A Recombinant Human scFv Anti-Rh(D) Antibody With Multiple Valences Using a C-Terminal Fragment of C4-Binding Protein

Abstract

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.

A novel C3d-containing oligomeric vaccine provides insight into the viability of testing human C3d-based vaccines in mice

“…Taking into account previous oligomerisation approaches ( Libyh et al, 1997 ; Christiansen et al, 2000 ), we used PCR to join cDNAs such that an N-terminal hC3d was fused to the well characterised model antigen tetanus toxin C fragment (TTCF), followed by SCRs 5–8 (to act as spacers) and the C-terminal oligomerisation domain from hC4BPα chain (hC3d-TTCF-hC4BP). Various hC4BPα chain-containing control proteins were also generated ( Fig.…”

“…The oligomerisation domain on the complement regulator C4BP ( Scharfstein et al, 1978 ) has previously been exploited to increase valency of either recombinant antibodies ( Libyh et al, 1997 ; Oudin et al, 2000 ) or a viral receptor ( Christiansen et al, 2000 ). This led us to conceive an oligomeric vaccine structure based on C4BP that would have three functionally established benefits.…”

Expression of Single Chain Variable Fragment (scFv) Molecules in Plants: A Comprehensive Update

“…Advances in molecular techniques further improved the prospect of engineering scFv to improve its specificity, avidity, affinity, and half-life. Multimerization of the variable domains using, the linker [22], the tetramerization domain of a native protein like p53 [7], the leucine zippers [23], or the C-terminal fragment of C4-binding protein [6] improved the affinity of the scFv to a great extent. The immunogenicity generated by the Fc portion of the antibody is absent in the conventional scFv molecule.…”

“…Multimeric scFv comprising of more than one pair of heavy and light chains can be generated by changing the length of peptide linker. Multimerization can also be made by using specific peptides which have the natural ability to induce it [6,7]. Multivalent scFv showing affinity to more than one protein at the same time can be generated through genetic engineering [8][9][10][11].…”

Solid-state NMR sequential assignments of the C-terminal oligomerization domain of human C4b-binding protein

“…It contains 2 cysteines, which stabilize the interaction between the chains by intermolecular disulfide bonds; however, these SS-bonds are not required for the oligomerization of the protein (Kask et al 2002). The C-terminal domain of C4bp was also shown to be able to oligomerize even in the presence of other proteins fused to it, with the potential to increase the half-lives or activity of the fusion protein (Libyh et al 1997;Shinya et al 1999;Oudin et al 2000;Dervillez et al 2006). Furthermore it has been observed that the murine and avian C-terminal domains of C4bp can act as an adjuvant for vaccines against malaria and tuberculosis when fused to the antigen (Draper et al 2008;Ogun et al 2008;Spencer et al 2012).…”