Enhanced exosome for expressing antigen-targeted antibody as well as preparation method and application of enhanced exosome

Abstract

The invention belongs to biological medicines, and discloses an enhanced exosome for expressing an antigen-targeted antibody as well as a preparation method and application of the enhanced exosome. An antigen targeting antibody is expressed on the surface of the exosome, and the antigen targeting antibody comprises: 1) a signal peptide; (2) a histidine tag (His-tag) and a Flag tag; 3) a single-chain antibody region of a targeted antigen; 4) a hinge area; 5) transmembrane area; and 6) a fluorescent protein. The enhanced exosome is prepared by a gene engineering method, and surface modification of the exosome is realized by utilizing a lentiviral vector, so that the enhanced exosome has definite antigen targeting property and preparation sustainability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an enhanced exosome expressing an antigen-targeting antibody and a preparation method and application thereof. BACKGROUND

[0002] Exosomes are nanoscale extracellular lipid bilayer vesicles of endocytic origin. Exosomes are 30-150 nm in diameter, secreted from intracellular multivesicular bodies (MVBs) or late endosomes Under physiological and pathological conditions, almost all cells secrete exosomes. Exosomes have physiological effects of immune response, neural communication, and participation in cell proliferation and homeostasis; their vesicular structure enables them to contain abundant proteins, lipids, mRNAs, microRNAs, etc., so they can act as mediators of cell communication or transmission of information to multiple cells and locations; the behavior of adjacent or distant cells can be changed by exosomes released from cells in a paracrine or endocrine manner There are three ways for receptor cells to uptake exosomes: receptor-ligand interaction, direct membrane fusion, and endocytosis / phagocytosis.

[0003] Exosomes, due to their physiological functions and physical properties, can be used as a new type of nanoparticle drug carrier. Compared with liposomes or other synthetic polymer-based drug carriers, exosomes have the key characteristics of nanoparticles, such as enhanced permeability and retention effect and passive targeting, as well as excellent biocompatibility and membrane permeability, can penetrate the blood-brain barrier, and also have low immunogenicity and good tolerance .

[0004] However, as a drug carrier, exosomes have the disadvantage of lacking targeting specificity. Although the proteins or lipids on the exosome membrane endow the exosomes with certain targeting specificity, the targeting specificity is still far from enough, and the receptor cells are widespread and uncontrollable. As mentioned earlier, receptor cells can uptake exosomes through receptor-ligand interaction; therefore, the exosomes are modified by modifying the targeting ligand on the surface of the exosomes, so that the exosomes can be applied to specific sites for administration.

[0005] The methods for surface modification of exosomes include genetic engineering, covalent modification and non-covalent modification. The exosomes modified by genetic engineering are derived from cells, which can reduce the immunogenicity of exosomes, reduce the reaction efficiency due to the complexity of the surface of exosomes, and reduce the structural damage of exosomes, compared with the methods of covalent and non-covalent modification. Studies have shown that exosomes expressing cardiac targeting peptide (CTP)-Lamp2b on the membrane of exosomes (CTP-Exo) through genetic engineering can enhance the delivery of exosomes to cardiac cells and cardiac tissues without cytotoxicity The chemical method of introducing ligands through covalent reaction may destroy the original active structure of the membrane due to relatively harsh conditions. During the modification process, the reaction conditions such as reaction temperature, special reagents or substrate concentration may damage the activity of the membrane. Similarly, genetic engineering modification also has its limitations, i.e., the process is complicated, and it is difficult to ensure the stable expression of the target gene.

[0006] Based on the above observations, in order to solve the problems of lack of targeting of exosomes and reduce the immunogenicity caused by protein modification, and ensure the stable expression of antigen targeting antibodies, it is necessary to provide enhanced exosomes expressing antigen targeting antibodies. SUMMARY

[0007] Therefore, the present application provides an enhanced exosome, which expresses an antigen targeting antibody on the surface.

[0008] Therefore, in one aspect, the present application provides an enhanced exosome, which expresses an antigen targeting antibody on the surface.

