• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present invention relates to an effective method of monoclonal antibody production directed against surface antigens of cells and viruses. This method includes antigens that are present in relatively small amounts, but only very small amounts of available antigens or antigens that readily lose their coordination in vivo. Thus, the method described herein comprises a series of methods comprising the step of aggregating B-cells derived from mammals injected with surface antigen-containing material against a relative number of specific B-cells and a small amount of fusion techniques. Steps.
    公开号:KR19980063909A
    申请号:KR1019970066848
    申请日:1997-12-05
    公开日:1998-10-07
    发明作者:페트루스제라두스안토니우스스틴바커스
    申请人:에프.지.엠.헤르만스；악조노벨엔.브이.；
    IPC主号:
    专利说明:

    Monoclonal antibodies, methods for their preparation, and pharmaceutical compositions and diagnostic reagents containing the same
    The present invention relates to a method for producing monoclonal antibodies against surface antigens, in particular surface antigens that occupy only a small portion of the total antigens injected into mammals and surface antigens which readily lose their coordination in vivo. Problems exhibited by antigens contained in small amounts relative to the total amount of antigens arise when, for example, the antigenic substance used for injection into a mammal is derived from another mammalian species.
    Problems associated with the aforementioned antigens are surfaced, for example, when there is a need to prepare monoclonal antibodies against specificity determining regions of the T-cell receptor (TCR). Such (monoclonal) antibodies are known as anti-clonal antibodies that recognize the antigen-specific moiety (or clonal structure) possessed by the T-cell receptor of one particular T-cell clone. Monoclonal anti-clonal antibodies to rat T-cell receptors (TCRs) have sometimes been reported, while known monoclonal anti-clonal antibodies to human T-cell receptors (rats) Much less has been reported. T-cells are difficult to culture, making it difficult to obtain and purify sufficient amounts of antigens, ie, T-cell receptor proteins. Therefore, it is difficult to obtain an immune response and selection is difficult. In one of the few cases in which monoclonal anti-clonal antibodies are effective against human TCR, this problem did not occur because the antigen was present on Jurkat leukemia cells (Ref. 1). Since leukemia cells can be cultured in an infinite amount, there is no problem of antigen deprivation. Thus, no useful method has been suggested that can produce monoclonal antibodies against trace and / or highly unstable antigens without the need to select a number of undesirable hybridoma clones.
    It is an object of the present invention to provide a method for preparing monoclonal antibodies against cell surface antigens under undesirable circumstances as described above, which significantly reduces the number of hybridoma clones to be screened, The present invention provides a more effective and successful method according to the present invention even if the method according to the technical level is not successful.
    1 is SDS / PAGE to identify H.243 T-cell surface molecules immunoprecipitated by MAbs directed against H.243.
    2 is Western blotting of anti-clone MAbs that recognize TCRαβ.
    3 shows proliferation stimulation of human T-cell clone H.243 by immobilized anti-TCR MAb.
    4 shows the inhibition or blocking of antigen-induced proliferation of human T-cell clone H.243 by pre-culture with anti-TCR MAb.
    5 shows anergy induction of human Th1-clone H.243 by anti-clonal MAb.
    FIG. 6 shows the inhibitory response of nonergic H.243 T cells by anergic H.243 T cells.
    In order to achieve this object the method according to the invention
    1) injecting a mammalian cell surface antigen-containing material selected from the group consisting of i) whole cells and ii) membrane fractions obtained by treating whole cells;
    2) separating the B-cell containing cell fraction from the spleen of the mammal;
    3) The cell fraction obtained in step 2) is brought into contact with the carrier-binding material of the cell associated with the whole cell lacking the cell surface antigen and integrated into B-cells specific for the cell surface antigen, and thus Separating the B-cells bound to the carrier-binding material of said related cells from the non-binding B-cell containing cell fraction to be used in the step;
    4) marginally diluting the integrated B-cell containing cell fraction obtained in the above step and then propagating the clone;
    5) selecting B-cell clones and proliferating the selected B-cell clones using a small amount of fusion techniques; And
    6) screening and cloning hybridomas capable of producing antibodies specifically binding to the cell surface antigen, and then separating the monoclonal antibody-containing fractions from the supernatants of the hybridomas.
    For clarity and clarity, the term cell used herein refers to mammalian cells as well as to viruses, specifically membrane-containing viruses.
    The term carrier-binding material of a cell includes the original cell or whole cell (and virus), a membrane fraction of such original whole cell (and virus) or an essentially purified surface antigen or a combination thereof.
    Unless otherwise stated, the term whole cell refers to a cell having the desired surface antigen. The term related cell preferably means a cell that differs only in that it lacks the desired surface-antigen or, more specifically, that there is at least no immunological determinant necessary to obtain a monoclonal antibody.
    Surprisingly, the isolated B-cell-containing cell fraction is obtained from the same species from which the cell surface antigen-containing material is derived or is contacted with a cell material that is free of cell surface antigens, thereby making the cell surface antigen specific. It is now possible to integrate cell fractions into B-cells. In other words, integration can be achieved by contacting B-cells with irrelevant related cells or cellular material and removing bound B-cells.
    Thus, it is possible to produce monoclonal antibodies directed against a small number of cell surface antigens even though cell surface antigens exhibit coordination instability.
    European Patent EP 0 488 470 and Reference 3 based on this European Patent Literature discloses: I) injecting an antigen into a mammal, II) isolating a B-cell containing fraction, III) B-cell specific for the antigen. Screening, IV) cloning the integrated fractions, V) screening B-cell clones and infinite propagation using a small amount of fusion techniques, and VI) selecting and cloning hybridomas followed by monoclonal A method consisting of separating a fraction containing a null antibody is described. In step III, the B-cells are selected by binding them to antigen-coated plastic surfaces or by rosetteing B-cells with antigen-coated paramagnetic beads. Nonspecific B-cells are washed away. Thus, apart from other differences, this method depends on the high availability and use of the antigen (when sorting, an antigen of 2 μg / ml is used to coat the plate with the antigen), while the present invention provides a usable antigen. This is to solve the problem of this very small case and very impure case.
    One preferred aspect of the present invention is characterized in that the mammal injected with the surface antigen is a different species from the mammalian species from which the surface antigen was obtained.
    The method according to the invention is well suited for the preparation of monoclonal antibodies in the presence of a large number of antigens capable of inducing an immune response.
    This situation is particularly the case when the surface antigen has a constant portion and a variable portion, and at least a portion of the variable portion indicates the specificity determining portion of the surface antigen.
    According to one preferred embodiment, the receptor molecule-containing material is used as cell surface antigen-containing material.
    The method according to the invention is particularly suitable for preparing monoclonal antibodies produced against specificity determining portions of receptor molecules. The specificity determining portion of a receptor molecule is only a minor region of the receptor molecule and is essentially a subcomponent of all molecules present on the cell surface-the antigen.
    Preferred targets according to the invention are T-cell clones, which are used to prepare receptor molecule-containing materials.
    Whole cells may be used as T-cells, or membrane fraction preparations may be used. Thus, in the former case, the term prepare will mean either simple separation of T-cells or resuspension in other media.
