identification of melanoma antigens. ...
A bacterially expressed human antibody (Ab) library (diversity approximately 10(5)) was generated from tumor-infiltrating B lymphocytes present in tissue isolated from a colon tumor. Immunoglobulin (IgG) heavy and light chain variable regions were amplified without isolating or enriching B cells, cloned into a phage-expression vector, and soluble antigen-binding fragment (Fabs) from >10(5) members of the library were screened rapidly by two distinct and complementary methodologies. In the first approach, soluble Fabs were screened by enzyme-linked immunosorbent assay (ELISA) on tumor cell monolayers. Alternatively, tumor cell surface antigens were selectively biotinylated with a plasma membrane-impermeable reagent, solubilized with non-ionic detergent, and were used to screen >10(5) members of the Ab library by capture lift. Reactive Fabs were partially characterized for tumor cell specificity and cross-reactivity, resulting in the identification of multiple Abs that bind cultured tumor cells but not normal human fibroblasts. The Fabs clustered into at least three distinct epitope specificity groups based on multiple criteria, including differential reactivity on two tumor cell lines and distinct antigen recognition patterns on western blot and immunoprecipitation. Moreover, DNA sequencing of the Ab variable regions demonstrated that the majority of the tumor-reactive Fabs were distinct and substantially different from the corresponding most homologous Ab germline gene. The relatively small size of the tumor-derived library allowed direct screening of soluble Fab of every member of the library, permitting the characterization of multiple human monoclonal antibodies (mAbs) that might not be discovered using alternative approaches, such as hybridoma technology or phage-display.
As a consequence of the invention of the hybridoma technology by Köhler and Milstein (1), many monoclonal antibodies (MAbs) have been evaluated in clinical trials since the early 1980s. Clinical outcomes were generally poor (2–5), with the notable exception of marked tumor responses, including long-term remissions of patients with malignant B-cell lymphoma who were treated with patient-specific antiidiotypic antibodies (6–8). The main factors responsible for these initial shortcomings were related to the immunogenicity of the murine protein, to modulation of targeted antigens, and to the poor ability of these antibodies to sufficiently mediate antibody-dependent effector functions in humans.
Objectives:
To construct a phage display human antibody library (PDHAL) against pneumoconiosis for the diagnosis and treatment of coal worker pneumoconiosis (CWP).
Methods:
The PDHAL was established via CWP blood and six positive antibodies were discovered. 867 coal workers (558 CWP and 309 without CWP) and 393 controls were recruited to validate the results.
Results:
A PDHAL against CWP was established, from which six strong positive clones were selected, sequenced and identified as VEGF, interleukin-18, HSP70, HER3, Gz-B and RF. Logistic regression analysis revealed that VEGF (OR (95% CI), 0.02 (0.01to 0.07), P < 0.05), RF-Ab (OR (95% CI): 0.46 (0.28 to 0.73), P < 0.05) and HSP70/HSP-70-Ab (OR (95% CI): 0.71 (0.53 to 0.95), P < 0.05) were protective factors for CWP after adjustment of confounding factors.
Conclusion:
The serum VEGF, RF-Ab and HSP-70/HSP-70 antibodies were potential biomarkers for diagnosis and treatment of CWP.
Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.
Copyright © 2015. Published by Elsevier B.V.
