Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody-binding protein as an adapter can simplify antibody functionalization by forming a specific antibody-bound complex and introducing site-specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody-binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein ("lasso"), which binds human immunoglobulin G1 (IgG1) with K D = 0.53 nM and a dissociation rate that is 55to 84-fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme-linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12-fold. The small size of the lasso and a long half-life of dissociation make the peptide a useful tool in antibody detection and immobilization.
A new modality in antibody engineering has emerged in which the antigen affinity is designed to be pH dependent (PHD). In particular, combining high affinity binding at neutral pH with low affinity binding at acidic pH leads to a novel antibody that can more effectively neutralize the target antigen while avoiding antibody-mediated antigen accumulation. Here, we studied how the in vivo pharmacokinetics of the superantigen, Staphylococcal enterotoxin B (SEB), is affected by an engineered antibody with pH-dependent binding. PHD anti-SEB antibodies were engineered by introducing mutations into a high affinity anti-SEB antibody, 3E2, by rational design and directed evolution. Three antibody mutants engineered in the study have an affinity at pH 6.0 that is up to 68-fold weaker than the control antibody. The pH dependency of each mutant, measured as the pHdependent affinity ratio (PARratio of affinity at pH 7.4 and pH 6.0), ranged from 6.7-11.5 compared to 1.5 for the control antibody. The antibodies were characterized in mice by measuring their effects on the pharmacodynamics and pharmacokinetics (PK) of SEB after co-administration. All antibodies were effective in neutralizing the toxin and reducing the toxin-induced cytokine production. However, engineered PHD antibodies led to significantly faster elimination of the toxin from the circulation than wild type 3E2. The area under the curve computed from the SEB PK profile correlated well with the PAR value of antibody, indicating the importance of fine tuning the pH dependency of binding. These results suggest that a PHD recycling antibody may be useful to treat intoxication from a bacterial toxin by accelerating its clearance.
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