    According to a preferred embodiment, the membrane fraction in step 1) is obtained by mechanical treatment of whole cells and the cell surface antigen-containing material is injected into the mammal without adjuvant.
    Both means prevent surface antigens from losing coordination in vivo.
    In addition, the aggregated B-cell containing cell fraction comprises a carrier containing a substance selected from the population consisting of j) whole cells, jj) membrane fractions obtained from whole cells and jjj) essentially purified cell surface antigens. -Further accumulation (step 3) by contact with the bound cell surface antigen, followed by B- which is not bound to the carrier-binding material from B-cells bound to the carrier-binding material containing the further integrated cell fraction Isolate the cells.
    Such further integration may also be referred to as a positive selection technique that specifically selects specific B-cells. Thus, such additional integration can be performed even if some antigens are rarely available.
    According to one preferred aspect, it is preferable to use paramagnetic beads as a carrier.
    Using paramagnetic beads during integration facilitates the separation of necessary and unnecessary B-cells.
    In addition, small amounts of antigen present problems in the selection process. In a preferred embodiment, the selection process in at least one of steps 5 and 6 comprises antibody coated carrier and cell surface antigens capable of binding the supernatant of the B-cell clone to the antibodies of the injected mammalian species used in step 1 It is carried out using agglutination assay which is contacted with containing whole cells and detecting the agglutination reaction.
    In the above, simple mixing of antibody-coated carriers capable of binding to B-cell supernatants, whole cells and the antibodies of the mammalian species used in step 1 detects suitable clones quickly and with very high sensitivity without requiring washing. can do.
    Whole cells that are related to whole cells but lack surface antigens are preferably used as controls.
    This method saves time by eliminating false-positive clones and eliminating unnecessary minor fusion steps.
    In a preferred embodiment of the method according to the invention, the B-cell clone selected in step 5 is mixed with myeloma cells and treated by micro-electrofusion.
    Micro-electrofusion is an effective fusion method for very few (eg hundreds) of cells.
    The invention also relates to pharmaceutical compositions containing monoclonal antibodies prepared according to the invention in admixture with suitable excipients.
    The present invention also relates to monoclonal antibodies which react with the clonal structure of the T-cell receptor.
    Such monoclonal antibodies can be used for diagnostics as well as for the preparation of pharmaceutical compositions.
    More specifically, T-cell receptors are T-cell receptors associated with autoimmune diseases, especially rheumatoid arthritis.
    The monoclonal antibody preferably reacts with the T-cell receptor of HC gp-39 reactive T-cell clone, in particular T-cell clone H.243 (ECACC Accession No. 96103122).
    Specific examples of suitable monoclonal antibodies include high selected from the crowd consisting of TCR 69 (ECACC Accession No. 96103118), TCR 70 (ECACC Accession No. 96103119), TCR 72 (ECACC Accession No. 96103120) and TCR 83 (ECACC Accession No. 96103121). It was produced by bridoma.
    As mentioned above, the present invention also relates to a pharmaceutical composition comprising a monoclonal antibody according to the invention in admixture with a suitable excipient, which is suitable for treating rheumatoid arthritis.
    Finally, the present invention relates to the diagnostic use of a monoclonal antibody prepared using the method according to the invention and a monoclonal antibody selected from the group consisting of the monoclonal antibody according to the invention. Moreover, one aspect of this invention is a diagnostic reagent containing the said antibody.
    Hereinafter, the present invention will be described in more detail with reference to the following examples which present the best embodiments for carrying out the present invention used to prepare murine monoclonal antibodies specific for the cloned form of human T-cell receptor. do.
    Legend for Drawings
    1 is SDS / PAGE to identify H.243 T-cell surface molecules immunoprecipitated by MAbs directed against H.243. This SDS / PAGE was performed in non-reducing conditions (lanes 2, 3 and 4) or reducing conditions (lanes 5, 6 and 7) in a 10% gel. Lane 1: 10 kD ladder marker; Lanes 2 and 5: control MAb; Lanes 3 and 6: TCR 83; Lanes 4 and 7: anti-CD3, OKT3.
    2 is Western blotting of anti-clone MAbs that recognize TCRαβ. First, an immunoprecipitated TCR / CD3 complex of H.243 is isolated using a 10% SDS-PAGE gel under non-reducing conditions and then transferred to a PVDF membrane. This membrane was incubated with MAb and finally detected using alkaline phosphatase-bound goat anti-mouse Ig second antibody.
    Lane 1: 10 kD ladder marker, lane 2: TCR 64, lane 3: TCR 66, lane 4: TCR 69, lane 5: TCR 70, lane 6: TCR 72, lane 7: TCR 73, lane 8: TCR 76, Lane 9: TCR 78, lane 10: TCR 79, lane 11: TCR 83, lane 12: control MAb, lane 13: medium control.
    3 shows proliferation stimulation of human T-cell clone H.243 by immobilized anti-TCR MAb. Proliferation induced by MAbs directed against H.243 (TCR 64 to TCR 83) was compared with control MAb as indicated and TCR44, an anti-clone MAb directed against other TCRs. Each value is expressed as the mean count value ± standard deviation per 5 minutes of 4 replicate cultures.
    4 shows the inhibition or blocking of antigen-induced proliferation of human T-cell clone H.243 by pre-culture with anti-TCR MAb. a) compares the inhibition by MAb directed against H.243 (TCR 64 to TCR 83) with TCR 44, a control MAb as indicated and an anti-clone MAb directed against other TCRs. Each value represents the mean count value +/- standard deviation per 5 minutes of 4 replicate cultures. b) is the dose response curve of the strongly inhibitory MAb; TCR 64 (empty circle), TCR 70 (filled triangle), TCR 78 (empty rectangle), TCR 83 (filled rectangle), and control MAb 1 (filled circle). Each value represents the mean count per 5 minutes of three replicate cultures.
    5 shows anergy induction in human Th1-clone H.243 by anti-clonal MAb. Immobilized anti-clone MAb or control MAb 1 was incubated with H.243 T cells overnight. After isolation of cells from anergic stimulation, cells were subjected to complete stimulation with increasing peptide and DRB1 * 0401 combination APC concentrations. Proliferation was assessed by 3 H-thymidine infusion. Each value represents the mean count value ± standard deviation per 5 minutes of three replicate cultures.
    FIG. 6 shows the inhibitory response of nonergic H.243 T cells by anergic H.243 T cells. Immobilized TCR 76 or control MAb1 was incubated with H.243 T cells overnight. These two T-cell groups were then used for proliferation analysis for increasing peptide (or PHA) and APC concentrations using 2 x 10 4 T cell volume per well. In addition, various amounts of anergic cells obtained from incubation with TCR 76 were mixed with non-ergic cells 2 × 10 4 obtained from incubation with MAb 1 and analyzed for proliferation. Anergic cell ratio to nonnergic cell, A / N, is 1 = 2 × 10 4 cells. Proliferation was assessed by 3 H-thymidine infusion. Each value represents the mean count value + standard deviation per 5 minutes of three replicate cultures.
