Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to human glycosylation inhibiting factor (GIF) specific for an antigen and which can be used to suppress the human immune response to the antigen.
• GEF human glycosylation inhibiting factor
• the immune response is often seen as beneficial, in certain circum- stances the immune response to an antigen can actually be harmful to the animal in which the immune response occurs.
• An example where the immune response creates a condition wherein the host is subject to serious pathologic sequelae is in such autoimmune diseases as lupus erythemato- sus.
• lupus erythematosus antibodies are often present which react with determinants in the thyroid, erythrocytes, DNA, and platelets of the host.
• IgE antibodies against allergens cause hay fever, and are involved in the other allergic diseases such as extrinsic asthma.
• the crucial role of IgE antibodies in the allergic diseases raised the possibility that the regulation and suppression of the IgE antibody formation against allergens would be one of the fundamental treatments for allergic diseases.
• IgE antibodies against the allergens are always detected.
• the IgE antibody titer goes up after the pollen season, and then declines only slightly during the rest of the year. Since the half life of IgE in the serum is only 2 to 3 days, the persistence of the IgE antibody titer indicates that the antibodies are being synthesized continuously by the lymphoid cells of the patients in spite of the lack of allergen exposure.
• IgE antibody response in experimental animals was improved.
• One of the approaches was to improve classical immunotherapy or desensitization treatment, in which allergic patients receive repeated injections of a minute dose of allergen. It was shown that the desensitization treatment can improve clinical symptoms in some patients.
• the IgE antibody titer in the serum of hay fever patients did not decline after the treatment.
• the major immunological effects of the treatment is an enhancement of the IgG antibody formation, and the suppression of an increase in the IgE antibody titer after the pollen season.
• a limitation in the desensitization, or immunosuppression treatment is that patients cannot tolerate a large dose of allergen because of side effects.
• a chemically modified allergen such as urea-denatured antigen or polyethylene glycol (PEG) -conjugates of the antigen for the treatment. Since the modified antigens do not bind to antibodies against the native antigen, relatively large doses of the modified antigen can be injected without causing allergic symptoms. However, the modified antigen can stimulate antigen-specific T- cells. Evidence was obtained that intravenous injections of the modified antigen into mice resulted in the generation of antigen-specific suppressor T-cells which suppressed the primary IgE antibody response to the native antigen.
• IgE synthesis is selectively regulated in an immunoglobulin isotype-specific manner.
• two types of T-cell factors have been found which have affinity for IgE and selectively regulate IgE synthesis.
• IgE-BF IgE-binding factors
• IgE-BF IgE-binding factor
• the major difference between the IgE-potentiating factors and IgE-suppressive factors appears to be carbohydrate moieties in the molecules.
• IgE- potentiating factors bind to lentil lectin and concanavalin A, while IgE- suppressive factors fail to bind to these lectins (Yodoi, et al., J. Immunol., 128: 289, 1982).
• IgE-potentiating factors or IgE-suppressive factors as well as gene cloning of the factors, indicated that the IgE-potentiating factor and IgE-suppressive factor share a common structural gene and that the nature of the carbohydrate moieties and biologic activities of the factors are established during the post-translational glycosylation process (Martens, et al, Proc. Nat'l Acad.
• glycosylation inhibiting factor GEF
• GEF glycosylation inhibiting factor
• GIF GIF-specific suppressor T-cells
• HVG host versus graft
• GVH graft versus host rejection
• An improved way to suppress an immune response to an antigen in a human would be to administer an immunosuppressively effective amount of human GIF which can specifically bind to the antigen. In so doing, the concentration of T suppressor factor is favored and, as a result, the immune response to the antigen is decreased.
• the present invention provides a means for accomplishing this result.
• the inventor has substantially purified human antigen-specific GIF which suppresses the immune response to the homologus antigen.
• the human antigen-specific GIF if desired, can be used therapeutically to suppress the human immune response to the antigen.
• the present invention relates to substantially pure human antigen-specific GIF with specificity for an antigen associated with an undesirable immune response.
• This human antigen-specific GIF is highly useful for the immunosuppression of the undesirable immune response in an antigen- specific manner.
• Preferred in the present invention are human antigen-specific GIFs which can specifically bind allergens.
• a human antigen-specific GIF is disclosed which binds to an epitope on phospholipase A ⁇ PLA ⁇ , the major allergen in honey bee venom.
• GIFs with the same specificity, to be used to suppress the human immune response to PLA ⁇
• a human antigen-specific GIF which binds to an epitope on Japanese cedar pollen which can be used to suppress the human immune response to this antigen.
• GIF with antigen-specificity to P rV can be readily extended to other antigens by those of skill in the art to prepare and purify other GIF molecules with antigenic specificity for those other antigens without undue experimentation.
• the broad pioneering nature of the invention enables the preparation of human antigen-specific GIFs for other allergens which can be used to suppress such immune response mediated disorders as autoimmune disease and allergy.
• the production of various human antigen-specific GIFs is especially facilitated where the antigen of the undesirable immune response is known such as with most allergies and various autoimmune diseases.
• the human PU ⁇ -specific GIF of the invention is obtained from, or has the identifying characteristics of, an antigen-specific GIF obtained from the cell line AC5 having ATCC accession number HB 10473.
• the human Japanese cedar pollen-specific GIF of the invention is obtained from, or has the identifying characteristics of, an antigen-specific GIF obtained from the cell line 31 E9.
• PBMC peripheral blood mononuclear cells
• the invention is directed to a method of producing a continuous hybridoma cell line which produces human antigen-specific GIF comprising:
• the antigen-primed T-cells can be obtained from any sample, including the mononuclear cell fraction of peripheral blood.
• the antigen-primed T-cells can then be activated by culturing in the presence of the antigen to which they have been primed, followed by expanding the activated T-cells in the presence of interieukin-2 (IL-2) and a phospholipase inhibitor.
