The Recombinant and Reconstituted Novel AlbuminâLidamycin

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The recombinant and reconstituted novel albumin-lidamycin conjugate shows lasting tumor imaging and intensively enhanced therapeutic efficacy liang li, lei hu, chunyanzhao zhao, sheng-hua zhang, rong wang, yi li, Rong-Guang Shao, and Yongsu Zhen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00456 • Publication Date (Web): 14 Aug 2018 Downloaded from http://pubs.acs.org on August 18, 2018

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Bioconjugate Chemistry

The Recombinant and Reconstituted Novel Albumin-lidamycin Conjugate Shows Lasting Tumor Imaging and Intensively Enhanced Therapeutic Efficacy

Liang Li†, Lei Hu†, Chun-yan Zhao†, Sheng-hua Zhang†, Rong Wang†, Yi Li†, Rong-guang Shao†, Yong-su Zhen†

†Institute

of Medicinal Biotechnology, Chinese Academy of Medical

Sciences and Peking Union Medical College, Beijing, 100050, P.R. China.

* To whom the correspondence should be addressed. Yong-su Zhen, Laboratory of Cancer, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, P.R. China. 1 Tian Tan Xi Li, Beijing, China E-mail:

[email protected].

Tel:

+86-10-83158065.

+86-10-6131808.

ACS Paragon Plus Environment

Fax:

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Depending on increasing extracellular proteins utilization and altering metabolic

programs,

cancer

cell

could

proliferate

and

survive

unrestrictedly by ingesting human serum albumin (HSA) to serve as nutritional amino acids. Here, we hypothesize that the consumption of albumin by cancer cells could be utilized as an efficient approach to targeted drug delivery. Lidamycin (LDM), an antitumor antibiotic with extremely potent cytotoxicity to cultured cancer cells, consists of an apoprotein (LDP) and an active enediyne chromophore (AE). In the present study, a novel albumin-lidamycin conjugate was prepared by DNA recombination and molecular reconstitution. Results shown, the IC50 values of albumin-lidamycin conjugate (HSA-LDP-AE) for a variety of tested cancer cells were at sub-nanomolar levels. At tolerated dose, the albumin-lidamycin conjugate significantly inhibited the growth of lung carcinoma PG-BE1 xenografts by 97.8%. The therapeutic efficacy of the albumin-lidamycin conjugate was much stronger than that of free lidamycin. Meanwhile, the images of albumin-lidamycin conjugate showed obvious and lasting tumor localization and fluorescence enrichment and there was no detectable signal in non-tumor locations. Taken together, albumin-lidamycin conjugate, a new format of lidamycin, could

be

a

promising

antitumor

therapeutic

agent

and

albumin-integration might be a feasible approach to targeted antitumor


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drug delivery.

INTRODUCTION: Cancer is defined as the uncontrolled proliferation of cells. As shown, cancer cells demand for more nutrient and cellular building blocks which could enable them to grow and divide rapidly and unrestrictedly. In order to sustain these limitless replicate potential, cancer cell could increase nutrient utilization and alter the metabolic programs to fuel quickly proliferation and survival1. Amino acid and glucose represent two essential consumed nutrients in cancer cell culture

2, 3.

As for the amino

acids, the extracellular proteins can be actively scavenged or ingested via macropinocytosis by cancer cells

4, 5.

Indeed, the human serum albumin,

the most abundant protein in human blood plasma, has been verified that it can be enormously internalized into the cancer cell. The breakdown of albumin contributes to the supply of free amino acids in tumors and serves as an important nutritional source for cancer cells6. Apparently, this altered extracellular protein metabolic status of cancer cells could be utilized for targeted delivery of antitumor drugs and the nutrient substance such as albumin could be acting as an ideal carrier in the biological process mimetic delivery. In preclinical studies, albumin has been used for targeted delivery of various anti-tumor drugs, such as chemotherapeutics, tyrosine kinase inhibitors, nanomedicines, and



peptide drugs7-9. Meanwhile, a variety of albumin-based drugs have been used in tumor therapy or under clinical trials10-12. Lidamycin (LDM), a 9-membered cyclic enediyne antibiotic produced by Streptomyces globisporus C-1027, is composed of an active enediyne chromophore (AE, MW:843 Da) and a lidamycin apoprotein (LDP, MW:10489 Da). AE exerts extremely potent cytotoxicity that could inhibit cancer cell proliferation at sub-nanomolar concentration and LDP could bind with AE and stabilize the enediyne structure with hydrophobic interaction13-15（Figure 1）. Once inside the cell, the AE of LDM interacts in the DNA minor groove and cleaves double-helical DNA, causing single-strand and double-strand breaks through radical-mediated hydrogen abstraction16. The major mechanisms of action of LDM include DNA damage, cell cycle arrests and induction of apoptosis and mitotic cell death. The previous study shows that the cytotoxicity of LDM is about 1000-fold more potent than that of doxorubicin in various cultured cancer cells17. Moreover, LDM could suppress angiogenesis with low doses and show synergistic antitumor effect in combination with first-line chemotherapeutics and molecular targeted drugs such as cisplatin, gemcitabine, gefitinib, and bortezomid18-21. Here, we hypothesize that the uptake and consumption of extracellular albumin by cancer cells could be utilized as a targeting process to deliver precisely and massively the highly potent LDM to tumor location,


Page 4 of 33

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resulting in higher therapeutic efficacy of LDM. Aiming to reduce unwanted side effects and accumulate the drug at cancer site, a novel recombinant conjugate consisting of human serum albumin (HSA) and LDM was prepared by genetic engineering and molecular reconstitution. The cytotoxicity, therapeutic efficacy and in vivo tumor targeting of the albumin-lidamycin conjugate have been demonstrated. It provides evidence that the uptake of extracellular albumin-integrated LDM could intensively improve tumor targeting and enhanced therapeutic efficacy.

