Site-Specific Conjugation Strategy for Dual Antibody–Drug Conjugates Using Aerobic Formylglycine-Generating Enzymes

Abstract

Multiple, site-specific protein conjugation is increasingly attractive for the generation of antibody−drug conjugates (ADCs). As it is important to control the number and position of cargoes in an ADC, position-selective generation of reactive sites in the protein of interest is required. Formylglycine (FGly) residues are generated by enzymatic conversion of cysteine residues embedded in a certain amino acid sequence motif with a formylglycine-generating enzyme (FGE). The addition of copper ions increases FGE activity leading to the conversion of cysteines within less readily accepted sequences. With this tuned enzyme activity, it is possible to address two different recognition sequences using two aerobic formylglycine-generating enzymes. We demonstrate an improved and facile strategy for the functionalization of a DARPin (designed ankyrin repeat protein) and the single-chain antibody scFv425-Fc, both directed against the epidermal growth factor receptor (EGFR). The single-chain antibody was conjugated with monomethyl auristatin E (MMAE) and carboxyfluorescein (CF) and successfully tested for receptor binding, internalization, and cytotoxicity in cell culture, respectively.

Bivalent EGFR-Targeting DARPin-MMAE Conjugates

“…Existing HIPS and Knoevenagel ligations have been further optimized by utilizing bifunctional HIPS and tandem Knoevenagel reagents in combination with highly efficient strain-promoted azide-alkyne cycloadditions (SPAAC) [ 39 , 40 ]. The new coupling strategy was tested with mono- and bivalent DARPins and a scFv construct targeting the EGFR.…”

“…Design of scFvFc: The scFv425 N-terminally fused to the human IgG1 Fc antibody fragment containing two FGE recognition motifs at the C-terminus is named Sc2 [ 39 ]. The Sc2 was modified by introducing the point mutation C487S inside the CTAGR-tag of the two C-terminal FGE recognition motifs to get the scFvFc .…”

“…For scFvFc , a C-terminal fragment of the Fc part comprising the FGE dual-tag of the Sc2 from the plasmid pcDNA5/FRT_Sp-RAGE_scFv425_IgG1-Fc_dualFGE-tag (pZMB0520) [ 39 ] was PCR-amplified using the primers 5′-GGCGGGGAGCAAAGTTCTACCGCAGGTCGTGCTGCATTCATAACTGGGCAGGGTCTTTGCACACCATCACGTACCGGTTG-3′ and 5′-AGCCCCGGGAACCTCAGG-3′ (point mutation C487S) and cloned in the backbone of pZMB0520 using XmaI and AgeI resulting in the plasmid pZMB0720, which enables expression of the scFvFc with only one C-terminal aldehyde-tag.…”

“…FGE oxidizes cysteine within the consensus sequence CxPxR to the non-canonical, electrophilic amino acid C α -formylglycine (FGly) [ 28 , 29 , 30 , 31 , 32 , 33 , 34 ], which can be addressed selectively by nucleophiles. In particular, hydrazine-functionalized indoles (Hydrazino- iso -Pictet-Spengler ligation) [ 35 , 36 , 37 , 38 , 39 ] or pyrazolones ( trapped and tandem Knoevenagel ligation) [ 40 , 41 , 42 , 43 ] are used in combination with FGE to label proteins or to produce antibody-drug conjugates.…”

Efficient Site‐Selective Immobilization of Aldehyde‐Tagged Peptides and Proteins by Knoevenagel Ligation

“…[19] In addition, the formylglycine-generating enzyme (FGE) can be used in vivo or in vitro to selectively oxidize a cysteine within the consensus sequence CxPxR (aldehyde tag) to C αformylglycine (FGly). [20][21][22][23][24][25] Aldehydes introduced by one of these methods can be addressed by nucleophiles like amines, [26][27][28][29] hydroxylamines, [30][31][32] hydrazines [33][34][35] or carbon nucleophiles [36][37][38][39] under slightly acidic to neutral conditions, forming an imine (pH < 5), oxime (pH < 5), hydrazone (pH [5][6] or CÀ C double bond (pH 6-7.2) (Scheme 1). Although oximes and hydrazones have higher stabilities compared to imines, reduction with NaBH 3 CN is still required for full stability.…”