• IL-2 interieukin-2
• An especially useful phospholipase A g inhibitor for such purposes is lipocortin.
• synthetic compounds with inhibitory activity can be used such as 2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, (ONO-RS-082, ONO Pharmaceutical Co.).
• agents useful for such modification include guanidine HCI and cyanogen bromide, but those of skill in the art can easily ascertain similar agents without undue experimentation.
• agents which do not destroy the external structure of the antigen since it is thought that such external structures are important in suppressor T-cell epitopic recognition of the antigen.
• this issue is not significant for most antigens, such as many allergens, which are not cytotoxic. Consequently, with typical allergens, the native molecules can be used to stimulate the T-cells.
• the present invention is directed to a method for generating antigen-specific human T-cells and T-cell hybridomas which produce human antigen-specific GIF, which are specifically reactive with an antigen which is associated with an immune response to be immunosuppressed.
• the isolation of T-cell hybridomas producing a human antigen-specific GIF with the antigenic specificity of the human antigen-specific GIF of the invention can be accomplished using routine screening techniques to determine the elementary reaction pattern of the human antigen-specific GIF of interest.
• Still another way to determine whether a human antigen-specific GIF has the specificity of a human antigen-specific GIF of the invention is to pre-incubate the human antigen-specific GIF of the invention with the antigen with which it is normally reactive (for example, bee venom PLA 2 ), and determine if the human antigen-specific GIF being tested is inhibited in its ability to bind the antigen. If the human antigen-specific GIF being tested is inhibited then, in all likelihood, it has the same epitopic specificity as the human antigen- specific GIF of the invention.
• epitopic determinants are meant to include any determinant capable of specific interaction with a human antigen-specific GIF or the monoclonal antibodies of the invention.
• Epitopic determinants usually consist of chemically active groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
• the invention relates to a method of producing substantially pure human antigen-specific GIF comprising:
• the continuous hybridoma cell lines so used can themselves be produced as described above.
• the hybridoma cell line is preferably stimulated to produce human antigen-specific GIF by exposing the hybridoma cells to syngeneic macrophages which have been pulsed with the antigen to which the antigen-specific GIF binds, or with antibodies to the CD3 or T-cell receptor.
• a particularly useful technique is affinity purification using the antigen, for example attached to a solid phase, to which the antigen-specific GIF binds.
• a modification of this technique is to use two affinity absorption steps, if desirable, to substantially purify the human antigen-specific GIF.
• the step of isolating substantially pure human antigen-specific GIF includes:
• the step of isolating substantially pure human antigen-specific GIF includes: (i) reacting the hybridoma cell line culture with the antigen to which the human antigen-specific GIF binds; (ii) eluting the human GIF from the antigen; (iii) reacting the eluted GIF with a monoclonal antibody specifically reactive with human GIF; (iv) eluting the human antigen-specific GIF from the monoclonal antibody; and (v) recovering the human antigen-specific GIF.
• antigen-binding GIF in culture supernatants can be purified by ion-exchange chromatography, described above. Under such conditions, the process of isolating substantially pure human antigen-specific GIF includes:
• ion-exchange chromatographic purification is used in combination with affinity-purification to isolate human antigen-specific GIF, for example, by using the antigen to which the antigen-specific GIF binds or by using an antibody specifically reactive with human GIF, or both, as described above.
• DEAE diethylaminoethyl Sepharose
• Other ion- exchange materials which can be utilized include virtually any of the commercially available anion exchange agaroses and celluloses, such as polysulfated agaroses, specifically including but not limited to QAE (quaternary amine) derivatives, ecteola (epichlorohydrintri-ethanolamine), TEAE (triethylaminoethyl) derivatives, and AE (aminoethyl) cellulose.
• QAE quaternary amine
• ecteola epichlorohydrintri-ethanolamine
• TEAE triethylaminoethyl
• AE aminoethyl
• the hybridoma cell line culture supernatant is added to the anion- exchange matrix equilibrated with about 20mM salt, e.g., NaCI, much of the GIF will pass through the column and the remainder are eluted with salt concentrations up to about 60mM.
• concentrations of NaCI from about 20mM to about 60mM contained in 10mM Tris.
• a monoclonal antibody which is particularly useful in the affinity purification of human GIF is the monoclonal antibody produced by a cell line 388F 1 or monoclonal antibodies having the specificity of a monoclonal antibody produced by cell line 388F r
• suppressive denotes a lessening of the detrimental effect of the undesirable immune response in the human receiving therapy.
• immunosuppressively effective means that the amount of human antigen-specific GIF used is of sufficient quantity to suppress the cause of disease or symptoms due to the undesirable immune response.
• the dosage ranges for the administration of the human antigen-specific GIF of the invention are those large enough to produce the desired effect in which the symptoms of the immune response show some degree of suppression.
• the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
• the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
• the dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary from about 0.001 mg/kg/dose to about 2 mg/kg/dose, preferably about 0.001 mg/kg/dose to about 0.2 mg/kg/dose, in one or more dose administrations daily, for one or several days.
• the human antigen-specific GIF of the invention can be administered parenterally by injection or by gradual perfusion over time.
• the human antigen-specific GIF of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermaliy.
• Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions.
• non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
• Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
• Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on
• Ringer's dextrose and the like.
• Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
• the invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the human antigen-specific GIF of the invention, the medicament being used for therapy of an undesirable immune response to an antigen wherein the antigen is capable of binding by the human antigen-specific GIF of the invention.
• the present invention is also directed to monoclonal antibodies, and B-cell hybridomas which produce them, which are specifically reactive with human GIF.
