RTRAIL mutant and monomethyl auristatin E conjugate thereof

Abstract

Disclosed are an rTRAIL mutant and monomethyl auristatin E (MMAE) conjugate thereof. The amino acid sequence thereof is as represented by SEQ ID No.1. Also disclosed are a coding gene of the rTRAIL mutant and expression system, recombinant vector and expression unit containing the coding gene. Also disclosed are an rTRAIL mutant-vcMMAE conjugate and preparation and uses thereof. The conjugate of the present invention has the biological functions of both the rTRAIL mutant and the MMAE; the MMAE is directionally transferred to a tumor cell through the specific binding between the rTRAIL mutant and a death receptor on the surface of the tumor cell, and is released and takes effect in the tumor cell, thus killing the tumor cells less sensitive or even resistant to TRAIL, and reducing the toxicity generated by the separate administration of the MMAE.

Claims

1. An rTRAIL mutant, characterized in that the amino acid sequence is represented by SEQ ID No.1.

2. A gene encoding the rTRAIL mutant according to claim 1, characterized in that the base sequence is represented by SEQ ID NO.2.

3. The polynucleotide of claim 2 in operable association with a promoter, or a recombinant vector or an expression system containing the polynucleotide according to claim 2.

4. An rTRAIL mutant-MMAE conjugate, characterized in that it is formed by conjugating monomethyl auristatin E (MMAE) with an rTRAIL mutant trimer through a linker, the amino acid sequence of the rTRAIL mutant is represented by SEQ ID No.1, and said linker is a maleimide-modified valine-citrulline dipeptide.

5. An use of the rTRAIL mutant-MMAE conjugate according to claim 4 in preparation of anti-tumor drugs.

6. A method of preparing an rTRAIL mutant-MMAE conjugate, comprising: (1) An rTRAIL mutant polymer is depolymerizd and re-polymerized to generate an rTRAIL mutant trimer; (2) The rTRAIL mutant trimer is mixed with a MMAE having a linker to perform a coupling reaction; (3) After completion of the reaction, the rTRAIL mutant-MMAE conjugate is obtained by separation and purification; wherein, the amino acid sequence of the rTRAIL mutant is represented by SEQ ID No.1; said linker is a maleimide-modified valine-citrulline dipeptide.

7. The method according to claim 6, wherein said rTRAIL mutant trimer is prepared by a method comprising: dissolving the rTRAIL mutant in a buffer containing zinc ion, adding tris(-chloroethyl) phosphate thereinto, and reacting under water-bath for 13 hours at 30 C.40 C.

8. The method according to claim 6, wherein said coupling reaction is performed at a temperature of no higher than 4 C. for 3060 min.

9. The method according to claim 6, wherein said separation and purification includes: removing the substances with molecular weights below 10 kDa from the reaction solution by ultrafiltration, removing the precipitate by centrifugation to give a supernatant, and sterilizing the supernatant by filtration to obtain the rTRAIL mutant-MMAE conjugate.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the electrophoretogram of rTRAIL mutant N109C expressed by E. coli. M is the low molecular weight marker; lane 1 is the non-induced bacterial liquid; lanes 2 and 3 are the induced bacterial liquid; lane 4 is the supernatant (soluble fraction) obtained by centrifuging lysed non-induced bacterial liquid; lanes 5 and 6 are the supernatant (soluble fraction) obtained by centrifuging lysed induced bacteria; lanes 7, 8 and 9 are the samples of induced and non-induced precipitation reconstituted with 8M urea.

(2) FIG. 2 is the electrophoretogram showing the affinity purification result of rTRAIL mutant N109C. M is the protein molecular weight marker; lane 1 is the bacterial liquid induced with IPTG; lane 2 is the supernatant after lysing the bacteria; lane 3 is the flow-through of Ni column; lane 4 is the eluate of imidazole (10 mM); lane 5 is the eluate of imidazole (60 mM); lane 6 is the eluate of imidazole (500 mM).

