Cell lines and cell culture
The hybridoma cell line HB-8696 was purchased from ATCC and grown in Dulbecco's modified Eagle Medium (DMEM) and fortified with penicillin-streptomycin (100 U/ml, 100 μg/ml respectively) and 10% fetal bovine serum (FBS). Medium was changed every 2–3 days. The breast cancer cell lines, Zr-75-30 and MCF-7, and the Burkitt's Lymphoma cell line, Raji (obtained from the Laboratory of Transplant Immunology and the Department of Laboratory Medicine, Division of Clinical Immunology, West China Hospital) were grown in RPMI 1640 medium containing double antibiotics and 10% FBS. Medium was changed every 2–3 days. All cell lines were incubated at 37°C in 5% CO2 incubator (Sanyo Electro. Biomed. Japan).
The preparation of parental antibody 520C5 and toxicin colicin Ia
HB-8696 murine hybridoma cells were grown to a density of 107 cells/ml. Under sterility and 4°C, the cells were removed from the medium by centrifugation at 1000 rpm, and the supernatant (containing the original mAbs 520C9 that are the parental antibody of the mimetic peptide molecules) was further purified by centrifugation at 10,000 g. The following purification procedure was done according to purification kit' protocol (Millipore). The purified antibodies were stored at -20°C for subsequent experiments.
The pSELECT-1 plasmids (from the Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, China) that contain the colicin Ia gene and the reversed direction immunity protein gene of colicin Ia were transformed into competent TG1 cells. Then spread those TG1 cells on FB agar medium containing 25 μg/ml ampicillin and cultivated at 37°C for 12–16 hours under humidity and screening TG1 cells containing pSELECT-1 plasmids, and the positive clones were selected to cultivate rotatorily at 180 rpm in 2 ml FB medium containing 50 μg/ml ampicillin under the same condition as above mentioned for overnight, then carefully dumped to 60 ml FB medium for continuous cultivation for 5–6 hours. Until the total volume of medium reached 8 × 600 ml and the OD for TG1 cells reached 0.5 under same culture condition, centrifuged those cells at 6,000 g for 17 minutes under 4°C, and resuspended precipitate in 60–80 ml borate buffer (50 mM borate buffer, pH 9.0, with 2 mM EDTA) containing 0.5 mM phenylmethylsulfonyl fluoride. The cells were sonicated and debris removed by centrifugation for 90 min at 75,000 g under 4°C. Nucleic acids were precipitated by addition of 1/5 volume streptomycin sulfate (25%). Supernatants were dialyzed against borate buffer for 12 hours (changing the buffer every 5–6 hours) at 4°C then applied to the ÄKTA™ prime protein purification system (2.5 × 12 cm CM-Sepharose column, Amersham Pharmacia Biocech). Proteins were recovered at 4°C by gradient elution with 0.1, 0.2 and 0.3 M NaCl in borate buffer and collected in 0.5 ml fractions. The harvested colicin Ia was dialyzed against PBS (pH 7.4–7.5) for 12 hours at 4°C, and stored at -80°C freezer for subsequent experiments.
The scanning of VH and VL domain DNA sequences of original antibody
VH and VL domain genes for mAb A520C9 IgG were isolated from HB-8696 mouse hybridoma cell. Total RNA was extracted and amplified by RT-PCR (Takara RNA PCR Kit (AMV Ver.3.0)) using the following primers: H-chain: 5'-ACTAGTCGACATGGCTGTCYTRGBGCTGYTCY TCTG-3'and 5'-CCCAAGCTTCCAGGGRCCARKGGATARACWGRTGG-3'; L-chain: 5'-GGGAATTCATGGAGACAGACACACTCCTGCTAT-3'and 5'-CCCAAGCTTACTGGA TGGTGGGAAGATGGA-3', purified RT-PCR products were ligated into the plasmids pMD18-T, purchased from Takara. The DNA sequences of plasmids were isolated and analyzed to determine the genes of VH and VL domains of mAb.