• the B-cell hybridomas of the invention were prepared by immunizing BALB/c mice with affinity-purified human GIF and later boosted. Two weeks after the last immunization, spleen cells were obtained from the animals and transferred to syngeneic BALB/c mice which had been lethally irradiated. The syngeneic recipients were immunized twice with purified human GIF and 2 weeks after the last immunization the spleen cells were fused with SP 2/0-14AG myeloma cell line. Hybridomas were screened for monoclonal antibody production to human GIF.
• the isolation of hybridomas producing monoclonal antibodies with the reactivity of the monoclonal antibodies of the invention can be accomplished using routine screening techniques to determine the elementary reaction pattern of the monoclonal antibody of interest. Thus, if a monoclonal antibody being tested reacts with human GIF, but does not react with mouse GIF, then the antibody being tested and the antibody produced by the hybridomas of the invention are equivalent.
• the isolation of other hybridomas producing monoclonal antibodies with the specificity of monoclonal antibody 388F, or any other monoclonal antibody of the invention can be accomplished by one of ordinary skill in the art by producing anti-idiotypic antibodies (Herlyn, et al., Science, 232: 100, 1986).
• An anti-idiotypic antibody is an antibody which recognizes unique determinants present on the monoclonal antibody produced by the hybridoma of interest. These determinants are located in the hypervariable region of the antibody. It is this region which binds to a given epitope and, thus, is responsible for the specificity of the antibody.
• the anti-idiotypic antibody can be prepared by immunizing an animal with the monoclonal antibody of interest.
• the animal immunized will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants.
• anti-idiotypic antibodies produced by the second animal which are specific for the monoclonal antibodies produced by a single hybridoma which was used to immunize the second animal, it is now possible to identify other clones with the same idiotype as the antibody of the hybridoma used for immunization and thereby greatly simplify and reduce the amount of screening needed to find other hybridomas producing monoclonal antibodies with the specificity of the monoclonal antibodies of the invention.
• Idiotypic identity between monoclonal antibodies of two hybridomas demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant.
• antibodies to the epitopic determinants on a monoclonal antibody it is possible to identify other hybridomas expressing monoclonal antibodies of the same epitopic specificity.
• a monoclonal antibody it is possible to evaluate, without undue experimentation, a monoclonal antibody to determine whether it has the same specificity as 388F 1 of the invention by determining whether the monoclonal antibody being tested prevents 388F 1 from binding to a particular antigen, for example human GIF, with which 388F, is normally reactive. If the monoclonal antibody being tested competes with 388F 1( as shown by a decrease in binding by 388F 1t then it is likely that the two monoclonal antibodies bind to the same epitope.
• Still another way to determine whether a monoclonal antibody has the specificity of 388F is to pre-incubate 388F., with an antigen with which it is normally reactive, for example, human GIF, and determine if the monoclonal antibody being tested is inhibited in its ability to find the antigen. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same epitopic specificity as the monoclonal antibody of the invention.
• monoclonal antibodies of one isotype might be more preferable than those of another in terms of their diagnostic or therapeutic efficacy.
• Particular isotypes of a monoclonal antibody can be prepared either directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma producing monoclonal antibody of different isotype, by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proceedings of National Academy of Sciences, USA, 82: 888653, 1985; Spira, et al., Journal of Immunological Methods, 74: 307, 1984).
• the monoclonal antibodies of the invention would include class-switch variants having the specificity of monoclonal antibody 388F, which is produced by ATCC HB 10472. This cell line was placed on deposit for 30 years at the American Type Culture Collection (ATCC) in Rockville, Maryland prior to June 4, 1990.
• ATCC American Type Culture Collection
• antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as, for example, Fab and F(ab 1 ) 2 , which are capable of binding the epitopic determinant.
• the monoclonal antibodies of the invention can also be used in immunoaffinity chromatography for the purification of the various types of human GIF mentioned herein.
• immunoaffinity chromatography One way by which such immunoaffinity chromatography can be utilized is througn the use of, for example, the binding of the monoclonal antibodies of the invention to CNBr-Sepharose-4B or Tresyl-activated Sepharose (Pharmacia).
• These solid phase-bound monoclonal antibodies can then be used to specifically bind human GIF from mixtures of other proteins to enable its isolation and purification.
• the bound IFN-gamma can be eluted from the affinity chromatographic material using techniques known to those of ordinary skill in the art such as, for example, chaotropic agents, low pH, or urea.
• Lyophilized phospholipase A ⁇ PLA from bee venom was purchased from Sigma Chemical Co., St. Louis, MO.
• Denatured PLA 2 (-PLA and cyanogen bromide-treated PLA 2 were prepared by the method described by King, et al., Arch. Biochem and Biophys., 172: 661 , 1976.
• D- PLA > 5 mg of PLA 2 were dissolved in 0.1 M Tris HCI buffer, pH 8.6, and denatured in 6M guanidine HCI in the presence of 5 mg/ml dithiothreitol. After 18 hours at room temperature, sulfhydryl groups were carboxymethylated with iodoacetic acid.
• the denatured protein was diaiyzed against 0.02 M acetic acid and kept at -40 ° C until use.
• 10 mg bee venom PLA 2 was dissolved in 0.4 ml distilled water, and 1.2 ml formic acid containing 100 mg CNBr were added.
• mice monoclonal antibody against rabbit lipomodulin 141B9 (Iwata, et al., J. Immunol., 132: 1286, 1984) was the same preparation as that described in a previous article (Askasaki, et al., J. Immunol., 131 : 3172, 1986). Specifically-purified goat antibodies against mouse IgG, which contained both anti-heavy ( ⁇ ) chain and anti-light chain, were previously described (Suemura, et al. J. Immunol., 125: 148, 1980). Fluoresceinated goat anti-mouse IgG antibodies were purchased from Cappel. Human IgE and anti-Iipomodulin antibody (141 B9) were coupled to CL-Sepharose 4B; approximately 5 mg of protein were coupled to 1 ml Sepharose.
