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    公开号:ES2553129T9
    申请号:ES05807990.6T
    申请日:2005-11-24
    公开日:2016-02-22
    发明作者:Derek Thomas Brown；Hishani Kirby；Helene Margaret Finney；Alastair David Griffiths Lawson
    申请人:UCB Biopharma SRL；
    IPC主号:
    专利说明:

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    DESCRIPTION
    Anti-TNFα antibodies that selectively inhibit TNFα signaling by means of p55R
    The present invention relates to antibodies to TNFα. In particular, the present invention relates to antibodies that selectively inhibit TNFα signaling by means of p55R with respect to p75R, for example by selectively inhibiting TNFα binding to the p55 receptor.
    Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that is released by and interacts with cells of the immune system. TNFα has been shown to be increased in several human diseases, including chronic diseases such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
    Human TNF-α is a 17 kDa protein, and the active form exists as a homotimer (Pennica et al., 1984, Nature, 312, 724-729; Davis et al., 1987, Biochemistry, 26, 1322-1326 ; Jones et al., 1989, Nature, 338, 225-228). TNFα exerts its biological effects through interaction with two structurally related but functionally different cell surface receptors, p55R and p75R, which are co-expressed in most cell types (Loetscher et al., 1990, Cell, 61, 351; Smith et al., 1990, Science, 248, 1019). The p55R is also known as p55TNFR; CD120a; TNFR I; TNFR 1 and TNFRSF1a. The p75R is also known as p75TNFR; CD120b; TNFR II; TNFR 2 and TNFRSF1b. Both receptors are also released proteolytically in the form of soluble molecules capable of binding to TNFα. The extracellular domains of the two receptors exhibit sequence similarity, consisting of four repetitive cysteine-rich motifs containing four to six cysteines in conserved positions. In contrast, their sequences of cytoplasmic signaling regions are unrelated, suggesting different modes of signaling and function.
    The different roles of the two receptors were demonstrated by generating genetically deficient mice of one or both receptors (Peschon et al., 1998, J. Immunol., 160, 943-952). This study demonstrated that p55R is responsible for the majority of inflammatory responses mediated by TNFα, and that p75R can act in certain circumstances to inhibit inflammatory responses mediated by TNFα, and that the two receptors can act as a system that balances the action. of TNFα.
    Inhibition of TNFα activity as a method of treating a disease, in particular rheumatoid arthritis, has been achieved by different means with the use of inhibitors such as antibodies and soluble receptors. Examples include etanercept, marketed by Immunex Corporation as Enbrel ™, which is a recombinant fusion protein comprising two soluble TNF p75 receptor domains bound to the Fc portion of a human immunoglobulin. Infliximab, marketed by Centocor Corporation as Remicade ™, is a chimeric antibody that has murine anti-TNFα variable domains and human IgG 1 constant domains. Adalimumab, marketed by Abbott Laboratories as Humira ™, is a completely human, recombinant anti-TNFα antibody (Tussirot and Wendling, 2004, Expert Opin. Pharmacother., 5, 581-594). Other inhibitors include modified TNFα molecules that form trimers with native TNFα and that prevent receptor binding (Steed et al., 2003, Science, 301, 1895-1898; WO03033720; WO0164889). Sandborn et al. 2002 Biologic Therapy of IBD Gastroenterology 122: 1592-1608 describes the anti-TNF alpha CDP571 antibody in the treatment of Crohn's disease.
    These current methods for inhibiting TNFα activity block the binding of TNFα to the p55 and p75 receptors (see, for example, Mease, 2005, Expert Opin. Biol. Therapy, 5, 11, 1491-1504). Interestingly, it has been shown that Lenercept and Infliximab exacerbate multiple sclerosis, suggesting that there is also a beneficial role for TNFα in MS (Wiendl and Hohlfeld, 2002, Biodrugs, 16, 183-200). It is currently believed that, although TNFα signaling by means of p55R is necessary for the harmful effects of TNFα during the acute phase of MS, TNFα signaling by means of p75R can lead to beneficial effects, such as the removal of inflammatory infiltrates. . This immunosuppressive role of TNFα has also been proposed in other autoimmune diseases (Cope, 1998, Current Opinion in Immunology, 10, 669-676). In fact, it has been proposed that p75R agonists could be used to treat allergic conditions, such as allergic bronchial asthma (WO99 / 59632).
