Abstract 2396: Development of a new generation of dimeric, highly cytotoxic fusion proteins containing active GrB and VEGF targeting tumor neovasculature: Incorporating IgG heavy chain Fc fragments improves cytotoxicity, stability and pharmacokinetics

The Kar3 protein (Kar3p), a protein related to kinesin heavy chain, and the Cik1 protein (Cik1p) appear to participate in the same cellular processes in S. cerevisiae. Phenotypic analysis of mutants indicates that both CIK1 and KAR3 participate in spindle formation and karyogamy. In addition, the expression of both genes is induced by pheromone treatment. In vegetatively growing cells, both Cik1::beta-gal and Kar3::beta-gal fusions localize to the spindle pole body (SPB), and after pheromone treatment both fusion proteins localize to the spindle pole body and cytoplasmic microtubules. The dependence of Cik1p and Kar3p localization upon one another was investigated by indirect immunofluorescence of fusion proteins in pheromone-treated cells. The Cik1p::beta-gal fusion does not localize to the SPB or microtubules in a kar3 delta strain, and the Kar3p::beta-gal fusion protein does not localize to microtubule-associated structures in a cik1 delta strain. Thus, these proteins appear to be interdependent for localization to the SPB and microtubules. Analysis by both the two-hybrid system and co-immunoprecipitation experiments indicates that Cik1p and kar3p interact, suggesting that they are part of the same protein complex. These data indicate that interaction between a putative kinesin heavy chain-related protein and another protein can determine the localization of motor activity and thereby affect the functional specificity of the motor complex.

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Bactericidal Effects of a Fusion Protein of Llama Heavy-Chain Antibodies Coupled to Glucose Oxidase on Oral Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.9.3390-3395.2004 ◽

2004 ◽

Vol 48 (9) ◽

pp. 3390-3395 ◽

Cited By ~ 20

Author(s):

A. Szynol ◽

J. J. de Soet ◽

E. Sieben-van Tuyl ◽

J. W. Bos ◽

L. G. Frenken

Keyword(s):

Fusion Protein ◽

Glucose Oxidase ◽

Heavy Chain ◽

Fusion Proteins ◽

Antimicrobial Agents ◽

Oral Care ◽

Oral Bacteria ◽

Reverse Transcription Pcr ◽

Bactericidal Effects ◽

Variable Domains

ABSTRACT Enzymes such as lactoperoxidase and glucose oxidase (GOx) are used as antimicrobial agents in oral care products. Their low specificities and substantiveness can be reduced by covalent coupling of antimicrobial molecules to antibodies. Variable domains (VHH) derived from llama heavy-chain antibodies are particularly suited for such an approach. The antibodies are composed solely of heavy-chain dimers; therefore, production of active fusion proteins by using molecular biology-based techniques is less complicated than production by use of conventional antibodies. In this study, a fusion protein consisting of VHH and GOx was constructed and expressed by Saccharomyces cerevisiae. A llama was immunized with Streptococcus mutans strain HG982. Subsequently, B lymphocytes were isolated and cDNA fragments encoding the VHH fragments were obtained by reverse transcription-PCR. After construction of a VHH library in Escherichia coli and screening of the library against mutans group streptococci and Streptococcus sanguinis strains, we found two VHH fragments with high specificities for S. mutans strains. A GOx gene was linked to the two VHH genes and cloned into S. cerevisiae yeasts. The yeasts expressed and secreted the recombinant proteins into the growth medium. The test of binding of fusion proteins to oral bacteria through their VHH fragments showed that S. mutans had been specifically targeted by GOx-S120, one of the fusion protein constructs. A low concentration of the fusion protein was also able to selectively kill S. mutans within 20 min in the presence of lactoperoxidase and potassium iodide. These findings demonstrate that the fusion protein GOx-VHH is potentially valuable in the selective killing of target bacteria such as S. mutans.

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Single-domain antibody fragments derived from heavy-chain antibodies: a review

Veterinární Medicína ◽

10.17221/6336-vetmed ◽

2012 ◽

Vol 57 (No. 9) ◽

pp. 439-513 ◽

Cited By ~ 40

Author(s):

L. Eyer ◽

K. Hruska

Keyword(s):

Heavy Chain ◽

Water Solubility ◽

Review Article ◽

Single Domain ◽

Single Domain Antibody ◽

Therapeutic Drugs ◽

Domain Antibody ◽

Application Potential ◽

New Generation ◽

Recombinant Fragments

Single-domain antibody (sdAb) fragments derived from heavy-chain antibodies of camelids and cartilaginous fish represent a new generation of therapeutic agents and immunoreagents. Due to their unique characteristics, such as low molecular weight, high physical-chemical stability, good water solubility, and the ability to bind antigens inaccessible to conventional antibodies, they could potentially act as a substitute for conventional therapeutic drugs in the treatment of serious human diseases, and, moreover, could be broadly used in analyses and diagnostics. In this review article, an analysis of 826 publications oriented to heavy-chain antibodies and their sdAb fragments indexed in the Web of Science<sup>®</sup> database since 1993 has been carried out. Attention has predominantly been paid to papers published from 2010 to June 2012. Key publications are presented in tables and are characterised by descriptive words, abstracts and references. The presented publications have been sorted according to seven basic criteria: review articles and monographs, heavy-chain antibodies of camelids and sharks, production of sdAb fragments using recombinant technology, characteristic properties of sdAb fragments, application of sdAb fragments in therapy, application of sdAb fragments in diagnostic and immunoanalytical methods and other prospective uses of sdAb fragments. This review article should highlight the typical properties of heavy-chain antibodies and sdAb fragments which differentiate them from conventional antibodies and other available recombinant fragments, and also emphasize their extremely broad application potential, mainly in human disease therapy. At the same time it allows an easy and rapid orientation in numerous publications written on this subject, and facilitates the search for the required data.

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