Figure 1. Structure of lidamycin. (A) Active enediyne chromophore of lidamycin. (B) Apoprotein of lidamycin

RESULTS: Construction, expression, purification of HSA-LDP conjugate After overlap PCR and DNA cloning process, DNA fragments encoding for HSA-LDP conjugate with (G4S)3 linker between HSA and LDP was obtained and inserted into the expression vector (Figure S1). A (His)6 tag



was introduced at the COOH terminal of conjugate to facilitate purification through Ni2+ affinity chromatography. The HSA-LDP conjugate was successfully expressed in Pichia pastoris and secreted into the broth in a soluble form (Figure S2 and 3). The purity of engineered protein was over 95% when analyzed by SDS-PAGE and SEC-HPLC (Figure 2 and 3).

Figure 2. The SDS-PAGE analysis of HSA-LDP conjugate. (M: marker, Lane 1: HSA-LDP conjugate)

Figure 3. The HPLC analysis of HSA-LDP conjugate. (Blue: HSA-LDP


Page 6 of 33

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conjugate (tR=8.9 min), Orange: HSA (tR=9.3min), Green: LDP (tR =10.6 min)) (Equipment: aglient 1200 LC system, column: phenomenex, BioSep-SEC-S3000 300×7.8 mm, mobile phase: 0.1M PBS, flow rate: 1ml/min)

The reconstitution of HSA-LDP conjugate and AE To obtain HSA-LDP conjugate with the highly potent antitumor activity, the HSA-LDP conjugate was assembled with AE, which was separated from natural lidamycin produced by Streptomyces globisporus C-1027 (Figure S4), to form the enediyne-energized HSA-LDP-AE. After removing the free AE by centrifugal filter, the active HSA-LDP-AE conjugate was confirmed by HPLC. As shown, the HPLC chromatogram of HSA-LDP-AE displayed an AE peak, which suggested that HSA-LDP conjugate successfully assembled with AE and the HSA-LDP conjugate retained the binding activity with AE (Figure 4 and Figure 5). Meanwhile, AE could not assemble with HSA which does not contain the structure of LDP (Figure S5).

Figure 4. Schematic diagram of the reconstituted HSA-LDP-AE.



Figure 5. Chromatograph of HSA-LDP-AE on RP-HPLC after assembly of HSA-LDP with AE (tR=10.74min).

Cytotoxicity of the albumin-lidamycin conjugate HSA-LDP-AE The inhibition of various cancer cells proliferation by HSA-LDP-AE was determined by MTT assay. HSA-LDP-AE and LDM markedly inhibited the proliferation of various cancer cell lines at sub-nanomole levels. As shown (Table 1 and Figure S6), the IC50 values of HSA-LDP-AE for PG-BE1, HT-1080, MCF-7, SKOV3, A549, and C26 cells were 2.54×10-4 µM, 5.51×10-4 µM, 1.03×10-3 µM, 3.34×10-5 µM, 2.34×10-5 µM and 3.91×10-4 µM, respectively. The IC50 values of HSA-LDP-AE for all tested cancer cells were slightly lower than that of LDM, indicating that cancer cells were rather sensitive to HSA-LDP-AE than LDM.


Page 8 of 33

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Table 1 The IC50 values (µM) of LDM and HSA-LDP-AE to various cancer cell lines Group

HT-1080

C26

MCF-7

PG-BE1

SKOV3

A549

LDM

9.82×10-4

5.42×10-4

4.24×10-3

5.62×10-4

9.76×10-5

9.58×10-5

HSA-LDP-AE

5.51×10-4

3.91×10-4

1.03×10-3

2.54×10-4

3.34×10-5

2.34×10-5

Enhanced therapeutic efficacy of the albumin-lidamycin conjugate HSA-LDP-AE In mouse hepatoma H22 model, the antitumor efficacy of HSA-LDP-AE was compared with LDM and HSA-LDP conjugate (0.8mg HSA-LDP-AE is equivalent to 0.1mg LDM). After 3 days of tumor implantation, HSA-LDP-AE

(0.8 mg/Kg, 0.4 mg/Kg), LDM (0.05 mg/Kg), HSA-LDP conjugate (20 mg/Kg) were respectively injected via tail vein. Twice injections with a week interval were administered. At day 12, the mice were sacrificed, and the tumor weights were measured. The tumor growth inhibition rates of HSA-LDP-AE, LDM, and HSA-LDP were 90.8%, 69.9% and 34.8%, respectively. The HSA-LDP-AE treated group showed statistically significant differences compared with control group. More importantly, the therapeutic efficacy of HSA-LDP-AE treated group was markedly superior to that of LDM treated group, and there was statistically significant difference between these two treatment groups (Table 2).



Page 10 of 33

Table 2 The tumor growth inhibition efficacy of HSA-LDP-AE, LDM, and HSA-LDP on hepatoma 22 in mice Groups

Dose

Mouse

(mg/kg)

number

Control

BWC(g)

10/10

+ 7.66

Tumor

Inhibition

weight(g)

rate (%)

2.14±0.89

LDM

0.05

10/10

+5.12

0.65±0.27

69.9*

HSA-LDP-AE

0.8

10/10

+0.87

0.20±0.06

90.8*, **

0.4

10/10

+3.09

0.32±0.03

84.9*

20

10/10

+7.20

1.39±0.46

34.8*

HSA-LDP

* P

The Recombinant and Reconstituted Novel AlbuminâLidamycin

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