Phage antibodies: filamentous phage displaying antibody variable domains

McCafferty, John; Griffiths, Andrew D.; Winter, Greg; Chiswell, David J.

doi:10.1038/348552a0

Cited by 2,177 publications

(1,065 citation statements)

References 17 publications

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“…However, in the last 2-3 years we and others utilized the selection power of the phage display approach [51][52][53][54] together with the availability of very large human naive antibody libraries. These enabled us to screen these large libraries of such vast diversity and size in order to isolate these rare and unique TCR-like recombinant antibodies targeting human tumor and viral epitopes [21,22,[24][25][26].…”

Section: Discussionmentioning

confidence: 99%

Targeting TARP, a novel breast and prostate tumor‐associated antigen, with T cell receptor‐like human recombinant antibodies

et al. 2008

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MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/ peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptidespecific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRc alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/ TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes. Key words: Immunotoxin Á MHC Á Recombinant antibody Á T-cell receptor IntroductionMost patients with metastatic prostate and breast cancers are provided with the limited benefits from standard chemo-and hormone-based therapies. During the recent years, much effort has been invested in developing new approaches, such as immunotherapy, to improve the therapeutic abilities, by combining the tumor specificity of cell-mediated immunity with the freedom from toxic chemotherapies.Recent immunotherapy approaches employ the principle that CD8 + CTL recognize and kill tumor cells that display peptides from tumor-associated antigens presented by MHC class I molecules. Several tumor antigens and HLA allele-specific peptides from prostate cancer-associated antigens have been identified as CD8 + T cell epitopes, including HLA-A2-binding peptides derived from prostate-specific antigen (PSA) [1,2], prostate-specific membrane antigen (PSMA) [3], prostate stem cell antigen (PSCA) [4,5], and prostate acid phosphatase 6, which are all now components of current vaccine trials [5][6][7][8][9][10][11].Identification of new tumor-specific antigens is an essential step for the successful development of immunotherapy ap- [12][13][14]. One of these, T cell receptor gamma alternate reading frame protein (TARP), is expressed on breast and prostate cancer cells [15,16]. It was shown that TARP was expressed on >90% of cancer specimens examined [9,15]. In order to define new breast and prostate CD8 + T cell tumor antigens, two wild-type HLA-A2 epitopes from TARP were identified [17]. The wild-type sequences were also improved by sequence modifications to produce epitopeenha...

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Section: Discussionmentioning

confidence: 99%

Targeting TARP, a novel breast and prostate tumor‐associated antigen, with T cell receptor‐like human recombinant antibodies

et al. 2008

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“…Here, the genotype and phenotype of peptides were linked by fusing their short gene fragments to the minor coat protein III gene of the filamentous bacteriophage M13, resulting in the expression of this fusion protein on the surface of phage which allowed affinity purification of the gene by peptide binding. In a similar way, antibody fragments, fused to pIII, can be presented on the surface of phage M13 (McCafferty et al, 1990;Barbas et al, 1991;Breitling et al, 1991;Clackson et al, 1991;Hoogenboom et al, 1991). Regardless the production and presentation of complete IgG molecules (including the Fc part) are not possible due to limitations of the folding machinery of E. coli, this is not necessary as the Fv-part of an IgG provides the complete antigen specificity and can be easily produced in E. coli.…”

Section: Phage Displaymentioning

confidence: 99%

Perspectives for systematic in vitro antibody generation

Konthur

Hust

Dübel

2005

Gene

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After the completion and refinement of the human genome, the characterization of individual gene products in respect of their functions, their modifications, their cellular localization and regulation in both space and time has generated an increased demand for antibodies for their analysis. Taking into account that the human genome contains¨25,000 genes, and that their products are found in different splice variants and produce proteins with post-translational modifications, it can be estimated that at least 100,000 different protein products have to be investigated to gain a complete picture of what's going on in the proteome of a cell.Antibodies are preferred tools helping with the characterization and detection of proteins as well as with elucidating their individual functions. The generation of antibodies to all available human protein products by immunization and/or the hybridoma technology is not only logistically and financially enduring, but may prove to be a difficult task, as quite a number of interesting targets may evade the immune response of experimental animals, for example, allosteric variants dependent on fragile interactions to cofactors, highly conserved antigens etc. For this reason, alternative methods for the generation of antibodies have to supplement these approaches. In vitro methods for antibody generation are seen to offer this capability. In addition, they may provide a cost effective and large scale production alternative for detection reagents for the research community in their own right. Among in vitro techniques, phage display has been evolved as the most efficient option for tackling this problem and approaches optimised for automation are emerging.Maximum benefit for proteomic research could be generated by judicious and preferably international coordination of the ongoing efforts to combine the strengths of the well established animal based approaches and the novel opportunities offered by in vitro methods. D