    Example
    Materials and methods
    reagent
    Culture medium: sodium bicarbonate, 2500 mg / l, 2- mercaptoethanol, 2.3 mg / l, sodium pyruvate 55 mg / l, ethanol amine 1.22 mg / l, L- glutamine, 360 mg / l, 4.5 x 10 Ah selenite Sodium F12 (Gibco Listing No. 32500) of DMEM / HAM supplemented with 4 mg / l, 62.5 mg / l penicillin sodium 62.5 mg / l and streptomycin sulfate. In the fusion experiment, this medium was supplemented with 13.61 mg / l hypoxanthine and 3.83 mg / l thymidine. This medium is also called DMEM / HAM F12 / HT. Selection of the hybridomas was performed in DMEM / HAM F12 / HT supplemented with 1% (v / v) of IL-6 containing supernatant of human bladder sarcoma cell line T24 (T24CM) and 0.4 μΜ of aminopterin.
    Fusion medium: inositol 280 mM, calcium acetate 0.1 mM, magnesium acetate 0.5 mM and histidine 1 mM; Resistivity: 1.11 · 10 4 Ωcm. The components were dissolved in milli-Q water and then the conductivity was adjusted to 90 μS / cm using a solution containing milli-Q water or 1 mM calcium acetate and 5 mM magnesium acetate.
    Cell culture
    T-cell clone H.243 was obtained from a patient suffering from rheumatoid arthritis. This Th1-like T-cell clone recognizes epitope RSFTLASSETGVG (SEQ ID NO: 1) derived from human cartilage gp-39 (HC gp-39) under the environment of DRB1 * 0401 (belonging to MHC group II). The TCR of this clone is characterized by being Vα8 and Vβ9 positive. Cells were cultured conventionally in DMEM / HAM F12 supplemented with 10% FCS, 20 U / ml IL-2, 5U / ml IL-4, and antigen- and tissue-compatible antigen expressing cells (APCs) or plant hemagglutinin ( PHA) and feeder cells were periodically restimulated. In the proliferation assay, normal serum (NHS) was used instead of fetal calf serum (FCS).
    Preparation of T-Cell Lysates and Loading of TCR / CD3 Complexes on Beads
    10-14 days after restimulation, T-cells were washed with PBS and the cells were extracted with extraction buffer (10 mM triethanolamine, 150 mM NaCl, 1 mM Na 2 EDTA, 1% Digitonin according to Wetgen et al. , 10㎍ / ml leupeptin, 10㎍ / nl aprotinin, 1㎍ / ml pepstatin, 1 mM Pefabloc (registered trade name) AEBSF and 1.8 mg / ml iodo-acetamido amine pH 7.8) of 10 8 cells / ml concentration of The solution was incubated at 30 ° C. (0 ° C.) for dissolution. Soft detergent Digitonin can natively extract the TCR / CD3 complex. Nuclei and cell residues were removed by centrifugation at 16,000 g for 15 minutes at 4 ° C. and the supernatants were stored at −20 ° C. in constant amounts until used for B-cell selection or immunoprecipitation.
    To select specific B-cells, paramagnetic beads were loaded with the TCR / CD3 complex.
    Briefly, 100 μl of positive anti-mouse Ig bound paramagnetic beads (SAM-beads; Dynabeads® 110.02) were incubated with 22 μg of anti-CD3, OKT3 in PBS containing 0.1% BSA ( Overnight, 4 ° C.). After washing the beads with PBS / BSA, PBS / BSA containing 1.4 × 10 8 cell equivalents of T-cell lysate and equivalent (1.4 ml) of 1% normal mouse serum was added. PBS / BSA containing this 1% normal mouse serum was added to prevent B-cells from binding to the free SAM binding site on the beads. This bead suspension was incubated at 4 ° C. for 2-4 hours and washed with PBS / BSA before use.
    Immunization
    Six-week-old female BALB / C mice were loaded with 5 × 10 6 whole T-cells (invention) or 5 × 10 7 cells equivalent TCR / CD3 complexes at 3-7 week intervals (control experiment). Immunized with. The route of administration used several routes as shown in Table 1. In the case of adjuvant (control experiment), the complete Freund's supplement was used for the first infusion, the incomplete Freund's injection was not used for the next infusion, and the adjuvant was not used for the final additional antigen stimulation.
    Generation of anti-clone MAbs
    Six-week-old female BALB / C mice were injected intraperitoneally with resting T-cells 5 × 10 6 in PBS four times at three to seven week intervals. Five days after the last injection mice were killed and erythrocytes and monocyte-deficient splenic cell populations were prepared as previously described (Refs. 23, 24). This resulted in a cell fraction in which B-cells were accumulated, but did not accumulate B-cells specific for the cell surface antigen as compared to other non-specific B-cells according to step 2 of the present invention.
    In order to preclean the splenic cell population against B-cells reactive to the constant regions of human CD3 or TCRαβ, T-cell clones (Vα3.Vβ14 positive T-cell clones, SCRO.08A) unrelated to about 3 × 10 7 cells However, 20 μl of SAM beads loaded with the TCR / CD3 complex of any other irrelevant T-cell may be incubated twice. The cell suspension thus obtained was incubated with SAM beads loaded with the TCR / CD3 complex of the desired T-cell clone (H.243) to select B-cells specific for the variable region of TCR. Each incubation was performed for 90 minutes at room temperature in DMEM / HAM F12 supplemented with 10% calf serum (Hyclone®) and 0.2% normal rat serum. During this incubation, the suspension was carefully homogenized every 5 minutes. After the final incubation, the beads were carefully washed 5 times with DMEM / HAM F12, 10% calf serum and resuspended in the same medium.
    The B-cells thus selected were cloned and micro-electrofused as previously described (23) to produce monoclonal antibody producing hybridomas. To summarize this briefly, first selected B-cells were 10% FCS in 96 well flat bottom tissue culture plates with T-cell suspension (TSN) and 50,000 irradiated (2500 rad) EL-4 B5-cells. DMEM / HAM F12 supplemented with a final volume of 200μl was mixed. After 8 days, the supernatants were tested in a one-step T-cell aggregation assay using the desired T-cell clones or unrelated T-cell clones as described below. B-cell cultures producing the discriminating MAbs were subjected to micro-electrofusion procedures. The contents of this B-cell culture were mixed with 10 6 NS-1 myeloma cells (25) and washed with DMEM / HAM F12 / HT to remove serum. Cells were then treated with Pronase solution for 3 minutes and then washed with fusion medium. Electrofusion is carried out in an alternating electric field of 30 s, 2 MHz, 400 V / cm in a 50 µl fusion chamber, followed by an alternating electric field of 30 s, 2 MHz, 400 V / cm, orthogonal high-pass pulses of 10 μs, 3 kV / cm It was. Finally, the contents of the fusion chamber were transferred to 20 ml sorting medium and plated on 96 well microtiter plates. After 8 days of fusion, cultures were examined for hybridoma proliferation and again selected by a one-step T-cell aggregation assay.