    The exact mechanism by which the two receptors bind to TNFα is unknown, but a report suggests that both TNFα receptors bind to TNFα through the use of similar interaction sites (Banner et al., 1993, Cell, 73, 431-445). Several studies that used point mutations in the TNFα polypeptide have shown that certain small areas of surface loops located in the final part of the subunit are the most functionally relevant. In the trimer, these areas face each other through the surface groove between two subunits. This suggests that a receptor interacts with the sites of two adjacent subunits, and that the TNFα trimer has three spatially different but equivalent receptor binding sites. It is not believed that it is possible for both receptors to bind to the same trimer at the same time (Barbara et al., 1994, EMBO, 13, 843-850).
    However, it has been possible to create TNFα mutants that selectively bind to the p75 or p55 receptor. It has been shown that TNFα mutants that do not bind to p55R but that do bind to p75R retain activity
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    antibodies that interact with TNFα and subsequently testing those antibodies to identify those that selectively inhibit TNFα signaling by means of p55R. In another example, the antibodies are identified by first identifying the antibodies that interact with TNFα and subsequently testing those antibodies to identify those that selectively inhibit the binding of TNFα to p55R and optionally also screening those antibodies based on selective inhibition of signaling. . Alternatively, antibodies can be screened directly to identify those that selectively inhibit TNFα signaling by means of p55R with respect to p75R, for example by directly screening in signaling and / or binding assays.
    Antibodies that interact with TNFα can be identified by the use of any suitable method, for example by the use of an assay system in which the TNFα polypeptide is contacted with a candidate antibody and the capacity of the candidate antibody is determined. of interacting with the TNFα polypeptide. Preferably, the ability of a candidate antibody to interact with a TNFα polypeptide is compared with a reference or control range. If desired, this assay can be used to screen a variety of candidate antibodies by using a variety of TNFα polypeptide samples. In one example, a first and second sample comprising a native or recombinant TNFα polypeptide is contacted with a candidate antibody or a control agent, and the ability of the candidate antibody to interact with the TNFα polypeptide is determined by comparing the difference. of the interaction between the candidate antibody and the control agent. Preferably, the TNFα polypeptide is first immobilized, for example, by contacting the polypeptide with an immobilized antibody that specifically recognizes and binds to it, or by contacting a purified preparation of TNFα polypeptide with a surface designed to bind proteins The TNFα polypeptide can be partially or completely purified (eg partially or completely free of other polypeptides) or part of a cell lysate. In addition, the polypeptide may be a fusion protein comprising the TNFα polypeptide or a biologically active portion thereof and a domain such as glutathione-S-transferase or the Fc region of IgG1. Alternatively, the polypeptide can be biotinylated by the use of methods well known to those skilled in the art (eg, biotinylation equipment, Pierce Chemicals; Rockford, IL). In certain cases, the TNFα polypeptide or candidate antibody is labeled, for example, with a radioactive label (such as 32P, 35S or 125I) or a fluorescent label (such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to enable the detection of an interaction between the TNFα polypeptide and a candidate antibody. The ability of the candidate antibody to interact with the TNFα polypeptide can be determined by methods known to those skilled in the art, for example, ELISA, BIAcore ™, flow cytometry or fluorescent microvolume assay technology (FMAT).
    As described above, antibodies can be pre-screened to identify antibodies that bind to TNFα before screening for antibodies that bind for their ability to selectively inhibit TNFα signaling by means of p55R.
    In one embodiment, the antibodies of the present invention selectively inhibit TNFα signaling by means of p55R by inhibiting TNFα binding to p55R. Antibodies that selectively inhibit the binding of TNFα to p55R can be identified by any suitable method, for example:
    (i)  comparing the binding of TNFα to p55R in the presence of a candidate antibody with the binding of TNFα to p55R in the absence of the candidate antibody or in the presence of a control agent; Y
    (ii)  comparing the binding of TNFα to p75R in the presence of the candidate antibody with the binding of TNFα to p75R in the absence of the candidate antibody or in the presence of a control agent; Y
    (iii) determining whether the candidate antibody substantially inhibits the binding of TNFα to p55R with respect to p75R.
    Such assays can be used to screen candidate agents in clinical monitoring and / or drug development.
