CN102199218A - Fusion protein of Her2 antibody and interleukin 2 and application thereof - Google Patents
Fusion protein of Her2 antibody and interleukin 2 and application thereof Download PDFInfo
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Abstract
The invention discloses a fusion protein of Her2 antibody and interleukin 2 and application thereof, and belongs to the technical fields of medical science and oncology phymatology. The fusion protein comprises CD16 monoclonal antibody heavy chain signal peptide, ErbB2 antibody heavy chain variable region, a Linker 1, a light chain variable region, a human antibody Fc segment, a Linker 2 and IL-2 mature peptide, and has the amino acid sequence shown as SEQ ID No.1. The fusion protein can inhibit Herceptin resistant breast cancer cell proliferation, and can kill Her2 high-expression breast cancer cells. The invention lays a foundation for clinical application of the fusion protein of the Her2 antibody and the interleukin 2 in the anti-tumor aspect.
Description
Technical field
The invention belongs to medical science oncology technical field, be specifically related to fusion rotein of a kind of anti-Her2 antibody and interleukin II and uses thereof.
Background technology
The natural effector function of antibody is very weak, and the existence of circulation middle and high concentration IgG can be disturbed combining of antibody and effector cell surface FcR.Therefore, the effector function of enhancing antibody is the important directions of antibody research field always.From the eighties, successively a series of research work of taking the antibody of effector molecule have been carried out both at home and abroad, to attempt to strengthen the effector function of antibody.In the later stage eighties, immune-regulating factor strengthens cell immune response and the effect of antiidiotype immune response in immunotherapy of tumors received great concern.Interleukin-22 (IL-2) is the important reporter molecule in the immune response, also is one of the most strong known antitumor cell factor, has biologic activity widely.After people such as Rosenberg report the anti-tumour effect of IL-2 in renal cell carcinoma and melanoma clinical treatment, adopt IL-2 and lymphokine activated killer cell (LAK cell) combination therapy malignant tumour to obtain clinically to use widely.Yet the systemic administration of IL-2 and other cytokine is everlasting and is produced high density in the circulation away from tumor locus, and does not reach the optimum concn of treatment in the microenvironment of tumor by local.Because the systemic administration of IL-2 can cause fatal side effect, this makes the therapeutic dose of IL-2 restricted greatly.
People focus onto how to improve the effective concentration of cytokine at tumor by local rapidly, thereby avoid the general toxic reaction.For palp tumour, cytokine is injected directly into the way that tumor by local is a solution, but for the treatment of tumour micro metastasis, the method for direct injection is obviously infeasible.Adopt the method for transgenosis, recombinant cytokine is imported patient self tumour cell or fibroblast, react to induce local inflammation, can produce the general anti tumor immune response in some cases by the emiocytosis cytokine of genetic modification.Yet malignant tumor patient has often experienced radiotherapy, chemotherapy or marrow, autologous peripheral blood stemcell transplant, and high-dose chemotherapy can cause serious immunosuppression.Particularly when allogeneic bone marrow transplantation for after preventing that graft versus host disease (GVH disease) (GVHD) from using powerful T cytostatics cyclosporine A (cyclosporin A), make the immunotherapy of this dependence ctl response be difficult to prove effective.In addition, this method is a patient-specific, needs to adopt patient's self tumour cell, at the external genetic modification that carries out, and then feeds back and gives patient, and complicated operation expends time in, the expense costliness.Up to now, valuable clinical data is limited.Some investigators begin to attempt utilizing antibody target tropism ground premunition regulatory factor, the immunotherapy strategy of cytokine is combined with the anti-tumour effect that strengthens antibody, to obtain the cytokine of high density at tumor by local, irritation cell immune response effectively, thereby removing tumour.In theory, compare with the scheme of cytokine gene treatment, the technology of preparing of antibody-cytokine fusion protein (immune cell factor) is easier, is not subjected to the restriction of patient-specific.And compare with immunotoxin, adopt chimeric antibody or humanized antibody to merge humanized's toxic protein, then can reduce immunogenicity effectively.In recent years, the investigator has made up the immune cell factor of the different tumor associated antigens of several targets.Experimentation on animals shows, the anti-tumour effect of antibody cell factor fusion protein significantly is better than waiting the application that mixes of dosage antibody and cytokine.At present existing two kinds of immune cell factors have entered clinical experimental stage
