Synthesis of Multiple Bispecific Antibody Formats with Only One Single Enzyme Based on Enhanced Trypsiligase

Abstract

Bispecific antibodies (bsAbs) were first developed in the 1960s and are now emerging as a leading class of immunotherapies for cancer treatment with the potential to further improve clinical efficacy and safety. Many different formats of bsAbs have been established in the last few years, mainly generated genetically. Here we report on a novel, flexible, and fast chemo-enzymatic, as well as purely enzymatic strategies, for generating bispecific antibody fragments by covalent fusion of two functional antibody Fab fragments (Fabs). For the chemo-enzymatic approach, we first modified the single Fabs site-specifically with click anchors using an enhanced Trypsiligase variant (eTl) and afterward converted the modified Fabs into the final heterodimers via click chemistry. Regarding the latter, we used the strain-promoted alkyne-azide cycloaddition (SPAAC) and inverse electron-demand Diels-Alder reaction (IEDDA) click approaches well known for their fast reaction kinetics and fewer side reactions. For applications where the non-natural linkages or hydrophobic click chemistry products might interfere, we developed two purely enzymatic alternatives enabling C- to C- and C- to N-terminal coupling of the two Fabs via a native peptide bond. This simple system could be expanded into a modular system, eliminating the need for extensive genetic engineering. The bispecific Fab fragments (bsFabs) produced here to bind the growth factors ErbB2 and ErbB3 with similar K_D values, such as the sole Fabs. Tested in breast cancer cell lines, we obtained biologically active bsFabs with improved properties compared to its single Fab counterparts.

Keywords: Trypsiligase; antibody engineering; biorthogonal chemistry; bispecific antibody; click chemistry.

Scheme 1

Synthesis and structure of anti-ErbB2-anti-Erb3-bsFab formats. (A) Enzymatic synthesis of C- to N-linked anti-ErbB3-YRH-anti-ErbB2-bsFab via eTl-catalysis from anti-ErbB3-Fab-YRH and anti-ErbB2-RH-Fab; (B) Chemo–enzymatic synthesis of C- to C-linked anti-ErbB3-anti-ErbB2-bsFab via eTl-mediated coupling of click anchors to both Fabs (reaction 1a and 1b), followed by click coupling of the purified intermediate Fab products (reaction 2); (C) Enzymatic synthesis of C- to C-linked anti-ErbB3-anti-ErbB2-bsFab via a two-step eTl-catalysis initiated by the enzymatic coupling of anti-ErbB2-Fab-YRH to linker 5, followed by a second enzymatic coupling of the purified intermediate to anti-ErbB3-Fab-YRH. Light grey Fab: anti-ErbB3-Fab-YRH, dark grey Fab: anti-ErbB2-Fab; eTl: enhanced Trypsiligase.

Figure 1

Structures of click linkers used. H-RHAC(Mal-PEG₃-TCO)-OH (1) and H-RHAC(Mal-PEG₄-MeTz)-OH (2) were used for IEDDA; H-RHAK(PSA)-OH (3) and H-RHAC(Mal-PEG₄-DBCO)-OH (4) were used for SPAAC; H-RHAGGK(H-RHAGG)GGWGGK(N₃)-OH (5) was used for enzymatic C- to C-terminal Fab coupling. Mal: maleiimide, TCO: trans-cyclooctene, MeTz: methyltetrazine, PAA: pentanoic acid azide, DBCO: dibenzocyclooctyne.

Figure 2

Synthesis of C- to N-terminal conjugated bsFab via direct eTl-coupling of anti-ErbB3-Fab-YRH and anti-ErbB2-RH-Fab. (A) Kinetics of product formation with reaction yields of about 60%, a: anti-ErbB3-YRH-anti-ErbB2-bsFab, b: anti-ErbB2-RH-Fab; (B) Time-resolved SDS-PAGE analysis of coupling reaction, c: anti-ErbB3-Fab-YRH, M: molecular marker; (C) MS analysis of the final bsFab product after purification by SEC, anti-ErbB3-YRH-anti-ErbB2-bsFab M_calcd: 94,804 Da, M_found: 94,805 Da. Light grey Fab: anti-ErbB3-Fab-YRH, dark grey Fab: anti-ErbB2-Fab-RH; eTl: enhanced Trypsiligase. Reaction conditions: 20 µM anti-ErbB2-RH-Fab, 100 µM anti-ErbB3-Fab-YRH, 2.5 µM eTl, 50 µM ZnCl₂, 100 mM HEPES/NaOH, pH 7.8, 100 mM NaCl, 10 mM CaCl₂.

