MMP-cleavable linker platform for tumour-responsive homo- and heterobivalent antibody-drug conjugates

Abstract

Herein we report the development of a novel linker platform for tumour-responsive antibody-drug conjugates. A series of functionalised homo- and heterobivalent BisFab conjugates have been synthesised, comprising an MMP-cleavable peptide sequence which facilitates selective monomerisation in the tumour microenvironment of the BisFab into two smaller cytotoxic species. The platform was then expanded to produce bispecific BisFab conjugates.

Fig. 1. Overview of the cleavable BisFab concept. Sequential disulfide re-bridging of two Fab units to a peptide linker creates a BisFab construct that can be functionalised with payloads-of-interest via ‘click’ chemistry. The full conjugate is stable in plasma but will fragment in the presence of MMPs to release two smaller monospecific conjugates. If a selective drug release is desired, self-immolative linker systems can be incorporated.

Fig. 2. Synthetic approach towards unfunctionalised BisFabs: (A) two-stage synthesis of BisDVP linkers B1 and B2 comprising SPPS (solid phase peptide synthesis) and HATU-mediated solution phase DVP coupling; (B) trastuzumab Fab dimerisation with BisDVP linkers to produce BisFabs D1 and D2, respectively; (C) cleavage studies using MMP2. (Left) Scheme of D1 showing MMP2/9 cleavage site. BisFab D1 showed full cleavage with MMP2. D1 or D2 (5 µM) were incubated at 37 °C with 0.01 equivalents of MMP2 in TCN buffer (50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5) over 8 hours, with aliquots taken at 1, 2, 4 and 8 hours; (Middle) SDS-PAGE analysis from BisFab D1 and D2 cleavage studies with MMP2; lanes: MW = molecular weight marker, time points indicated above lane marker; (Right) deconvoluted MS of Fab monomer species produced upon MMP2-mediated cleavage of D1: observed 48 424 Da and 48 596 Da, expected 48 421 Da and 48 594 Da. PG = Alloc, X = variable letter, Oeg = 8-amino-3,6-dioxaoctanoic acid, Ahx = 6-aminohexanoic acid.

Fig. 3. Homo- and heterobifunctional BisFabs. (A) Cleavable D3 and non-cleavable D4 BisFabs (synthesised via SPPS, DVP coupling, and bioconjugation) were reacted with AF488 or MMAE via click chemistry to generate homobifunctional BisFabs D3-MMAE, D3-AF488, D4-MMAE, or D4-AF488; (B) cleavable D5 and non-cleavable D6 BisFabs (synthesised via SPPS, DVP coupling, and bioconjugation) were reacted with the TAMRA and AF488 FRET pair via consecutive SPAAC and CuAAC to generate heterobifunctional BisFabs D5-FRET and D6-FRET; (C) fluorometric measurement of Alexa Fluor® 488 quenching in dimers D5-FRET and D6-FRET: (i) incubation of D5-FRET in the presence (green) and absence (red) of MMP2; (ii) incubation of D5-FRET with MMP2 in the presence (orange) and absence (green) of the MMP inhibitor (2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid; (iii) incubation of non-cleavable D6-FRET in the presence (green) or absence (orange) of MMP2. λ_ex = 495 nm, λ_em = 535 nm. Each point is the average of three technical replicates. THPTA = tris(3-hydroxypropyltriazolylmethyl)amine; MMAE = monomethyl auristatin E; AF488 = Alexa Fluor® 488; DBCO = dibenzylcyclooctyne; TAMRA = carboxytetramethylrhodamine; Oeg = 8-amino-3,6-dioxaoctanoic acid.

Fig. 4. ELISA, microscopy and cytotoxicity data for the functionalised BisFabs. (A) ELISA binding affinity comparison between trastuzumab IgG, trastuzumab Fab, and conjugates D6-MMAE and D7-MMAE. All conjugates exhibited concentration-dependent binding to HER2. Error bars represent the standard deviation of three technical replicates; (B) live cell microscopy images of HER2-positive (SKBR3 and BT474) and HER2-negative (MDA-MB-468 and MCF7) cell lines following incubation for either 1 or 4 hours with fluorescent BisFabs D3-AF488 and D4-AF488. Scale bar represents 100 µm. Alexa Fluor® 488 and Hoechst channels have been merged in all images; (C) cell viability of HER2-positive (i) BT474 and (ii) SKBR3, and HER2-negative (iii) MCF7 and (iv) MDA-MB-468 cell lines, after 96-hour treatment with trastuzumab Fab, D3, D4, D3-MMAE, D4-MMAE. Data is reported as the mean of three biological replicates, and error bars represent the standard error of the mean.

Fig. 5. Bispecific BisFabs. (A) Synthesis of cleavable and non-cleavable bispecific BisFabs from cetuximab, durvalumab and trastuzumab Fabs; (B) click functionalisation of BisFabs D7 and D8 to with MMAE and AF488 to give D7-MMAE, D7-AF488, D8-MMAE, and D8-AF488. THPTA = Tris(3-hydroxypropyltriazolylmethyl)amine; MMAE = monomethyl auristatin E; AF488 = Alexa Fluor® 488.

Fig. 6. ELISA, microscopy and cytotoxicity of the bispecific BisFabs. (A) HER2 × EGFR sandwich ELISA analysis of trastuzumab Fab, cetuximab Fab, D7-MMAE and D8-MMAE. All α-HER2 × α-EGFR conjugates displayed concentration-dependent binding, whilst the monovalent controls exhibited no significant absorbance. Error bars represent the standard deviation of biological triplicates; (B) live cell microscopy images of SKBR3 (HER2-positive/EGFR-positive) and BT474 (HER2-positive/EGFR-negative) and MDA-MB-468 (HER2-negative/EGFR-positive) and MCF7 (HER2-negative/EGFR-negative) cell lines following incubation for either 1 or 4 h with fluorescent BisFabs D7-AF488 and D8-AF488. Scale bar represents 100 µm; (C) cell viability of (i) BT474 (HER2+/EGFR−), (ii) SKBR3 (HER2+/EGFR+), (iii) MCF7 (HER2-/EGFR−) and (iv) MDA-MB-468 (HER2−/EGFR+) cell lines after 96-hour treatment with trastuzumab Fab, cetuximab Fab, D3-MMAE, D4-MMAE, D7-MMAE, or D8-MMAE. Data is reported as the mean of three independent biological replicates, and error bars represent the standard error of the mean.