ErbB-2 is the transmembrane glycoprotein with tyrosine kinase activity, belongs to I type growth factor receptors family member.ErbB-2 crosses in the malignant tumour in multiple epithelial cell such as mammary cancer, ovarian cancer, prostate cancer, cancer of the stomach, lung cancer source and expresses, and very low in the normal tissue expression level.Therefore, ErbB-2 is the desirable target molecule of immunotherapy of tumors.The anti-ErbB-2 monoclonal antibody of drugs approved by FDA humanization in 1998
Be used for ErbB-2 and cross the clinical treatment of expressing mammary cancer, become breast cancer treatment means new behind operation, radiotherapy, chemotherapy and endocrine therapy.Yet use separately
The treatment advanced breast cancer only is 15% as the total effective rate of two wires medication, is 23% as the total effective rate of a line medication.But, the patient who is not all Her2 high expression levels can be benefited from Antybody therapy, and patient wherein over half is insensitive to Trastuzumab treatment, and in medication after 1 year, the Trastuzumab of larger proportion treats effective patient and resistance occurs, causes the treatment failure.Trastuzumab fails to respond to any medical treatment and drug-fast mechanism it be unclear that, but studies show that, development has the medicine of the novel targeted Her2 of different mechanism of action, will fill up the blank of currently available products treatment spectrum, selects for more patient provides new medication.
Adopt gene engineering method, successfully design, make up the Fc section that contains anti-ErbB-2 scFv, human IgG1, the fusion rotein (HF) of IL-2.HF has kept the biologic activity of identification antigenic ability of ErbB-2 and IL-2.Yet there is following defective in HF: 1, biologic activity is low; 2, expression level is low, can not be used for large-scale production.These defectives make the clinical application of HF be subjected to very big restriction, are badly in need of HF is carried out molecular modification to overcome the bottleneck of large-scale production, make HF can be applied to the clinical treatment of Her2 male mammary cancer.
Summary of the invention
The object of the present invention is to provide a kind of anti-Her2 antibody-interleukins 2 fusion protein.
The present invention also aims to provide the aminoacid sequence of a Linker, the aminoacid sequence of described Linker 2 is shown in SEQ ID No.3 in the sequence table, and its superior part is for can significantly improve output and the biologic activity of fusion rotein in mammalian cell expression system.
The present invention also aims to provide a kind of recombinant vectors and amplimer thereof that contains anti-Her2 antibody-interleukins 2 fusion protein gene.
The present invention also aims to provide a kind of clone and host bacterium of expressing anti-Her2 antibody-interleukins 2 fusion protein.
The present invention also aims to provide a kind of Her2 of treatment antibody class medicine of positive mammary cancer.
The present invention also aims to provide the antibody class medicine of a kind of Her2 of treatment positive, the drug-fast mammary cancer of Herceptin.
A kind of anti-Her2 antibody-interleukins 2 fusion protein, it is characterized in that described fusion rotein comprises people CD16 monoclonal antibody heavy chain signal peptide, ErbB2 antibody heavy chain variable region, Linker 1, variable region of light chain, people's antibody Fc fragment, Linker 2 and IL-2 mature peptide.
The aminoacid sequence of described fusion rotein is shown in SEQ ID No.1 in the sequence table, and the aminoacid sequence of described Linker 1 is shown in SEQ ID No.2 in the sequence table, and the aminoacid sequence of described Linker 2 is shown in SEQ ID No.3 in the sequence table.
The nucleotide sequence of fusion rotein encoding gene is shown in SEQ ID No.4 in the sequence table in the described genophore.
The clone that contains the described fusion rotein encoding gene of claim 3.
The host bacterium that contains the described fusion rotein encoding gene of claim 3.
Arbitrary segmental primer in the described fusion rotein encoding gene of amplification claim 3.
A kind of antibody class medicine for the treatment of the positive mammary cancer of Her2 is characterized in that its activeconstituents is the described fusion rotein of claim 1.
A kind of antibody class medicine for the treatment of the Her2 positive, the drug-fast mammary cancer of Herceptin is characterized in that, its activeconstituents is the described fusion rotein of claim 1.