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“…Phage display of antibody fragments has been widely used as a platform for rapid identification of antibody fragments that bind to targets with therapeutic, diagnostic, and research reagent applications. [20][21][22][23][24] These libraries have been engineered to display the highly variable antigen-binding regions of human immunoglobins: the hypervariable domain of the light chain (V L ) is linked to that of the heavy chain (V H ) to form a scFv of V L -linker-V H . 25 The complementarity determining regions (CDRs) in both V L and V H regions determine the scFv specificity.…”

Section: Introductionmentioning

confidence: 99%

Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

et al. 2011

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We have screened a human immunoglobulin single-chain variable fragment (scFv) phage library against the C-terminal tetramerization regions of erythroid and nonerythroid beta spectrin (bI-C1 and bII-C1, respectively) to explore the structural uniqueness of erythroid and nonerythroid b-spectrin isoforms. We have identified interacting scFvs, with clones ''G5'' and ''A2'' binding only to bI-C1, and clone ''F11'' binding only to bII-C1. The K d values, estimated by competitive enzyme-linked immunosorbent assay, of these scFvs with their target spectrin proteins were 0.1-0.3 lM. A more quantitative K d value from isothermal titration calorimetry experiments with the recombinant G5 and bI-C1 was 0.15 lM. The a-spectrin fragments (model proteins), aI-N1 and aII-N1, competed with the bI-C1, or bII-C1, binding scFvs, with inhibitory concentration (IC 50 ) values of~50 lM for aI-N1, and 0.5 lM for aII-N1. Our predicted structures of bI-C1 and bII-C1 suggest that the Helix B 0 of the Cterminal partial domain of bI differs from that of bII. Consequently, an unstructured region downstream of Helix B 0 in bI may interact specifically with the unstructured, complementarity determining region H1 of G5 or A2 scFv. The corresponding region in bII was helical, and bII did not bind G5 scFv. Our results suggest that it is possible for cellular proteins to differentially associate with the C-termini of different b-spectrin isoforms to regulate a-and b-spectrin association to form functional spectrin tetramers, and may sort b-spectrin isoforms to their specific cellular localizations.Keywords: beta spectrin; erythroid and nonerythroid; scFv; structural difference; affinity difference Abbreviations: aI-N1, a recombinant protein of aI-spectrin segment with residues 1-156; aI-spectrin, erythroid a-spectrin; aII-N1, a GST fusion protein of aII-spectrin segment with residues 1-147; aII-spectrin, nonerythroid a-spectrin; bI-C1, a GST fusion protein of bI-spectrin segment with residues 1898-2083; bI-C1D, a GST fusion protein of bI-spectrin segment with residues 1898-2070, that is, the residues 2071-2083 in bI-C1 were deleted; bI-spectrin, erythroid b-spectrin; bII-C1, a GST fusion protein of bII-spectrin segment with residues 1906-2093; bII-spectrin, nonerythroid b-spectrin; CD, circular dichroism; CDRs, complementarity determining regions; ELISA, enzyme-linked immunosorbent assay; EPR, electron paramagnetic resonance; GST, glutathione S-transferase; HRP, horseradish peroxidase; ITC, isothermal titration calorimetry; PBS, phosphate buffered saline at pH 7.4; rG5, recombinant G5, a single-chain variable fragment found to bind bI-C1; scFv, single-chain variable fragment.Yuanli Song's current address is

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Phage antibodies: filamentous phage displaying antibody variable domains

Cited by 2,177 publications

References 17 publications

Targeting TARP, a novel breast and prostate tumor‐associated antigen, with T cell receptor‐like human recombinant antibodies

Targeting TARP, a novel breast and prostate tumor‐associated antigen, with T cell receptor‐like human recombinant antibodies

Perspectives for systematic in vitro antibody generation

Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

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