Half-life extension of a single-chain diabody by fusion to domain B of staphylococcal protein A

Abstract

Binding of a therapeutic protein to a long-circulating plasma protein can result in a strongly extended half-life. Among these plasma proteins, albumin and immunoglobulins are of special interest because of their exceptionally long half-life, which is to a great extent determined by recycling through the neonatal Fc receptor (FcRn). Many strategies have been established employing reversible binding to albumin, e.g. using an albumin-binding domain from streptococcal protein G. We show here that the half-life of a recombinant antibody molecule can also be prolonged by fusion to a single immunoglobulin-binding domain (IgBD) from staphylococcal protein A. This domain (domain B, SpA(B)) is composed of 56 amino acid residues and was fused to the C-terminus of a bispecific single-chain diabody (scDb). The scDb-SpA(B) fusion protein was produced in HEK293 cells and retained its antigen-binding activity as shown by enzyme-linked immunosorbent assay and flow cytometry. Furthermore, the fusion protein was capable of binding to human and mouse IgG in a pH-dependent manner. In mice, the terminal half-life of the fusion protein was improved from ∼1-2 h of the unmodified scDb to 11.8 h. Although the fusion protein did not reach the long half-life seen for IgG, our results established the applicability of a single bacterial IgBD for half-life extension purposes.

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

“…Site-directed mutagenesis studies show how modulated binding of IgGs to FcRs or C1q can alter their biological effect. 5,32,40,70,[77][78][79] Because our characterization of the CODV format documents the excellent conservation of the properties of natural IgG, we anticipate that these mutations equivalently affect CODV-Ig functionalities, and that multi-parametric drug optimizations benefit from such mutations.…”

“…Molecular weights range from »55 kDa to over 300 kDa, their valences for antigen binding are from two to six, and serum half-lives are reported to be between »0.5 hours and »2 weeks. 1,2,[5][6][7] Each format presents a particular strength in physicochemical properties, manufacturing, formulation, pharmacokinetics, or pharmacodynamics while being limited in the other evaluation criteria. 1 Sustained structural and functional homogeneity throughout manufacture, storage, and in vivo exposure is critical to their successful therapeutic application, but recognized in many bispecific formats as limiting factor.…”

Plasma Half-life Extension of Small Recombinant Antibodies by Fusion to Immunoglobulin-binding Domains

“…Recently, we have shown that fusion of an scDb 2 to the immunoglobulin-binding domain B (IgBD) from protein A (SpA B ) also results in a prolonged plasma half-life (5). Immunoglobulin-binding domains (IgBDs) are known from various bacterial proteins, including staphylococcal protein A (SpA), streptococcal protein G (SpG), and peptostreptococcal protein L (PpL) (6,7).…”

Anti-PSMA/CD3 Bispecific Antibody Delivery and Antitumor Activity Using a Polymeric Depot Formulation

“…Several strategies for half-life extension have been proposed using genetic fusion or chemical conjugation of the antibody fragment to an IgG Fc, human serum albumin, or polyethylene glycol (26). Some of these strategies have been applied to small bispecific antibody constructs (27)(28)(29)(30)(31)(32)(33) with preclinical reported success, but they all imply a modification of the original protein and a plasma exposure of a higher quantity of drug at the beginning of the administration, which could become a disadvantage regarding its potential toxicity. Concerning blinatumomab, infusion of a constant concentration of the drug using a pump enables to keep the drug level between the therapeutic and toxic range but can become challenging for the comfort and mobility of the patient.…”