    Examples of inhibition assays for binding of suitable TNFα receptors (p55R and p75R) have been described, see for example US 5,606,023 and Loetscher et al., 1993, The Journal of Biological Chemistry, 268, 26350-26357 . Additional examples of suitable cell-free and cell-based assays are provided in the Examples.
    Preferably, the ability of a candidate antibody to selectively inhibit TNFα binding to p55R is compared against a reference or control range. If desired, this assay can be used to screen a variety of candidate antibodies by using a variety of receptor binding inhibition assays. In an example of a cell-free assay, a first and second samples comprising native or recombinant TNFα polypeptide are contacted with a candidate antibody or a control agent, and the ability of the candidate antibody to inhibit binding of the polypeptide is determined. from TNFα to p55R or p75R comparing the difference in TNFα binding to each receptor in the presence of the candidate antibody and a control agent. In an example of such an assay, the extracellular domain of the receptor polypeptide is first immobilized, for example, by contacting the extracellular domain of the appropriate receptor with an immobilized antibody that specifically recognizes and binds to it, or by contacting a preparation. purified polypeptide
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    p55R, which cross-blocks the binding of the '462' antibody or the '463' antibody or an antibody comprising one or more of the CDRs provided in SEQ ID Nos. 9, 10, 11, 12, 13, 14 and 21 a Human TNFα. Cross-blocking antibodies may inhibit the binding of the '462' antibody or the '463' antibody or an antibody comprising one or more of the CDRs provided in SEQ ID Nos. 9, 10, 11, 12, 13, 14 and 21 a Human TNFα by 80% or more.
    Alternatively or additionally, the antibodies can be cross-blocked at the binding to human TNFα by any '462' antibody or '463' antibody or an antibody comprising one or more of the CDRs provided in SEQ ID Nos. 9, 10 , 11, 12, 13, 14 and 21. An anti-TNFα antibody that selectively inhibits TNFα signaling by means of p55R that is cross-blocked in human TNFα binding by the '462' antibody or the '463' antibody or an antibody comprising one or more of the CDRs provided in SEQ ID Nos. 9, 10, 11, 12, 13, 14 and 21. Cross-blocking antibodies can be inhibited in binding to human TNFα by the '462' antibody or the '463' antibody or an antibody comprising one or more of the CDRs provided in SEQ ID Nos. 9, 10, 11, 12, 13, 14 and 21 by 80% or more.
    If desired, an antibody for use in the present invention can be conjugated to an effector molecule. The term "effector molecule" as used herein includes, for example, antineoplastic agents, drugs, toxins, biologically active proteins, for example enzymes, other antibodies or antibody fragments, synthetic or natural polymers, nucleic acids and fragments thereof. same, eg, DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and indicator groups such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy. In one example, anti-TNFα antibodies can be conjugated to an effector molecule, such as a cytotoxic agent, a radionuclide or a drug moiety to modify a given biological response. For example, the therapeutic agent can be a drug residue that can be a protein or polypeptide that possesses a desired biological activity. Such moieties may include, for example and without limitation, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, a protein such as tumor necrosis factor, interferon α, interferon β, nerve growth factor , platelet-derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin, or a biological response modifier such as a lymphokine, interleukin 1 (IL-1 ), interleukin 2 (IL-2), interleukin 6 (IL-6), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF ) or other growth factor.
    In another example, the effector molecules may be cytotoxins or cytotoxic agents that include any agent that is harmful to the cells (eg destroying them). Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mitramycin, dynostosteric, actinomycortin, 1-dihydrocortin, glutathine, dynostosteric, actinomycortin procaine, tetracaine, lidocaine, propranolol, and puromycin, and the analogs or homologs thereof. The effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbacin), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (carmustine) BSNU) and lomustine (CCNU), cyclotosfamide, busulfan, dibromomanitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin, antibiotics) eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, antramycin (AMC), calicheamycins or duocarmycins), and anti-mitotic agents (eg, vincristine and vinblastine).
    Other effector molecules may include radionuclides such as 111In and 90Y, Lu177, Bismuth213, Californium252, Iridium192 and Tungsten188 / Renium188; or drugs such as, but not limited to, alkyl phosphocholines, topoisomerase I inhibitors, taxoids and suramin.
