HUT67010A - Supressor t-cell hybridoma - Google Patents

Abstract

Human antigen-specific glycosylation inhibition factor (GIF) and method for immunosuppression of a specific immune response.

Description

The present invention relates to a human antigen-specific glycosylation inhibitory factor (GIF) which can be used to suppress an immune response to the antigen of interest.

Although immune responses are often beneficial, in some circumstances an immune response against an antigen may be detrimental to the animal in which the reaction occurs. For example, in autoimmune diseases such as lupus erythematosus, the immune response creates a condition in which the host is subjected to a serious pathological aftermath. In lupus erythematosus, antibodies that respond to determinants in the host's thyroid, erythrocytes, platelets, and DNA are often present.

Another example of an adverse effect of the immune response is the treatment of various allergic diseases. IgE antibodies against allergens have previously been shown to cause hay fever (carbon fever) and to play a role in other allergic diseases (such as exogenous asthma). The crucial role of IgE antibodies in allergic diseases has raised the possibility that regulating and suppressing IgE antibody production against allergens could be one of the basic treatments for allergic diseases. For example, IgE antibodies to allergens can always be detected in the plasma of patients sensitive to ragweed allergens. IgE antibody titer increases after the pollen-producing period and then decreases slowly for the remainder of the year. Because the

- ··> Plasma IgE has a response time of only two-narom days, and IgE antibody titer stability indicates that oetegex's lymphocytes continuously synthesize antibodies in the absence of allergy.

Over the past twenty years, there have been several attempts to control the IgE antibody response in experimental animals. One approach was to classify conventional immunotherapy or desensitization treatment, in which allergic patients were repeatedly inoculated with very small amounts of allergen.

It has been shown that desensitization treatment may relieve symptoms in some patients, but that titer is present in the plasma of senate patients. The major immunological effect of IgE antibody after treatment was not to increase IgG antibody formation and - to reduce the increase in IgE antibody titer after pollen-producing period.

Desensitization. and the applicability of immunosuppression treatment is limited by the fact that patients are unable to tolerate higher doses of allergens due to side effects. To overcome this difficulty, attempts have been made to treat chemically modified antigens (such as urea denatured antigen or polyethylene glycol conjugate of antigen). Because the modified antigens do not bind to antibodies against the original antigens, a relatively high dose of modified antigen can be injected without causing allergic symptoms. for this

- 4 • 5, the modified antigen can stimulate antigen-specific T cells. Modified antigen injections into mice have been shown to result in antigen-specific suppressor T cells that suppressed the primary IgE antibody response to the original antigen. However, if treatment is started after the IgE antibody titer has reached its maximum, then it is only

minimum six was exerted by continuous IgE antibody formation (Takatsu and Ishizaka, J. Immunol., 11Z, 1211, 1976). The in accordance with studies in mice,

Clinical trials with polyethylene glycol-conjugated allergens in patients with hay fever showed that the treatment did not reduce IgE antibody titer. The ineffectiveness of repeated antigen injections to suppress continuous IgE antibody formation is likely due to the presence of a relatively large population of antigen-specific helper T cells in allergic patients. Because the modified antigen not only induces the formation of antigen-specific suppressor T cells, but also increases the population of helper T cells, the latter effect of the treatment may outweigh the effect on suppressor T cells. The correctness of this explanation is supported by the fact that the transfer of antigen-specific T cells into immunized mice resulted in suppression of continuous IgE antibody formation (Takatsu and Ishizaka, J. Immunol. 117, 1211, 1976). Taken together, these results suggest that, if the formation of antigen-specific suppressor T cells is possible without the growth of helper T cell populations, continuous IgE antibody production may be suppressed in patients with hay fever.

Since 1980, researchers have been exploring various options for selective regulation of IgE synthesis by immunoglobulin in an isotype-specific manner. As a result of the research, two types of T-cell factors have been found which show affinity for IgE and selectively regulate its synthesis. One type of IgE-binding factor (IgE-BF) selectively enhances the IgE response, while another type of IgE-BF selectively suppresses it. The main difference between IgE stimulating and IgE suppressing factors is the carbohydrate constituents in the molecules. IgE-stimulating factors bind to lens lectin and concanavalin A, while IgE-suppressor factors do not bind to these lectins (Yodoi et al., J. Immunol., 128, 289, 1982). Investigation of the cellular mechanism of selective formation of both IgE-stimulating factors and IgE-suppressor factors, and gene cloning of these factors, showed that IgE-stimulating and / or IgE-stimulating factors. suppressor factor is encoded by a common structural gene and that the characteristics of the carbohydrate constituents and the biological activity of the factors are formed during post-translational glycosylation (Martens et al., Proc. Natl. Acad. Sci., U.S.A., 84,809, 1987). Under physiological conditions, the said glycosylation process is regulated by two T-cell factors that enhance or inhibit it. These two factors are glycosylation inhibitory factor (GIF) and glycosylation

enhancer factor (GABB>.

