Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin

Abstract

We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH 6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly 2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography--utilizing the Strep-tag II appended to gelonin--and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC 50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins.

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

“…There are several examples of SortA ligations in the development of next-generation ADCs (28)(29)(30). NBE-Therapeutics, for example, recently announced a patent-pending Sortase-mediated antibody conjugation (SMAC TM )-Technology.…”

“…This technique has been used to conjugate biotin functional handles to the C-terminus of a single-chain Fv fragment derived from an anti-EGFR antibody (27). A more recent account demonstrates the fusion between the heavychain C terminus of an anti-Her2 Fab and the 30-kDA plant toxin gelonin (29). Levary and co-workers verified the flexibility of SortA by ligating a variety of oligo-glycinetagged biomolecules to IgGs without disrupting antigen binding (28).…”

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates

“…The feasibility of such a peptide-tagged ADC approach has recently been proven by the introduction of small substrate sequences for enzyme-promoted conjugation at different constant region loops positions of trastuzumab for efficient site-specific ADC preparation16. The possibility to place the peptide-tags at multiple sites within the antibody scaffold would clearly provide an advantage compared to covalent peptide labeling via sortases that exclusively allow for C -terminal payload conjugation17293031. In addition to microbial transglutaminase our approach offers the opportunity for site-specific and efficient antibody conjugation based on a stable isopeptide bond.…”

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

“…The antibodies to be joined can be expressed separately as full-length antibodies, and the coupling reactions occur under physiological conditions. Likewise, sortase-catalyzed reactions enable largescale preparation of modified biomolecules (49) and immunotoxins (50). A panel of sortase-modified antibodies equipped with click handles far more readily facilitates combinatorial exploration of many different combinations of C-C-linked bispecific antibodies than genetic fusions would allow.…”