[0009] In an embodiment of the present application, the antigen targeting antibody comprises the following elements:

[0010] 1) signal peptide region; 2) histidine tag (His-tag) and Flag tag; 3) single-chain antibody region targeting antigen; 4) hinge region; 5) transmembrane region; and 6) fluorescent protein.

[0011] In an embodiment of the present application, the signal peptide can be any signal peptide of a mammalian cell surface protein, such as human albumin signal peptide, human insulin signal peptide or mouse IgG kappa signal peptide, preferably, the signal peptide is mouse IgG kappa signal peptide, and the amino acid sequence is shown as SEQ ID NO: 1.

[0012] In embodiments of the present application, the positions of the histidine tag (His-tag) and the Flag tag can be interchanged. In specific embodiments, the amino acid sequences of the histidine tag (His-tag) and the Flag tag are as shown in SEQ ID NO: 2.

[0013] In embodiments of the present application, the single-chain antibody region targeting an antigen can be any single-chain antibody targeting a cell surface protein. In specific embodiments, the single-chain antibody region targeting an antigen is an antibody targeting the cell surface Her2 of breast cancer cells, and the amino acid sequence thereof is as shown in SEQ ID NO: 3.

[0014] In embodiments of the present application, the amino acid sequence of the hinge region is as shown in SEQ ID NO: 4.

[0015] In embodiments of the present application, the transmembrane region can be the transmembrane region of human CD8, human CD28, human IL-15R, or human PDGFR, preferably the transmembrane region of human PDGFR, and the amino acid sequence thereof is as shown in SEQ ID NO: 5.

[0016] In embodiments of the present application, any fluorescent protein can be selected according to experimental needs, and in specific embodiments of the present application, the amino acid sequence of the fluorescent protein is as shown in SEQ ID NO: 6.

[0017] In a second aspect, the present application provides a nucleic acid molecule encoding the antigen-targeting antibody described above.

[0018] In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule described above encoding the antigen-targeting antibody.

[0019] In a fourth aspect, the present application provides a recombinant lentivirus comprising the nucleic acid molecule of the second aspect or prepared by the expression vector of the third aspect.

[0020] In a fifth aspect, the present application provides a method for preparing the recombinant lentivirus of the fourth aspect, comprising co-transfecting a mammalian host cell with the expression vector of the third aspect and a helper plasmid to obtain the recombinant lentivirus.

[0021] In embodiments of the fifth aspect, the helper plasmid can be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0022] In embodiments of the fifth aspect, the mammalian host cell can be, but is not limited to, HEK293 cells, human PER.C6 cells, human Hela cells, and mouse CHO cells, and preferably HEK293 cells.

[0023] In a sixth aspect, the present application provides a cell secreting the enhanced exosome as described above.

[0024] In a seventh aspect, the present application provides a method for preparing the cell of the sixth aspect, comprising transfecting a host cell with the recombinant lentivirus of the fifth aspect.

[0025] In an embodiment of the seventh aspect of the present application, the host cell can be HEK293, T cell, mesenchymal stem cell, etc.

[0026] In a specific embodiment of the seventh aspect, the method for preparing the cell of the sixth aspect specifically comprises the following steps:

[0027] (1) constructing a recombinant lentivirus plasmid carrying the nucleic acid molecule of the second aspect;

[0028] (2) transfecting the host cell with the recombinant lentivirus plasmid and the helper plasmid to prepare a recombinant lentivirus that can infect cells;

[0029] (3) transfecting the recombinant lentivirus obtained in step (2) into the cell to produce a cell secreting the enhanced exosome expressing the antigen-targeting antibody.

[0030] In a specific embodiment, the helper plasmid is pMDLG-pRRE-Kana, pMD2G-Kana and pRSV-REV-Kana.

[0031] In an eighth aspect, the present application provides a method for preparing the enhanced exosome of the first aspect, comprising the following steps:

[0032] (1) culturing and isolating the cell of the sixth aspect;

[0033] (2) collecting the culture supernatant of the cell secreting the enhanced exosome expressing the antigen-targeting antibody, and centrifuging and filtering to remove impurities;

[0034] (3) purifying and concentrating with a hollow fiber column to obtain the enhanced exosome expressing the antigen-targeting antibody.