    T-cell aggregation assay
    Selection of hybridoma cultures against anti-T-cell antibodies and measurement of cross-reactivity of other T-cells with MAb were performed by one step aggregation assay. 50 μl of hybridoma supernatant using half-section of the microtiter plate, paramagnetic beads covalently bound to 5 × 10 4 desired T-cells and 2 × 10 5 anti-mouse Ig in DMEM / HAM F12 Was mixed with 20 μl of bead / cell suspension containing. After incubation at 37 ° C. for 2 to 3 hours, the aggregates of cells and beads were found and examined for the aggregation reaction under a microscope.
    Immunoprecipitation and Western Blotting
    After 10-14 days of restimulation, T-cells were washed and cell surface proteins were labeled with biotin. Briefly, T-cells were incubated with 0.5 mg / ml ImmunoPure® sulfo-NHS-biotin in PBS at a concentration of 5 x 10 6 cells / ml for 30 minutes at room temperature. The cells were then washed again and lysed as described above. Cell lysates were immediately precleaned by incubating with SAM-paramagnetic beads prior to use in immunoprecipitation experiments. Immunoprecipitation was performed by incubating the precleaned lysates of 10 7 cell equivalents with 15 μl of SAM-paramagnetic beads preloaded with MAb (1 ml hybridoma supernatant) for 3-4 hours. The immunoprecipitate was then washed three times with PBS and heated in sample buffer and then subjected to SDS-PAGE on a 10% gel under reducing or non-reducing conditions (Remley method (Ref. 5) and Mini-Protean II system ( Biorad)). Immunoblotting was performed according to Tobin et al. (Ref. 6) using PVDF membranes. Nonspecific binding sites were blocked by incubation of the membrane with 5% skim milk in PBS supplemented with 0.5% Tween 20. After washing this, the blots were probed for 1 hour at room temperature with streptavidin-alkaline phosphatase in PBS, 0.5% Tween 20, 1% BSA, 1% normal goat serum. Finally, blots were developed using BCIP and NBT as chromophore substrates. In Western blot studies, TCR / CD3 complexes as described above were loaded onto paramagnetic SAM-beads using OKT3 and normal T-cell lysates as immunoprecipitating antibodies. After SDS-PAGE on 10% PAA-gel under reducing or non-reducing conditions, the blots were incubated with 2.5 ml of hybridoma supernatant. Binding antibodies were detected using alkaline phosphatase-bound goat anti-mouse Ig and BCIP / NBT as described above.
    Antibody-Mediated T-Cell Proliferation
    Flat bottom microtiter wells were coated with 100 μl of anti-mouse Ig in PBS at a concentration of 40 μg / ml (overnight, room temperature). The wells were immediately washed and then incubated for 2 hours with 100 μl of MAb contained at 2 μg / ml in PBS. Excess free MAb was washed off and 2.5 × 10 4 resting T-cells were added in 200 μl of DMEM / HAM F12 supplemented with 10% NHS. After incubating for 2 days at 37 ° C., the cells were pulsed with 0.5 μCi [ 3 H] -thymidine and further incubated for 16-18 hours. Finally, cells were harvested on a glass fiber filter and [ 3 H] -thymidine infusions were determined by beta counting on Matrix 96 ™ (packcard). Each variable was tested in four replicate experiments.
    Antibody-mediated Inhibition of Antigen-Induced Proliferation
    Into the microtiter wells of the flat bottom, 20 μl hybridoma supernatant contained in 150 μl of DMEM / HAM F12 supplemented with 10% NHS, 2 × 10 4 resting T-cells and 1 × 10 5 tissue affinity MNC It was. After 3 hours of incubation at 37 ° C., 50 μl of peptide RSFTLASSETGVG (16 μg / ml) was added to the culture. This culture was incubated for another 2 days at 37 ° C. and the [ 3 H] -thymidine infusion was measured as described above. Each variable was tested in four replicate experiments.
    Anergy Induction by Clonal-specific MAbs
    24 well culture plates were coated (1 night, room temperature) with 1 ml of anti-mouse Ig dissolved in 40 μg / ml in PBS. The wells were immediately washed and then incubated for 2 hours with 1 ml of MAb contained in PBS at a concentration of 2 μg / ml. Excess free MAb was washed off and 2 × 10 6 resting T-cells thus washed were added in 2 ml of DMEM / HAM F12, 10% NHS. Then, cyclosporin A (Sandos, Basel, Switzerland), rIL-2, cycloheximide (Sigma, St. Louis, MO) or HLA-DRB1 * 0401 combination APC (300 rad irradiation) Is added at the beginning of incubation where indicated. After incubation overnight the culture plates were cooled on ice and T cells resuspended by pipetting. These cells were washed once with complete culture and used for T-cell proliferation assay, cytokine assay and FACS assay as described below.
    Antigen-specific proliferative responses of T cells were 200 μl of DMEM / HAM F12, 2 × 10 4 T cells contained in 10% NHS, 1 × 10 5 HLA-DRB1 * 0401 combination, irradiated (3000 rad) PBMC And flat bottomed microwell cultures containing various concentrations of antigen. After incubation at 37 ° C. for 2 days, the cells were pulsed with 0.5 μCi of [ 3 H] -thymidine and incubated for an additional 16-18 hours. Finally, cells were harvested on glass fiber filters and [ 3 H] -thymidine bottling was measured by gas scintillation on Matrix 96 (Packard, Connecticut, Conn.). Each variable was tested in three replicates.
    result
    Immunization
    Immunization with Adjuvant (Control Experiment) Produces Any Serum Containing Antibodies That Can Distinguish Between H.243 and Unrelated T-Cells (Table 1; Immunization Groups II, III, and IV) Did not do it. Serum titers in group V were low (1: 100 in aggregation analysis), while serum titers in group I and II were very high (1: 1200).
    Generation of anti-clone MAbs
    Incubation with irrelevant T-cell clones, here, H.258-derived TCR / CD3 complexes using splenocytes from group I, resulted in a lot of rosette-like cells loaded with paramagnetic beads. And removal of TCRαβ-reactive B-cells is successful. When the non-roset-type cells were subjected to secondary precleaning with irrelevant TCR / CD3-complexes derived from the same T-cell clones, little rosette-type cells were observed.
    Microscopic examination of the desired T-cell clones and paramagnetic beads showed no visible rosette formation. This is not surprising if certain B-cells of the expected frequency have been provided. After limiting dilution and clone propagation, 36 B-cell supernatants were observed to aggregate H.243 but not to irrelevant T-cell clone H.258 (Table 2). Some B-cell supernatants can aggregate both clones, thus eliminating the need for precleaning procedures.
    In addition, it can be seen from Table 2 that most of the specific B-cell cultures are not clonal when B-cells proliferate and do not react with any T-cells. However, this is not a problem since the selection of the hybridomas of the nonspecific B-cells takes place after micro-electrofusion. Eighteen of the specific B-cell cultures were treated by micro-electrofusion. Some fusions did not produce T-cell specific hybridomas and some other fusions could not be cloned into stable cell lines. As a result, stable hybridomas were obtained in 11 of 18 micro-electrofusions. These 11 MAbs were further characterized. The isotypes of these MAbs are shown in Table 3.