    Techniques for conjugating said effector molecules to antibodies are well known in the art (see, Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., Eds., 1987, p. 623-53; Thorpe et al. ., 1982, Immunol. Rev., 62: 119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). In one example, the antibody or fragment thereof is fused by means of a covalent bond (eg, a peptide bond), optionally at the N-terminus or the C-terminus, to an amino acid sequence of another protein. (or portion thereof; preferably, at least a 10, 20 or 50 amino acid portion of the protein). Preferably, the antibody, or fragment thereof, is linked to the other protein at the N-terminal end of the constant domain of the antibody. Recombinant DNA methods can be used to create such fusions, for example as described in WO 86/01533 and EP 0392745.
    In another example, the effector molecule can increase the half-life in vivo, and / or increase the transport of an antibody through an epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds.
    In one example, the antibodies of the present invention may be linked to poly (ethylene glycol) (PEG) moieties. In a particular example, the antibody is an antibody fragment and PEG molecules may be bound to
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    use. Oral liquid preparations may contain suspending agents as is known in the art.
    In the case of oral solid preparations, such as powders, capsules and tablets, vehicles such as starches, sugars, microcrystalline cellulose, granulating agents, lubricants, binders, disintegrating agents, and the like can be included. Due to their ease of administration, tablets and capsules represent the most advantageous oral unit dosage form, in which case solid pharmaceutical vehicles are generally used. In addition to the usual pharmaceutical forms set forth above, the active agents of the invention can also be administered using controlled release administration means and / or devices. The tablets and capsules may comprise conventional carriers or excipients such as binding agents, for example, syrup, gum arabic, gelatin, sorbitol, gum tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; lubricants for tablets, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated by usual aqueous or non-aqueous techniques according to methods well known in normal pharmaceutical practice.
    The pharmaceutical compositions of the present invention suitable for oral administration may be presented in the form of discrete units such as capsules, wafers or tablets, and each contains a predetermined amount of the active agent, in the form of a powder or granules, or in the form of a solution or suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the pharmacy methods, but all methods include the step of contacting the active agent with the vehicle, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately mixing the active agent with liquid vehicles or finely divided solid vehicles or both, and then, if necessary, shaping the product until the desired presentation. For example, a tablet can be prepared by compression or molding, optionally with one or more secondary ingredients.
    Pharmaceutical compositions suitable for parenteral administration may be prepared in the form of solutions or suspensions of the active agents of the invention in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under usual conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
    Pharmaceutical forms suitable for injectable use include sterile aqueous or nonaqueous injection solutions that may contain antioxidants, buffers, bacteriostats and solutes that make the composition isotonic with the blood of the desired recipient, and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. Solutions, dispersions and suspensions for improvised injection can be prepared from sterile powders, granules and tablets.
    The pharmaceutical compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices described in US 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: US 4,487,603, which describes an implantable microinfusion pump for dispensing the medication at a controlled rate; US 4,486,194, which describes a therapeutic device for administering medications through the skin; US 4,447,233, which describes a medication infusion pump for administering the medication at a precise infusion rate; US 4,447,224, which describes an implantable variable flow infusion apparatus for continuous drug administration; US 4,439,196, which describes an osmotic drug delivery system that has multi-chamber compartments; and US 4,475,196, which describes an osmotic drug delivery system. Those skilled in the art know many other implants, administration systems, and modules.
    Pharmaceutical compositions adapted for topical administration may be formulated in the form of ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated bandages, sprays, aerosols or oils, transdermal devices, dusting powders, and the like. These compositions can be prepared by conventional methods containing the active agent. Thus, they can also comprise compatible conventional carriers and additives, such as preservatives, solvents to aid drug penetration, emollients in creams or ointments and ethanol or oleyl alcohol for lotions. Such vehicles may be present in the form of about 1% to about 98% of the composition. More normally, they will constitute about 80% of the composition. As an illustration only, a cream or ointment is prepared by mixing sufficient amounts of hydrophilic material and water, containing about 5-10% by weight of the compound, in amounts sufficient to produce a cream or ointment having the desired consistency.
    Pharmaceutical compositions adapted for transdermal administration may be presented in the form of discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active agent can be administered from the patch by iontophoresis.
