Human antibodies by design

Vaughan, Tristan J.; Osbourn, Jane; Tempest, Philip R.

doi:10.1038/nbt0698-535

Cited by 141 publications

(79 citation statements)

References 53 publications

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“…In contrast, the folding of larger proteins tends to be multistate (17), which would likely preclude the sharp on͞off transition required for our approach. Despite these limitations, however, the availability of well established immunochemical and in vitro selection techniques (4)(5)(6)28) for the production of novel, single-domain proteins of arbitrary specificity suggests that our approach to protein-based optical biosensors may be fairly general.…”

Section: Discussionmentioning

confidence: 99%

Engineering a signal transduction mechanism for protein-based biosensors

Kohn

Plaxco

2005

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

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Hybridization-induced conformational changes have been successfully used in biosensors for the transduction of DNA-binding events into readily observable optical or electronic signals. Similar signal transduction has not, however, proven of equal utility in proteinbased biosensors. The discrepancy arises because, unlike ssDNA, most proteins do not undergo significant conformational changes upon ligand binding. Here, we describe a solution to this problem. We show that an arbitrarily selected, normally well folded protein can be rationally engineered such that it undergoes ligand-induced folding. The engineered protein responds rapidly (milliseconds) and selectively to its target, and it couples recognition with the largest possible conformational change: folding. These traits suggest that ligand-induced folding could serve as an ideal signaltransduction mechanism. Consistent with this claim, we demonstrate a label-free optical biosensor based on the effect that is sufficiently selective to detect its target even in complex, contaminant-ridden samples such as blood serum.fluorescence ͉ molecular beacon ͉ natively unfolded ͉ quenching

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Section: Discussionmentioning

confidence: 99%

Engineering a signal transduction mechanism for protein-based biosensors

Kohn

Plaxco

2005

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

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“…First, the primer design was optimized for amplification of variable region gene pools to maintain maximum diversity. Second, the efficiency of scFv gene assembly was increased by exploiting the presence of the DNA encoding the (G 4 S) 3 peptide linker included at the end of the 3Ј-primer of the V H gene and 5Ј-primer of the V L gene. This design allowed us to assemble scFv genes from only two DNA fragments.…”

Section: Construction Of the Scfv Librarymentioning

confidence: 99%

“…Both pIII and pVIII have been used to display peptide and antibody libraries (1)(2)(3)(4)(5)(6)(7)(8), and pVI has been used to display fusion proteins at its C terminus that makes it amenable for the display of cDNA-encoded libraries (25)(26)(27). Significantly, large, nonimmune or ''naïve'' antibody-phage libraries displayed on pIII have proven to be a general method to readily isolate high-affinity and specific human antibodies against a variety of target antigens (28)(29)(30)(31)(32).…”

mentioning

confidence: 99%

“…ombinatorial antibody library technology represents a powerful tool for discovering and designing antibodies that bind targets with high affinity and specificity (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Antibody phagedisplay libraries obviate the need for immunization and the concomitant laborious hybridoma protocols for obtaining mAbs, directly afford the cloned antibody genes in single-chain Fv (scFv) or Fab format for convenient manipulation, and, importantly, can be derived from the human antibody repertoire.…”

mentioning

confidence: 99%

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A method for the generation of combinatorial antibody libraries using pIX phage display

Gao

Mao

Kaufmann

et al. 2002

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

117

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For more than a decade, phage displayed combinatorial antibody libraries have been used to generate and select a wide variety of antibodies. We previously reported that the phage coat proteins pVII and pIX could be used to display the heterodimeric structure of the antibody Fv region. Herein, aspects of this technology were invoked and extended to construct a large, human single-chain Fv (scFv) library of 4.5 ؋ 10 9 members displayed on pIX of filamentous bacteriophage. Furthermore, the diversity, quality, and utility of the library were demonstrated by the selection of scFv clones against six different protein antigens. Notably, more than 90% of the selected clones showed positive binding for their respective antigens after as few as three rounds of panning. Analyzed scFvs were also found to be of high affinity. For example, kinetic analysis (BIAcore) revealed that scFvs against staphylococcal enterotoxin B and cholera toxin B subunit had a nanomolar and subnanomolar dissociation constant, respectively, affording affinities comparable to, or exceeding that, of mAbs obtained from immunization. High specificity was also attained, not only between very distinct proteins, but also in the case of the Ricinus communis (''ricin'') agglutinins (RCA60 and RCA120), despite >80% sequence homology between the two. The results suggested that the performance of pIX-display libraries can potentially exceed that of the pIII-display format and make it ideally suited for panning a wide variety of target antigens.

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“…However, murine monoclonal antibodies can cause serious immune responses in patients, which has led to the study of antibody humanization technologies (2). ReoPro is the first humanized antibody that was authorized for clinical treatment (3).…”

Section: Introductionmentioning

confidence: 99%

Screening for antibodies against intact cancer cells with a naï¿½ve large phage antibody library

Zhou²,

Wang³

et al. 2011

Int J Mol Med

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Abstract. Large phage antibody libraries are expected to be an efficient technology for obtaining humanized anti-tumor antibodies. However, few reports have been concerned about the selection strategies for intact cancer cells as targets. In this study, a 2x1011 large naïve scFv library from blood B lymphocytes of normal adult donors and neonatal cord blood was constructed using the LoxP-cre system. Three human esophageal cancer cell lines were equally mixed for use as the target. These intact cells were divided into two groups, PF and NF, according to the treatment method of the cells (fixed with 2% PFA or not, respectively). Positive phage antibodies were identified following 4 panning cycles of adhesion, elution and amplification. Using a cell ELISA assay, it was found that the additional procedure of directly infecting XL1-Blue bacteria with the cell pellet following washing with an acidic solution can effectively decrease the loss of positive phage, yielding a positive screening rate of 11.6% (61/525). Most of the phage antibodies displayed binding activity with all three esophageal cancer cell lines. Moreover, other phages (such as NFc53a and NFc70a) appeared to be specific for certain cell lines. Regarding the method used to treat the target cells, both the positive screening rate and the genetic diversity of the antibody variable region were significantly higher in the NF group (12.9 and 71.4%, respectively) than in the PF group (10.5 and 14.2%, respectively). Immunohistochemical analysis demonstrated that the scFv phages have good specificity for esophageal cancer. This technology is helpful for developing small molecular tracers and targeted therapies for malignant tumors.

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Human antibodies by design

Cited by 141 publications

References 53 publications

Engineering a signal transduction mechanism for protein-based biosensors

Engineering a signal transduction mechanism for protein-based biosensors

A method for the generation of combinatorial antibody libraries using pIX phage display

Screening for antibodies against intact cancer cells with a naï¿½ve large phage antibody library

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