(3) FIG. 3 is the electrophoretogram showing the SP strong cation exchange column purification result of rTRAIL mutant N109C. Lane 1 is the protein solution desalted with an imidazole eluent (10 mM); lanes 2 and 3 are the protein eluted with SP column, namely purified N109C; lane 4 shows the result of non-denaturing electrophoresis of N109C.

(4) FIG. 4 is the electrophoretogram of N109C and its MMAE conjugate. Lane 1 is N109C; lane 2 is the N109C-vcMMAE conjugate; lane 3 is the desalted N109C-vcMMAE conjugate; lanes 4, 5 and 6 show the electrophoresis results of the samples in lanes 1, 2, and 3 under non-denatured condition, respectively.

(5) FIG. 5 is the schematic diagram showing the connection relationship among various portions of the rTRAIL mutant-vcMMAE conjugate.

(6) FIG. 6 shows the killing effect of various TRAIL elements on different cell lines.

(7) FIG. 7 is the distribution of N109C-vcMMAE conjugate in tumor-bearing mice.

(8) FIG. 8 is a photomicrograph showing the experiment of cell endocytosis of N109C-vcMMAE conjugate by tumor cells.

(9) FIG. 9 is the schematic diagram showing the apoptosis-inducing mechanism of the rTRAIL mutant-vcMMAE conjugate.

DETAILED DESCRIPTION

(10) The present invention is further illustrated in detail hereinafter with reference to the accompanying drawings and the embodiments.

(11) 1. Establishment of Human Prostate cDNA Library

(12) (1) Extraction of Total mRNA from Human Prostate

(13) Frozen small pieces of human prostate tissue (about 100 mg) were ground in liquid nitrogen into powders, into which TRIZOL reagent (1 mL) was added to grind repeatedly, and transferred to 1.5 ml RNase-free eppendorf (EP) tube. The tube was placed at room temperature for 5 min, followed by addition of 0.2 mL chloroform to shake vigorously for 15 s and placed at room temperature for 3 min. Centrifuge at 4 C. for 15 min at 12000 g. The upper aqueous phase was transferred into another RNase-free EP tube. Isopropanol (0.5 mL) was added and kept at room temperature for 10 min to precipitate RNA. Centrifuge at 4 C. for 10 min at 12000 g and the supernatant was removed. The precipitate obtained was washed with 75% ethanol. Centrifuge at 4 C. for 5 min at 12000 g and remove the supernatant. The pellet was dried and then dissolved with 50 L DEPC water, and stored at 70 C.

(14) (2) Reverse Transcription (RT) PCR

(15) With oligodT as a primer, human prostate total RNA as a template, AMV reverse transcriptase was added to conduct Reverse-PCR to obtain a cDNA library, including the following steps:

(16) 1) RNA pre-denaturation: 5 g human prostate RNA+25 g OligodT was made up to 10 L with 01% DEPC, and subjected to 70 C. water-bath for 5 min and cooled to room temperature to destroy the secondary structure of mRNA.

(17) 2) RT: the synthesis system was as follows:

(18) TABLE-US-00003 Ingredient Volume Said RNA mixed solution 10 L dNTP (each10 mM) 3 L RNase inhibitor (40 U/L) 20 U Reverse transcriptase 15 U 5 Buffer 4 L 0.1% DEPC Make up to 20 L The PCR program was: reverse transcription process 42 C. 60 min inactivation of reverse transcriptase 70 C. 10 min

(19) The obtained cDNA was stored at 20 C. for later use.

(20) 2. Obtaining TRAIL Coding Sequence

(21) With said cDNA as a template, P1 and P2 as the upstream and downstream primers, the coding sequence of TRAIL was synthesized and amplified under the catalysis of Taq DNA polymerase. Sequence analysis confirmed that the DNA sequence obtained was identical with the TRAIL coding sequence registered in GenBank (NM_003842.4), i.e., the TRAIL coding sequence was obtained. The sequences of the primers were as follows:

(22) TABLE-US-00004 P1: (SEQIDNO:5) 5-ATGGCTATGATGGAGGTCCAGG-3; P2: (SEQIDNO:6) 5-TTAGCCAACTAAAAAGGCCCCG-3.