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    Supernatants from these plates were screened by ELISA for anti-TNFα antibodies by using human TNFα (50 ng / ml) captured with a sheep polyclonal antibody coated on immunoplates. The supernatants from the positive wells were then further tested in the L929 bioassay and in the specific protein assays of the p55 and p75 receptors described below.
    Example 2. Inhibition assays of TNFα receptor binding
    L929 trial
    L929 cells (a mouse fibroblast cell line) expressing the mouse p55TNFα receptor, but not the p75TNFα receptor, were used to test the anti-TNFα antibodies that block the binding of TNFα to this receptor. These cells are destroyed by human TNFα if they are sensitized with a protein synthesis inhibitor.
    Cells were grown in standard tissue culture medium and seeded in 96-well tissue culture plates the day before they were needed for the assay. The culture medium was removed, and the test supernatants were added to individual wells. Human recombinant TNFα was then added to each well at 200400 pg / ml in the presence of 1 µg / ml (final concentration) of actinomycin D. The plates were then incubated overnight at 37 ° C.
    The next day the plates were washed gently in PBS and the cells were fixed in methanol. They were then stained with 1% crystal violet (living cells remain attached to the plates and absorb the dye). Excess staining was removed by washing, and the remaining stained cells were solubilized in 30% acetic acid. The plates were then read at 570/405 nM.
    Wells containing antibodies that block the binding of TNFα to mouse p55TNFR protect cells from cytotoxicity mediated by TNFα, and show an increased signal compared to negative / control wells.
    Positive wells were further tested in the p55R and p75R assays.
    P55TNFR and p75TNFR binding inhibition assay
    Standard ELISA plates were coated with a human anti-TNFα polyclonal antibody diluted 1 / 10,000. The plates were then blocked with PBS + 1% BSA. Human TNFα was then added to each well at 25-50 ng / ml. After 1 hour, unbound TNFα was removed by washing. Supernatants containing anti-TNFα antibodies were then added to duplicate wells. In addition, a human p55TNFR human-Fc fusion protein or human p75TNFR human-Fc fusion protein was added to a well of each duplicate. These were incubated for 1 hour, and then washed to remove the unbound receptor. Following this step, a peroxidase-conjugated human IgG polyclonal antibody (Stratech Scientific) was added at a 1/2000 dilution. The plates were left for 1 hr and then washed to remove the unbound conjugate. The TMB substrate was then added to each well, and the color was allowed to develop. Therefore, wells in which anti-TNFα antibodies have blocked the binding of the receptor (s) can be visualized.
    Figure 1 shows the percentage inhibition of TNFα binding to p55TNFR and p75TNFR by four different anti-TNF antibodies. The '3D6' antibody inhibited the binding of TNFα to p55TNFR in 49.3%, but only inhibited the binding of TNFα to p75TNFR in 14.6%. In contrast, the 22H3 antibody, for example, inhibited the binding of TNFα to p55R and p75R by 78.9 and 71.9%, respectively. The 3D6 antibody, therefore, selectively blocks the binding of TNFα to p55R.
    Example 3 Isolation of additional selective antibodies
    By using the same population of rats as in Example 1, cultured B cells were screened to identify selective antibodies to TNFα.
    Human TNFα (Strathman Biotech GmbH) was biotinylated with a 10-fold molar excess of Sulfo-NHS-LC-LCbiotin (Pierce) for 1 hour at room temperature following the manufacturer's protocol. 5 µg of biotinylated TNFα was mixed with 50 µl of 9.95 micron supervidin coated microspheres (Bangs Beads) for 1 hour at room temperature in a volume of 500 µl (mix for 1 x 384 well plate). The microspheres were then washed 5 times in PEG blocking buffer (1% PEG / 0.1% tween / PBS) to remove unbound TNFα. The TNFα coated microspheres were then resuspended in approx. 4 ml of PEG blocking buffer, and 10 µl was added to each well of a 384 well plate. 10 µl of B-cell culture containing rat antibody and 10 µl of goat anti-Fc gamma specific IgG rat-Cy5 antibody conjugate was added at a 1: 1666 dilution to the well containing the microspheres. The plates were incubated at room temperature in the dark for 1 hour, and then read on an Applied Biosystems 8200 apparatus. An Applied Biosystems software was used to identify positive wells.
    Approximately 1400 B cell culture plates were screened, which are approximately 140000 wells that
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    they represent approximately 2x108 B cells.
