Fig. 4: Characterizations of CASTi and antitumor activity evaluation of Tra-CASTi-MMAE in vitro and in vivo.
From: Copper assisted sequence-specific chemical protein conjugation at a single backbone amide
a CAST peptide (-FFKKDDHAA-) and CASTi peptide (-IAPDDHAA-) in vitro stability in mouse plasma. b 1 modified CASTi stability comparison with cysteine-maleimide conjugate, incubation conditions: 37 °C, 20 mM glutathione, PBS (pH 7.4), 22 h. Peaks at * are the maleimide hydrolysis byproducts, and the peak at ♦ corresponds to the released free peptide WCGIAPDDHAA. Source data are provided as a Source Data file. c Tra-CASTi-MMAE stability test in mouse plasma using anti-human IgG or anti-MMAE. n = 3 independent experiments, error bars represent s.e.m. d Tra-CASTi-MMAE (red diamond) effectively killed HER2-positive SK-BR-3 (HER2 + + + ), SK-OV-3 (HER2 + ++) and JIMT-1 (HER2 + +) cells but only showed minimal toxicity against HER2-negative MCF7 (HER2-) cells, Tra-CASTi (blue square) displayed no obvious toxicity towards all cells, DBCO-MMAE (green triangle) showed minimal toxicity towards all cells. n = 3 independent experiments, error bars represent s.e.m. e In vivo efficacy study on SK-OV-3 xenograft tumor models. Left: tumor volume measurement; right: percent survival (female Balb/c nude mice, n = 5 for 12 mg/kg Fc isotype control, n = 5 for 12 mg/kg Tra-CASTi, n = 4 for 6 mg/kg Tra-CASTi-MMAE, n = 6 for 12 mg/kg Tra-CASTi-MMAE), black arrow indicates i.v. drug administration, error bars represent s.e.m. f In vivo efficacy study on JIMT-1 xenograft tumor models. Left: tumor volume measurement; right: percent survival (female NSG mice, n = 6 for Tra-CASTi-MMAE, n = 6 for Tra-CASTi), black arrow indicates i.p. drug administration, error bars represent s.e.m.
