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A method to confer Protein L binding ability to any antibody fragment.

ABSTRACT: Recombinant antibody single-chain variable fragments (scFv) are difficult to purify homogeneously from a protein complex mixture. The most effective, specific and fastest method of purification is an affinity chromatography on Protein L (PpL) matrix. This protein is a multi-domain bacterial surface protein that is able to interact with conformational patterns on kappa light chains. It mainly recognizes amino acid residues located at the VL FR1 and some residues in the variable and constant (CL) domain. Not all kappa chains are recognized, however, and the lack of CL can reduce the interaction. From a scFv composed of IGKV10-94 according to IMGT®, it is possible, with several mutations, to transfer the motif from the IGKV12-46 naturally recognized by the PpL, and, with the single mutation T8P, to confer PpL recognition with a higher affinity. A second mutation S24R greatly improves the affinity, in particular by modifying the dissociation rate (kd). The equilibrium dissociation constant (KD) was measured at 7.2 10(-11) M by surface plasmon resonance. It was possible to confer PpL recognition to all kappa chains. This protein interaction can be modulated according to the characteristics of scFv (e.g., stability) and their use with conjugated PpL. This work could be extrapolated to recombinant monoclonal antibodies, and offers an alternative for protein A purification and detection.

SUBMITTER: Lakhrif Z

PROVIDER: S-EPMC4966575 | biostudies-literature | 2016 Feb-Mar

REPOSITORIES: biostudies-literature

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A method to confer Protein L binding ability to any antibody fragment.

Lakhrif Zineb Z Pugnière Martine M Henriquet Corinne C di Tommaso Anne A Dimier-Poisson Isabelle I Billiald Philippe P Juste Matthieu O MO Aubrey Nicolas N

mAbs 20151218 2

Recombinant antibody single-chain variable fragments (scFv) are difficult to purify homogeneously from a protein complex mixture. The most effective, specific and fastest method of purification is an affinity chromatography on Protein L (PpL) matrix. This protein is a multi-domain bacterial surface protein that is able to interact with conformational patterns on kappa light chains. It mainly recognizes amino acid residues located at the VL FR1 and some residues in the variable and constant (CL) ...[more]

PMID: 26683650

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Project description:Efgartigimod is a human IgG1 antibody Fc-fragment that lowers IgG levels through blockade of the neonatal Fc receptor (FcRn) and is being evaluated for the treatment of patients with severe autoimmune diseases mediated by pathogenic IgG autoantibodies. Engineered for increased FcRn affinity at both acidic and physiological pH, efgartigimod can outcompete endogenous IgG binding, preventing FcRn-mediated recycling of IgGs and resulting in increased lysosomal degradation. A population pharmacokinetic-pharmacodynamic (PKPD) model including FcRn binding was developed based on data from two healthy volunteer studies after single and repeated administration of efgartigimod. This model was able to simultaneously describe the serum efgartigimod and total IgG profiles across dose groups, using drug-induced FcRn receptor occupancy as driver of total IgG suppression. The model was expanded to describe the PKPD of efgartigimod in cynomolgus monkeys, rabbits, rats and mice. Most species differences were explainable by including the species-specific in vitro affinity for FcRn binding at pH 7.4 and by allometric scaling of the physiological parameters. In vitro-in vivo scaling proved crucial for translation success: the drug effect was over/underpredicted in rabbits/mice when ignoring the lower/higher binding affinity of efgartigimod for these species versus human, respectively. Given the successful model prediction of the PK and total IgG dynamics across species, it was concluded that the PKPD of efgartigimod can be characterized by target binding. From the model, it is suggested that the initial fast decrease of measurable unbound efgartigimod following dosing is the result of combined clearance of free drug and high affinity target binding, while the relatively slow terminal PK phase reflects release of bound drug from the receptor. High affinity target binding protects the drug from elimination and results in a sustained PD effect characterized by an increase in the IgG degradation rate constant with increasing target receptor occupancy.

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A method to confer Protein L binding ability to any antibody fragment.

Publications

A method to confer Protein L binding ability to any antibody fragment.

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