The unique property of GIF lies in its biochemical activity. This lymphokine binds to monoclonal antibodies to lipomodulin (a phospholipase inhibitor protein) and thus appears to be a phosphorylated derivative of the phospholipase inhibitor protein (Uede et al., J. Immunol., 130, 878, 1983). In mice, the major source of GIF has also been shown to be the antigen-specific suppressor T cell (Ts) (Jadieu et al., J. Immunol., 133, 3266, 1984). Experiments with Ovalbumin (OVA) -specific suppressor T-cell hybridomas have shown that stimulation of hybridoma cells with syngeneic macrocells treated with antigen (ovalbumin) resulted in the production of GIF (antigen-binding GIF) that exhibits affinity for ovalbumin.

On the other hand, the same hybridomas also secreted significant amounts of non-specific (non-specific) GIF. Non-specific GIF and OVA binder

Studies on the relationship between GIFs have shown that antigen-binding GIF is composed of an antigen-binding polypeptide chain and a non-specific GIF (Jardieu and Ishazaka, Immune Regulation By Characterized Polypeptides; Goldstein et al., Editors), Alan R. Liss, Inc., NY, p595, 1987). It has also been shown that antigen-binding GIF recognizes antigenic determinants common to antigen-specific suppressor T-cell factors, described by other researchers, and that it suppresses the antibody response in an antigen (carrier) -specific manner. Moreover, not only antigen-specific GIF but also antigen-specific TsF, described by other researchers, binds to the immunoreactor linked to monoclonal anti-lipomodulin (141-B9) and is eluted from it by acidic pH η.

The disadvantage of treating desensitization allergy is to use this method. Consequently, there is a need for a technique in which the antigen-specific butter antibody does not cause side effects such as existing desensitization methods.

The suppression of the immune response is critically important for the prevention of host versus graft versus graft (HVG) and graft versus host (GVH) rejection. In both HVG and GVH autoimmune disorders, the immune response is suppressed by highly toxic drugs that have a limited effect and are more general than specific. The limiting factors of such treatments have highlighted the need to develop immunosuppressive agents with lower toxicity and higher specificity.

Against an antigen

A more perfect way to suppress an immune response in a human being is to administer to the patient an immunosuppressively effective amount of human GIF that specifically binds to the antigen of interest.

in this case, the concentration of the T suppressor factor develops favorably and the immune response to the antigen is reduced.

Our invention provides a means to achieve this result.

purified human antigen-specific GIF which suppresses the immune response to homologous antigens. Suppressing the human immune response to a human antigen-specific GIF against a pharmaceutically usable antigen.

The present invention relates to a substantially pure human antigen specific GIF which is specific for an antigen associated with an unwanted immune response. This human antigen-specific GIF is very useful in antigen-specific immunosuppression of an unwanted immune response.

Of particular importance for the present invention are those which are human antigen specific

GIFs that specifically bind allergens.

In a particularly preferred embodiment of the invention, there is provided a human antigen-specific GIF that binds to an epitope of phospholipase A2 (.PLA2, the major allergen of the honeybee venom). This binding specificity makes it suitable for said antigen specific

GIF and other GIFs of similar specificity for use in suppressing the human immune response to PLA2. In another particularly preferred embodiment of the invention, a human antigen-specific epitope is provided

GIF is provided which binds to Japanese cedar pollen and can be used to suppress a human immune response to this antigen.

The experience of generating PL1A2 antigen-specific GIF can be easily extended by those skilled in the art to the production and purification of other antigen-specific GIF molecules without undue experimentation. The broad pioneering nature of the invention makes it possible to produce human antigen-specific GIF molecules that bind to non-allergens and can be used to suppress immune-related diseases such as autoimmune diseases or allergies. The production of various human antigen-specific GIF molecules is particularly facilitated by knowledge of the antigen of the non-invasive immune response, as in most allergies and various autoimmune diseases.