Beneficial effect of the present invention: 1. anti-Her2 antibody of the present invention-interleukins 2 fusion protein biologic activity and output obviously improve.2. anti-Her2 antibody-interleukins 2 fusion protein of the present invention can effectively suppress the tumor cell proliferation of Her2 high expression level in vivo and in vitro.3. anti-Her2 antibody-interleukins 2 fusion protein of the present invention can effectively suppress the drug-fast breast cancer cell propagation of Herceptin in vivo and in vitro.4. but the breast cancer cell of anti-Her2 antibody of the present invention-interleukins 2 fusion protein direct killing Her2 high expression level.
Description of drawings
Fig. 1 is the genophore plasmid figure that contains anti-Her2 antibody-interleukins 2 fusion protein.
Fig. 2 is the content of target protein in HFI and the HF cells and supernatant.
The HFI of Fig. 3 different concns and HF albumen and the antigenic activity that combines.
Fig. 4 is the relative reactivity of IL-2 in HFI and the HF cell.
Fig. 5 HFI is in the external influence that the breast cancer cell of high expression level Her2 is bred.
The influence that Fig. 6 HFI breeds the breast cancer transplantable tumor of high expression level Her2 in vivo.
Fig. 7 HFI is in external influence to Herceptin resistance breast cancer cell BT474, MDA-453 and SKBR3 propagation.
The influence that Fig. 8 HFI breeds Herceptin resistance breast cancer cell transplanted tumor in vivo.
Embodiment
The present invention will be further described below in conjunction with the drawings and specific embodiments.
Following examples do not limit the present invention, and unspecified operation steps please refer to " molecular cloning experiment guide " third edition corresponding section (J. Sa nurse Brooker E.F. is Ritchie etc. not, Science Press) or consults the specification sheets of used kit among the embodiment.
The experiment material source that following examples adopt: E.coli JM109 engineering strain is available from sky root bio tech ltd; CHO, BT474, MDA-453, SKBR3, MCF-7 cell are available from ATCC; The T4DNA ligase enzyme is a Gibco BRL company product; Restriction enzyme is a Biolab company product; Taq archaeal dna polymerase, cloning vector pGEM-T Easy are Promega company product; Ex Taq archaeal dna polymerase is a TaKaRa company product; Plasmid extraction kit, dna fragmentation reclaim test kit available from QIAGEN company; Protein A affinity chromatography medium is available from this yuan Zhenyang company; Foetal calf serum (FCS), DMEM substratum, 1640 substratum are HyClone company product; MTX (methotrexate) and MTT (four tetrazolium bromides) are Sigma company product.
The structure of embodiment 1 anti-Her2 antibody-interleukins 2 fusion protein expression vector
To contain anti-ErbB-2 scFv, human IgG1's Fc section, the antigen-4 fusion protein gene carrier pCID/HF of IL-2 is template (nucleotide sequence of fusion rotein is shown in SEQ ID No.5 in the sequence table in the carrier), contain the anti-people CD16 monoclonal antibody heavy chain signal peptide gene sequence of coding with primer P1 (its nucleotide sequence is shown in SEQ ID No.6 in the sequence table) and P2 (its nucleotide sequence is shown in SEQ ID No.7 in the sequence table) pcr amplification, the ErbB2 antibody heavy chain variable region gene, Linker and chain variable region gene and people's antibody Fc gene fragment, condition is: 95 ℃ of sex change 2min; 94 ℃ of sex change 1min, 58 ℃ of annealing 1min, 72 ℃ of extension 2min carry out 30 circulations then; Last 72 ℃ are extended 10min.Insert among the expression vector pCID with NheI and XhoI site, obtain carrier pCID/ScFv-Fc.
Contain the coding N-terminal with primer P3 (its nucleotide sequence is shown in SEQ ID No.8 in the sequence table) and P4 (its nucleotide sequence is shown in SEQ ID No.9 in the sequence table) pcr amplification and contain the decapeptide Linker with good characteristic after the optimization and the gene fragment of IL-2 mature peptide, condition is: 95 ℃ of sex change 2min; 94 ℃ of sex change 1min, 56 ℃ of annealing 1min, 72 ℃ of extension 1min carry out 30 circulations then; Last 72 ℃ are extended 10min.Insert among the expression vector pCID/ScFv-Fc with XhoI and MluI site, obtain carrier for expression of eukaryon pCID/HFI.