    Specificity of MAb
    To further study the specificity of MAbs, Western blots as well as immunoprecipitation and T-cell aggregation tests were performed.
    SDS / PAGE under non-reducing and denaturing conditions showed that all MAbs were able to immunoprecipitate approximately 85 kD major band and 21 kD minor band from the digittonin lysate of the T-cell clone H.243. These results were consistent with the molecular weights of the TCRαβ-complex and CD3 chain ε / δ, respectively. Under reducing conditions, the 85 kD band dissociated into two bands, 40 kD and 50 kD, which matched the molecular weights of the TCR α and β chains, respectively. An immunoblot with one of the anti-H.243 specific MAbs is shown in FIG. 1, using OKT3 as a positive control and an irrelevant MAb as a negative control.
    Table 3 shows the cross reactivity of the MAb with six Vβ9 positive T-cell clones, two Vα8 positive T-cell clones, and eight other T-cell clones with different Vα and Vβ. Antibody TCR 74 should be considered to have Vβ9 specificity such as to aggregate all Vβ9 positive T-cells but not the next cells. Other TCR antibodies only react with H.243, which is therefore likely to be directed against the antigen-binding portion of H.243, and thus the constant regions of antigens Vα8, Vβ9, TCRαβ, CD3 and other common T- It does not react with cell surface molecules. FIG. 2 shows that antibodies from hybridoma clones TCR64, TCR66, TCR69, TCR70, TCR73 and TCR 76 MAb can stain TCR (85 kD band) on non-reducing western blots. The nonspecific heavy chain band at the top in this figure is developed from the immunoprecipitating antibodies SAM and OKT3. Western blots run under reducing conditions were not stained with anticlonal MAb at all, thus suggesting that the MAb recognizes the coordination epitopes originally formed by the TCRαβ complex.
    MAb-induced T-cell proliferative
    To investigate whether MAb can induce proliferation of H.243 T-cells, MAb was immobilized on microtiter plates in the flat bottom and incubated with T-cells. 2 shows that there is no significant difference between MAbs, but all of them can induce proliferation of H.243 cells. Proliferation similar to anti-CD3 (OKT3) was also obtained with TCR 64, TCR 66, TCR 70, TCR 76 and TCR 79. No proliferation occurred with TCR 44, an anti-clone MAb of other TCRs.
    MAb-mediated Inhibition of Antigen-Induced Proliferation by T-cells
    In addition, it was investigated whether a panel of MAbs could inhibit antigen-induced proliferation of H.243 T-cells. In this experiment, MAb was pre-incubated with T-cells and APC and pulsed with peptides of various concentrations after 3 hours. 4 shows that TCR 64, TCR 70, TCR 76, TCR 78 and TCR 83 strongly inhibit antigen-induced proliferation. Up to 98% inhibition was obtained with TCR 83. Dose response curves show that maximum inhibition can be achieved with small amounts of TCR 64, TCR 70 and TCR 83 by 100 ng / ml. TCR 78 had a titer of approximately 10 times less. The inhibition pattern is irrelevant whether peptides of suboptimal or saturated concentrations are added (results not shown). Anti-clone MAbs directed against other TCRs (TCR 44) did not show any inhibition.
    Nonresponsive induction of human T-cell clone H.243 upon continuous stimulation with antigen and APC by anti-clonal MAb
    Occupation of T-cell receptors without costimulation is known to induce anergi. As previously described, the inventors have discovered that anti-clonal MAbs immobilized against H.243 can functionally stimulate the TCR of this clone. Therefore, it has been of interest whether the same antibody can induce anergy. For this purpose, ten different anti-clone MAbs were fixed in 24 well plates and incubated with H.243 T cells overnight to give anergic stimulation. Next, the T cells were separated from the plate and tested to see if they could respond to increasing concentrations of HC gp-39 262-277 (SEQ ID NO: 3) represented by irradiated DRB1 * 0401-combined APC. It was. FIG. 5 shows that this response completely disappeared in 8 out of 10 MAbs, whereas control MAb 1 and H.243 cells incubated with still respond well to peptides expressed by APC. The response of T cells incubated with TCR 69 and TCR 83 was significantly reduced but did not disappear completely at higher peptide concentrations.
    Inhibition of response of nonergic cells by anergic cells
    We also studied whether anergic H.243 cells could inhibit the response of nonergic H.243 cells. Anergi was induced by TCR 76 and nonergic cells were obtained by incubation with control MAb 1. Subsequently, several T cell populations, namely 2 × 10 4 anergic T cells / well, 2 × 10 4 nonergic T cells / well, or anergic T cells at reduced concentrations (4 × 10 4 , 2 × 10) Proliferation assay was performed for peptide HC gp-39 261-275 (SEQ ID NO: 2) using a mixture / well of 4 , 1 × 10 4 , and 0.5 × 10 4 ) and 2 × 10 4 nonergic T cells. . The response of nonergic cells was markedly reduced in a dose related manner when anergic cells were added (FIG. 6). A reduction in proliferation of about 90% was obtained using an antigen concentration of 1 μg / ml and the ratio of anergic to nonnergic cells 2: 1. As the antigen concentration was further increased, the rate of proliferation decreased (72% at 5 μg / ml and 39% at 25 μg / ml).
    Thus, this example shows that anti-clonal monoclonal antibodies against human T-cell receptors can be produced. This experiment produced 10 hybridomas, 4 of which were deposited in ECACC (Salbury, UK) as described in Table 3. This method does not require many T-cells or large amounts of purified TCR. The method according to the present invention is a preferred alternative to an unnecessarily long recombination procedure that requires the production of soluble TCRs of TCRs expressed on syngeneic mouse cells for use in immunization procedures. Thus, it is possible to overcome possible problems associated with coordination stability. In tests for antibody-induced T-cell proliferation and tests for antibody mediated inhibition of antigen-induced T-cell proliferation (up to 98% inhibition was observed), the monoclonal antibodies showed different responsiveness. This suggests that several epitopes recognize the same clonal structure.
    All anti-clonal monoclonal antibodies could induce proliferation of H.243 T-cells if crosslinked. In autoreactive clones it has been demonstrated that small amounts of immobilized anti-clonal MAb can induce anergi. After anergy stimulation, T cells did not proliferate due to lack of IL-2 gene transcription upon restimulation. It was also observed that IFNg production was also reduced.
    FACS analysis of anergi cells showed that anergi did not result in TCR downregulation or the absence of free TCR. In coculture experiments, anergic T cells were found to inhibit the response response of reactive cells from the same clone. This bystander inhibition resulted in 90% inhibition of growth. This data demonstrates the anergic potential of in vitro cloned-specific antibodies. Thus, such MAbs can be used to induce and maintain antigen-specific T-cell resistance in rheumatoid arthritis.