• RPMI 8866 lymphoblastoid cells were cultured in RPMI 1640 medium enriched with 10% fetal calf serum, 2 mM L-giutamine, 50 ⁇ M 2- mercaptoethanol and antibiotics (RPMI 1640 culture medium).
• the mouse T-cell hybridoma 12H5 cells (Iwata, et al., J. Immunol., 140: 2534, 1988) were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) described in a previous article (Huff, et al., Proc. Natl. Acad. Sci., USA, 129: 509, 1982).
• DMEM Dulbecco's modified Eagle's medium
• a hypoxanthine guanine phosphoribosyltransferase- def icient mutant of the human lymphoblastoid cell line CEM (BUC) cells were previously described (Huff & Ishizaka, Proc. Natl. Acad. Sci., USA, 8 .: 1514, 1984).
• Peripheral blood was obtained from a patient allergic to honey bee venom, and mononuclear cells of the blood (PBMC) were obtained by centrifugation on Ficoll-Pague (Pharmacia).
• PBMC mononuclear cells of the blood
• Ficoll-Pague Pharmacia
• Non-adherent cells were recovered, resuspended in fresh culture medium (2x10 5 cells/ml), and then cultured for four days with 60 units/ml purified IL-2 (chromatographically purified human IL-2, Electro-nucleonics, Silverspring, MD), in the presence of 3 ⁇ g/ml recombinant human lipocortin 1 , which was kindly supplied by Drs. J. Browning and B. Pepinsky, Biogen.
• IL-2 chromatographically purified human IL-2, Electro-nucleonics, Silverspring, MD
• T-cell hybridomas 1.2x10 7 T-cells, which had been propagated by IL-2, were mixed with twice the number of BUC cells. Mixed cells were pelleted together and fused by using polyethylene glycol (1300 - 1600 MW.,
• the cells were treated with 8 ⁇ g/ml OKT 3 for 40 minutes at 0° C, and the antibody-treated cells (1x10 6 /ml) were seeded in Limbro tissue culture wells (Flow Labs, McLean, VA) which had been coated with 10 ⁇ g/ml anti-MGG. Culture supematants were obtained after 24 hour culture.
• hybridoma cells (1x10 6 /sample) were incubated 40 minutes at 0°C with 8 ⁇ g/ml OKT 3 or a 1:1000 dilution of anti-TcR ⁇ (WT31)-containing ascitic fluid in RPMI 1640 medium supplemented with 5% FCS and 10 ⁇ M NaN3.
• As controls aliquots of the same cells were treated with the same concentration of mouse lgG 2a (Becton-Dickinson, isotype control). Cells were washed twice with PBS containing 5% FCS, and then incubated with fluoresceinated anti-mouse IgG for 40 minutes. After washings, cell- associated fluorescence was analyzed by using FACScan from Becton- Dickinson.
• the CD3 + hybridoma cells were identified by rosetting using ox erythrocytes coated with anti-mouse IgG.
• the antibodies were coupled to erythrocytes by the method of Wilhelm, ef al., (J. Immunol. Methods, 90: 89, 1986).
• erythrocytes were washed 4 times with saline, and resuspended in 0.75 ml of 0.5 ⁇ g/ml purified anti-MGG; 25 ⁇ l of CrCI 3 (16.5 mg CrCI 3 dissolved in 5 ml saline) were added to the cell suspension under gentle mixing, and the cell suspension was incubated for 1 hour at 30° C.
• the anti-MGG-coupied erythrocytes were washed 4 times with saline and resuspended in 5 ml FCS (approximately 1x10 9 erythrocytes/ml).
• pellets of 10 6 hybridoma cells were suspended in 80 ⁇ l of DPBS containing 5% FCS and 8 ⁇ g/ml OKT 3. After 45 minutes at 0° C, the cells were washed twice and resuspended in 80 ⁇ l DPBS-5% FCS, and 20 ⁇ l of a suspension of anti-MGG-coated erythrocytes and crystal violet were added to the cell suspension. The mixtures were centrifuged at 200g for 5 minutes and tubes were incubated for 2 hours at 0 e C. The pellets were gently resuspended and examined for rosetting cells under microscope.
• Hybridoma cells treated with 8 ⁇ g/ml OKT 3 were mixed with anti-MGG coupled erythrocytes (ca 4x10 8 erythrocytes) to form rosettes by the procedures described above.
• Pellets were resuspended and applied to the top of Percoll gradient consisting of 60% and 50% Percoll layers. Tubes were centrifuged for 20 minutes at 1200 RPM (700g) at room temperature. The pelleted cells were washed twice with culture medium, and the erythrocytes were lysed by treatment with 0.83% NH 4 CI buffer for 1 minute at 0°C. The cells were washed with and resuspended in DME culture medium and cultured to expand the cell population.
• Hybridoma cells were treated with OKT 3 and stained with fluoresceinated anti-MGG. The positively stained cells were selected by sorting the cells by using FACSTAR (Becton-Dickinson).
• IgE-BF The presence of IgE-BF in culture filtrates or acid eluate fraction from IgE- Sepharose was assessed by inhibition of rosette formation of FceR + B lymphoblastoid cell line, RPMI 8866 cells with human IgE-coated ox erythrocytes (E-lgE) by the procedures previously described (Kisaki, et al., J. Immunol., 138: 3345, 1987). The proportion of rosette forming ceils (RFC) in 300 RPMI 8866 cells was determined in triplicate and was expressed as the average ⁇ SD.
• FRC rosette forming ceils
• Rodent IgE-BF formed by the 12H5 cells were detected by the same procedure, except that rat IgE-coated ox erythrocytes were employed as indicator cells, and mesenteric lymph node cells of Lewis strain rate infected with Nipportronngylus brasiliensis were used as a source of FceR + B cells (Yodoi & Ishizaka, J. Immunol., 124: 1322, 1980).
