Targeted Peptide-based Energized Fusion Protein And Chemical Conjugate: Preparation And Antitumor Activity

Targeting tumor cells or tumor vasculature by peptides is apromising strategy fordelivering cytotoxic drugs in cancer therapy. Targeted peptide, such as NGR and RGD,bind to surface molecules specific to tumor cells; therefore, they ca serve as theguided carrier molecules for effector agents. In the first part of this study, theenergized fusion protein of NGR and LDP was prepared by recombinant DNAtechnology. In the second part, the conjugate of RGD and Yunnamycin wassynthesized. The antitumor activity of the peptide-based protein and conjugate werestudied.1. Preparation and antitumor activity of the energized fusion proteinNGR-LDP-AENGR/CD13 is a good tumor vascular-targeted system. The NGR motif canselectively recongnize the CD13 isoform expressed in tumor blood vessels and inseveral cancer cells. Lidamycin (LDM, also called C-1027) is a macromolecularpeptide antitumor antibiotic which consists of an apoprotein (LDP) and an activeenedinye chromophore (AE).The AE has exetremely potent cytotoxicity and LDPprotects AE. LDP and AE can be separated and reconstituted. Therefore, the fusionprotein of NGR and LDP can reload the AE and form an energized fusion protein.The gene sngrldp was obtained by PCR on the pEFL plasmid which contained theldp gene. The primers were designed to include the Ndeâ… and Xhoâ… restriction sitesfor cloning in pET30a(+). The coding sequences of the signal peptide pelB andCNGRC were added to the forward primer. The pelB could make the recombinantprotein secreted into the periplasm. The product of PCR was about 0.4kb. The sngrldpfragment was cloned in T-vector by T4 ligase and sequenced. Then the T-vector wasdigested with Ndeâ… and Xhoâ… , and the digested fragment was cloned in plasmidpET30a(+) which was digested with the same enzymes. The recombinant expressingplasmid pET30sngrldp was transformed into E.coli BL21 (DE3) star^TM cells. The expression of protein was induced by addition of 0.05mM IPTG. The resultsof SDS-PAGE and Western-blot showed that NGR-LDP existed in the fermentionbroth, periplasm and cytoplasm. Because the proprotrein containing the signal peptidealso existed in the cytoplasm, only the protein in the fermentation broth was yield.The product was purified from the fermentation broth by ammonium sulfate (60%)precipitation and Ni-NTA His·Bind resin. Protein concentration was measured usingthe BCA reagent. Finally about 10 mg of purified protein was recovered from 1 L ofE.coli culture medium. Reducing and non-reducing SDS-PAGE of NGR-LDP showeda single band. The content of free sulfhydryl groups in NGR-LDP was about 0.687%.These results suggest that the recombinant protein is properly folded.The functional properties of the targeting domain of NGR-LDP fusion proteinwere investigated using ELISA and competitive ELISA. The results showed thatNGR-LDP could bind to HT-1080 cells and MCF-7 cells, but rLDP which contains noNGR motif could not bind to these cells. The binding of NGR-LDP to tumor cellscould be blocked by free NGR peptide in a dose-dependent manner. These results,collectively, suggest that NGR-LDP can bind with tumor cells and the bindingsequence was NGR domain. Western-blot showed that CD13 was expressed inHT-1080 and MCF-7 cells.The energized fusion protein NGR-LDP-AE was prepared by mixingchromophore AE and NGR-LDP with the mole ratio of 5:1 in PBS. 12 h later the freechromophore was removed from NGR-LDP-AE using PD-10(sephadex G-25 column).Full wave-length scan showed that NGR-LDP-AE had absorption both at 340 nm and280 nm. By contrast, NGR-LDP had absorption only at 280nm. Because chromophorehad absorption at 340nm, NGR-LDP-AE had been reconstituted successfully. TheA₂₈₀/A₃₄₀ ratio of purified LDM was 2:1. The ration of energized product was 3.1:1.It demonstrates that NGR-LDP was not