Toward Selection of Internalizing Antibodies from Phage Libraries

Becerril, Baltazar; Poul, Marie‐Alix; Marks, James D.

doi:10.1006/bbrc.1999.0177

Cited by 143 publications

(100 citation statements)

References 27 publications

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“…Known methods for stabilizing Fv fragments are as singlechain Fvs (scFvs) (1,27,28) and as disulfide-stabilized Fvs (dsFvs) (29)(30)(31)). Yet, scFvs still are often unstable (29,32,33) and can have lower affinities compared with Fabs and whole IgG because the linker interferes with binding or does not sufficiently stabilize the heterodimer (30,34,35). Although the dsFvs generally are more stable and do not have a linker, they require the incorporation of a disulfide into the library construction.…”

Section: Discussionmentioning

confidence: 99%

Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays

Gao

Mao

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

151

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The gene VII protein (pVII) and gene IX protein (pIX) are associated closely on the surface of filamentous bacteriophage that is opposite of the end harboring the widely exploited pIII protein. We developed a phagemid format wherein antibody heavy-and light-chain variable regions were fused to the amino termini of pVII and pIX, respectively. Significantly, the fusion proteins interacted to form a functional Fv-binding domain on the phage surface. Our approach will be applicable to the display of generic peptide and protein libraries that can form combinatorial heterodimeric arrays. Consequently, it represents a first step toward artificial antibodies and the selection of novel biological activities.Now that combinatorial antibody libraries have been secured (1-9), a powerful next step is the evolution toward artificial antibody constructs. Artificial antibodies are defined here as protein motifs of large diversity that use the functional strategy of the antibody molecule, but can be free of loop and framework structural constraints. When reduced to its essence, the antibody molecule is a biological device for the presentation of a combinatorial array of peptide elements in threedimensional space. The essential feature is that while CDRs (complementarity-determining regions) cooperate to form a binding site, their interaction is dynamic and functional with little structural association between the CDRs themselves. In this way, the full complement of amino acid residues is available for antigen recognition at a minimum energetic cost for binding. We propose that the ability to control the combinatorial design not only of sequence space but also of three-dimensional space would recapitulate and ultimately transcend the natural design of the immune repertoire.Although phage display has been investigated intensively, many details of the phage particle itself have not been fully elucidated, and the possibility of alternative display formats also remain to be explored. The filamentous bacteriophage fd, and, similarly, M13, consists of a circular, single-stranded DNA molecule surrounded by a cylinder of coat proteins (Fig. 1). The molecular mass of a particle is about 1.6 ϫ 10 7 Da, of which 88% is protein and 12% is DNA (10). There are about 2,700 molecules of the major coat protein pVIII that envelope the phage. At one end of the particle, there are five copies each of gene VII and VI proteins (pIII and pVI) that are involved in host-cell binding and in the termination of the assembly process. The other end contains five copies each of pVII and pIX that are actually hydrophobic peptides of 33 and 32 aa, respectively, required for the initiation of assembly and for maintenance of virion stability. Whereas pIII, pVI, and pVIII have been used to display biological molecules, pVII and pIX have not been utilized (1, 11).Substantial information has been accumulated about the structural and the phage display characteristics of pIII and pVIII. Yet, the data concerning the minor proteins encoded by genes VII and IX are...

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Section: Discussionmentioning

confidence: 99%

Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays

Gao

Mao

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

151

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“…Several approaches have been reported for identification of cell markers such as selection using activated cell sorting (10,11), selection directly on cells in suspension (12), selections directly on adherent cells (13,14), and selection on tumor tissue sections (15). Recently another approach has been described enabling the identification of cell surface components capable of internalizing binding ligands (16).…”

Section: Molecular and Cellular Proteomics 2: 61-69 2003mentioning

confidence: 99%

Identification of Keratinocyte-specific Markers Using Phage Display and Mass Spectrometry

Jensen

Ravn

et al. 2003

Molecular & Cellular Proteomics

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Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which assures a high diversity of binders. The method demonstrates, for the first time, the ability to detect, identify, and analyze both secreted and membrane-associated extracellular proteins as well as a variety of different cellular structures including proteins and carbohydrates. The optimized phage display method was applied to analysis of human skin keratinocytes resulting in the isolation of a panel of antibodies. Fourteen of these antibodies were further characterized, half of which predominantly recognized keratinocytes in a screen of a range of different cell types. Three cognate keratinocyte antigens were subsequently identified by mass spectrometry as laminin-5, plectin, and fibronectin. The combination of phage display technology with mass spectrometry methods for protein identification is a general and promising approach for proteomic analysis of cell surface complexity.

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“…We observed this, and have isolated and identified such monoclonal anti-Polysorp scFvs (sequences shown in Table 4). This problem has also been found by using large na ve libraries for panning against complex antigen targets, such as cells [37][38][39]. Table 4.…”

Section: Cdrl3mentioning

confidence: 95%

An Efficient Method for Isolating Antibody Fragments Against Small Peptides by Antibody Phage Display

Zhi¹,

Siegumfeldt²

2010

CCHTS

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Abstract:We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1 -casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do not always bind efficiently to passive adsorption surfaces, and we developed a simple method to quantify the binding capacity of surfaces with the peptides. Background binding (the binding of scFvs to the background matrix) is an obstacle for successful selection, and we evaluated two methods that drastically reduced the background binding. An optimized method therefore enabled a panning procedure where the specific (peptide binding) scFv-phages were always dominant. Using 15-mer peptides immobilized on Nunc Immobilizer Streptavidin plates, we successfully generated scFvs specifically against them. The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides.

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Toward Selection of Internalizing Antibodies from Phage Libraries

Cited by 143 publications

References 27 publications

Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays

Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays

Identification of Keratinocyte-specific Markers Using Phage Display and Mass Spectrometry

An Efficient Method for Isolating Antibody Fragments Against Small Peptides by Antibody Phage Display

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