(23) The PCR reaction system was as follows:

(24) TABLE-US-00005 Ingredients Volume 5 PCR buffer (containing magnesium ions) 10 L dNTP Mixture (each 2.5 mM) 4 L P1 1 L P2 1 L cDNA 2 L Prime Star High-Fidelity enzyme (2.5 U/L) 0.5 L ddH.sub.2O 31.5 L

(25) The PCR reaction program is as follows:

(26) TABLE-US-00006 Pre-denaturation 95 C. 3 min Denaturation 95 C. 30 seconds Annealing 55 C. 30 seconds {close oversize brace} 30 cycles Extension 72 C. 1 min Extension 72 C. 10 min Hold 4 C. hold
3. Obtaining Mutant Sequence of TRAIL 95-281 (rTRAIL)

(27) (1) Obtaining Mutant Sequence of rTRAIL N109C

(28) With the TRAIL coding sequence as a template, P3 and P4 as the upstream and downstream primers, the coding sequence of rTRAIL N109C mutant was synthesized and amplified under the catalysis of Taq DNA polymerase. Sequence analysis confirmed that the DNA sequence obtained was shown in SEQ ID No.2, which was consistent with expectation. The sequences of the primers were as follows:

(29) TABLE-US-00007 P3: (SEQIDNO:7) 5-TATACCATGGGCACCTCTGAGGAAACCATT TCTACAGTTCAAGAAAAGCAACAATGTATTTCT-3; P4: (SEQIDNO:8) 5-TTCTCGAGTTAGCCAACTAAAAAGGCCCC GAAAAAACTGGCTTCATGGTCCATGTCCATGTC-3.

(30) Except for the primers and the template, the PCR reaction system and program were the same as that of step 2.

(31) (2) Obtaining Mutant Sequence of rTRAIL S96C

(32) With the TRAIL coding sequence as a template, P5 and P6 as the upstream and downstream primers, the coding sequence of rTRAIL N109C mutant was synthesized and amplified under the catalysis of Taq DNA polymerase. Sequence analysis confirmed that the DNA sequence obtained was shown in SEQ ID No.4, which was consistent with expectation. The sequences of the primers were as follows:

(33) TABLE-US-00008 P5: (SEQIDNO:9) 5-TATACCATGGGCACCTGTGAGGAAACCATT TCTACAGTTCAAGAAAAGCAACAAAATATTTCT-3; P6: (SEQIDNO:10) 5-TTCTCGAGTTAGCCAACTAAAAAGGCCCC GAAAAAACTGGCTTCATGGTCCATGTCCATGTC-3.

(34) Except for the primers and the template, the PCR reaction system and program were the same as that of step 2.

(35) Hereinafter, the rTRAIL N109C mutant sequence was taken as an example to further illustrate preparation of rTRAIL mutant and construction of conjugates. The preparation of rTRAIL S96C mutant and construction of conjugates were the same.

(36) 4. Construction of Expression Vector Comprising the rTRAIL Mutant Sequence

(37) The rTRAIL N109C mutant sequence and pET28a(+) were double-digested with NcoI/XhoI respectively, and then were ligated at a molar ratio of 3:1 with T4 ligase (Takara). The ligation products were transformed into E. coli DH5a competent cells, and positive clones were selected and cultured. After extraction of the plasmid, NcoI/XhoI double-digestion was used to verify their ligation, and sequencing was used to make further verification, resulting in that rTRAIL mutant expression vectors were obtained: pET28a(+)-rTRAIL N109C.

(38) 5. Transformation of Expression Vector into E. Coli to Establish Engineered Bacteria

(39) The expression vector pET28a(+)-rTRAIL N109C was transformed into E. coli expression host BL21 (DE3) (purchased from Novagen), and monoclone was picked from kanamycin plate and cultured overnight at 37 C., 160 rpm. The bacterial liquid PCR method was used to determine whether the rTRAIL mutant expression vector had been transformed into the expression host. With bacterial liquid as a template, P3 and P4 as primers, PCR was performed. Verified positive monoclones were identified as the engineered bacteria of interest, and the bacterial liquid was kept at 80 C. after addition of 10-15% glycerol.

