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.
Monomeric recombinant molecules prove generally unsatisfactory for in vivo use. Most biological systems are indeed multivalent either structurally, associating different chains, or functionally, when cross-linked by their ligands. Mimicking natural molecules for immune intervention implies the need for multimerizing systems to create multivalent molecules capable of interfering with physiological processing. A multivalent anti-Rh(D) recombinant protein has been designed by reconstructing the antibody binding site of a human monoclonal anti-Rh(D) antibody as a single chain Fv mini antibody, then multimerizing it by inserting at its C-terminal end the C-terminal part of the C4 binding protein (C4bp) alpha chain, which is responsible for the octamer multimerization of that molecule. This soluble multivalent recombinant molecule was functional, bound red blood cells (RBCs), agglutinated them, and did not activate complement. This demonstration model opens the way for future in vivo use of multivalent molecules associating antibody valences and other functional molecules for cell targeting, imaging, or removal of cells such as Rh(D)-positive RBCs for preventing Rh alloimmunization.
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