reconstituted with AE completely. About 65%of NGR-LDP was reconstituted with chromophore.The cytotoxicity of the energized product was examed by MTT. The resultsshowed that the IC₅₀ values of NGR-LDP-AE and LDM were 1.94×10^-9 M and1.08×10^-10 M for HT-1080 cells as well as 2.67×10^-9M and 2.44×10^-10 M for MCF-7 cells,respectively. NGR-LDP did not show any cytotoxicity. The apoprotein couldinhibit the cytotoxicity of LDM or NGR-LDP-AE. After a 30 min incubation at 1 nMLDM followed by a 48 h drug-free period, the survival rate of HT-1080 cells was62.2%. When 10 nM rLDP was added, it increased to 77.4%. After a 48 h incubationat 1 nM LDM alone or at 1 nM LDM and 10 nM rLDP, the survival rates were 43.3%and 58.8%. After a 30min incubation at 5 nM NGR-LDP-AE followed by a 48 hdrug-free period, the survival of HT-1080 was 32.6%. When 10 nM rLDP orNGR-LDP were added, the survival rates were 64.5% and 62.32%. Though 100 nMBSA also could make the survival rate increase to 55.7%, it was lower than rLDP orNGR-LDP at lower concentration. These results demonstrate that LDP can inhibit thecytotoxciity of LDM specifically.In vivo anti-tumor activity and NGR-LDP-AE and LDM against murinehepatoma 22 (H22) was investigated in KunMing mice. Administration of 0.2 mg/kg,0.4 mg/kg and 0.8 mg/kg of NGR-LDP-AE, iv, on day 3 and 10 after inoculation,exerted significant antitumor effects. On day 16 the inhibition rates were 59.8%,77.8% and 94.8%, respectively. To LDM at 0.05mg/kg, the inhibition rate was 66.9%.No significant antitumor effects were observed when NGR-LDP was given at 2 mg/kg.No body weight loss or other severe side-effects were found during treatment. Itsuggests that NGR-LDP-AE shows remarkable antitumor effects without causingmajor toxicity.2. Synthesis and anti-tumor activity study of the conjugate ofYunnanmycin and RGD peptideRGD (Arg-Gly-Asp) motif which presents in many extracellular matrixcomponents such as fibronectin and vitronectin binds to integrins is related to celladhesion and motility. Yunnanmycin is a new cytosine nucleoside antitumor antibiotic.In order to obtain a conjugate which has the bioactivity of both yunnanmycin (YNM)and RGD, we couple YNM with RGD by chemical conjugation.According to the structure characteristics of YNM and RGD, the carboxy-group of YNM was conjugated to the amino-group of RGD with EEDQ after protecting theamino-group of YNM with Boc and the carboxyl-group of RGD with OMe. Thinlayer chromatography was used to monitor the reaction process. The molecular weight(MW) of the product was analyzed with ESI-MS. The product purity was analyzedwith HPLC. The cytotoxicity of YNM-RGD conjugate to PG cells and Bel-7402 cellswas determined by MTT assay. Boyden chamber system was used to evaluate theanti-migration activity. ESI-MS showed that the conjugate product containedYNM-RGD, YNM-RGD(OMe)₂ and YNM(Boc)-RGD(OMe)₂ with MW 773Da,801Da and 901Da. The overall content of the three compounds was 87.16% by HPLCassay. MTT showed that the IC₅₀ values of the conjugate and YNM was 0.129 mMand 0.01 mM to PG cells, 0.135 mM and 0.029 mM to Bel-7402 cells, respectively. At0.1 mM RGD did not show obvious cytotoxicity to PG cells and Bel-7402 cells. At 1mM the inhibitory rates of the conjugate, YNM and RGD were 73.1%, 91.8%and16.9%, respectively. Boyden chamber assay showed that the conjugate could inhibitthe invasion capacity of PG cells. The inhibitory rates of YNM-RGD conjugate andfree RGD were 50.9%and 40.8% at 0.1 mM; 66.4%and 56.2%at 0.2 mM; 66.6%and 57.1% at 0.4 mM. At 0.1 mM YNM could not inhibit the cells invasion capacity.All of these results demonstrate that YNM-RGD conjugate has the biological activityof both YNM and RGD. Though the cytotoxicity of YNM-RGD conjugate was lowerthan that of YNM, it was much higher than that of RGD. YNM-RGD conjugate couldinhibit the invasion capacity of tumor cells in a concentration dependent manner.