Site-Specific Conjugation of an Enzyme and an Antibody Fragment

Werlen, R C; Lankinen, Mervi; Rose, Keith; Blakey, David C.; Shuttleworth, Helen; Melton, Roger G.; Offord, Robin E.

doi:10.1021/bc00029a006

Cited by 25 publications

(17 citation statements)

References 11 publications

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“…24 Thus, all the protein are expected to be immobilized in an ''end-on'' orientation on the surface. This condition is fulfilled by the enzymatic degradation of the whole protein leading to monovalent FabЈ fragments prepared with a single thiol group ͑noted HS-FabЈ͒, which are biologically active in solution.…”

Section: B Fabј Immobilization Through Single Sulphydrylmentioning

confidence: 99%

Covalent immobilization of immunoglobulins G and Fab′ fragments on gold substrates for scanning force microscopy imaging in liquids

Droz¹

1996

Journal of Vacuum Science &Amp; Technology B: Microelectronics and Nanometer Structures Processing, Measurement, and Phenomena

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Scanning force microscopy offers the possibility of observing protein molecules under liquid environment. The main difficulty in obtaining reproducible images is given by the low adhesion of the molecules to the substrate. Physisorbed molecules are displaced by the scanning tip or are resuspended in the medium. We have therefore performed a covalent immobilization of immunoglobulin G ͑IgG͒ or its monovalent FabЈ fragment on gold surfaces thanks to thiol groups. For this purpose, multiple thiol groups were chemically introduced into the IgG molecule by treatment with Traut's reagent. As an alternative, for a FabЈ fragment, we prepared molecules with a single thiol group located close to the C terminus of the truncated heavy chain. Both immobilization techniques enable us to observe clearly discernable individual molecules in liquid media. The grafting of FabЈ fragments on gold surface open new opportunities to study protein interactions.

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Section: B Fabј Immobilization Through Single Sulphydrylmentioning

confidence: 99%

Covalent immobilization of immunoglobulins G and Fab′ fragments on gold substrates for scanning force microscopy imaging in liquids

Droz¹

1996

Journal of Vacuum Science &Amp; Technology B: Microelectronics and Nanometer Structures Processing, Measurement, and Phenomena

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“…First, we needed antibodies that could operate on carbamates derived from L-glutamic acid (Fig. 2) to permit direct comparisons of antibody performance with those of the bacterial CPG2 exopeptidase enzyme previously reported to convert this prodrug and give antitumor activity (10). Second, the locus of the nitrogen mustard in prodrug 1 was selected to define the position of the linker arm in order to minimize prodrug alkylation of the abzymes.…”

mentioning

confidence: 99%

Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Wentworth¹,

Datta²,

Blakey³

et al. 1996

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

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Antibody-directed enzyme prodrug therapy, ADEPT, is a recent approach to targeted cancer chemotherapy intended to diminish the nonspecific toxicity associated with many commonly used Antibody-directed enzyme prodrug therapy, ADEPT, has been developed to overcome the unwanted nonspecific toxicity associated with anticancer agents (1-5). There are two components to such therapy: an antibody-enzyme conjugate and an anticancer prodrug of low toxicity. The conjugate is administered first and accumulates predominantly at the tumor site through antibody binding to tumor-associated antigenic determinants. Once the conjugate has been cleared from the plasma, the prodrug is administered to the patient. Cleavage of the prodrug to generate the active cytotoxic agent by the enzyme component of the conjugate occurs selectively at the tumor site and so leads both to enhanced efficacy of the anticancer agent and to reduced peripheral cytotoxicity (Fig. 1).To diminish the extent of peripheral hydrolysis of the prodrug, the enzyme component selected has commonly been of bacterial origin in order to achieve the necessary degree of specificity of activation (3): a human enzyme would be less specific. Unfortunately, such a choice imposes a dose-limiting immunogenicity on the ADEPT conjugate which could reduce the clinical potential of this therapy (6).Antibodies generated against appropriate transition-state analogues (TSAs) can catalyze a variety of chemical transfor-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. mations (7,8). Furthermore, murine antibodies can be "humanized" by existing technologies to reduce their inherent in vivo mammalian immunogenicity (9). These concepts have been juxtaposed by Bagshawe (6), who has proposed that humanized catalytic antibodies ("abzymes") could replace the enzyme component of ADEPT in an improved targeted therapy. We here demonstrate the feasibility of that proposition, which we have named antibody-directed abzyme prodrug therapy, ADAPT. We have identified an antibody that can efficiently hydrolyze a carbamate prodrug and so effect cytotoxicity in vitro. This provides a major extension to the potential for application of catalytic antibodies to clinical therapy.Our TSA design takes account of three requirements. First, we needed antibodies that could operate on carbamates derived from L-glutamic acid (Fig. 2) to permit direct comparisons of antibody performance with those of the bacterial CPG2 exopeptidase enzyme previously reported to convert this prodrug and give antitumor activity (10). Second, the locus of the nitrogen mustard in prodrug 1 was selected to define the position of the linker arm in order to minimize prodrug alkylation of the abzymes. Third, we chose to promote the disfavored BAC2 mechanisms of carbamate hydrolysis rather than the spontaneous E1cB process to obtain the maximum catalytic rate enhancement f...

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“…Yield: 2.5 g, 50%. Synthesis of tris(2-(4-formylbenzamide) ethyl) amine [21][22][23][24] TAA (243.7 mg, 5/3 mmol) was completely dissolved in 25 mL DMSO. The reaction at room temperature was initiated by adding another 25 mL DMSO solution of N-succinimidyl 4-formylbenzoate (1.236 g, 5 mmol).…”

Section: Synthesis Of N-succinimidyl 4-formylbenzoate 20mentioning

confidence: 99%

Synthesis and self-assembly of nonamphiphilic hyperbranched polyoximes

et al. 2012

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Nonamphiphilic hyperbranched polyoximes (HPOXs) were successfully synthesized by the polycondensation of trialdehyde and bis-aminooxy monomers with different molar feeding ratios.Various characterization techniques, such as 1D and 2D nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and multi-detector gel permeation chromatography (GPC) were used to identify the highly branched structure of the HPOXs. Despite there being no amphiphilic block segments, HPOXs with a torispherical structure could self-assemble into nanoparticles in a mixed solution of dimethyl sulfoxide and H 2 O. Besides, the modulation of the degree of branching (DB) and the terminal groups resulted in the appearance of spherical and bowl-shaped morphologies due to the change of the intra-and inter-molecular interactions. Accordingly, dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), scanning electron microscopy (SEM), fluorescence spectroscopy (FL) together with ultraviolet and visible spectrometery (UV-vis) were employed to unravel the tentative mechanism of the formation of the HPOX selfassemblies. Moreover, dynamic oxime linkages and hydrogen bonds endowed the HPOX nanoparticles with pH and thermal dual responsiveness, which was confirmed by TEM measurements. HPOXs are developed to offer a novel pathway for the design of nonamphiphilic self-assemblies with dual responsiveness.

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Site-Specific Conjugation of an Enzyme and an Antibody Fragment

Cited by 25 publications

References 11 publications

Covalent immobilization of immunoglobulins G and Fab′ fragments on gold substrates for scanning force microscopy imaging in liquids

Covalent immobilization of immunoglobulins G and Fab′ fragments on gold substrates for scanning force microscopy imaging in liquids

Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Synthesis and self-assembly of nonamphiphilic hyperbranched polyoximes

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