(40) 6. Preparation and Purification of rTRAIL Mutant

(41) The constructed engineered bacteria were inoculated in LB culture medium (200 mL, containing 15 g/mL kanamycin), cultured in a shaker with a rotating speed of 160 rpm at 37 C. until the bacterial liquid OD600 reached about 0.8. IPTG (final concentration: 1 mM) was added to induce BL21 (DE3) to express fusion protein, with an induction time of 12 h. BL21 bacteria were obtained by centrifuging at 7200 g for 10 min.

(42) Said bacteria was resuspended with loading buffer A and disrupted by a French press. After 7200 g centrifugation for 30 min, the supernatant was filtered through a 0.22 m water membrane and was subjected to metal affinity chromatography with a Ni-NTA affinity column (GE). Imidazole (10 mM) was used to elute other proteins as impurities, and imidazole (60 mM) was used to get target protein. The fraction eluted with imidazole (60 mM) was treated by a desalinization column (GE) to change its buffer to buffer B, which was further subjected to ion exchange chromatography with a SP strong cation exchange column (GE). Buffer C was used for eluting the protein on the ion exchange column.

(43) Wherein, the ingredients of each buffer were as follows: Buffer A: 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, pH 7.4 Buffer B: 20 mM NaH.sub.2PO.sub.4, pH 6.0 Buffer C: 20 mM NaH.sub.2PO.sub.4, 1 M NaCl, pH 6.0

(44) FIG. 1, FIG. 2 and FIG. 3 illustrated the electrophoretogram of prokaryotic expression and purification of the rTRAIL N109C mutant. As shown in FIG. 1, the rTRAIL N109C mutant achieved soluble expression in E. coli successfully. As shown in FIG. 2, the rTRAIL N109C mutant began to be eluted with imidazole (10 mM), and its purity was no less than 85% after preliminary purification. As shown in FIG. 3, part of other proteins could be removed after purification by SP column, yet 3 slight undesired bands were found. Meanwhile, according to the results of non-denaturing gel electrophoresis, dimmers, tetramers etc were still found in the substantially purified rTRAIL N109C mutant, which was caused by the disulfide bond between the mutant protein molecules.

(45) The amino acid sequence of the N109C mutant obtained is shown in SEQ ID No.1, and the amino acid sequence of the S96C mutant obtained is shown in SEQ ID No.3.

(46) 7. Conjugation of rTRAIL Mutant and MMAE

(47) 0.5 mg rTRAIL N109C mutant was dissolved in 0.8 mL PBS (pH 6.0, containing 10 M ZnCl.sub.2), into which 6 L TCEP was added, and water bath was maintained for 2 hours at 37 C. While stirring, 10-fold excessive amount of vcMMAE dissolved with 50 L 30% acetonitrile/water was added to react at 4 C. for 40 min, and the reaction was ended by addition of excessive amount of cysteine. (Said vcMMAE was synthesized by Jiangyin Concortis Biotechnology Co., Ltd, and 10-fold excessive amount meant that the molar quantity of vcMMAE was greater than or equal to 10-fold of that of N109C in the reaction system.) Ultrafiltration tube with 10 kDa MWCO (Millipore) was used to remove the small molecules in the reaction system. The obtained conjugate was filtered through 0.22 m (pore size) water membrane to remove bacteria and stored at 20 C. for later use.

(48) FIG. 4 illustrated the electrophoretogram for the conjugate of N109C and MMAE. As shown in this figure, the protein band apparently shifted after a conjugate was formed, indicating that the small molecule, MMAE, had been conjugated to the protein, such that the electrophoretic behavior thereof was changed. By comparing lane 1 with lane 4, it can be seen that polymers existed since one cysteine residue was mutated in N109C. Further, by comparing lane 3 with lane 6, during conjugation, the rTRAIL N109C polymer was depolymerized by TCEP, and the zinc ion facilitated N109C monomer to form a stable trimer. Between its cysteine sulfhydryl group and the maleimide group of vcMMAE, an alkylation reaction took place to generate conjugates coupled with different numbers of MMAE. The connection relationship among various portions of the rTRAIL mutant-vcMMAE conjugate was shown in FIG. 5.

