Deriving Topological Constraints from Functional Data for the Design of Reagentless Fluorescent Immunosensors

Renard, Martial; Belkadi, Laurent; Bedouelle, Hugues

doi:10.1016/s0022-2836(02)01334-7

Cited by 24 publications

(22 citation statements)

References 35 publications

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“…This suggests that the 2 PS on the scFv are well-placed for coupling, but their positioning adversely affects their photophysics leading to insubstantial cell kills. Research by Bedouelle and coworkers 39 has also shown that the position of fluorophores coupled to scFvs is critical to the function of immunosensors with quenching by protein-protein interaction being required for an optical readout. The IC 50 s are expressed in terms of scFv protein concentration and effective photosensitizer concentration.…”

Section: Discussionmentioning

confidence: 99%

Targeted photodynamic therapy with multiply‐loaded recombinant antibody fragments

Bhatti

Yahioglu

Milgrom

et al. 2007

Intl Journal of Cancer

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Current photodynamic therapy (PDT) of cancer is limited by inefficiencies involved in specifically targeting photosensitizers to tumors. Although antibodies are being explored as targeting vehicles, they present significant challenges, particularly in terms of pharmacokinetics and drug-coupling. We describe here a novel and effective system to covalently attach multiple photosensitizer molecules (both preclinical, pyropheophorbide-a and clinically approved, verteporfin photosensitizers) to single-chain Fvs. Further, we demonstrate that not only do the resulting photoimmunoconjugates retain photophysical functionality, they are more potent than either free photosensitizer, effectively killing tumor cells in vitro and in vivo. For example, treatment of human breast cancer xenografts with a photoimmunoconjugate comprising an anti-HER-2 scFv linked to 8-10 molecules of pyropheophorbide-a leads to significant tumor regression. These results give an insight into the important features that make scFvs good carriers for PDT drugs and provide proof of concept of our unique approach to targeted photodynamic therapy (tPDT). This promises to significantly improve on current photodynamic therapies for the treatment of cancer. ' 2007 Wiley-Liss, Inc.Key words: photodynamic therapy; single chain Fv; pyropheophorbide-a; verteporfin Photodynamic therapy (PDT) is a minimally invasive procedure used in a range of conditions where superficially localized lesions such as age-related macular degeneration (AMD) or tumors need to be treated. 1 PDT is typically a 2-step process that involves the administration of a photosensitizer (PS) leading to marginal accumulation in the tumor. Following this, the PS is activated by exposure to light of an appropriate wavelength. This ultimately leads to the conversion of molecular oxygen into reactive oxygen species (ROS), primarily singlet oxygen, leading to tumor cell death via irreversible damage to cellular components such as proteins, lipids and DNA. 2 Current clinical use of PDT achieves efficacies similar to conventional therapies but with lower morbidity, simplicity of use and improved functional and cosmetic outcome. 3,4 PDT has mainly been used where conventional approaches have failed or were unsuitable. These include premalignant dysplastic lesions and noninvasive cancers, which are commonly found in the mucosa of the aerodigestive 5 and urinary tracts. 6 Success in treating these types of cancers has been achieved using Photofrin 1 , 7 Levulan 18 and Foscan 1 . 9 The most successful application of PDT has been for wet age-related macular degeneration (AMD) for which the photosensitizer verteporfin (Visudyne 1 ) has been used to destroy ocular neovasculature. 10 PDT has also had great successes in dermatology because of its impressive cosmetic outcome. Methyl 5-aminolaevulinate (Metvix 1 ) has been used to treat actinic keratosis with up to 90% cure. 11 Although PSs accumulate in cancer cells, the tumor specificity ratios are low and their inherent hydrophobicity, causes them to persist...

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Section: Discussionmentioning

confidence: 99%

Targeted photodynamic therapy with multiply‐loaded recombinant antibody fragments

Bhatti

Yahioglu

Milgrom

et al. 2007

Intl Journal of Cancer

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“…Selection of peptides from a random library by the method of phage display has shown that residues 306-314 of gpE from the DENV1 serotype (gpE.DEN1) constitute a mimotope of the mAb4E11 epitope (Thullier et al, 2001). Its paratope has been partially characterized by alanine-scanning mutagenesis of its third hypervariable loops (Bedouelle et al, 2006;Renard et al, 2003). This mutagenesis study of the Fab fragment of mAb4E11 has shown that the two residues that come from its diversity segment, H-Trp96 and H-Glu97 (Kabat's numbering), together provide .85 % of the free energy of interaction with the ED3.DEN1 antigen.…”