• GIF was detected by using T-cell hybridoma 12H5 cells (Iwata, et al., J. Immunol., 140: 2534, 1988).
• a suspension of the hybridoma cells was mixed with an equal volume of a test sample, and the cell suspensions were cultured for 24 hours with 10 ⁇ g/ml mouse IgE.
• Culture supernatants were filtered through CF50A membranes, and filtrates containing IgE-BF were fractionated on lentil lectin Sepharose (Yodoi, et al., J. Immunol., 125: 1436, 1980).
• GIF was taken as (+), if the ratio of the percent rosette inhibition between the effluent/eluate fraction were 3.0 or higher.
• Hybridoma cells were treated with OKT 3 antibody (8 ⁇ g/ml) and 8 ml aliquots of the antibody treated or untreated cell suspension (1.5x10 6 cells/ml) were cultured in anti-MGG-coated tissue culture flasks. Culture supernatants were concentrated four-fold, and a 2 ml sample was absorbed with 0.4 ml IgE-Sepharose. The effluent fraction was mixed with 0.5 ml PLA ⁇ -Sepharose overnight, and immunosorbent was packed into a small column.
• Affinity-purified GIF was treated with alkaline phosphatase as previously described (Uede, et al, J. Immunol., 139: 898, 1983). Briefly 1 ml of the preparation was diaiyzed against Tris-HCl buffer, pH 8.2 and was mixed with 1 unit of insoluble alkaline phosphatase (calf intestinal, Sigma) for 2 hours at room temperature. After centrifugation, the supernatant was diaiyzed against 0.1 M Tris-HCl buffer, pH 8.0.
• Phospholipase A 2 inhibitory activity of the alkaline-phosphatase treated samples was determined using E coll which were biosynthetically labeled with 3 H-oleic acid and porcine pancreatic PLA ⁇ (Sigma) (Rothut, et al., Biochem. Biophys. Res. Commun., V ⁇ . 878, 1983). Detailed procedures were described in Ohno, et al. (Intemat.
• porcine pancreatic PLA (1x10 "5 units) was mixed with GIF in a total volume of 150 ⁇ l. After 5 minutes at 25 °C, 50 ⁇ l of a suspension of 3 H-labeled E coli (5000 cpm) was added, and the mixtures were incubated for 5 minutes at 25 ° C. The reaction was stopped by the addition of 50 ⁇ l 2 M HCI, and 50 ⁇ l of 100 mg/ml BSA was added to the mixtures. The suspensions were centrifuged for 1 minute at 5500 g, and radioactivity in 250 ⁇ l of supernatant was measured in a scintillation 5 spectrometer.
• Culture supernatant of AC5 cells in serum-free medium was concentrated 25 to 100 fold by ultrafiltration. After centrifugation at 10,000 rpm for 20 min, the supernatant was diluted 8-fold with distilled water, adjusted to pH 8.0 o with Tris, and immediately applied to a DEAE-Sepharose CL-6B (Pharmacia) column (3 ml volume) which was equilibrated with 10mM Tris HCI buffer, pH 8.0. After effluent (passed-through) fraction was recovered, the column was washed with 4 column volumes of 10 mM Tris-HCl buffer containing 20mM NaCI, and the washing was combined to the passed through fraction.
• the supernatant was concentrated 50 to 100-fold by ultrafiltration.
• An appropriate fraction of the supernatant from a DEAE-Sepharose column was concentrated to 5-6 ml and mixed overnight at 4° C with 1.0 to 1.5 ml of Affigel 10-immunosorbent coupled with monoclonal anti-GIF antibody.
• the suspension was packed into a small column and the immunosorbent was washed with 40 column volumes of DPBS.
• the immunosorbent was washed with 40 column volumes of DPBS and 20 column volumes of PBS containing 0.5 M NaCI. Proteins bound to the immunosorbent were eluted with 0.05 M glycine HCI buffer containing 0.15 M NaCI, pH 3.0-3.2.
• Affinity-purified GIF was diaiyzed against 0.01 % SDS in deionized water, and lyophilized in a Speed vac (Savant Instruments, Hicksville, NY). Samples were then analyzed by SDS gel electrophoresis in 15% polyacrylamide slab gel by using the Laemmli system (Laemmli, U.K., Nature, 227:680. 1970). Gels were fixed and protein bands were detected by silver staining (Ochs, et al., Electrophoresis, 2:304, 1981). Molecular weight standards were obtained from Pharmacia.
• Binding of mouse Ig to the plate was detected by using alkaline phosphatase-coupled goat anti-mouse Ig (Zymed Lab, So. San Francisco, CA) and alkaline phosphatase substrate (Sigma).
• ELISA signal was read in a microplate reader MR 5000 (Dynatech Lab) with a 410 nm filter 30 min after the addition of substrate.
• Isotype of monoclonal antibodies was determined with ELISA assay by using an isotyping kit for mouse mAb (Zymed Lab).
• the plate was washed and 50 ⁇ l of a 1 :1500 dilution of streptavidin-alkaline phosphatase conjugate (Zymed Lab) were added to each well. After incubation for 1 hour at 37 ° C, quantities of alkaline phosphatase bound to the wells were measured by amplification system (Stanley, ef al., J.lmmunol. Methods, 83:89, 1985), (GIBCO-BRL, Bethesda, MD). ELISA signal was determined at 490 nm.
• PBMC of a bee venom-sensitive patient were cultured for three days in the presence of 10 ⁇ g/ml D-PLA ⁇ and activated T-cells were propagated by IL-2 for four days in the presence of 3 ⁇ l/ml recombinant lipocortin. T-cells were then fused with BUC cells to construct hybridomas.
• 4 hybridoma clones were obtained. Each hybridoma clone was cultured in complete DMEM and culture supematants were filtered through YM100 membranes. Filtrates were concentrated ten-fold and assessed for the presence of GIF by using the 12H5 cells.
