Divinylpyrimidine reagents generate antibody-drug conjugates with excellent in vivo efficacy and tolerability

Abstract

The development of divinylpyrimidine (DVP) reagents for the synthesis of antibody-drug conjugates (ADCs) with in vivo efficacy and tolerability is reported. Detailed structural characterisation of the synthesised ADCs was first conducted followed by in vitro and in vivo evaluation of the ADCs' ability to safely and selectively eradicate target-positive tumours.

Fig. 1. Modification of trastuzumab with DVP reagents generates either a non-cleavable or cleavable ADC. TBS = Tris-buffered saline (25 mM TrisHCl, 25 mM NaCl, 0.5 mM EDTA (pH 8)).

Fig. 2. In vitro characterisation of DVP ADCs. (a) HIC trace of C-ADC, (b) HIC trace of NC-ADC, (c) SEC traces of trastuzumab, NC-ADC and C-ADC, (d) HER2 ELISA of trastuzumab, NC-ADC and C-ADC. Error bars represent the standard deviation of biological quadruplicates, (e) cell viability of C-ADC against HER2-positive (+ve) and HER2-negative (−ve) cell lines, and (f) cell viability of NC-ADC against the same cell lines. Viability data shows the mean of three independent replicates and error bars represent S.E.M.

Fig. 3. In vivo evaluation of DVP ADCs. Effect of BT474 cell line xenograft in NSG mice (n = 3) up to 60 days post-treatment with (a) C-ADC and (b) NC-ADC. (c) Changes in mouse bodyweight up to 60 days post-treatment with C-ADC or NC-ADC. H&E staining, anti-human IgG (α-hIgG), TUNEL and Ki67 IHC of (d) BT474 and (e) MCF7 mouse xenograft tumours 72 hours post-treatment with C-ADC, NC-ADC, trastuzumab or vehicle (PBS). Quantification of Ki67 levels from IHC analysis of (f) BT474 and (g) MCF7 mouse xenograft tumours (n = 3) 72 hours post-treatment with C-ADC, NC-ADC, trastuzumab or vehicle (PBS). Statistical significance calculated using a two-tailed paired t test by comparison to PBS treated animals. Not significant (ns) p > 0.05, *p < 0.05, **p < 0.005. (h) Pharmacokinetic analysis of the in vivo mouse plasma half-life of NC-ADC and C-ADC.