    Mouse serum was immunized by injecting a somewhat pure TCR / CD3 immune complex bound to paramagnetic beads (Table 1; immunization group IV). This mouse serum was tested to be more positive for specific T-cells used for immunization than unrelated T-cells. However, it was not possible to obtain hybridomas that produce anti-clonal monoclonal antibodies. Although no specificity could be demonstrated for the target T-cell versus unrelated T-cells in the sera of mice immunized with a small amount of total T-cells, the method according to the present invention is directed to anti-clonal monoclonal antibodies. Obtain hybridomas that can be produced.
    Thus, it goes without saying that antigen binding fragments can be prepared using the monoclonal antibodies according to the invention, such as papain. In addition, the monoclonal antibody or antigen-binding fragment thereof can be labeled to facilitate its detection, all of which are within the scope of the present invention.
    It is also natural that unrelated cells used to remove nonspecific B-cells are preferably very similar to cell surface antigen-carrying cells.
    Immunization Schemes and Serum Characteristics Immunization groupantigenSupplementsRoute of administrationCohesive / FACSProliferation inductionAg- induced proliferative # Western Blot T cells 1 * (immunized)T cell 2 * (unrelated) IT cellradiship (5x)++++++-++- IIT cells; TCR / CD3Uip; im; im; ip++++++-++- IIITCR / CD3Uip; im; im; im; ip--+/--- IVTCR / CD3Usc; sc; sc; ip--+-- VTCR / CD3radishis (5x)++/-+--Normal mouse serum----- Two BALB / C mouse groups were immunized by administration of TCR / CD3 complexes bound to total T cells or paramagnetic beads. Immunization was performed in the presence or absence of adjuvant as indicated. Immunization was carried out in several ways. Ip = intraperitoneal; im = intramuscular; sc = transdermal; is = serum from spleen mice was characterized by binding, FACS-staining and Western blot in T-cell aggregation assays. Functional characterization was performed by antibody-mediated T-cell proliferation assay and antibody-mediated inhibition of antigen-induced T-cell proliferation assay. * T 1 = T- cell-cell clones used for immunization; T cells, 2 = no associated T- cell clones. # + Means T-cell proliferation inhibition.
    Overgrowth of anti-T-cell specific B cells in EL-4 B-cell culture Inoculum well count dSmear densityB-cell overproliferated wellsCohesion with H.243Cohesion with H.258 384x3846840 1921/6 x171137 1921/36 x813One 28 x 10 6 lymphocytes were used in the immunobead selection procedure for selecting anti-clone specific B cells. Selected B cells were propagated using different smear densities in an EL-4 B-cell culture system and antibody cohesion with target T cells (H.243) and unrelated T cells (H.258) at 8 days using supernatant. Was tested against. x: undetermined number of selected cells
    Cohesion of Anti-TCR mAbs with Different Types of T Cells 6 mAbIsomorphismT-cell cohesion H.243 1) Six Vβ9 Positive T Cells2 Vα8 positive T cells8 different T cells TCR 64IgG2a+--- TCR 66IgG1+--- TCR 67n.d.+--- TCR 69IgG1+--- TCR 70IgG2a+--- TCR 72IgM+--- TCR 73IgG1+--- TCR 74n.d.++-- TCR 76IgG2a+--- TCR 78IgM+--- TCR 79IgG1+--- TCR 83IgM+---
    The method according to the present invention is a preferred alternative to an unnecessarily long recombination procedure that requires the production of soluble TCRs of TCRs expressed on syngeneic mouse cells for use in immunization procedures. Thus, it is possible to overcome possible problems associated with coordination stability.
    Reference
    1. Moreta, A., et al. (1985) Screening and characterization of monoclonal antibodies against genotype-like structures of interleukin-2-producing human leukemia T-cell lines, Int. J. Cancer 36: p. 253-259.
    2. Wetgen, H. Et al. (1986) T3-like protein complexes bound to antigen receptors for rat T cells, Nature 320: p. 272-275.
    3. Steinbeckers, P.A. Et al. (1994) Effective generation of monoclonal antibodies from certain antigen-specific B cells. Molecular Biology Reports 19, p. 125-134.
    4. Steinbeckers, P.A. Et al. (1992) New attempts at producing human and rat antibody-producing hybridomas. J. Immunol. Methods 152, p. 69-77.
    5. Ramley, U.K. (1970) Cleavage of structural proteins during assembly of the bacteriophage T4 head. Nature 227: p. 680-685.
    6. Tobin, H. Et al. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some uses, Proc. Natl. Acad. Sci. USA 76: p. 4350-4354.
    Abbreviation
    PBS Phosphate Buffered Saline
    BCIP 5-Bromo-4-chloro-3-indolylphosphate p-toluidine salt
    NBT p-nitro blue tetrazolium chloride
    BSA Bovine Serum Albumin
    FCS fetal calf serum
    SAM positive anti mouse
    TSN T-Cell Supernatant
    MAb monoclonal antibodies (antibodies)
    Manufacturer
    Biorad, Richmond, CA, USA
    Gibco, paisley, scotland
    Hyclone, Logan, USA
    Packard, Maryland, Connecticut, USA
    Sequence Listing
    (1) General Information:
    (i) Applicant:
    (A) Name: Akzo Nobel N. V.
    (b) Street: Belperberg 76
    (C) City: Arnhem
    (E) Country: Netherlands
    (F) ZIP Code: 6824
    (G) Phone: 0412 666379
    (H) Fax number: 0412 650592
    (ii) Name of the Invention: Monoclonal Antibodies, Methods for Making the Same, and Pharmaceutical Compositions and Diagnostic Reagents Containing the Same
    (iii) number of sequences: 3
    (v) computer readable form:
    (A) Media type: floppy disk
    (B) Computer: IBM PC compatible model
    (C) operating system: PC-DOS / MS-DOS
    (D) Software: Patented Release # 1.0, Version # 1.30 (EPO)
    (2) information for SEQ ID NO:
    (i) sequence properties:
    (A) Length: 13 amino acids
    (B) Type: Amino Acid
    (C) Number of chains: 1 chain
    (D) mode: linear
    (ii) Type of sequence: protein
    (v) Fragment: Middle Fragment
    (xi) SEQ ID NO 1:
    (2) Information about SEQ ID NO:
    (i) sequence properties:
    (A) Length: 15 amino acids
    (B) Type: Amino Acid
    (C) Number of chains: 1 chain
    (D) mode: linear
    (ii) Type of sequence: protein
    (v) Fragment: Middle Fragment
    (xi) SEQ ID NO: 2:
    (2) Information about SEQ ID NO:
    (i) sequence properties:
    (A) Length: 16 amino acids
    (B) Type: Amino Acid
    (C) Number of chains: 1 chain
    (D) mode: linear
    (ii) Type of sequence: protein
    (v) Fragment: Middle Fragment
    (ix) sequence characteristics:
    (xi) SEQ ID NO: 3:
    权利要求:
    Claims (41)
    [1" claim-type="Currently amended] 1) injecting a mammalian cell surface antigen-containing material selected from the group consisting of i) whole cells and ii) membrane fractions obtained by treating the whole cells;
    2) separating the B-cell containing cell fraction from the spleen of the mammal;
    3) in the cell fraction obtained in step 2), contacting with a carrier-binding material of a cell associated with the whole cell lacking a cell surface antigen to accumulate specific B-cells on the cell surface, the next step thus integrated Separating the B-cells bound to the carrier-binding material of the relevant cells from the unbound B-cell-containing cell fraction to be used in the method;
    4) limit diluting the cloned B-cell-containing cell fraction obtained in the above step and then propagating the clone;
    5) selecting B-cell clones and infinitely multiplying the selected B-cell clones using a small amount of fusion techniques; And
    6) screening and cloning the hybridomas capable of producing antibodies specifically binding to the cell surface antigen, and then separating the monoclonal antibody-containing fractions obtained from the supernatants of the hybridomas Method for producing monoclonal antibodies against cell surface antigens.