[0035] In a ninth aspect, the present application provides a drug delivery tool comprising the enhanced exosome of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect and the cell of the sixth aspect.

[0036] In a tenth aspect, the present application provides the use of the enhanced exosome, the nucleic acid molecule, the expression vector, the recombinant lentivirus or the cell for preparing a drug for treating a disease as a delivery tool.

[0037] In the embodiments of the present application, the disease refers to a disease characterized by high expression of Her2 protein on the cell surface. In specific embodiments, the disease can be breast cancer, liver cancer, etc.

[0038] In the embodiments of the present application, Her2-scFv represents an expressed Her2-targeting antibody, Her2-scFv-293F cell represents a cell that can secrete exosomes expressing Her2-targeting antibody, and Her2-scFv exosome represents an exosome expressing Her2-targeting antibody.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] At present, there is a lack of enhanced exosomes targeting Her2 protein for clinical treatment. The present application uses a lentiviral vector to perform surface engineering on specific exosomes by a genetic engineering method, so that the exosomes have antigen targeting and preparation sustainability. The prepared enhanced exosomes have clear targeting, can further encapsulate drugs for delivery to target positions, and provide a new technical solution and platform for precise treatment of diseases, and have great potential for transformation and application in clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is a schematic diagram of the vector structure constructed in the present embodiment.

[0042] Figure 2 is a schematic diagram of the exosome expressing the antigen-targeting antibody in the present embodiment.

[0043] Figure 3 is a chart of the number and particle size of Her2-scFv exosomes and a qualitative analysis of screening in the present embodiment.

[0044] Figure 4 is a comparison chart of Her2-scFv exosome uptake by different cells in the present embodiment. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present application are described in detail below to make the advantages and features of the present application more easily understood by those skilled in the art, so as to make the scope of protection of the present application more clear and explicit.

[0046] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the examples are carried out according to the conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2012), or according to the conditions suggested by the manufacturer's instructions.

[0047] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained through commercial channels unless otherwise specified.

[0048] Example 1: Design and construction of plasmid encoding antigen-targeting antibody

[0049] In this example, the plasmid encoding the antigen-targeting antibody was synthesized by Nanjing Baao De Biotechnology Co., Ltd., and the antigen-targeting antibody was an antibody targeting HER2 on the surface of breast cancer cells (Her2-scFv). The plasmid contains nucleic acid sequences encoding the following elements from 5' to 3' direction: signal peptide (SEQ ID NO: 1), histidine tag (His-tag) and Flag tag (SEQ ID NO: 2), single-chain antibody region targeting antigen (SEQ ID NO: 3), hinge region (SEQ ID NO: 4), transmembrane region (SEQ ID NO: 5), and fluorescent protein (SEQ ID NO: 6). The plasmid was cloned into the lentivirus master plasmid pMSGV-IRES (Shanghai Nuopu Biotechnology Co., Ltd., Catalog No: V000092) to obtain a plasmid expressing Her2-scFv, which is called pMSGV-Her2-scFv and is the master plasmid for lentivirus packaging. The structure of pMSGV-Her2-scFv is shown in Figure 1 .

[0050] Example 2: Preparation of recombinant lentivirus

[0051] The master plasmid pMSGV-Her2-scFv was co-transfected with three helper plasmids pMDLG-pRRE-Kana (Changsha Aibive Biotechnology Co., Ltd., Catalog No: HG-VMA0374), pMD2G-Kana (Changsha Aibive Biotechnology Co., Ltd., Catalog No: HG-VMA0648), and pRSV-REV-Kana (Changsha Aibive Biotechnology Co., Ltd., Catalog No: HG-VMA0370) into Wayne LV Pro HEK293 cell line (Chinese Academy of Sciences Typical Culture Collection Cell Bank, Catalog No: SCSP-5207, hereinafter referred to as 293F cells) to prepare recombinant lentivirus that can infect host cells. The specific operation steps are as follows:

[0052] 1. Adjust the density of 293F cells to 0.5×10 6 6×10 6 cells / mL, and culture in SMM 293-TII Expression Medium (Yiqiao Shenzhou Biotechnology Co., Ltd., Catalog No: M293TII) medium at 37 ℃, 5% CO2, 160 rpm shaker for 48 h, and count 4×10

[0053] 2. Take 144 μL of PEI (Merck BioTech Co., Ltd., 937762) with a concentration of 1 mg / mL, add 1 mL of opti-MEM (Thermo Fisher Scientific, Inc., Cat. No. 11058021) and mix well, and then stand at room temperature for 20 min.