PCID/HFI is transformed the JM109 competence bacteria, screening positive clone, amplification and plasmid DNA purification, concrete steps are:
1) 100 μ l competence bacterias place frozen water, add DNA 0.5 μ l, leave standstill 30 minutes;
2) 42 ℃ of water-baths are 2 minutes;
3) ice bath is 5 minutes;
4) add and not contain antibiotic LB substratum, under 37 ℃, 150rpm shaking culture 50 minutes.
5) transformed bacteria is inoculated in the LB agar plate that contains penbritin, puts 37 ℃ of incubator overnight incubation.
6) select 4 of positive colonies, be inoculated in the LB liquid nutrient medium respectively, put 37 ℃ of shaking table concussions and cultivate.
7) extract the positive colony plasmid DNA with test kit.
Identify plasmid DNA with Nhe I and MluI double digestion, send the order-checking of Invitrogen company, record nucleotide sequence shown in SEQ ID No.4 in the sequence table.
The double digestion system is:
37 ℃ of water-baths 4 hours.
The screening of embodiment 2 engineering cell strains and the expression of HFI
With the CHO/dhfr-cell cultures in the IMDM substratum that contains 10%FCS, 0.1mol/L xanthoglobulin, 0.016mol/L Thymine deoxyriboside.According to Lipofectamine reagent specification sheets with recombinant vectors PCI/HFI transfection CHO/dhfr-cell.After transfectional cell is cultivated 48h, adopt the sandwich ELISA method to detect the transient expression of fusion rotein, method is the same.Adopt limiting dilution assay that cells transfected is carried out cloning and cultivate, substratum is for containing 10% dialysis FCS, 10
-6The DMEM substratum of M MTX.In the screening process,, adopt the sandwich ELISA method to detect the antibody expression level of each cell clone in different time points.Detailed process is as follows: by 96 orifice plates, 4 ℃ are spent the night with goat anti-human igg (2 μ g/ml) bag.Behind 2% bovine serum albumin sealing 1h, add the Chinese hamster ovary celI culture supernatant of transfection, 37 ℃ of incubation 1h.After washing 5 times, add goat anti-human igg's (dilution in 1: 5000) of horseradish peroxidase-labeled, 37 ℃ of incubation 1h.With the OPD colour developing, measure OD490 after washing 5 times.High-expression clone continues to go down to posterity in containing the MTX selective medium, and adopts the sandwich ELISA method to detect, and filters out high-expression clone, as engineering cell strain.
With HFI engineering cell and HF engineering cell all with 5 * 10
6Being inoculated in floorage is 150cm
2Culturing bottle in, under same culture conditions (30 ℃, 5%CO2), cultivated 6 days.
The results culture supernatant is got 5 μ l HFI and HF culture supernatant and is surveyed its protein content, and the result shows that protein content is 0.28mg/ml in (as shown in Figure 2) HFI culture supernatant, and protein content is 0.18mg/ml in the HF culture supernatant.
Get culture supernatant by protein A affinitive layer purification, with the sample behind the purifying and culture supernatant by SDS-PAGE and coomassie brilliant blue staining post analysis.The result shows that the purity of HFI is greater than 90%.
Embodiment 3HFI and HF antigen-binding activity and biological activity assay experiment
Collect 1 * 10
6Individual SKBR3 cell, after the washing of PBS damping fluid, HFI and HF behind the adding purifying, 30min is hatched in the ice bath concussion, washes 3 times with PBS, adds the goat anti-human igg of FITC mark, ice bath concussion is down hatched 30min, wash 3 times with PBS, last machine (Becton Dickinson company) is analyzed, with the cell of not hatching with the transfectional cell culture supernatant as negative control.
The CTLL-2 cell of cultivating is washed 3 times with 1640 substratum, behind the counting, with 3 * 10
4/ hole is inoculated in 96 orifice plates, adds HFI and HF behind the purifying simultaneously, with the IL-2 standard substance of doubling dilution as positive control.After cultivating 18h, add 10 μ l MTT (5mg/ml), continue to cultivate 4h.With 10%SDS-0.01mol/L HCl lysing cell, survey the A490 value.
The result shows (as shown in Figure 3), and the antigen-binding activity of HFI is a little more than HF, and the biologic activity of HFI is significantly higher than HF (as shown in Figure 4).