    [2" claim-type="Currently amended] The production method according to claim 1, wherein the mammal into which the surface antigen is injected is a different species from the mammalian species from which the surface antigen is obtained.
    [3" claim-type="Currently amended] The method according to claim 1, wherein the surface antigen has a constant portion and a variable portion, and at least a portion of the variable portion represents the specificity determining portion of the surface antigen.
    [4" claim-type="Currently amended] The method according to claim 2, wherein the surface antigen has a constant portion and a variable portion, and at least a portion of the variable portion represents the specificity determining portion of the surface antigen.
    [5" claim-type="Currently amended] 4. A process according to claim 3, wherein the receptor molecule-containing material is used as the surface antigen-containing material.
    [6" claim-type="Currently amended] The method of claim 4, wherein the receptor molecule-containing material is used as the surface antigen-containing material.
    [7" claim-type="Currently amended] The method of claim 5, wherein the T-cell clone is used to prepare a receptor molecule-containing material.
    [8" claim-type="Currently amended] The method of claim 6, wherein the T-cell clone is used to prepare a receptor molecule-containing material.
    [9" claim-type="Currently amended] The production method according to any one of claims 1 to 8, wherein the membrane fraction obtained in step 1) is obtained by mechanical treatment of whole cells.
    [10" claim-type="Currently amended] The method of any one of claims 1 to 8, wherein the cell surface antigen-containing material is injected into the mammal without an adjuvant.
    [11" claim-type="Currently amended] The method of claim 9, wherein the cell surface antigen-containing material is injected into the mammal without an adjuvant.
    [12" claim-type="Currently amended] 12. The cell fraction according to any one of claims 1 to 8 and 11, in order to further integrate the integrated B-cell-containing cell fraction of step 3) used in step 4), j) Whole cells, jj) contact with a carrier-binding cell surface antigen containing a material selected from the population consisting of membrane fractions obtained from whole cells, and jjj) essentially purified cell surface antigens, followed by binding to this carrier-binding material By separating unB-cells from B-cells bound to the carrier-binding material, the B-cells bound to the carrier-binding material will contain the more integrated cell fraction used in step 4). Manufacturing method.
    [13" claim-type="Currently amended] 10. The membrane fraction of claim 9, wherein the cell fraction is obtained from j) whole cells, jj) whole cells, in order to further integrate the integrated B-cell-containing cell fraction of step 3) used in step 4). And jjj) contacting with a carrier-binding cell surface antigen containing a substance selected from the group consisting essentially of purified cell surface antigens, followed by binding B-cells not bound to the carrier-binding material to the carrier-binding material Separation from the B-cells, wherein the B-cells bound to the carrier-binding material contain the more integrated cell fraction used in step 4).
    [14" claim-type="Currently amended] The membrane fraction obtained according to claim 10, in order to further integrate the integrated B-cell-containing cell fraction of step 3) used in step 4), the cell fraction obtained from j) whole cells, jj) whole cells, And jjj) contacting with a carrier-binding cell surface antigen containing a substance selected from the group consisting essentially of purified cell surface antigens, followed by binding B-cells not bound to the carrier-binding material to the carrier-binding material Separation from the B-cells, wherein the B-cells bound to the carrier-binding material contain the more integrated cell fraction used in step 4).
    [15" claim-type="Currently amended] The manufacturing method according to any one of claims 1 to 8, 11, 13, and 14, wherein paramagnetic beads are used as carriers.
    [16" claim-type="Currently amended] The manufacturing method according to claim 9, wherein paramagnetic beads are used as carriers.
    [17" claim-type="Currently amended] The manufacturing method according to claim 10, wherein paramagnetic beads are used as carriers.
    [18" claim-type="Currently amended] The manufacturing method according to claim 12, wherein paramagnetic beads are used as carriers.
    [19" claim-type="Currently amended] The method according to any one of claims 1 to 8, 11, 13, 14, 16 to 18, wherein the selection in one or more of steps 5) and 6) is performed by B. Agglutination assay consisting of contacting the supernatant of the cell clone with a cell surface antigen-containing whole cell and a carrier coated with an antibody capable of binding to the antibodies of the immunized mammalian species used in step 1) and then detecting the aggregation reaction. Process for producing, characterized in that carried out as.
    [20" claim-type="Currently amended] The method of claim 9, wherein the selection in one or more of steps 5) and 6) binds the supernatant of the B-cell clone to the cell surface antigen-containing whole cells and the antibodies of the immunized mammalian species used in step 1). And contacting the carrier coated with an antibody which is capable of contacting, followed by an aggregation assay consisting of detecting the aggregation reaction.
    [21" claim-type="Currently amended] The method of claim 10, wherein the selection in one or more of steps 5) and 6) binds the supernatant of the B-cell clone to the cell surface antigen-containing whole cells and the antibodies of the immunized mammalian species used in step 1). And contacting the carrier coated with an antibody which is capable of contacting, followed by an aggregation assay consisting of detecting the aggregation reaction.
    [22" claim-type="Currently amended] The method of claim 12, wherein the selection in one or more of steps 5) and 6) binds the supernatant of the B-cell clone to the cell surface antigen-containing whole cells and the antibodies of the immunized mammalian species used in step 1). And contacting the carrier coated with an antibody which is capable of contacting, followed by an aggregation assay consisting of detecting the aggregation reaction.
    [23" claim-type="Currently amended] The method of claim 15, wherein the selection in one or more of steps 5) and 6) binds the supernatant of the B-cell clone to the cell surface antigen-containing whole cells and the antibodies of the immunized mammalian species used in step 1). And contacting the carrier coated with an antibody which is capable of contacting, followed by an aggregation assay consisting of detecting the aggregation reaction.
    [24" claim-type="Currently amended] 20. The method of claim 19, wherein all cells that are free of cell surface antigens but are related to whole cells are used as controls.
    [25" claim-type="Currently amended] 24. The method according to any one of claims 20 to 23, wherein whole cells which do not have cell surface antigens but which are related to whole cells are used as a control.
    [26" claim-type="Currently amended] The method according to any one of claims 1 to 8, 11, 13, 14, 16 to 18, and 20 to 24, B- selected in step 5). The cell clone is mixed with myeloma cells and then subjected to micro-electrofusion method.
    [27" claim-type="Currently amended] The method according to claim 9, wherein the B-cell clone selected in step 5) is mixed with myeloma cells and then treated by micro-electrofusion method.