The human PLA2-specific GIF of the present invention was obtained from the AC5 cell line deposited under ATCC Accession No. HB 10473 (or having the identity characteristics of an antigen-specific GIF produced by this cell line). The Japanese cedar pollen-specific GIF of the present invention was obtained from the 31E9 cell line (or has the identity of an antigen-specific GIF molecule produced by this cell line).

Methods of producing and characterizing hybridomas

General methods for producing hybridomas are well known (Kohler et al., European J. Imm., 292, 1976). Short:

mononuclear peripheral poems (PBMCs) from human allergic to bee venom were cultured in the presence of chemically modified PLAz. Non-adherent cells were harvested and cultured with IL-2 and lipocortin-1 prior to fusion with the BUC lymphoblast cell line. The hybridomas were fused to produce human PLA2-specific GIF.

More generally, the present invention relates to a process for the production of a continuous hybridoma cell line producing a human antigen-specific GIF, comprising the steps of:

(a) recovering human antigen-mobilized T cells, activated for said antigen and cultured in the presence of IL-2 and phospholipase A2 inhibitor; and (b) fusing the activated T cells with a fusion partner cell line to generate hybridomas capable of producing human antigen-specific GIF.

Antigen-mobilized T-cells can be obtained from any sample containing a mononuclear cell fraction of peripheral blood. The antigen-mobilized T cells are then activated by culturing in the presence of the antigen to which they have been mobilized, and then the activated T-cells are cultured in the presence of interleukin-2 (IL-2) and phospholipase A2 inhibitor. A particularly useful phospholipase for this purpose is the inhibitor lipocortin. Other synthetic compounds having PLA2 inhibitory activity may also be used,

1.1 such as 2- (p-amylcinnamoyl) amino-4-chlorobenzoic acid (0NO-RS-082, ONO Pharmaceutical Co.).

In some cases, such as when the primary antigen is toxic to T cells, chemical modification of the antigen may be desirable. Suitable agents for such modifications are, for example, guanidine hydrochloride and cyanobromide, but one skilled in the art can readily ascertain the utility of other agents. Generally speaking, agents that do not disrupt the surface structure of the antigen are desirable, since it is believed that this surface structure is essential for antigenic epitope recognition by suppressor T cells. However, for most antigens, such as many non-cytotoxic allergens, this problem is not significant. Consequently, in the case of typical allergens, the original molecules can be used to stimulate T cells.

The present invention relates to the production of antigen-specific human T cells and T cell hybridomas producing a human antigen-specific GIF. The said GIF molecules exhibit specific reactivity to the antigen that elicits an immune response to be immunosuppressed.

Isolation of the human antigen-specific GIF producing T cell hybridomas having the antigen specificity of the human antigen-specific GIF of the present invention can be accomplished by conventional screening techniques to determine the elemental response mode of the human antigen L2-specific GIF. For example, in the case of human PLA2-specific GIF, if the tested human antigen-specific GIF suppresses the Immune response in cells derived from a patient allergic to PLAz, then the human PLAz-specific GIF produced by the human antigen-specific GIF tested and with each other.

Whether the human antigen-specific GIF molecules of the present invention have the specificity of the human antigen-specific GIF of the present invention can also be determined by preincubating the human antigen-specific GIF molecule of the invention with the antigen under normal conditions. reactive (e.g., honeybee venom PLAz) and determining whether the tested human antigen-specific GIF antigen binding activity is inhibited. If the human antigen-specific GIF tested is inhibited, it will most likely have the same epitope specificity as the human antigen-specific GIF of the invention.

As used herein, the term "epitope" refers to a determinant capable of specific interaction with the human antigen-specific GIF or monoclonal antibodies of the invention. Epitope determinants are usually chemically active moieties, such as amino acid or sugar side chains, and generally have specific three-dimensional structural properties and specific charge characteristics.

• ·

In another aspect, the invention relates to a process for the preparation of a substantially pure human antigen-specific GIF comprising the steps of:

(a) culturing a continuous hybridoma cell line capable of producing human antigen-specific GIF to produce human antigen-specific GIF; and (b) isolating the substantially pure human antigen-specific GIF from the culture.