Amino acid sequences of peptides from parental antibody
The aa sequences of all six CDRs in the parental antibody 520C9 Fab are:
VH: H2N-EMQLVESGPEVKKPGASVKVSCKASGYTFTNYGMN WVRQAPGQGLEWM GWINTYTGQSTYADDFKE RVTMTTDTSTSTAYDMLRSLRSDDTAVYYCARRFGFAY WQ GTLVVSS-COOH (bold letters represent CDR domain)
VL: H2N-DIQMTQSPSSLSASVGDRVTITCRASQDIGNSLT WYQQKPGKTPKLLIYATS SLDS GVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYAIFPYT FGQGTRLEIK-COOH (bold letters represent CDR domain)
The sequence of the single-chain Fv (ScFv) for the parental antibody 520C9 is:
The sequence of the VHFR1C-10-VHCDR1-VHFR2-VLCDR3-VLFR4N-10mimetic of the parental antibody is:
Preparation of the mimetic moiety and conjugated peptide
The DNA sequences for the VHFR1C-10, VHCDR1, VHFR2, VLCDR3 and VLFR4N-10 regions of the 520C9 Fab were conjugated to follow position I626 of colicin Ia by double-stranded oligonucleotide mutagenesis (QuickChange kit, Stratagene) using the pSELECT-1 plasmid that contains the colicin Ia gene and the reversed direction immunity protein gene of colicin Ia to form colicin Ia-VHFR1C-10-VHCDR1-VHFR2-VLCDR3-VLFR4N-10 (Fig. 1). The oligonucleotides used contained the desired mutations for SCKASGYTFTNYGMNWVRQAPGQGLEWMGLQYAI FPYTFGQGTRLEIK were 5'-GCG AAT AAG TTC TGG GGT ATT TCC TGC AAG GCT TCT GGT TAC ACC TTT ACC TAA ATA AAA TAT AAG ACA GGC-3', 5'-GCT TCT GGT TAC ACC TTT ACC AAC TAT GGA ATG AAC TGG GTG CGA CAG GCC TAA ATA AAA TAT AAG ACA GGC-3', 5'-ATG AAC TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT GAG TGG ATG GGA CTA TAA ATA AAA TAT AAG ACA GGC-3', 5'-GGG CTT GAG TGG ATG GGA CTA CAA TAT GCT ATT TTT CCG TAC ACG TTC GGC TAA ATA AAA TAT AAG ACA GGC-3' and 5'-ATT TTT CCG TAC ACG TTC GGC CAA GGG ACA CGA CTG GAG ATT AAA TAA ATA AAA TAT AAG ACA GGC-3' (boldface triplets represent inserted sites).
Plasmids containing inserted DNA sequences were transformed into competent TG1 E. coli, and cells were grown in FB medium containing 50 μg/ml ampicillin. The procedures of cultivating TG1 cells and purifying conjugated peptides were the same as that of preparing colicin Ia protein.
In vitro killing activity, Immunolabeling and affinity assays
ZR-75-30, MCF-7, and Raji cells were grown in the Falcon 3046 six-well cell culture plates (Becton Dickinson Co.) under the same condition as that of above described. 24 hours later, 5–125 μg/ml PMN, wild type colicin Ia (wt Ia), parental antibody-colicin Ia fusion protein (Fab-Ia), single-chain antibody-colicin Ia fusion protein (Sc-Ia) (CL(Xi'an) Bio-scientific) and nonrelative control protein, low molecular weight marker protein (LWMP, purchased from Takara) were respectively added to the cell culture wells. After co-incubating for 24 hours, the living and dead cells were stained with 50 nM acridine orange and 600 nM propidium iodide and staining was imaged using a digital data collection system under an inverted fluorescent microscope (IX-71, Olympus) using U-MWU2, U-MNB2 and U-MNG2 filters. For the comparison of killing competency presented by those agents with each other, we selected five image fields to respectively count the number of dead and living cells in every culture well after 24, 48 and 72 hours.
MCF-7 cell were grown in 1640 medium for 72 h, fixed in 10% paraformaldehyde for 40 min at room temperature, then 100 μl fixed cells (106/ml) were incubated with 10 μl PBS, LWMP, Fab, Sc (CL(Xi'an) Bio-Scientific) and PMN respectively with different concentration (102-10-1nM) for 1 hr at 37°C, then incubated with parental antibody for 40 min at 37°C and fluorescein isothiocyanate (FITC) -labeled second antibody (Pierce) for 30 min at 37°C. After incubating with DAPI for 25 minutes at 37°C, the mean fluorescent intensity of per 1,500 cells was measured by BD FACSCanto Flow Cytometer (BD Biosciences). For concentration-dependent inhibitory experiments against the killing activity of PMN, different concentrations of either parental A520C9 mAbs, or synthetic VHFR1C-10-VHCDR1-VHFR2-VLCDR3-VLFR4N-10 (South West University) were added with PMN (75 μg/ml) to incubate with MCF-7, Zr-75-30 or Raji cells, respectively (102-10-1nM), then living and dead cells were counted with 0.2% Trypan blue under an inverted microscope (IX-71, Olympus).