[0054] 3. Add 16 μg of the main plasmid pMSGV-Her2-scFv and 12 μg of the pMDLG-pRRE-Kana, 4 μg of pMD2G-Kana and 4 μg of pRSV-REV-Kana auxiliary plasmids into 1 mL of SMM 293-TII Expression Medium, and mix well.

[0055] 4. Add the PEI mixed solution in step 2 and 1 mL of opti-MEM into the solution in step 3, mix well, and stand at room temperature for 20 min.

[0056] 5. Take the mixed solution in step 4, add it to the cells prepared in step 1, and mix well.

[0057] 6. After 6-8 h, add 2% LV Feed 04 feed (Zhongshan Kangsheng Biological Technology Co., Ltd., Cat. No. QRD003).

[0058] 7. After continuous culture for 48 h, collect the virus-containing culture medium supernatant in the culture dish, filter it with a 0.22 μm filter cup, incubate it at 4°C for 2 h, transfer it to a centrifuge tube, weigh it to balance, centrifuge it at 20000 g at 4°C for 2 h. After centrifugation, carefully aspirate the liquid in the centrifuge tube in a biological safety cabinet, add 120 μL of PBS buffer to resuspend the precipitate, and then obtain the recombinant lentivirus, which is stored at -80°C.

[0059] Example 3: Lentivirus titer detection

[0060] Take Jurkat cells (ATCC, Cat. No. MD130) that have been cultured for 2 generations and recovered, centrifuge at 300 g for 10 min, resuspend the Jurkat cells to 2×10 6Example 3: Preparation of Her2-scFv-293F cells

[0061] Example 4: Preparation of Her2-scFv-293F cells

[0062] Exosomes derived from HEK-293 cells have extremely low immunogenicity and exhibit excellent preclinical safety in in vitro and in vivo tests 293F cells are derived from human embryonic kidney cells HEK-293, so 293F cells are used as host cells.

[0063] 1. Calculate the required virus amount according to MOI = 3. The calculation formula is as follows: required virus amount (mL) = (MOI * cell number) / virus titer.

[0064] 2. Transfect the virus particles into 293F cells (Chinese Academy of Sciences Typical Culture Collection Cell Bank: item number: SCSP-5207) to obtain Her2-scFv-293F cells.

[0065] 3. Take 1 x 10 6 Her2-scFv-293F cells, and incubate the Her2-scFv-293F cells with anti-Flag antibody (Bi Yun Tian Biotechnology Co., Ltd., item number: AG8050) for 30 min; then incubate the Her2-scFv-293F cells with FITC-labeled goat anti-rabbit IgG (H+L) (Bi Yun Tian Biotechnology Co., Ltd., item number: A0562) for 20 min; and detect after incubation.

[0066] 4. Take 1 x 10 6 Her2-scFv-293F cells, and incubate the Her2-scFv-293F cells with FITC-labeled anti-His antibody (Wuhan San Ying Biotechnology Co., Ltd., item number: FITC-66008) for 20 min; and detect after incubation.

[0067] 5. Use a cell flow cytometer to detect the positive rate of Flag and His in Her2-scFv-293F cells to measure the positive rate of Her2-scFv. The flow cytometry detection results show that 99% of Her2-scFv-293F cells express Her2-scFv.