The continuous hybridoma cell lines used in the above manner are capable of self-production. During the cultivation of the hybridoma cell line, the hybridoma cells - antigen specific for antigen-specific GIF or CD3 and / or CD3 - are used. T cell receptor antibodies - stimulated with pulse-treated syngeneic macrophages to produce human antigen-specific GIF.

Isolation and purification of human antigen-specific GIF from culture can be accomplished by a variety of methods. An especially suitable method is affinity purification using an antigen (e.g., solid phase coupled) to which the antigen-specific GIF binds. To modify this procedure, two affinity absorption steps are used to thoroughly purify human antigen-specific GIF, if necessary. The isolation of substantially pure, human, antigen-specific GIF consists of the following steps:

(i) reacting the hybridoma cell line culture with a monoclonal antibody specifically reactive with human GlF;

(ii) eluting the human GIF from the monoclonal antibody;

(iii) reacting the eluted GIF with the antigen to which the human antigen-specific GIF binds;

(iv) eluting the human antigen-specific GIF from the antigen; and (v) recovering the human antigen-specific GIF.

Alternatively, the order of the two absorption steps can be reversed, in which case the isolation of the substantially pure human antigen-specific GIF consists of the following steps:

(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 reactive with human GIF;

(iv) eluting the human antigen-specific GIF from the monoclonal antibody; and (v) recovering the human antigen-specific GIF.

Culturing the hybridoma cells in serum-free medium (e.g., ABC) facilitates purification of human antigen-specific GIF. After treatment of the subclone with anti-CD3 antibody, the antigen binding in the supernatant of the culture is followed by hybridoma cell culture in a Protein A coated tissue culture dish.

GIF ion exchanger

Purification by LÖ chromatography. In this case, the isolation of substantially pure, human, antigen-specific GIF consists of the following steps:

(i) contacting the culture supernatant of the hybridoma cell line with an ion exchange material;

(ii) eluting the human GIF from the ion exchange material;

(iii) reacting the eluted GIF with a monoclonal antibody specifically reactive with human GIF, or with the antigen to which the human antigen-specific GIF binds, or both;

(iv) eluting human antigen-specific GIF; and (v) recovering the human antigen-specific GIF.

As such, ion exchange chromatography is used in conjunction with affinity purification to isolate human antigen-specific GIF. For isolation, as described above, either an antigen to which the antigen-specific GIF binds or an antibody reactive with human GIF is used, or both.

DEAE (diethylaminoethyl) Sepharose is used as the ion exchange matrix for purification of antigen-specific human GIF. Virtually all commercially available agarose and cellulose gels, such as polysulphate agarose, quaternary amine (QAE) derivatives, ecteola (epichlorohydrin triethanolamine), TEAE (triethylaminoethyl) derivatives, and aminoethyl (I) are used as ion exchangers. AE) cellulose. (The ion-exchange materials used are not · ·

- 1.6 would be limited to the materials listed below.) The specific binding and elution characteristics of ten different ion exchange matrices are known to those skilled in the art and can be readily determined.

When the culture supernatant of the hybridoma cell line was c. It is added to an anion exchange matrix equilibrated with 20 mM saline (e.g., NaCl), most of the GIF passes through the column and the residue is eluted with up to 60 mM saline. For elution from the DEAE column, a concentration of 20-60 mM NaCl (10 mM Tris) is most preferred.

Particularly useful for affinity purification of human GIF is the monoclonal antibody produced by the 388F1 cell line or other monoclonal antibodies having the specificity of the monoclonal antibody produced by said cell line.

Medical applications of human antigen-specific GIF

The term suppressive means reducing the leverage effect of an unwanted immune response in patients undergoing treatment. An immunosuppressively effective term means that the amount of antigen-specific human GIF used is sufficient to reduce the causes of the disease or symptom resulting from the unwanted immune response.

The applied doses of the human antigen-specific GIF of the invention are high enough to reduce to some extent

I

symptoms of the immune response. Doses should not be high enough to cause disturbing side effects (eg side effects, anaphylactic reactions and the like). Generally, the dosage should be determined by one of ordinary skill in the art, taking into consideration the age, condition, sex and progression of the patient.

In case of any contraindication, the dosage should be determined by the attending physician. Dosage administration is generally - for one or several days at one or more doses per day, from 0.001 to 2 mg / kg / dose, but more preferably from 0.001 to 0.2 mg / kg / dose.