• Table I indicate that two of the four hybridoma clones constitutively secrete GIF.
• the 12H5 cells were cultured with 10 ⁇ g/ml mouse IgE alone, and IgE-BF in culture filtrates were fractionated on lentil lectin Sepharose.
• Culture filtrates were fractionated on 141B9-Sepharose, and acid eluates from the immunosorbent were concentrated to 1/100 volume of the original culture supernatant.
• the samples were treated with alkaline phosphatase, and dephosphorylated materials were assessed for the ability to inhibit pancreatic phospholipase A2.
• the presence of CD3 determinants on the hybridoma clone CL3 was assessed by fluorocytometry and the rosetting technique.
• the cells were treated with 8 ⁇ g/ml monoclonal antibody OKT3 and then stained with fluoresceinated goat anti-mouse Ig. Less than 10% of the total cells were stained.
• the CD3 + cell population was further enriched by cell sorting and expanding the cells by culture. After repeating the cell sorting twice, a CL3 population was obtained which stably expressed CD3. Fluorescent staining of the population with OKT3 and WT31 (anti-TcR ⁇ j3) indicated that essentially 100% of the cells expressed CD3 and the majority of the cells expressed TcR ⁇ /3.
• the CD3 + cell population and CD3 ' population were cultured and culture filtrates were assessed for the presence of GIF by using the 12H5 cells. The GIF activity was detected in culture filtrates of CD3 + cells, but not in the culture filtrates of CD3 ' population. The results indicated that the source of GIF is CD3 + cells.
• mouse GIF Since one of the unique properties of mouse GIF is that the monoclonal anti-lipomodulin (141 B9) binds the lymphokine, it was decided to determine whether human GIF from the CL3 cells would be absorbed with 141 B9- coupled Sepharose.
• the CD3 + , CL3 clone was cultured to yield 1 liter of culture supernatant. After filtration through a YM100 membrane, the filtrates were concentrated to 5 ml, and fractionated on 1 ml 141-B9 Sepharose. After recovering the effluent fraction, the immunosorbent was washed with 10 column volumes of DPBS, and then eluted with 5 column volumes of glycine-HCI buffer, pH 3.0.
• the immunosorbent was washed with 5 column volumes of DPBS, and then eluted with 5 column volumes of glycine HCI buffer, pH 3.0.
• GIF activity was assessed by using the 12H5 cells by the same procedures described in Table I. Numbers in the column represent percent rosette inhibition by the effluent/eluate fractions from lentil lectin Sepharose. The proportion of IgE-RFC in the absence of IgE-BF was 22.9 ⁇ 0.6 (SD)% in this assay. (+) indicated the presence of GIF.
• Previous experiments provided evidence that murine GIF is a phosphorylated derivative of a phospholipase inhibitory protein (Uede, et al., J. Immunol., 139: 898, 1983).
• GIF in the culture filtrates of CL3 clone was purified by using the 141 B9-Sepharose. Culture filtrate of the 5 three other clones, CL1 , CL2, and CL7 were fractionated on the 141 B9-
• Sepharose in a similar manner.
• the acid eluates from the immunosorbent were treated with alkaline phosphatase, and assessed for the ability to inhibit the release of 3 H-oleic acid from biosynthetically labeled E. coli by pancreatic phospholipase A 2 (Rothut, et al., Biochem. Biophys. Res. o Commun., 117: 878, 1983).
• the results included in Table I indicate that the affinity-purified GIF from CL3 and CL7 exerted phospholipase inhibiting activity, while the same fraction from CL1 and CL2 failed to inhibit phospholipase A ⁇ .
• Culture supernatants were filtered through YM100 membranes and concentrated seven-fold by ultra-filtration.
• the concentrated culture filtrates were absorbed overnight with 1 ml IgE-Sepharose, and unbound protein fraction and 2 ml of washings were combined.
• the IgE-Sepharose was thoroughly washed, and eluted with glycine HCI buffer.
• the eluate fractions from IgE-Sepharose were assessed for the presence of IgE-BF by using RPMI 8866 cells as the source of FceR + cells.
• IgE-BF was 37.7 ⁇ 1.0%.
• the effluent fraction from IgE-Sepharose was concentrated two-fold and 1 ml samples were fractionated on 0.25 ml PLA,-Sepharose. The effluent fraction, washing, and eluate fraction were adjusted to 1.5 ml, and the samples assessed for GIF activity. As shown in Table III, GIF from both unstimulated and anti-CD3 treated cells failed to bind to PLA,- Sepharose.
• PBMC of a bee venom sensitive patient were constructed.
• the protocol for the construction of T-cell hybridomas was exactly the same as that described above, except that PBMC were stimulated with 10 ⁇ g/ml CNBr- treated PLA ⁇ instead of D-PLA ⁇ .
• 22 hybridoma clones were obtained.
• the GIF assay of culture filtrates of each clone indicated that 10 out of 22 clones constitutively formed GIF (results not shown).
• Seven GIF-secreting clones were treated with OKT3 and the antibody-treated cells were cultured in anti-MGG-coated dishes. Culture filtrates were concentrated four-fold and absorbed with IgE-Sepharose.
• GIF activity was determined by using the 12H5 cells. Numbers represent the percent rosette inhibition by the effluent/eluate fractions from lentil lectin Sepharose. Proportion of IgE-RFC in the absence of IgE-BF was 26.0 ⁇ 0.7 (SD)%. (+) indicates the presence of GIF. As shown in Table IV, acid eluate fraction from IgE-Sepharose of 6 out of 7 clones contained detachable amounts of IgE-BF. The effluent fractions from IgE-Sepharose were then fractionated on PLA ⁇ -Sepharose, and the effluent and eluate fractions from the immunosorbent were assessed for GIF activity.