[0068] Example 5: Pretreatment of Her2-scFv exosomes

[0069] 1. The Her2-scFv-293F cells were suspended in serum-free medium, i.e. SMM 293-TII Expression Medium (Yiqiao Biotechnology Co., Ltd., Catalog No.: M293TII) and OPM-293 CD05 Medium (Shanghai Oupaimai Biological Technology Co., Ltd., Catalog No.: 81075-001) mixed at a volume ratio of 1:1, and cultured at a temperature of 37°C and 5% CO2, with 160 rpm shaking, and the serum-free medium was replaced every 1-2 days; the collected medium contained Her2-scFv exosomes.

[0070] 2. The collected medium in step 1 was centrifuged at 12000 g and 4°C for 30 min, and the supernatant was filtered with a 0.22 µm filter; as needed, it can be stored at 4°C, -20°C, or -80°C.

[0071] Example 6: Purification and concentration of Her2-scFv exosomes.

[0072] In this example, a tangential flow filtration system with a hollow fiber column was used to prepare, purify, and concentrate Her2-scFv-CD9-VSVG exosomes. The hollow fiber pore size in the hollow fiber column was 300 kD. The shear force generated by the hollow fiber module is smaller, which is beneficial to protect the integrity of the exosomes; secondly, the hollow fiber column can efficiently concentrate large volumes of fermentation supernatant, which is beneficial to the subsequent operation of anion complex chromatography and other steps; in addition, the pore size of the hollow fiber column can effectively filter out most impurities such as cell debris, proteins, and nucleic acids, while the exosomes are retained, achieving efficient separation and concentration of exosomes; more importantly, the hollow fiber column is simple to operate, easy to control and maintain compared with other concentration techniques, providing convenience for experimental operation.

[0073] 1. Assemble the hollow fiber column with its sample inlet pipeline and backflow pipeline (Hangzhou Kebaiter Filter Material Co., Ltd., Catalog No.: HFEMI03000530P), and rinse the entire circuit with water for injection for 3-5 min, and keep the pressure at the sample inlet below 5 psi.

[0074] 2. Rinse the circuit with 0.5 M NaOH for 30 min, adjust the flow rate to keep the shear force at 6000 s -1 , and keep the pressure at the backflow end and the sample inlet below 5 psi; adjust the pressure to make the flow rates at the permeation end and the backflow end basically consistent, and after the rinsing is completed, empty the NaOH in the system.

[0075] 3. Rinse the entire circuit with water for injection to neutralize the pH of the liquid in the circuit, and keep the sample end pressure below 5 psi.

[0076] 4. Take 4-6 times the dead volume of PBS to rinse, and empty the PBS in the system;

[0077] 5. Close the permeate end, and put the sample tube and the return tube into the sample bottle, and take 1 L of the culture medium supernatant containing Her2-scFv exosomes obtained in Example 4 into the sample bottle; adjust the sample end pressure to be less than 5 psi, and adjust the flow rate to keep the shear force less than 6000 s -1 ; record the permeate end flow rate; after concentration to 15 mL, close the permeate end, and circulate the sample in the system for 3-5 min for gel removal; empty the system, and connect the return end sample to the concentrated sample.

[0078] 6. Close the permeate end, and wash the pipeline with 1-2 times the dead volume of PBS for 3-5 min; empty the system, and mix the sample with the concentrated sample in step 5 to obtain Her2-scFv exosomes (Her2-scFv exo), and the schematic diagram of the exosomes expressing the antibody targeting antibody is shown in Figure 2 .

[0079] Example 7: Detection of the targeting property of Her2-scFv exosomes

[0080] 100 μL of Her2-scFv exosomes were sent to Rui Be Hongda Biotechnology Co., Ltd. to detect the number and particle size of Her2-scFv exosomes by a nano-flow cytometer, and the results are shown in Figure 3 (a).

[0081] Figure 3 The results in (a) show that the Her2-scFv exosomes secreted by 293F cells account for nearly 1 / 4 of the total vesicles, and the particle size is 50 nm-100 nm.

[0082] The exosome concentration and purification process is consistent with the concentration multiple, and 20 μL of the obtained exosomes were taken for Western Blot detection, and the exosome marker and Flag tag were detected by CD81 antibody (Bi Yun Tian Biotechnology Co., Ltd., product number: A5270) and FITC antibody (Bi Yun Tian Biotechnology Co., Ltd., product number: A8050) respectively to measure the exosome secretion ability and exosome structure of Her2-scFv-293F cell exosomes, and to determine the 293F cell strain stably expressing Her2-scFv (Her2-scFv-3), and the results are shown in Figure 3 (b).