Embodiment 4HFI kills and wounds the application aspect the medicine of Her2 high expression level breast cancer cell in preparation
1, the external breast cancer cell that kills and wounds high expression level Her2
MCF-7 cell and MCF-7/Her2 (the MCF-7 cell of high expression level Her2) are with 3 * 10
496 orifice plates are inoculated in/hole, add the HFI behind the purifying simultaneously, with commercialization Herceptin medicine in contrast.Add 10 μ l MTT (5mg/ml) after cultivating 96h, continue to cultivate 4h.With 10%SDS-0.01mol/L HCl lysing cell, survey the A490 value.
The result shows (as shown in Figure 5), and HFI can be at the external breast cancer cell that effectively kills and wounds high expression level Her2.
2, kill and wound the breast cancer cell of high expression level Her2 in the body
4 the week age nude mice, back subcutaneous implantation oestrogenic hormon slow release tablet.Next day, logarithmic phase BT474 cell is pressed 1 * 10
7/ only to be inoculated in the right armpit of nude mice subcutaneous.Laboratory animal is 3 groups at random, 6/group.Beginning in the 5th day administration of transplanted tumor inoculation back.Control group (control): physiological saline, 0.1ml/ time/only, twice weekly; The Herceptin group: commercialization Herceptin, according to 3mg/kg/ time/, volume injected is 0.1ml, twice weekly; The HFI group: the HFI purification of samples, according to 0.5mg/kg/ time/, volume injected is 0.1ml, twice weekly.From the transplanted tumor inoculation, measured the transplanted tumor volume in per two days, calculation formula is: transplanted tumor volume=major diameter * minor axis
2/ 2.
The result shows that HFI can effectively suppress the growth of transplanted tumor in nude mouse, and its curative effect obviously is better than Herceptin treatment group (as shown in Figure 6).
The application of embodiment 5HFI aspect the medicine of the anti-Herceptin resistance breast cancer cell propagation of preparation
1, the propagation of HFI vitro inhibition Herceptin resistance breast cancer cell
Employing progressively improves the screening method of Herceptin concentration in culture system, breast cancer cell BT474, MDA-453 and the SKBR3 cell of high expression level Her2 are tamed.Obtained the drug-fast cell strain of three strain Herceptin (BT474-HercepR, MDA-453-HercepR and SKBR3-HercepR) through 6 months screenings.
With above-mentioned three kinds of Herceptin mdr cells with 3 * 10
496 orifice plates are inoculated in/hole, add the HFI behind the purifying simultaneously, with commercialization Herceptin in contrast.Add 10 μ l MTT (5mg/ml) after cultivating 96h, continue to cultivate 4h.With 10%SDS-0.01mol/L HCl lysing cell, survey the A490 value.
The result shows that HFI has stronger restraining effect (as shown in Figure 7) external to mammary cancer mdr cell BT474, the MDA-453 of high expression level Her2 and the propagation of SKBR3.
2, the propagation that suppresses Herceptin resistance breast cancer cell in the HFI body
4 the week age nude mice, back subcutaneous implantation oestrogenic hormon slow release tablet.Next day, logarithmic phase BT474-HercepR cell is pressed 1 * 10
7/ only to be inoculated in the right armpit of nude mice subcutaneous.Laboratory animal is 3 groups at random, 6/group.Beginning in the 5th day administration of transplanted tumor inoculation back.Control group: physiological saline, 0.1ml/ time/only, twice weekly; The Herceptin group: commercialization Herceptin, according to 3mg/kg/ time/, volume injected is 0.1ml, twice weekly; The HFI group: the HFI purification of samples, according to 0.5mg/kg/ time/, volume injected is 0.1ml, twice weekly.From the transplanted tumor inoculation, measured the transplanted tumor volume in per two days, calculation formula is: transplanted tumor volume=major diameter * minor axis
2/ 2.
The result shows, HFI has stronger restraining effect (as shown in Figure 8) to the propagation of the mammary cancer mdr cell BT474 of high expression level Her2 in vivo.
Claims (8)
1. anti-Her2 antibody-interleukins 2 fusion protein, it is characterized in that described fusion rotein comprises people CD16 monoclonal antibody heavy chain signal peptide, ErbB2 antibody heavy chain variable region, Linker 1, variable region of light chain, people's antibody Fc fragment, Linker 2 and IL-2 mature peptide.
