Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange

Abstract

A series of single-chain anti-CD20 antibodies was produced by fusing single-chain Fv (scFv) with human IgG1 hinge and Fc regions, designated scFv-Fc. The initial scFv-Fc construct was assembled using an 18 amino acid (aa) linker between the antibody light- and heavy-chain variable regions, with the Cys residue in the upper hinge region (Kabat 233) mutagenized to Ser. Anti-CD20 scFv-Fc retained specific binding to CD20-positive cells and was active in mediating complement-dependent cytolysis. Size-exclusion HPLC analysis revealed that the purified scFv-Fc included multimeric as well as monomeric components. Variant scFv-Fcs were constructed incorporating four different hinges between the scFv and Fc regions, or three different linkers in the scFv domain. All formed multimers, with the highest level of multimerization found in the scFv-Fc with the shortest linker (8 aa). Elimination of an unusual salt bridge between residues L38 and H89 in the V(L)-V(H) domain interface failed to reduce the formation of higher order forms. Structural analysis of the scFv-Fc constructed with 18 or 8 aa linkers by pepsin or papain cleavage suggested the proteins contained a form in which scFv units had cross-paired to form a 'diabody'. Thus, domain exchange or cross-pairing appears to be the basis of the observed multimerization.

Construction and Expression of a Novel Anti-CD14 Human-Mouse Chimeric Antibody Hm2F9

“…The dimeric form decreased after the purified Hm2F9 was stored at 4°C longer than 2 weeks, and a novel 200-kDa multimer component was generated (data not shown). This observation was consistent with previous studies of an scFv-Fc fusion protein using anti-CD20 antibody (Wu et al, 2001;Cang et al, 2012). Another study of an anti-CEA antibody has shown a concentration dependence of scFv-diabody interconversion (Wu et al, 1996).…”

An anti-CD20–IL-2 immunocytokine is highly efficacious in a SCID mouse model of established human B lymphoma

“…15 The strong preclinical data and preliminary clinical results with other IL-2-based immunocytokines have encouraged us to apply this technology to the lymphoma setting where a minimal residual disease setting is possible in a large majority of patients following C2B8 therapy. Specifically, we have genetically engineered the variable (V) regions of the mouse Leu16 anti-CD20 16 antibody, which binds to the same Ala-X-Pro motif as rituximab (and several other anti-CD20 antibodies) and causes distribution of CD20 into the Triton X-insoluble fraction of lipid rafts, but does not cause homotypic cell aggregation. 17 The antibody was humanized by first removing potential helper T-cell epitopes (deimmunization) and second, by combining these V regions with human light (L) chain and heavy (H) chain constant regions.…”

Pretargeting of Carcinoembryonic Antigen–Expressing Cancers with a Trivalent Bispecific Fusion Protein Produced in Myeloma Cells

“…A further complication is the tendency of scFv-based constructs to form multimers via domain swapping, resulting in a heterogeneous product consisting of monomers, dimers, trimers, etc. (24,25). The molecular design of hBS14 favors the formation of an 80 kDa heterodimer having one h679 and two hMN-14 Fvs.…”