[0083] Her2-scFv exosomes carrying fluorescence groups excited by 405 nm excitation light; each take 10 8 ParticlesHer2-scFv exosomes, respectively added to 293T cells (Kangshen Biotechnology Co., Ltd., Cat. No. A23109), HepG2 cells (Chinese Academy of Sciences Typical Culture Collection Cell Library, Cat. No. TCHu72) and SKBR3 cells (Chinese Academy of Sciences Typical Culture Collection Cell Library, Cat. No. TCHu225), and after 2h of co-incubation, fluorescence microscopy was performed on each cell and the number of positive cells was counted, as shown in Figure 4 .

[0084] Figure 4 The results show that after 2h of co-incubation of Her2-scFv exosomes with different cells, the number of enhanced exosome positive cells in the field of view of HepG2 cells and SKBR3 cells is significantly higher than that of 293T cells; that is, compared with 293T cells which are negative for Her2 protein, Her2-scFv exosomes are taken up faster by HepG2 cells and SKBR3 cells which highly express Her2 protein; the targeted enhanced exosomes exhibit stronger Her2 positive cell targeting in vitro.

[0085] The above is only an embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent structural transformation or direct or indirect application in other related technical fields using the content of the present application is also included in the patent protection scope of the present application.

[0086] References

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[0088] KIM, H., YUN, N., MUN, D., KANG, J., LEE, S., PARK, H., PARK, H. & JOUNG, B. (2018), "Cardiac-specific delivery by cardiac tissue-targeting peptide-expressing exosomes.", Biochemical and biophysical research communications, Vol. 499 No. 4, pp. 803-808.

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[0091] ZHAO, H., YANG, L., BADDOUR, J., ACHREJA, A., BERNARD, V., MOSS, T., MARINI, J. C., TUDAWE, T., SEVIOUR, E. G., SAN LUCAS, F. A., ALVAREZ, H., GUPTA, S., MAITI, S. N., COOPER, L., PEEHL, D., RAM, P. T., MAITRA, A. & NAGRATH, D. (2016), "Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism.", eLife, Vol. 5 e10250.

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Claims

1. An enhanced exosome, characterized in that, The exosome has antigen targeting property and expresses antigen targeting antibody on the surface.

2. The enhanced exosome according to claim 1, characterized by The antigen targeting antibody comprises the following elements: 1) signal peptide; 2) histidine tag (His-tag) and Flag tag; 3) single-chain antibody region targeting antigen; 4) hinge region; 5) transmembrane region; and 6) fluorescent protein, wherein the amino acid sequence of the single-chain antibody region targeting antigen is shown in SEQ ID NO:

3.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antigen targeting antibody of claim 2.

4. An expression vector, characterized in that, The expression vector comprises the nucleic acid molecule of claim 3.

5. A recombinant lentivirus characterized in that The recombinant lentivirus is prepared from the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. A method of producing the recombinant lentivirus according to claim 5, characterized in that, The method comprises the following steps: co-transfecting a mammalian host cell with the vector of claim 4 and a helper plasmid.

7. A cell characterized by, The cell expresses the antigen targeting antibody of claim 2 and secretes the enhanced exosome of claim 1.

8. A method of producing the cell of claim 7, characterized in that, The method comprises the following steps: infecting a host cell with the recombinant lentivirus of claim 5.

9. The method of producing the enhanced exosome of claim 1, wherein, The method comprises the following steps: (1) culturing and isolating the cell of claim 7; (2) collecting the culture supernatant of the cell capable of secreting enhanced exosome, centrifuging and filtering to remove impurities; (3) purifying and concentrating the supernatant with a hollow fiber column to obtain the enhanced exosome.

10. A drug delivery tool characterized by, The delivery tool comprises the enhanced exosome of claim 1, the nucleic acid molecule of claim 3, the expression vector of claim 4 or the cell of claim 7.

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