Section: Introductionmentioning

confidence: 99%

Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus

Lisova

Hardy

Petit

et al. 2007

Journal of General Virology

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Dengue is caused by a taxonomic group of four viruses, dengue virus types 1-4 (DENV1-DENV4). A molecular understanding of the antibody-mediated protection against this disease is critical to design safe vaccines and therapeutics. Here, the energetic epitope of antibody mAb4E11, which neutralizes the four serotypes of DENV but no other flavivirus, and binds domain 3 (ED3) of their envelope glycoprotein, was characterized. Alanine-scanning mutagenesis of the ED3 domain from serotype DENV1 was performed and the affinities between the mutant domains and the Fab fragment of mAb4E11 were measured. The epitope residues (307-312, 387, 389 and 391) were at the edges of two distinct b-sheets. Four residues constituted hot spots of binding energy. They were aliphatic and contributed to form a hydrophobic pocket (Leu308, Leu389), or were positively charged (Lys307, Lys310). They may bind the diversity residues of mAb4E11, H-Trp96-Glu97. Remarkably, cyclic residues occupy and block the hydrophobic pocket in all unrelated flaviviruses. Transplanting the epitope from the ED3 domain of DENV into those of other flaviviruses restored affinity. The epitope straddles residues of ED3 that are involved in virulence, e.g. Asn/Asp390. These results define the epitope of mAb4E11 as an antigenic signature of the DENV group and suggest mechanisms for its neutralization potency. INTRODUCTIONDengue is a re-emerging viral disease. It is caused by four types of virus, dengue virus types 1-4 (DENV1-DENV4), which belong to the species Dengue virus in the genus Flavivirus (Heinz et al., 2000). They are transmitted by Aedes mosquitoes and infect between 50 million and 100 million persons each year. The disease generally takes a mild form, dengue fever, but severe forms, dengue haemorragic fever and dengue shock syndrome, have recently become epidemic (Gubler, 2002).The immune response against an infection by DENV involves both a humoral component, in the form of neutralizing antibodies, and a cell-mediated component (Guzman & Kouri, 2002). The preferential reactivation of the memory B cells that correspond to a primary infection, and an antibody-dependent enhancement of infection, might constitute triggering mechanisms of the severe forms during a secondary infection by a different viral serotype (Halstead, 2003; Mongkolsapaya et al., 2003). A molecular understanding of the events that lead to antibody neutralization, enhancement or escape is critical to the development of efficient and secure vaccines and therapeutics. DENV1-DENV4 are enveloped RNA viruses, like all flaviviruses. The structures of the whole virus and of the envelope glycoprotein E (gpE) have been solved by electron cryomicroscopy and X-ray crystallography (Modis et al., 2003(Modis et al., , 2005 Zhang et al., 2003). Ninety dimers of gpE cover the surface of the virus. Each monomer comprises three ectodomains, ED1-ED3, and a transmembrane segment. ED2 includes the dimerization interface, a glycosylation site and the peptide of fusion with the cellular membrane. ED3 is continuou...

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“…The values of f b and f c varied between different conjugates, 5.6-and 3.7-fold, respectively. These variations suggested that the accessibility of the fluorescent group to the solvent varied widely with its coupling site, as observed for another antibody (4). The values of ∆f c and ∆f c /f b were the highest for the three conjugates that we constructed from residues L-Tyr49, L-Ser93, and L-Thr94.…”

Section: Molar Fluorescences Of Singlementioning

confidence: 68%

Improving the Sensitivity and Dynamic Range of Reagentless Fluorescent Immunosensors by Knowledge-Based Design

Renard

Bedouelle

2004

Biochemistry

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The variable fragment (Fv) of an antibody can be transformed into a reagentless fluorescent biosensor by mutating a residue into a cysteine in the neighborhood of the paratope (antigen-binding site) and then coupling an environment-sensitive fluorophore, e.g., N- ((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester), to the mutant cysteine. For some residues, named operational, the formation of the conjugate does not affect the affinity of the Fv fragment for the antigen, and the binding of the antigen generates a measurable variation in the fluorescence intensity of the conjugate. We tested if this signal variation could be increased by coupling several molecules of fluorophores to the same molecule of Fv. Seven operational residues have been previously identified in the single-chain Fv (scFv) of monoclonal antibody D1.3 (mAbD1.3), directed against lysozyme. Ten double mutants of scFvD1.3, involving these residues, were constructed and coupled to the IANBD ester. The fluorescence of the double conjugates revealed a transfer of resonance energy between the two identical fluorescent groups. This homotranfer could be more important in the free state of the conjugate than in its antigenbound state and increase its sensitivity for the detection of the antigen by up to 2.9-fold. A poorly sensitive conjugate could be improved by coupling a second molecule of fluorophore to residues located far from the paratope. Mutations altering the affinity of scFvD1.3 for lysozyme were introduced into one of its fluorescent conjugates. Using a mixture of three mutant derivatives of this unique conjugate, we could titrate lysozyme with precision in a concentration range encompassing 3 orders of magnitude.A signal-transducing receptor generally comprises an extracellular domain that recognizes a specific molecular signal, a transmembrane domain through which the signal is transmitted to the interior of the cell and an intracellular domain through which the signal is transformed and amplified, eventually in the form of second messenger molecules. These three domains are assembled in a single-receptor macromolecule (1). Similarly, a biosensor transforms a specific molecular signal into an electrical signal and comprises several modules: a recognition module, which can be biological or biomimetic; a transduction module, which tranforms the recognition event into a measurable signal; and a module of data evaluation. The recognition and transduction modules are integrated into a compact device. A biosensor can function without additional reagent, provide quantitative analytical information, and follow the concentration of an analyte continuously. Important characteristics of a biosensor are its specificity and selectivity, the sensitivity, linearity and speed of the response, the dynamic range of the measurements, the possibility of calibration and accuracy (2).In a previous work, we have shown that it is possible to transform any antibody into a reagentless fluorescent immunosensor. The antibody is...

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Deriving Topological Constraints from Functional Data for the Design of Reagentless Fluorescent Immunosensors

Cited by 24 publications

References 35 publications

Targeted photodynamic therapy with multiply‐loaded recombinant antibody fragments

Targeted photodynamic therapy with multiply‐loaded recombinant antibody fragments

Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus

Improving the Sensitivity and Dynamic Range of Reagentless Fluorescent Immunosensors by Knowledge-Based Design

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