The human antigen-specific GIF of the present invention can be administered parenterally by injection or gradual perfusion. The human antigen-specific GIF of the present invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, transdermally, or into the body cavity.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil) and injectable organic esters such as ethyl oleate. The water

carriers include water, alcoholic / aqueous solutions. emulsions and suspensions containing saline and buffer They contain. Used for parenteral administration carriers a sodium chloride solution, Ringer's dextrose

solution, dextrose and sodium chloride, lactate Ringer, Ill.

·· · * · «. ···· • · · · »<·· · ♦« • · · * «·

- 1.8 greased (saturated) oils. Intravenous vehicles include fluid and diluent supplements, electrolyte supplements (based on Ringer's dextrose solution) and the like. The above materials may also contain preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases and the like.

The invention also relates to a process for the preparation of a human antigen-specific GIF comprising a human antigen-specific GIF which is capable of binding to the antigen.

The invention further relates to monoclonal antibodies specifically reactive with human GIF and to B cell hybridomas that produce them.

As described previously, methods for preparing hybridomas are well known to those skilled in the art. Short; the B cell hybridomas of the invention are prepared by immunizing BALB / c mice with affinity purified human GIF. Following the final immunization, spleen cells are obtained from the animals and transferred to previously lethal irradiated syngeneic BALB / c mice. Recipient syngeneic mice were immunized twice with purified human GIF, and two weeks after the last immunization, spleen cells were fused with the SP 2 / 0-14AG myeloma cell line. The hybridomas are:

screened for the production of monoclonal antibodies reactive with human GIF.

In order to determine the elemental response mode of a given monoclonal antibody, the isolation of hybridomas producing the monoclonal antibodies having the reactivity of the monoclonal antibodies of the present invention may be accomplished by standard shear ligation techniques.

Thus, if the monoclonal antibody tested reacts to human GIF but does not react to mouse GIF, then the antibody tested and the antibody produced by the hybridomas of the invention are equivalent.

Isolation of the 388F1 monoclonal antibody or other hybridomas producing the monoclonal antibodies having the specificity of any of the monoclonal antibodies of the invention can be accomplished by those skilled in the art by producing anti-idiotypic antibodies (Herlyn et al., Science, 232, 100, 1986). An anti-idiotype antibody is an antibody that recognizes unique determinants on the monoclonal antibody produced by a given hybridoma. These determinants are located in the hypervariable region of the antibody. This is the region that binds to a given epitope and thus determines the specificity of the antibody. An anti-idiotype antibody can be produced by immunizing an animal with that antibody. The immune system of the immunized animal recognizes the idiotypic determinants of the immunizing antibody and responds by producing antibodies against these determinants. Used to immunize the next animal by hybridoma

by using anti-idiotypic antibodies specific for the monoclonal antibodies produced by the second animal, it is possible to identify other clones having the same idiotype as the antibody produced by the hybridoma used for immunization, thereby greatly simplifying the production of monoclonal antibodies to the monoclonal antibody producing monoclonal antibodies. screening required.

The idiotypic identity of the monoclonal antibodies produced by two hybridomas proves that the two monoclonal antibodies are equivalent in recognition of epitope determinants. Thus, by using antibodies specific for epitope determinants on monoclonal antibodies, it is possible to identify other hybridomas expressing monoclonal antibodies with similar epitope specificities.

Without undue experimentation, it can be determined whether a monoclonal antibody has the specificity of the 388F1 monoclonal antibody of the invention by determining whether the monoclonal antibody tested inhibits the 388F1 antibody to a specific antigen, such as human GIF (which is normally reactive with 388F1). ). If the monoclonal antibody tested is competent with the 388F1 monoclonal antibody, which manifests itself in a

388F1 binding to a given antigen - it is likely that the two monoclonal antibodies bind to the same epitope.

- 21 Whether a monoclonal antibody has the specificity of the 388F1 monoclonal antibody can also be determined by preincubating 388F with an antigen that is normally reactive (e.g., human GIF) and found to be limiting is the antigen binding ability of the monoclonal antibody tested. If the monoclonal antibody tested is inhibited, it is likely to have the same epitope specificity as the monoclonal antibody of the invention.