(49) 8. Measurement of In Vitro Anti-Tumor Activity of the Conjugate

(50) 4 types of cells were selected to measure in vitro anti-tumor activity of the rTRAIL mutant and the MMAE conjugate: TRAIL sensitive type (NCI-H460), TRAIL insensitive type (Hela), TRAIL resistant type (MCF-7) and normal cells (HEK293). All of these 4 types of cells had TRAIL death receptor on their surfaces.

(51) Hereinafter, TRAIL sensitive type NCI-H460 cell was taken as an example to give detailed description of the test procedure, including the following steps:

(52) (1) Digesting the NCI-H460 cells into individual cells, followed by diluting to 110.sup.4 cells/mL, spreading the cells on 96-well plate (100 L for each well), and culturing the cells for 24 hours under normal condition.

(53) (2) Diluting N109C, N109C-vcMMAE and S96C-vcMMAE with culture medium respectively, and adding them into the cell plate with final concentrations of 32, 63, 125, 250, 500, 1000 ng/mL for serving as the test groups, while using standard TRAIL (114-281) as the positive control group, using the buffer (used for dissolving the test sample, diluted with the culture medium in a same manner) as the negative control group, and using the culture medium (without adding any solution) as the blank control group. All the control groups and test groups were tested in triplicate.

(54) (3) Adding the sample in control group and test group into the cell plate, culturing for 96 hours at 37 C., and observing the killing ability on target cells of the control group and test group.

(55) (4) Adding 10 L CCK-8 color-developing solution into each well after completion of the culture, incubating the samples for color-developing for 1 hour in an incubator, and taking them out to measure at dual-wavelength of 450 nm and 630 nm.

(56) (5) Calculation of result: the samples in the test group having an OD value greater than the one of equivalent dilution in the negative group were deemed as positive (t-test, P<0.01). The calculation result was shown in FIG. 6.

(57) As shown in FIG. 6, as for the four different cells, the rTRAIL mutant-vcMMAE conjugate always exhibited stronger killing ability to tumor cells than the rTRAIL mutant.

(58) As for NCI-H460 cells, the activities of the rTRAIL mutant and its MMAE conjugate were not better than that of TRAIL (114-281).

(59) As for Hela and MCF-7 cells, the rTRAIL mutant-vcMMAE conjugate showed stronger killing ability at higher concentration, but was slightly weaker than TRAIL (114-281) at lower concentration (<250 ng/mL). It was likely that the rTRAIL mutant-vcMMAE conjugate endocytosed by cells did not accumulate sufficient amount of MMAE to induce apoptosis at lower concentration. Thus, higher concentration brought better killing effect. Nevertheless, N109C-vcMMAE had stronger killing ability than S96C-vcMMAE at both low and high concentrations.

(60) As for the normal HEK293 cells, all of TRAIL (114-281), the rTRAIL mutant and its MMAE conjugate were confirmed to have considerably low toxicity.

(61) 9. Distribution of the Conjugate in Tumor-Bearing Animal Model

(62) N109C-vcMMAE was taken as an example, and the conjugate was tracked in real-time in mice by Fluorescent labeling method to observe tumor targeting ability of the conjugate.

(63) (1) Establishment of Tumor-Bearing Animal Model

(64) Athymic mice weighing 20 g were selected and 10.sup.7 NCI-H460 cells were injected subcutaneously in the armpit of one side of the forelegs. 3 days later, tumor formation can be clearly observed.

(65) (2) Fluorescent Labeling of the Conjugate

(66) Cy5, as a Near-IR-activated dye, was used as a fluorescent label, so as to facilitate the fluorescence to pass through animal skin and achieve the goal of real time vital observation. In terms of concrete method of Cy5 labeling, please refer to the instruction offered by the manufacturer (Lumiprobe).

(67) (3) Administration and Real Time Observation

(68) 200 L sample containing about 50 g Cy5-labeled conjugate was injected into the tumor-bearing mice via tail vein, while equal amount of normal saline was injected into the blank control group.