Figure 3

Coupling of anti-ErbB2-Fab-YRH and anti-ErbB3-Fab-YRH to click linkers 1 and 2, respectively, via eTl-catalysis. (A) Reaction kinetics with maximum product yields higher than 72%, light grey line: anti-ErbB2-Fab-TCO, grey line: anti-ErbB3-Fab-MeTz; (B) HPLC analysis of the reaction mixture of anti-ErbB2-Fab-YRH with TCO click linker 1 at 0 min (light grey) and 75 min (dark grey) reaction times; (C) Mass spectrogram of the reaction mixture of anti-ErbB2-Fab-YRH with TCO click linker 1 after 75 min reaction time and after separation of excess click anchor via affinity chromatography (a: anti-ErbB2-Fab-TCO M_calcd: 48,860 Da, M_found: 48,861 Da, b: anti-ErbB2-Fab-YRH M_calcd: 49,548 Da, M_found: 49,549 Da, c: anti-ErbB2-Fab-Y-OH M_calcd: 47,870 Da, M_found: 47,871 Da). Light grey Fab: anti-ErbB3-Fab-YRH, dark grey Fab: anti-ErbB2-Fab-YRH; eTl: enhanced Trypsiligase. Reaction conditions: 100 µM anti-ErbB2-Fab-YRH and anti-ErbB3-Fab-YRH, 500 µM click linker 1 and 2, 10 µM eTl, 100 µM ZnCl₂, 100 mM HEPES/NaOH, pH 7.8, 100 mM NaCl, 10 mM CaCl₂.

Figure 4

Course and analysis of the click reaction of eTl synthesized anti-ErbB3-Fab-MeTz with anti-ErbB2-Fab-TCO forming anti-ErbB3-Fab-IEDDA-anti-ErbB2-bsFab. (A) Time-resolved UPLC analysis of the click reaction showing a complete conversion within 0.5 h of the reaction time, a: anti-ErbB3-Fab-IEDDA-anti-ErbB2-bsFab, b: anti-ErbB3-Fab-MeTz, c: anti-ErbB2-Fab-TCO; (B) MS analysis of the click reaction after reaction start, 2 h reaction time and, of the final conjugation product (a: anti-ErbB3-Fab-IEDDA-anti-ErbB2-bsFab M_calcd: 96,536 Da, M_found: 96,537 Da, b: anti-ErbB3-Fab-MeTz M_calcd: 47,704 Da, M_found: 47,705 Da, c: anti-ErbB2-Fab-TCO M_calcd: 48,860 Da, M_found: 48,861 Da); (C) Time-resolved SDS-PAGE analysis of the click reaction (1: anti-ErbB3-Fab-MeTz, 2: anti-ErbB2-Fab-TCO, M: molecular marker); (D) UPLC profile of anti-ErbB3-Fab-IEDDA-anti-ErbB2-bsFab conjugate after purification via SEC. Light grey Fab: anti-ErbB3-Fab-MeTz, dark grey Fab: anti-ErbB2-Fab-TCO. Reaction conditions: 30 µM anti-ErbB3-Fab-MeTz, 60 µM anti-ErbB2-Fab-TCO in PBS, (A): 30–50% acetonitrile/ddH₂O in 10 min, (D): 10–80% acetonitrile/ddH₂O in 10 min.

Figure 5

Results of the enzymatic C- to C-terminal coupling of anti-ErbB2-Fab-YRH and anti-ErbB3-Fab-YRH via linker 5. (A) Reaction kinetics: the single modified product (black line) was mainly formed while only traces of the homodimeric product (dark grey line) were generated. a: anti-ErbB2-Fab-linker 5, b: anti-ErbB2-Fab-YRH, c: homodimeric anti-ErbB2-linker 5-anti-ErbB2-Fab; (B) MS analysis of the product purified by HIC, a: anti-ErbB2-Fab-linker 5 M_calcd: 49,538 Da, M_found: 49,539 Da; (C) Reaction course of anti-ErbB2-linker 5-anti-ErbB3-bsFab synthesis with yields of about 60% analyzed by HIC, d: anti-ErbB2-linker 5-anti-ErbB3-bsFab; (D) MS analysis of the purified bsFab, anti-ErbB2-linker 5-anti-ErbB3-bsFab M_calcd: 96,244 Da, M_found: 96,245 Da. Reaction conditions: (A): 100 µM anti-ErbB2-Fab-YRH, 500 µM linker 5, 10 µM eTl, 100 µM ZnCl₂; (C): 20 µM anti-ErbB2-Fab-linker 5, 100 µM anti-ErbB3-Fab-YRH, 2.5 µM eTl, 50 µM ZnCl₂; (A,C): 100 mM HEPES/NaOH, pH 7.8, 100 mM NaCl, 10 mM CaCl₂.

Figure 6

Internalization of anti-ErbB3-IEDDA-anti-ErbB2-bsFab compared to the single anti-ErbB2- and anti-ErbB3-Fab by lysosome-stained SKBR-3 cells after 24 h. Lysosomes were stained with LysoBriteBlue, single and bispecific Fabs were modified with AlexaFluor568 NHS ester. IEDDA: inverse electron-demand Diels–Alder reaction.

Figure 7

Internalization of anti-ErbB3-IEDDA-anti-ErbB2-bsFab compared to the single anti-ErbB2- and anti-ErbB3-Fab by lysosome-stained HCC-1954 cells after 24 h. Lysosomes were stained with LysoBriteBlue, single and bispecific Fabs were modified with AlexaFluor568 NHS ester. IEDDA: inverse electron-demand Diels–Alder reaction.

Figure 8

Internalization of anti-ErbB3-IEDDA-anti-ErbB2-bsFab compared to the single anti-ErbB2- and anti-ErbB3-Fab by cell nucleus-stained HCC-1954 cells after 24 h. Substantial portions of the bsFabs could be found in the cell nucleus. Cell nuclei were stained with HOECHST33342, single and bispecific Fabs were modified with AlexaFluor568 NHS ester. IEDDA: inverse electron-demand Diels–Alder reaction.