In some circumstances, one of the isotypes of monoclonal antibodies may be more suitable than the other isotypes for diagnostic or therapeutic efficacy. Specific isotypes of a monoclonal antibody can be generated directly by - selecting from parent cell fusion or, alternatively, isolating class-switch variants from a parent hybridoma producing a variety of monoclonal antibodies by the so-called sib selection method (Steplewski et al., Proceedings of National, Proceedings of 82. 888653, 1985; Spira et al., Journal of

Immunologocal Methods, Z4, 307, 1984). As such, the monoclonal antibodies of the invention may include classswitch variants having the specificity of the monoclonal antibody of the 388F1 cell line deposited under accession number HB 10472 ATCC. The 388F1 cell line was deposited with the American 'l'ype Culture Collection (ATCC, Rockville, Maryland) for 30 years prior to June 4, 1990.

• ·

As used herein, an antibody is defined as a molecule or fragment of a molecule, e.g. Fab and F (both) taste, which are capable of binding to epitope determinants.

The monoclonal antibodies of the invention can also be used for immuno affinity chromatography used to purify various human GIFs. In one embodiment of the immuno-affinity chromatography, the monoclonal antibodies of the invention are bound on a CNBr-Sepharose-4B or Tresyl-activated Sepharose column (Pharmacia). Solid phase bound monoclonal antibodies specifically bind to human GIF molecules, selecting from other proteins, and thus allow isolation and purification of human GIF. Bound IFN-gamma can be eluted from the affinity chromatography column by methods well known to those skilled in the art, for example using chaotropic agents, low pH or urea.

The foregoing generally describes the invention. The following examples serve to improve the understanding of the invention but do not limit the scope of the invention.

• * • 4 · · • · ι • · · · · · ♦

Example 1: Construction of human antigen-specific glycosylation inhibitor (GIF) producing hybridoma cell lines; cleaning methods

A) Antigens

Lyophilized phospholipase A2 (PLA2) from bee venom a

Purchased from Sigma Chemical Co., St. Louis, MO. Denatured PLA2 (D-PLA2) and Cyanogen-Treated PLA2

King et al., Arch. Biochem. And Biophys. 172. 661, 1976. D-PLA2 was prepared by dissolving 5 mg PLA2 in 0.1 M Tris HCl pH 8.6 and denaturing with 6 M guanidine hydrochloride in the presence of 5 mg / ml dithiothreitol.

After standing for 1 hour at room temperature, the sulfhydryl groups were carboxymethylated with iodoacetic acid. The denatured protein was dialyzed against 0.02 M acetic acid and kept at -40 ° C until use. To cleave the methionine bonds in PLAz, 10 mg of bee venom PLA2 was dissolved in 0.4 mL of distilled water and 1.2 mL of formic acid containing 100 mg of CNBr was added. After 2 hours at room temperature, the mixture was diluted twice with water and lyophilized in Speed Vac. The original PLA2 was bound on a Tresyl-activated Sepharose column (Pharmacia) according to the manufacturer's instructions.

Unless otherwise noted, the protein-Sepharose ratio is 1 mg / ml.

• ·

B) Antibodies

Purified human E myeloma PS protein, monoclonal mouse IgE derived from H1 DNP-E-26 hybridoma (Liu et al., J. Immunol., 124, 2728, 1980) and monoclonal anti-CD3 (OKT 3) preparations were prepared by Carini et al. (J. Immunol. Methods, 127, 221, 1990). Abdominal fluid containing αβ, WT 31C monoclonal anti-T cell receptor (Spits et al., J. Immunol., 135, 1922, 1985) was described by Dr. J. DeVries, DNA Institute of Molecular and Cellular Biology, Palo Alto, CA. made it available to us. A monoclonal mouse antibody preparation against rabbit lipomodulin 141B9 (Iwata et al., J. Immunol., 132, 1286, 1984) was described in a previous article by Askasaki et al., (J. Immunol., 131, 3172, 1986). Specifically purified goat antibodies against mouse IgG containing antinehesis (Γ) chain and anti-light chain have been previously described by Suemura et al., (J. Immunol., 125, 148, 1980). Fluorescein-treated goat anti-mouse IgG antibodies were obtained from Cappel. Human IgE and anti-lipomodulin antibody (141B9) were bound to CL-Sepharose 4B; Approximately 5 mg of protein was coupled to 1 ml of Sepharose.