The MCF-7 cells were grown and fixed as the above-mentioned procedure. Then original antibodies (OAbs) and the mimetic peptides were diluted to 100, 10, 1 and 0.1 μmol/L by PBS (pH7.45), respectively. The indirect enzyme-linked immunosorbent assays (ELISA) were introduced to analysis the relative affinity of the mimetics and OAbs to antigens. The value of absorbance at 490 nm wavelength was inspected by microplate reader (Bio-Rad), which was used to determine the concentration of the OAbs and the mimetics when the saturation of Abs to antigens reached to one percent. The relative affinity was compared between OAbs and the mimetics at 50% saturation of Abs to antigens.
In vivo activity and the biodistribution of PMN
MCF-7 cells were grown under the same condition as that of above described, and collected by centrifugation at 1,000 rpm. Cells were resuspended in FBS-free medium at a concentration of 108 cells/ml. Twenty-five 4–5-week-old female BALB/c athymic nude mice weighing 16–20 g were purchased from the Experimental Animal Center of West China Hospital. Before implanting tumor cells, mice were allowed to acclimatize for 3 days. A total of 6–7 × 107 MCF-7 cells were subcutaneously (s.c.) implanted into the left armpit of mice. Tumor growth was monitored daily until the average sizes of tumors reached 5 × 5 × 5 mm, then randomly separated those mice to the treatment group (PMN group; n = 5), wild type colicin Ia group (wt Ia group; n = 5), Fab-Ia group (n = 5), Sc-Ia group (n = 5) and the PBS control group (PBS group; n = 5), and the treatment course began. The PMN group was treated with intraperitoneal (i.p.) injection of PMN at 1,200 μg/mouse/day (400 μg/8 hours, tid; n = 5). The wt Ia group, Fab-Ia group, Sc-Ia group and the PBS group were injected with wt Ia protein, Fab-Ia protein, Sc-Ia protein (400 μg/8 hours, i.p. tid; n = 5) and PBS (450 μl/8 hours, i.p. tid; n = 5), respectively. Animals had free access to standard food and water throughout the treatment course. After 14 days, all mice were sacrificed to collect tumors and organs for weighing and for histopathological inspection.
150 μg PMN proteins labeled by FITC (EZ-labeled FITC protein labeling kit, pierce) were ip injected into BALB/c mice (n = 5), weighing 16–20 g, inoculated MCF-7 cells at armpit for 2 weeks. 2.5 hours later, the mice were fastened supinely on a black board under ether inhalation. When the in vivo inspections completed, the mice were sacrificed and the tumors and vital organs were sectioned. The images were observed with the LT-99D2 Illumatool Dual Light System (excitation 470 nm, emission 515 nm, Lightool Research) and recorded by a built-in camera.
Assessment of toxicity of PMN
Kunming normal mice (purchased from Experimental Animal Center of West China Hospital, Sichuan University, China), weighing 15–25 g were injected with either PMN (100–2,500 μg/mouse/day, n = 5) or PBS (n = 5) intraperitoneally each day. After 3 weeks of administration, mice were sacrificed for histopathological inspection and blood samples were collected for indirect enzyme-linked immunosorbent assay (ELISA) to screen potential antibodies.
The Institutional Animal Care and Use Committee of Sichuan University and Project of Sichuan Animal Experiment Committee (license 045) approved the animal use and in vivo experiments.
0.9% agarose electrophoresis was applied to authenticate the reconstructed plasmids and 15% sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) was applied to authenticate the harvested protein, respectively.
SPSS version 11.0.1 for Microsoft Windows was used for statistical analysis. Two-tailed t-tests were performed using GraphPad Prism for Windows version 4.00. P < 0.05 was considered to be a statistically significant difference.