(69) Small living animal imager (NightOWLN320) was used to observe distribution of fluorescence in mice every 24 hours. Before observation, after 12 hours fasting, the mice were anesthetized with ether and observed successively for 4 days until systemic fluorescence of the mice disappeared totally. The mice were killed after 4.sup.th day, and heart, liver, spleen, lung, kidney and tumor tissues were isolated. Specific distribution of the conjugate in each tissue of animals in test group was confirmed by small living animal imager. The imaging result was shown in FIG. 7. The white arrow indicates tumor site.

(70) As shown in FIG. 7, most of the fluorescence had gathered on tumor site at 3.sup.rd day, and only the tumor site emitted fluorescence at 4.sup.th day. After killing the animals, the result by fluorescence detection was consistent with that detected in vivo and in real time, showing that N109C-vcMMAE had excellent tumor-targeting effect.

(71) 10. Research on Molecular Mechanism of the Conjugate for Anti-Tumor Activity

(72) NCI-H460 cells were taken as an example to observe behaviors of rTRAIL mutant-vcMMAE conjugate in tumor cells by laser confocal technology, including the following steps:

(73) a) A coverslip was immersed with 1M HCl and 70% ethanol, subjected to ultrasonic treatment for 30 min, washed for 5-6 times with double distilled water and sterilized for later use;

(74) b) Well-sterilized coverslip was placed in 24-well plate, and cells were plated at 10.sup.4 cells/well and cultured overnight at 37 C.;

(75) c) Administration: N109C-vcMMAE and S96C-vcMMAE were diluted respectively to a final concentration of 1 g/mL, and the cells were treated according to step d) at different time points (1.sup.st, 4.sup.th, 8.sup.th and 12.sup.th hour), while drug-free culture medium was used for the blank control group;
d) The coverslip was washed with cold PBS for 3 times and then fixed with 4% paraformaldehyde for 10 min at room temperature;
e) PBS was used to wash the coverslip for another 3 times;
f) 0.1% Triton X-100 was added to permeabilize for 10 min at room temperature;
g) 2% BSA was added to block for 30 min after 3 times washing with PBS;
h) Primary antibody incubation: rabbit anti-human rTRAIL polyclonal antibody was diluted with 1% BSA/PBS to 1 g/mL and incubated for 45 min at room temperature;
i) Secondary antibody incubation: FITC-labeled goat anti-rabbit secondary antibody was diluted 500-fold with 1% BSA/PBS and incubated for 45 min at room temperature;
j) Staining cell nucleus with DAPI: after PBS washing (3 times), DAPI diluted 1000-fold with PBS was added dropwise on each coverslip (it was appropriate to just cover the cells), and the coverslip was incubated for about 2 min at 25 C.;
k) Mounting: after PBS washing, one drop of Anti-fade Fluorescence Mounting Medium was added on the coverslip, then the coverslip was placed upside down on a glass slide and then sealed with nail polish around the coverslip;
l) Cofocal detection: the detection result was shown in FIG. 8.

(76) FIG. 8 shows the cell nucleus and the rTRAIL mutant-vcMMAE conjugates with fluorescence. 1 hour after administration, a lot of N109C-vcMMAE had entered the cells. 4 hours later, more and more N109C-vcMMAE entered the cells and began to aggregate slowly. 8 hours later, there were more aggregating points emerging inside the cells. 12 hours later, the aggregating points inside the cells decreased gradually and cell apoptosis began to occur, evidenced by heterogeneity in cell nucleus.

(77) We could conclude that the action mechanism of said rTRAIL mutant-vcMMAE conjugates is: the conjugates bind with dearth receptors on the surface of tumor cells through the rTRAIL mutants, and are endocytosed by tumor cells to form a phagosome, which will further be fused with lysosomes. The dipeptide linker arm in the conjugate is hydrolysed by lysosomal cathepsin to release MMAE, which will then play its role in tumor cells, i.e., inducing apoptosis by inhibiting dimerization of tubulin. Its apoptosis-inducing mechanism is shown in FIG. 9.