Site-specific PEGylation of lidamycin and its antitumor activity

Abstract

In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (M w 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs.

Keywords: ADCs, antibody drug conjugates; AE, active enediyne; Anti-TNF Fab′, anti-tumor necrosis factor Fab′; DMSO, dimethyl sulfoxide; Enediyne antibiotic; G-CSF, granulocyte colony stimulating factor; IC50 values, half-inhibitory concentrations; IFN, interferon; IPTG, isopropyl-β-d-thiogalactoside; LB, Luria-Bertani; LDM, lidamycin; Lidamycin; PEG, polyethyleneglycol; Polyethylene glycol; SEC-HPLC, size-exclusion high-performance liquid chromatography; Site-specific PEGylation; mPEG-ALD, methoxy-PEG-propionaldehyde; rLDP, recombinant lidamycin apoprotein; rhG-CSF, recombinant human granulocyte colony stimulating factor.

Graphical abstract

Figure 1

The characterization of rLDP. (A) SDS-PAGE analysis of the mPEG-rLDP and rLDP samples stained with Coomassie Blue. (B) SEC-HPLC analysis of rLDP was performed on a TSK G2000SW_XL column at ambient temperature. The mobile phase was 20 mmol/L sodium phosphate (pH 7.4) with a flow rate of 0.8 mL/min and a detection wavelength of 280 nm. (C) MALDI-TOF spectra of rLDP. The molecular weight of rLDP was 11,692 Da.

Figure 2

The physicochemical characters of mPEG-rLDP. (A) SEC-HPLC analysis of mPEG and rLDP conjugated mixture was performed on a TSK G2000SW_XL column at ambient temperature. The mobile phase was 20 mmol/L sodium phosphate (pH 7.4) with a flow rate of 0.8 mL/min and a detection wavelength of 280 nm. (B) SEC-HPLC analysis of mPEG-rLDP after purification. (C) MALDI-TOF spectra of mPEG-rLDP. The molecular weight of mPEG-rLDP was 33,231 Da. (D) Analysis of mPEG-rLDP and mPEG-rLDP-AE determined by RP-HPLC on C4 300A column at 340 nm.

Figure 3

In vivo antitumor effect of mPEG-rLDP-AE on transplanted tumor. (A) Changes in tumor volume (P<0.05 vs. Control); (B) changes in body weight of human lung cancer cell PG-BE1 xenograft-bearing mice.