Xavier Dervillez

A Soluble Recombinant Multimeric Anti-Rh(D) Single-Chain Fv/CR1 Molecule Restores the Immune Complex Binding Ability of CR1-Deficient Erythrocytes

CR1 (CD35, the C3b/C4b receptor) is a widely distributed membrane glycoprotein with a unique cluster conformation on the surface of erythrocytes (E). CR1 on E is responsible for the transport of immune complexes (IC) to liver and spleen. As a cofactor of the C3b cleavage by factor I, CR1 is also a potent inhibitor of C activation and inflammation. In some diseases (systemic lupus erythematosus, hemolytic anemia, AIDS, etc.) an acquired low level of CR1 on E has been observed, leading to an impaired clearance of IC. The aim of this study was to design a heterofunctional molecule that will bind to E and restore a normal or a supranormal CR1 density on E that could mimic the unique distribution pattern of CR1 on normal E. For that purpose a new multimerizing system based on the properties of the C-terminal part of the α-chain of the C4 binding protein (C4bp) was used. We first produced a multimeric soluble CR1 that proved to be a better inhibitor of in vitro C activation than the monomeric form of CR1, then a heteromultimeric molecule made of CR1 and single-chain Fv anti-Rh(D) valences able to attach E and providing E with as much as a 10-fold increase in CR1 density with the same CR1 distribution pattern as native E. CR1/single-chain Fv anti-Rh(D)-treated E were able in vitro to attach as many opsonized IC as native E. These data open the way for future use of multimeric and heteromultimeric forms of soluble recombinant CR1 as therapy of IC diseases.

Stable expression of soluble therapeutic peptides in eukaryotic cells by multimerisation: application to the HIV-1 fusion inhibitory peptide C46.

A major drawback of therapeutic peptides is their short half‐life, which results in the need for multiple applications and high synthesis costs. To overcome this, we established a eukaryotic expression system that allows the stable expression of small therapeutic peptides by multimerisation. By inserting the sequence encoding the therapeutic peptide between a signal peptide and the multimerising domain of the α‐chain from the human C4bp plasma protein, therapeutic peptides as small as 5 kDa are secreted as multimers from transfected cells; this allows easy purification. As proof of principle, we show that the T20‐derived HIV‐1 fusion inhibitory peptide C46 in its multimeric form: i) was efficiently secreted, ii) was more stable than the current antiviral drug T20 in vitro and in vivo, and iii) inihibited HIV‐1 entry with similar efficiency in vitro. Besides the gain in stability, multimerisation also leads to increased valency and allows the combination of several therapeutic functions. Furthermore, by expressing the multimers from cells, post‐translational modifications could easily be introduced.

Catabolism of the human erythrocyte C3b/C4b receptor (CR1, CD35): vesiculation and/or proteolysis?

Human erythrocytes (E) react by exocytosis of membrane vesicles to various stresses including the fixation of the membrane attack complex of Complement. E from normal individuals loose a notable proportion of their initial number of surface CR1 molecules during the ageing process. An acquired decrease of CR1 on E also occurs in pathological conditions such as Systemic Lupus Erythematosus or AIDS. The present study investigated whether calcium ionophore A23187 (Ca-ion) induced vesicle formation of human E in vitro is responsible for a preferential loss of CR1 as well as whether CR1 molecules at the surface of Ca-ion treated E or vesicles are: (i) functional, (ii) native or protease degraded, or (iii) more clustered than CR1 on native E. A study of E from 137 normal individuals showed that a one-hour Ca-ion induced vesicle formation preferentially removed one third of E surface CR1. Kinetic experiments suggested that all surface CR1 could be removed from E upon longer incubation times. CR1 molecules on vesicles were still able to inhibit Complement activation, and were found in larger clusters than on native E. These data suggest that a significant part of surface CR1 molecules may be removed from E by vesicle formation during the life of E in normal individuals. This phenomenon could be exacerbated in pathological conditions.