C) Cell lines

RPMI 8866 lymphoblastoid cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 µM 2-mercaptoethanol and antibiotics. The 12H5 mouse T-cell hybridoma cells (Iwata • · · »* ♦« · «* ♦« «· · · ·

25 et al., J. Immunol., 140, 2534, 1988) in high glucose Dulbecco's modified Eagle's medium (DMEM - Huff et al., Proc. Natl. Acad. Sci. USA, 129, 509, 1982). . Hypoxanthine-guanine phosphoribosyl transferase-deficient mutants in CEM (BUC) human lymphoblastoid cell line cells have been previously described (Huff and Ishazaka, Proc. Natl. Acad. Sci., USA, 1514, 1984).

D) Cell culture and production of hybridomas

Peripheral blood was collected from patients with allergies to the uterine venom and collected by centrifugation (Ficoll-Pague apparatus, Pharmacia) to obtain mononuclear blood cells (PBMCs). To activate antigen-mobilized T cells, PBMC was suspended in RPMI 164u at 3 x 10 <RTI ID = 0.0> ^e / ml </RTI> and cultured for 3 days in the presence of 10 / ml D-PLA2 or CNBr-treated PLA2. The non-adherent cells were harvested, resuspended in fresh medium (2 x 10 ^B cells / ml) and then, in the presence of 3 ng / ml recombinant human lipocortin 1 (provided by Dr. J. Browning and Dr. B. Pepinsky). days were cultured with 60 units / ml purified IL-2 (chromatographically purified human IL-2; Electro-nucleonics, Silverspring, MD).

For the production of T-cell hybridomas, 1.2 x 10 ^T cells cultured with IL-2 were mixed with twice the amount of BUC cells. The mixed cells were fused to each other and polyethylene glycol (1300-1600 MW, Sigma)

- We used it to illusion using 2.

for a detailed description of cell fusion see Ref. Huff et al., Proc. Natl. Acad. Sci., USA, fiA, 1514, 1984. The cells were resuspended in hypoxanthine / aminopterin / thymidine (HAT) -containing DMEM and 5 x 10 ⁴ cells per pocket were placed in a 96-pocket plate. The hybrid clones were maintained in DMEM complete with two-week secondary culture. For T cell hybridomas stimulation the cells were treated with 8 ug / ml OKT 3 antibody 0 ^E C for 40 minutes, the antibody-treated cells (1 x 10 ^e pc / ml) 10 ug / ml anti-MGG- inoculated Limbro tissue culture pockets (Flow Labs, McLean, VA). The culture supernatant was removed after 24 hours.

E) CD3 and Tcr detection

Hybridoma cells (1 x 10 ® db / sample) in RPMI 1640 medium supplemented with 5% FCS and 10 µM NaN3 at 0 ° C for 40 min with 8 µg / ml OKT 3 or 1: 1000 dilution -TcRa13 (incubated with abdominal fluid containing WT31 /. As control, the same cells were treated with a similar concentration of mouse IgG2a antibody (Becton-Dickinson, isotype control). The cells were washed twice in PBS containing 5% FCS and then fluorescein-treated incubated for 40 min. After additional washes, cell fluorescence was analyzed with a FACScan (Becton-Dickinson).

· * ·· ♦♦ ·· ♦ · Λ * · · · «« · * · «♦ * ·«

- 27 CD3 · * · hybridoma cells were identified by rosette formation using buffalo erythrocytes coated with anti-mouse IgG (rosetting). The antibodies were described by Wilhelm et al. (J. Immunol. Methods, Sü, 89, 1986) were coupled to erythrocytes. Short; 0.5 ml of concentrated buffalo erythrocyte was washed four times with saline, and the cells were resuspended in 0.75 ml of 0.5 µg / ml purified anti-MGG. To the cell suspension, 25 µl of CrCl3 solution (16.5 mg of CrCl3 in 5 ml of brine) was added with gentle agitation and the suspension was incubated at 30 ° C for one hour. The anti-MGG coupled erythrocytes were washed four times With saline and resuspended in 5 ml FCS (approximately 1 x 10 ^ö erythrocytes / mL, respectively). To identify CD3 * cells, 10® hybridoma cells were suspended in 80 µl DPBS containing 5% FCS and 8 µg / ml OKT 3. Following a 45 minute wait at 0 ° C, the cells were washed twice, then resuspended in 80 µl DPBS containing 5% FCS and a 20 µl suspension of anti-MGG coated erythrocytes and crystal violet was added to the cell suspension. The mixture was centrifuged at 200 g for five minutes and the vials were incubated at 0 ° C for two hours. The compacted cells were carefully resuspended and examined under the microscope for rosette-forming cells.

