Oligomeric forms of single chain immunoglobulin (scIgG)

Abstract

Assembly of immunoglobulin G (IgG) molecules from two heavy and two light chains can be facilitated by connecting the light chain to the heavy chain by an oligopeptide linker. Production of the anti-lysozyme D1.3-single chain (sc) IgG1 in HEK293T cells yielded up to 8 mg/L functional scIgG polypeptide. Size exclusion chromatography of material purified by protein-A affinity chromatography revealed that the majority of the D1.3-scIgG1 molecules were 150 kDa monomers, with a K(D) of 1.8 x 10(-10) M measured by surface plasmon resonance; however, significant fractions of scIgG dimers and oligomers with molecular masses of 300 kDa and >600 kDa, respectively, were identified. The oligomerization resulted in an increased avidity. The observed oligomerization capability may allow new approaches for the generation of bispecific/multivalent antibodies.

Figure 1

(A) Schematic illustration of igG and its single chain derivatives. Variable immunoglobulin regions (V_L, V_H) are shown in red, constant regions (C_H1, C_H2, C_H3) including the hinge region are shown in blue. interchain disulphide bonds are coloured black and non-sequences (linkers) are colored green. (B) SDS-pAGe and immunoblot of D1.3-scigG. After protein A affinity purification, protein samples were prepared under reducing and non-reducing conditions and separated by SDS-pAGe. Gels were stained using Coomassie Blue or blotted onto a pVDf membrane which was stained with a goat-anti-human (fc-specific) alkaline phosphate antibody conjugate (NBt-BCip substrate). (C) Size exclusion chromatography of protein A purified D1.3-scigG1. peak positions found for the molecular weight calibration standards chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), albumin (67 kDa), Crp (115 kDa), igG (160 kDa) and Blue Dextran 2000 (2 MDa) are indicated in red. the dashed line indicates the deduced calibration curve. (D) Antigen eLiSA of D1.3-scigG1 SeC fractions. Serial concentrations of the D1.3-scigG1 were analysed with BSA as negative control antigen. proposed oligomerization forms suggested by the observed molecular mass are schematically illustrated.