2. according to the described a kind of anti-Her2 antibody-interleukins 2 fusion protein of claim 1, it is characterized in that, the aminoacid sequence of described fusion rotein is shown in SEQ ID No.1 in the sequence table, the aminoacid sequence of described Linker 1 is shown in SEQ ID No.2 in the sequence table, and the aminoacid sequence of described Linker 2 is shown in SEQ ID No.3 in the sequence table.
3. a genophore that contains anti-Her2 antibody-interleukins 2 fusion protein is characterized in that, the nucleotide sequence of fusion rotein encoding gene is shown in SEQ ID No.4 in the sequence table in the described genophore.
4. the clone that contains the described fusion rotein encoding gene of claim 3.
5. the host bacterium that contains the described fusion rotein encoding gene of claim 3.
6. arbitrary segmental primer in the amplification claim 3 described fusion rotein encoding gene.
7. an antibody class medicine for the treatment of the positive mammary cancer of Her2 is characterized in that its activeconstituents is the described fusion rotein of claim 1.
8. an antibody class medicine for the treatment of the Her2 positive, the drug-fast mammary cancer of Herceptin is characterized in that, its activeconstituents is the described fusion rotein of claim 1.
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| CN103193887A (en) * | 2013-04-03 | 2013-07-10 | 江苏众红生物工程创药研究院有限公司 | Recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein as well as encoding gene and expressing method of fusion protein |
| CN103214580A (en) * | 2013-03-07 | 2013-07-24 | 中国人民解放军军事医学科学院基础医学研究所 | Anti Her2 immune cytokine and application thereof |
| CN104853782A (en) * | 2012-12-13 | 2015-08-19 | 宾夕法尼亚大学理事会 | DNA antibody constructs and method of using same |
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| CN104853782A (en) * | 2012-12-13 | 2015-08-19 | 宾夕法尼亚大学理事会 | DNA antibody constructs and method of using same |
| CN103214580A (en) * | 2013-03-07 | 2013-07-24 | 中国人民解放军军事医学科学院基础医学研究所 | Anti Her2 immune cytokine and application thereof |
| CN103214580B (en) * | 2013-03-07 | 2014-12-17 | 中国人民解放军军事医学科学院基础医学研究所 | Anti Her2 immune cytokine and application thereof |
| CN103193887A (en) * | 2013-04-03 | 2013-07-10 | 江苏众红生物工程创药研究院有限公司 | Recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein as well as encoding gene and expressing method of fusion protein |
| CN103193887B (en) * | 2013-04-03 | 2015-02-04 | 江苏众红生物工程创药研究院有限公司 | Recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein as well as encoding gene and expressing method of fusion protein |
| CN106794245A (en) * | 2014-08-29 | 2017-05-31 | 豪夫迈·罗氏有限公司 | The combination treatment of the variant immune cell factors of tumor-targeting IL 2 and the antibody for people PD L1 |
| CN106794245B (en) * | 2014-08-29 | 2021-06-01 | 豪夫迈·罗氏有限公司 | Combination therapy of tumor-targeted IL-2 variant immunocytokines and antibodies against human PD-L1 |
| CN106220733A (en) * | 2016-08-17 | 2016-12-14 | 林志国 | A kind of single-chain antibody and application thereof |
| WO2022089601A1 (en) * | 2020-10-30 | 2022-05-05 | 中国科学院生物物理研究所 | Bifunctional fusion protein consisting of il-2 and antibody subunit |
| CN113621069A (en) * | 2021-09-06 | 2021-11-09 | 福州迈新生物技术开发有限公司 | anti-HER-2 protein monoclonal antibody, and preparation method and application thereof |
| CN113621069B (en) * | 2021-09-06 | 2023-03-10 | 福州迈新生物技术开发有限公司 | anti-HER-2 protein monoclonal antibody, and preparation method and application thereof |
| WO2023061005A1 (en) * | 2021-10-14 | 2023-04-20 | 徕特康(苏州)生物制药有限公司 | Novel antibody-cytokine fusion protein, preparation method therefor and use thereof |
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Application publication date: 20110928 |