    [28" claim-type="Currently amended] The method according to claim 10, wherein the B-cell clone selected in step 5) is mixed with myeloma cells and then treated by micro-electrofusion method.
    [29" claim-type="Currently amended] The method according to claim 12, wherein the B-cell clone selected in step 5) is mixed with myeloma cells and then treated by micro-electrofusion method.
    [30" claim-type="Currently amended] The method according to claim 15, wherein the B-cell clone selected in step 5) is mixed with myeloma cells and then treated by micro-electrofusion method.
    [31" claim-type="Currently amended] The method according to claim 19, wherein the B-cell clone selected in step 5) is mixed with myeloma cells and then treated by micro-electrofusion method.
    [32" claim-type="Currently amended] The method according to claim 25, wherein the B-cell clone selected in step 5) is mixed with myeloma cells and treated by micro-electrofusion method.
    [33" claim-type="Currently amended] Monoclonal antibodies that are reactive with the clonal structure of the T-cell receptor.
    [34" claim-type="Currently amended] 34. The monoclonal antibody of claim 33, wherein the T-cell receptor is a T-cell receptor associated with autoimmune disease.
    [35" claim-type="Currently amended] 35. The monoclonal antibody according to claim 34, wherein the autoimmune disease is rheumatoid arthritis.
    [36" claim-type="Currently amended] 36. The monoclonal antibody of claim 35, wherein the monoclonal antibody is reactive with the T-cell receptor of the HC gp-39 reactive T-cell clone.
    [37" claim-type="Currently amended] The monoclonal antibody according to claim 36, wherein the T-cell clone is H.243 (ECACC Accession No. 96103122).
    [38" claim-type="Currently amended] 38. The hybridoma of claim 37, wherein the hybridoma is selected from the group consisting of TCR 69 (ECACC Accession No. 96103118), TCR 70 (ECACC Accession No. 96103119), TCR 72 (ECACC Accession No. 96103120), and TCR 83 (ECACC Accession No. 96103121). Monoclonal antibody, characterized in that produced by.
    [39" claim-type="Currently amended] A pharmaceutical composition suitable for the treatment of rheumatoid arthritis, comprising a monoclonal antibody prepared as described in any one of claims 1 to 32 and a suitable excipient.
    [40" claim-type="Currently amended] A pharmaceutical composition suitable for the treatment of rheumatoid arthritis, comprising a suitable excipient together with the monoclonal antibody according to any one of claims 33 to 38.
    [41" claim-type="Currently amended] A monoclonal antibody selected from the group consisting of a monoclonal antibody prepared as described in any one of claims 1 to 32 and a monoclonal antibody according to any one of claims 33 to 38. Diagnostic reagents.
    类似技术:
    公开号 | 公开日 | 专利标题
    Di Zenzo et al.2012|Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface
    US8183014B2|2012-05-22|High affinity antibodies to human IL-6 receptor
    TWI660972B|2019-06-01|Anti-mcam antibodies and associated methods of use
    US9458234B2|2016-10-04|TLR2 antagonistic antibody and use thereof
    RU2599450C2|2016-10-10|Ilt17 antibody
    Rao et al.1987|Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE.
    EP0068763B1|1987-04-08|Recombinant monoclonal antibodies
    Unkeless1979|Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.
    EP2342228B1|2017-09-06|Pd-1 specific antibodies and uses thereof
    JP2648419B2|1997-08-27|Monoclonal antibody mixture
    Staerz et al.1985|Characterization of a murine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor.
    ES2211884T3|2004-07-16|Antibodies against cd40.
    ES2263204T3|2006-12-01|New protein and process to produce the same.
    JP5665702B2|2015-02-04|Methods and compositions for increasing the effectiveness of therapeutic antibodies using NK cell enhancing compounds
    US5223426A|1993-06-29|Monoclonal antibodies reactive with defined regions of the t-cell antigen receptor
    EP0018795B2|1999-04-07|Monoclonal-antibody-producing hybrid cell line, antibody and method of preparing it, therapeutic composition containing it and its diagnostic and therapeutic uses
    KR100214759B1|1999-08-02|Antibody to human interleukin-6 receptor
    Preud’Homme et al.1972|Surface bound immunoglobulins as a cell marker in human lymphoproliferative diseases
    EP0018794B1|1985-07-24|Monoclonal antibody to human helper t cells, method of preparing it, hybrid cell line producing it, its diagnostic and therapeutic uses and diagnostic and therapeutic compositions comprising it
    US5436319A|1995-07-25|Cell free T cell antigen receptor beta chain and its clinical utilities
    CA2530272C|2018-05-01|Antibodies that cross-react with two or more kir2dl nk cell receptors and potentiate nk cell cytotoxicity
    KR0151408B1|1998-08-17|In-vitro method for producing antigen specific human monoclonal antibodies
    WO2016087531A1|2016-06-09|Bispecific antibodies against cd3epsilon and bcma for use in treatment of diseases
    Bankhurst et al.1971|Surface immunoglobulins on thymus and thymus-derived lymphoid cells
    US7265212B2|2007-09-04|Anti-CD45RB antibodies
    同族专利:
    公开号 | 公开日
    HU9702359A3|2001-11-28|
    ES2262170T3|2006-11-16|
    AU733165B2|2001-05-10|
    DK0856520T3|2006-07-31|
    DE69735621D1|2006-05-18|
    BR9706236A|1999-05-04|
    MX9709446A|1998-07-31|
    EP0856520B1|2006-04-05|
    AU4690897A|1998-06-11|
    PT856520E|2006-08-31|
    CA2221682A1|1998-06-06|
    CA2221682C|2011-10-18|
    KR100543053B1|2006-06-13|
    US6020170A|2000-02-01|
    NO975714D0|1997-12-05|
    US6392020B1|2002-05-21|
    IL122233D0|1998-04-05|
    HU9702359A2|1998-09-28|
    PL193821B1|2007-03-30|
    IL122233A|2001-04-30|
    US20020143150A1|2002-10-03|
    PL323572A1|1998-06-08|
    NO975714L|1998-06-08|
    NZ329314A|1999-02-25|
    JPH10179160A|1998-07-07|
    AT322507T|2006-04-15|
    NO327047B1|2009-04-14|
    DE69735621T2|2006-08-24|
    EP0856520A1|1998-08-05|
    HU9702359D0|1998-03-02|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1996-12-06|Priority to EP96203465
    1996-12-06|Priority to EP96203465.8
    1997-06-27|Priority to EP97201972.3
    1997-06-27|Priority to EP97201972
    1997-12-05|Application filed by 에프.지.엠.헤르만스, 악조노벨엔.브이.
    1998-10-07|Publication of KR19980063909A
    2006-06-13|Application granted
    2006-06-13|Publication of KR100543053B1
    优先权:
    申请号 | 申请日 | 专利标题
    EP96203465|1996-12-06|
    EP96203465.8|1996-12-06|
    EP97201972.3|1997-06-27|
    EP97201972|1997-06-27|
    [返回顶部]