• the present invention provides a technique to allow the development of GIF-producing T-cell populations from PBMC of patients allergic to bee venom PI ⁇ , and to establish GIF-producing hybridomas from the T-cells.
• Representative hybridomas express CD3 determinants and TCR ⁇ /3, indicating that they are T-cell hybridomas.
• the TcR complex on the hybridomas appears to be functional. Both parent T-cells (Carini, et al., J. Immunol. Methods, 127: 221, 1990) and the majority of the GIF- producing hybridomas (Tables III, IV) produced IgE-BF upon cross-linking of CD3.
• T-cell hybridomas produced antige ⁇ PLA ⁇ -binding GIF upon cross-linking of CD3 on the cells.
• This finding is in agreement with the fact that representative murine GIF-forming hybridomas formed antigen-binding GIF upon stimulation with antigen-pulsed syngeneic macrophages or by cross-linking of CD3 on the cells (Iwata & Ishizaka, J. Immunol., 141: 3270, 1988, Iwata, et al., J. Immunol., 143: 3917, 1989), and suggested similarities between the antigen-binding GIFs from the two species.
• the antigen-binding GIF obtained from the hybridomas suppressed the in vivo antibody response in carrier (antigen)-specific manner. It was also found that the antigen-binding GIF from the hybridomas were composed of antigen-binding polypeptide chain and non-specific GIF (Jardieu and
• GIF may be a counterpart of murine TsiF.
• the typical murine helper T-cell clone D10.G4.1 can produce antigen-binding GIF, if the cells were precultured in the presence of a phospholipase A 2 inhibitor, and then stimulated with antigen(conalbumin)-pulsed antigen-presenting cells (Ohno, etal., Internal Immunol., 2: 257, 1990). It was also found that this antigen-binding GIF bound to the monoclonal antibody 14-30, which is specific for TsiF (Ferguson and Iverson, J. Immunol., 136: 2896, 1986), rather than the monoclonal antibody 14-12.
• Tse clones from PBMC of KLH-primed individuals, who had received repeated injections of a large dose of homologous antigen.
• Modulin, et al. (Nature, 322: 459, 1986) also established Ts clones from lesions of lepromatous leprosy patients.
• effector molecules mediating suppressor activity (TsF) from human Ts cells have not been identified. Similarities between human GIF and mouse GIF suggest that the PLA ⁇ -binding GIF from human T-cell hybridomas may represent TsF from human suppressor T-cells.
• the T-cell hybridomas which produce antigen-binding GIF, will facilitate biochemical characterization of the molecules. It has been repeatedly shown in the mouse that Ts as well as TsF (antigen-binding GIF) suppressed the in vivo IgE antibody response more effectively than the IgG antibody response (Ishizaka, et al., J. Immunol., 114: 110, 1975). If the allergen-binding GIF from the human T-cell hybridomas actually represent TsF, it is a reasonable expectation that the T-cell factor may suppress the IgE antibody response of the donor of parent T-cells.
• Japanese cedar pollen is a major allergen in Japan and causes seasonal allergic rhinitis and conjuctivitis in a large percentage of the population.
• the methods for generating antigen-specific GIF-producing T-cells and T-cell hybridomas were applied to peripheral blood mononuclear cells from patients allergic to Japanese cedar allergen.
• the major allergen in Japanese cedar is a 40kDa glycoprotein designated cryj-1 (Yasueda, et al., J. Allergy and Clin. Immunol., 71:77, 1983).
• cryj-1 The major allergen in Japanese cedar pollen is a 40kDa glycoprotein designated cryj-1 (Yasueda, et al., J. Allergy and Clin. Immunol., 71:77, 1983).
• the allergen was isolated from extracts of cedar pollen by this method with slight modifications. Briefly, pollen was defatted with ether, and extracted 3 times with 0.125M ammonium bicarbonate. Carbohydrate in the extracts were removed by hexadecyltrimethyl ammonium bromide. Proteins in the extracts were precipitated with 80% saturated ammonium sulfate, and the precipitate dissolved in 0.05M Tris-HCl buffer, pH 7.8.
• the protein fraction was applied to a DEAE cellulose column (DE-52, Whatman), and a flow-through fraction was obtained.
• the fraction was concentrated, diaiyzed against 0.01 M acetate buffer, pH 5.0, and applied to a CM cellulose column (CM-52, Whatman), which was equilibrated with the buffer.
• the column was washed with the buffer, and proteins retained in the column eluted with 0.1 M phosphate buffer containing 0.3M sodium chloride. Proteins in the eluate were further fractioned by gel filtration through a Sephacryl S-200 HR column to obtain a major protein fraction containing cryj-1.
• the major protein in the fraction was 42kDa as determined by SDS-polycrylamide gel electrophoresis, and N-terminal amino acid sequence of the protein was identical to that of cryj-1.
• the protein was conjugated to Affigel 10 at 1.5 mg/ml gel.
• a synthetic phospholipase A 2 inhibitor, 2-(p-amylcinnamoyl)-amino-4- chlorobenzoic acid, (ONO-RS-082, ONO Pharmaceutical Co.) was used instead of recombinant human lipocortin I.
• mononuclear cells were obtained from periheral blood of patients allergic to Japanese cedar pollen, and suspended in RPMI 1640 medium containing 10% fetal calf serum (FCS).
• FCS fetal calf serum
• a suspension of the mononuclear cells (3x10 6 cells/ml) were cultured for 3 days in the presence of 10 ⁇ g/ml cryj-1.
• Non-adherent cells were recovered, resuspended in RPMI medium containing 10% FCS, (3x10 5 cells/ml), and cultured for 4 days in the presence of 60 units/ml human IL-2 and 2 ⁇ M ONO-RS-082. Cells propagated in this manner were then recovered and fused with BUC cells to construct hybridomas.
