Biomedical subjects

Claire L Harris

Publications and source records attributed to Claire L Harris.

6 recordsLinked to original sources

PubMed2003

Generation of anti-complement "prodrugs": cleavable reagents for specific delivery of complement regulators to disease sites.

Expression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics.

ADAM Proteins↗

PubMed2003

Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of CD55 and CD59.

Exosomes are secreted nanometer-sized vesicles derived from antigen-presenting cells, which have attracted recent interest as they likely play important roles in immune regulation, and their use as cell-free tools for immunotherapy has been proposed. Liposomes used clinically as transport vehicles can activate the complement system, leading to their rapid degradation and significant inflammatory toxicity. The use of isolated exosomes in therapy, therefore, may also elicit complement activation, reducing their potential efficacy. We have examined the expression and functional roles of the membrane regulators of complement (CD46, CD55 and CD59) on antigen-presenting cell-derived exosomes. Exosomes express the glycosylphosphatidylinositol (GPI)-anchored regulators CD55 and CD59, but not the transmembrane protein CD46. Antibody blocking of CD55 in the presence of sensitizing antibody (w6/32) and human serum resulted in increased C3b deposition and significantly increased exosome lysis. Blockade of CD59 also resulted in significant lysis, while blocking both CD55 and CD59 increased lysis still further. We conclude that exosomes express GPI-anchored complement regulators in order to permit their survival in the extracellular environment.

Antibodies, Monoclonal↗

PubMed2003

Complement therapeutics; history and current progress.

Complement (C) performs vital roles in immune surveillance, from killing of bacteria to generation of an optimal antibody response. However, the mediators responsible for this protective role can inappropriately target self tissues and cause pathology in many inflammatory diseases, in ischaemia-reperfusion injuries and also as a result of therapeutic intervention, such as in cardiopulmonary bypass. Here we review the history of anti-complement therapeutics and describe the plethora of reagents that have evolved to treat complement-mediated pathologies. These agents range from small compounds, including natural products isolated from plants and synthetic peptides designed to target and inhibit the complement cascade, to large, intricately engineered biological reagents. Recombinant, humanised antibody fragments which inhibit at specific points in the complement cascade have been generated and used successfully in man. Other reagents, mimicking the action of the natural complement regulatory proteins present on the surface of self cells, have also been developed and extensively tested. We discuss the pros and cons of these different reagents and describe recent advances in the field, such as specific targeting of drugs to sites of inflammation, which have opened the door to the use of anti-complement therapy in both acute and chronic inflammatory conditions.

Animals↗

PubMed2003

Characterization of the mouse analogues of CD59 using novel monoclonal antibodies: tissue distribution and functional comparison.

CD59, the sole membrane regulator of the membrane attack complex of complement, is broadly and abundantly expressed in man and other mammals. In mouse, CD59 is encoded by two homologous genes. The expression patterns and functional roles of the proteins encoded by these genes, mCD59a and mCD59b, have not been well characterized. Here we describe the generation of monoclonal and polyclonal antibodies detecting specifically mCD59a and mCD59b. These reagents have been used to study function and to ascertain the cell and tissue distributions of mCD59a and mCD59b. mCD59a was broadly distributed on endothelia, erythrocytes, platelets, and on numerous other cell types in organs, a distribution pattern resembling that of CD59 in other species. In marked contrast, expression of mCD59b was restricted to germ cell elements in the testis and mature spermatozoa. Both mCD59a and CD59b inhibited human and rodent complement with similar efficiency. These findings demonstrate that the broadly distributed mCD59a is the key regulator of the terminal complement pathway in mice whereas CD59b, expressed only in testis and on sperm, probably plays other roles in vivo.

Animals↗

PubMed2002

Efficient generation of monoclonal antibodies for specific protein domains using recombinant immunoglobulin fusion proteins: pitfalls and solutions.

Monoclonal antibody production (mAb) first requires the availability of large amounts of pure immunogen for animal immunisation and fusion screening procedures. To overcome this obstacle, we have developed a simple method for rapid generation of pure antigen by generation of recombinant protein containing the antigen of interest fused to the hinge and Fc domains of human immunoglobulin (Ig). The Fc domain forms a convenient 'tag' to enable detection of the protein in supernatant of transfected cells and for purification of immunogen by protein A affinity chromatography. The only requirement for immunogen preparation using this methodology is that a DNA sequence encoding a portion of the molecule of interest is known and that a suitable PCR template is available. Antibody production can be tailored to specific protein domains, for example functional domains, by expressing solely those domains in the fusion protein. We illustrate the technique with two different fusions used to raise antibodies against the porcine and human analogues of a complement (C) regulatory protein, decay accelerating factor (DAF) (CD55). Use of the specific Ig-fusion protein and a control protein facilitated screening of fusions by ELISA. We demonstrate two expression systems used to generate Ig fusion proteins, the first utilised a commercial vector to incorporate an amino terminal leader sequence and carboxy terminal Ig domains. Low levels of expression required subcloning into a high expression vector and resulted in yields of fusion protein at between 2 and 10 mg per litre of supernatant. The second expression system utilised the high expression vector directly, Ig domains of the chosen immunoglobulin isotype were amplified from peripheral blood mononuclear cell (PBMC) RNA and ligated into the vector in frame with DNA encoding the antigen. We describe potential pitfalls that may be encountered while using Ig fusion proteins as immunogen and demonstrate ways in which to tailor their design for optimal mAb production.

Animals↗

PubMed2002

Bacterial expression and membrane targeting of the rat complement regulator Crry: a new model anticomplement therapeutic.

Inappropriate or unregulated activation of complement can contribute to pathology in inflammatory diseases. Previous studies have shown that soluble recombinant regulators of complement are effective in animal models and some human diseases. However, limitations include cost, rapid clearance, and unwanted systemic effects. To avoid some of these problems, bacterial expression of regulators has been optimized and methods for the addition of a membrane-targeting moiety to the complement regulator developed. When administered directly to sites of inflammation, membrane-targeted human regulators are retained and inhibit complement-activation locally. To test the efficacy of membrane-targeted complement regulators in vivo, we have undertaken the expression and membrane targeting of the rat-complement regulator Crry. A soluble recombinant form of Crry, containing only the first four short consensus repeats, was expressed in a mammalian expression system and shown to be functional as a fluid phase regulator. To generate the quantities required for testing in vivo, Crry was expressed in bacteria and refolded successfully. Refolded protein had full-complement regulatory activity in vitro. Attachment of a membrane address tag conferred membrane-binding capacity and greatly increased complement regulatory function in vitro. This novel anticomplement agent can now be applied to rat models of arthritis and other inflammatory diseases.

Animals↗