N-Sulfonyl-β-lactam hapten as an effective labeling reagent for aldolase mAb

Abstract

Utilization of chemically programmed antibodies (cpAbs) is regarded to be one of the most efficient methods for the development of therapeutic systems. cpAbs can extend the half-life of programming reagents, activate immune systems via the Fc region of antibodies and achieve universal vaccination by attaching varieties of small, programmed molecules. In the current study, we aimed to develop a novel labeling reagent for the preparation of cpAbs and found that N-sulfonyl-β-lactams (NSBLs) were optimal. NSBL can be synthesized from readily available 4-(bromomethyl)benzenesulfonyl chloride via few simple manipulations and can label the aldolase monoclonal antibody (mAb) 84G3, which could not be labeled effectively by the conventional labeling reagent, N-acyl-β-lactam (NABL). We also demonstrated that the conjugate, which consists of mAb 84G3 and an NSBL bearing a biotin moiety, maintained strong binding activity to streptavidin. In addition, the stability assay of NSBL revealed that NSBLs can tolerate aqueous media without significant decomposition over 24h.

Keywords: Aldolase antibody; Bioconjugation; Chemically programmed antibody; N-Sulfonyl-β-lactam.

Figure 1

Concept and expected benefits of cpAbs.

Figure 2

Small-molecule haptens that induce aldolase antibodies.

Figure 3

Labeling study of mAb 84G3 with lactam haptens 3a–3c. Reagents and conditions: (a) cpAb (0.3 mol %), PBS (pH 7.4), rt.

Figure 4

ELISA of the cp84G3s.

Figure 5

Chemical structure of 14 and 15.

Scheme 1

Labeling of mAb 38C2 by NABL.

Scheme 2

Synthesis of the N-sulfonyl lactam haptens 3a and 3b. Reagents and conditions: (a) Et₃N, MeOH, rt, 30 min (52%). (b) NaH, 6, THF, 0 °C to rt, 3 h (33%). (c) LiCl, acetone, 55 °C, 2 h then phosgene, DMF, CH₂Cl₂, 0 °C to rt, 4 h (74%). (d) NaHMDS, 2-azetidinone, THF, -78 °C, 2 h or n-BuLi, 2-pyrrolidinone, -78 °C, 1 h (16% for 3a, 12% for 3b).

Scheme 3

Labling of 84G3 with lactam haptens. Reagents and conditions: (a) 3a or 3b or 3c, PBS (pH 7.4), rt, 60 min.

Scheme 4

Preparation of 84G3-biotin 9d. Reagents and conditions: (a) CuSO₄ 5H₂O, Na-(L)-ascorbate, 12, tBuOH, H₂O, rt, 1 h (60%). (b) mAb 84G3, PBS (pH 7.4), rt, 60 min.