    Of the screens, 2,500 wells contained antibodies that bind to TNFα.
    These were further screened based on the ability to selectively block TNFα signaling by means of p55R with respect to p75R through the use of the assays described in Example 4. The antibodies were first screened based on blocking of signaling of p55R, and those that blocked signaling were then tested for the ability to block TNFα signaling by means of p75R. Antibodies that selectively blocked signaling by means of p55R were isolated by using the homogeneous fluorescence assay described in WO2004 / 051268, and the genes of the heavy and light chain variable region were cloned by transcription PCR. reverse of individual rat B cells. The variable regions were expressed in recombinant IgG format to confirm binding and activity in sub-cloning signaling assays in expression vectors containing the genes of the human antibody constant region (human kappa light chain and gamma-4 heavy chain in which serine from position 241 has been changed to proline as described in Angal et al., Molecular Immunology, 1993, 30 (1), 105-108) and a rat / human chimeric antibody expressed transiently in CHO cells. Transfections of CHO cells were carried out using the Lipofectamine procedure according to the manufacturer's instructions (InVitrogen, catalog no. 18324).
    Two antibody sequences were obtained, and these were called '462' and '463'.
    The sequences of the V region of '462' are provided in SEQ ID Nos: 1, 2, 3 and 4. The variable region sequences without the leader sequences are provided in SEQ ID Nos: 5, 6, 7 and 8. The sequences of the V region of '463' are provided in SEQ ID Nos: 15, 16, 17 and 18. The sequences of the variable region without the leader sequences are provided in SEQ ID Nos: 19, 20, 7 and 8.
    Example 4: TNFR signaling assays
    4.1 p55R signaling test
    Luciferase assay of p55 NFkB
    A549-ES-Luc cells were used for this indicator gene assay. A549 cells are a carcinoma of lung epithelial cells that express the p55 TNF receptor, and have been stably transfected with a vector comprising the E-selectin promoter (containing 3 x NFkB binding sites) linked to the gene of luciferase and a selectable marker for the generation of a stable cell line.
    A549-ES-Luc were grown in the following media:
    RPMI 1640 (Phenol Free)
    +  10% FCS
    +  2 mM glutamine
    +  1 mg / ml of G418 (Life Tech, 50 mg / ml stock solution)
    A549-ES-luc cells were placed in white opaque 96-well plates (Perkin Elmer) by using a 1.5x105 cell / ml cell suspension; 100 µl / well = 15,000 cells / well. The cells were allowed to adhere overnight at 37 ° C / 5% CO2. The next day, the media were aspirated and replaced with 100 µl of antibody in assay medium that had been pre-incubated for 30 minutes with human TNFα at a final concentration of 3 ng / ml. The cells were incubated for 5 hours at 37 ° C / 5% CO2. Luciferase expression was then tested by using a test kit with luciferase reporter gene (Luckin of Perkin Elmer). The plate was then read in a luminescence plate reader, the LJL Analyst.
    4.2 P75 signaling test
    Jurkat cells that had been stably transfected with a vector containing a cassette encoding the extracellular domain of p75R bound to the intracellular signaling regions of CD28 and TCR zeta were used to test p75 signaling. Within the same vector, there are 5 binding sites for NFκB with a minimal promoter region of E-selectin, and this controls the expression of the luciferase reporter gene, and a selectable marker for the generation of stable cell lines. Stimulation of the p75 bioassay receptor with its ligand, human TNFα, leads through the CD28 / zeta regions of the bioassay receptor at the start of a signaling cascade within the cell. The signaling cascade induces the activation of NFκB and allows transcription of the luciferase reporter gene. Activation levels can then be measured in a luciferase assay. Antibodies that can block this activation will prevent luciferase expression.
    Construction of a receptor expression cloning cassette and shuttle vector.
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    权利要求:
    Claims (1)
    [1]
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    GB0425972|2004-11-25|
    GBGB0425972.7A|GB0425972D0|2004-11-25|2004-11-25|Biological products|
    PCT/GB2005/004511|WO2006056779A2|2004-11-25|2005-11-24|ANTI-TNF ALPHA ANTIBODIES WHICH SELECTIVELY INHIBIT TNF ALPHA SIGNALLING THROUGH THE p55R|
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