• Hybridomas were treated with the monoclonal anti-CD3 antibody SPB-T3b (Spits, et al., Hybridoma 2:423, 1983), and the presence of CD3 on the cells were tested by immunofluorescence. Only CD3+ hybridomas were subcloned by limiting dilution.
• the CD3+ hybridoma clones were maintained in complete DME medium containing 10% FCS, and culture supernatant of each clone was assessed for the presence of GIF by using the 12H5 cells. Results obtained with hybridomas from one patient are shown in Table V. GIF activity was detected in culture supematants of three hybridomas; 31 E9, 31 B7, and 32B4. Supematants of the other two hybridomas, 31 H6 and 31 H3, appear to have weak GIF activity. Thus, the GIF-producing hybridomas were treated with anti-CD3 antibody followed by anti-mouse immunoglobulin, and the cells were cultured for 24 hr. Culture supematants were then fractionated on cryj-1 coupled immunosorbent.
• Hybridomas in this table were derived from two separate experiments. Culture supematants of unstimulated hybridomas were screened for the presence of GIF. Aliquots of 12H5 ceils were incubated with culture supernatant of each hybridoma in the presence of mouse IgE. Culture supematants of the 12H5 cells were filtered through CF50A to remove IgE, and filtrates were fractionated on lentil lectin Sepharose. IgE-BF in the effluent and eluate fractions was assessed by rosette inhibition. Numbers in the column represent the percent rosette inhibition by the effluent/eluate fractions from lentil lectin Sepharose. (+)(-) signs indicate the presence or absence of GIF, respectively.
• Representative hybridomas were treated with anti-CD3 antibody and culture supematants were fractionated on cryj-1 coupled Affigel.
• the presence of GIF activity in the flow-through (unbound) fraction, and acid eluate (bound) fraction was determined by using 12H5 cells.
• Culture filtrates of the 12H5 cells were fractionated on lentil lectin Sepharose. Numbers represent percent rosette inhibition by the effluent/eluate fractions from lentil lectin Sepharose.
• GIF from the 31 E9 cells bound to cryj-1 -Affigel and was recovered by elution at acid pH, while GIF from the
• Human GIF in culture supernatant of the T-cell hybridoma CL3 was purified by using anti-lipomodulin (141 -B9)-Sepharose.
• the affinity-purified GIF was mixed in complete Freund's adjuvant, and BALB/c mice were immunized by intraperitoneal injections of the antigen, given 3 times at 2 week intervals. Two weeks after the last immunization, spleen cells of the immunized mice were obtained, and 1x10 7 spleen cells were transferred into syngeneic BALB/c mice which had been irradiated with 625R ⁇ ray. The recipients were immunized immediately after cell transfer and 2 weeks later with purified GIF included in incomplete Freund's adjuvant.
• the presence of anti-GIF in the culture supematants of the 11 hybridoma clones was then determined by using the 12H5 cells (Iwata, et al., J. Immunol., 140: 2534, 1988).
• the globulin factor of culture supernatant from each clone was obtained by precipitation with 50% saturated ammonium sulfate. After dialysis against phosphate buffered saline, the fraction was adjusted to 1/5 volume of the original culture supernatant. Aliquots of an affinity-purified GIF prepared from CL3 clone using 141B9 Sepharose.
• GIF in culture supernatant was absorbed with 141 -B9-Sepharose, and was recovered by elution at acid pH.
• the affinity-purified GIF preparation was then fractionated with anti-GIF (S ⁇ F ⁇ - coupled Sepharose. After the effluent fraction was obtained, the immunosorbent column was washed with 10 column volumes of Dulbecco's phosphate buffered saline (DPBS), and then eluted with glycine HCI buffer, pH 3.0.
• DPBS Dulbecco's phosphate buffered saline
• GIF in culture supematants of CL-3 clone was purified by using the anti-iipomodulin Sepharose.
• the affinity purified GIF (1.5 ml) was fractionated on 0.75 ml of 388F coupled Sepharose. After recovering the effluent fraction, the column was washed with 10 column volumes of DPBS, and then eluted with 3 column volumes of glycine HCI, pH 3.0.
• GIF activity was assessed by using the 12H5 cells by the same procedures described in Table IV. Numbers in the column indicate the percentage rosette inhibition by the effluent/eluate fractions from lentil lectin Sepharose. (+) indicates the presence of GIF.
• mouse GIF from Ts hybridoma, 231 F cells were purified by using 141 B9- Sepharose, and aliquots of the purified mouse GIF were fractionated on either 141 B9-Sepharose or 388F, -Sepharose.
• AC5 cells were subcloned by limiting dilution and CD3 + clones obtained. These cells were then adjusted to serum-free ABC medium. Expression of CD3 on the subclones cultured in the medium was confirmed by fluorocytometry. Culture supematants of CD3 + subclones were concentrated 10-30 fold, and GIF activity in serial dilutions of the preparations was determined. Based on these results, subclone (AC5-23) was selected, since a 1 :3 dilution of the 10-fold concentrated supernatant of this subclone could switch the 12H5 cells from the formation of glycosylated IgE-BF to the formation of unglycosylated IgE-BF.
• the GIF activity was detected in the passed-through fraction and in the eluate with 50mM NaCI, but not in the other fractions. Titration of a serial dilutions of the first two fractions indicated that the pass-through fraction had higher GIF activity than the 50mM fraction.
• the major band in the affinity-purified material has the molecular mass of 14KDa under reduced conditions and 15KDa under non-reduced conditions. In addition, a 67KDa band was frequently observed.
• a portion of the affinity-purified preparation was diaiyzed against DPBS and the GIF activity in the preparation was titrated. Assuming 100% recovery of GIF during dialysis and lyophilization, the sample applied to SDS-PAGE should have a GIF titer of 1 :250.

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