F) Growth of CD3 · * · cells

Cells treated with 8 µg / ml OKT 3 antibody (1.5 x 10 x db) were treated with anti-MGG-bound erythrocytes (approximately 4 x

· ♦ 990 •

• · t ·

10 ^θ ) was treated to form rosettes in cells using the method described below. The compacted cells were resuspended and applied to the top of a Percoll density gradient centrifuge containing 60% and 50% Percoll layers. The vials were centrifuged at room temperature at 1200 rpm (700 g) for 20 minutes. The concentrated cells were washed twice in culture medium and the erythrocytes were lysed at 0 ° C, 0.63% NH 4 Cl buffer treatment for one minute. Cells were washed in DME medium, resuspended and cultured to increase cell population.

Further growth of CD3 * cells was performed by cell sorting. The hybridoma cells were treated with OKT 3 antibody and stained with fluorescein-treated anti-MGG. Positive staining cells were selected by cell sorting using FACSTAR (Becton-Dickinson).

G) Purification and detection of IgE-BF

The culture supernatant of the Ί'-cell hybridomas was filtered through a Diaflo YM 100 membrane filter (Amicon Corp., Lexington, MA) and the filtrate was concentrated to 10 times ultrafiltration through the YM5 membrane. The IgE-BF in the filtrate was purified on an IgE-loaded Sepharose column (Ishizaka and Sandberg, J. Immunol. 126, 1692, 1981). IgE-BF present in the culture filtrate or in the fraction eluted with acid from the IgE-Sepharose column was determined by measuring the FceR * B lymphoblastoid. «·

- Rosette formation of RPMI 8866 cells of the z9 cell line was inhibited by human IgE-coated buffalo erythrocytes (E-IgE) (Kisaki et al., J. Immunol., 138, 3345, 1987). The ratio of rosette-forming cells (RFCs) in 300 RPMI 8866 cells was determined three times and expressed as mean ± standard deviation (± SD).

Detection of murine IgE-BF produced by 12H5 cells was also performed according to the above procedure, except that rat IgE-coated buffalo erythrocytes were used as indicator cells and that Lewis strain infected with Nipportronngylus brasiliensis was used as the source of FceR * B cells. rats used mesenteric lymphoid cells (Yodoi and Ishazaka, J. Immunol. 124, 1322, 1980).

H) Detecting GIF

Detection of GIF was performed using 12H5 T cell hybridoma cells (Iwata et al., J. Immunol. 140: 2534, 1988). The hybridoma cell suspension was mixed with an equal volume of test sample and the cell suspensions were cultured with 10 µg / ml mouse IgE for 24 hours. The culture supernatant was filtered through a CF50A membrane filter and the IgE-BF containing filtrate was fractionated on lens lectin Sepharose (Yodol et al., J. Immunol. 125: 1436, 1980). Both unbound proteins (effluent fraction) and proteins eluted with 0.2 M α-methyl mannoside (eluate fraction) were assayed for the presence of IgE-BF (rosette formation.

- 30 • «ν <

inhibitory process). When 12H5 cells were alone cultured with mouse IgE, substantially all of the IgE-BF produced by the cells was bound to the lens lectin Sepharose and could be eluted with α-methyl mannoside. As such, the percent rosette inhibition ratio between effluent / eluate fraction is less than 0.2. When sufficient amount of GIF was added to mouse IgE-cultured 12Hb cells, most of the IgE-BF produced by the cells lost its affinity for lens lectin and appeared in the effluent fraction (Iwata and Ishizaka, J. Immunol. 198Θ). As such, GIF detection is positive when the percent rosette inhibition ratio between the effluent / eluate fraction is 3.0 or more.

I) GIF fractionation

In order to determine if the derived GIF has bee venom hybridoma cell culture filtrates from the hybridomas

It is linked to the antigen by PLA2 affinity

Fractionation on Sepharose. Hybridoma cells were treated with 8 µg / ml OKT 3 antibody and 8 ml (1.5 x 10 10 cells / ml) of the supernatant of antibody-treated and untreated cell suspensions was cultured in anti-MGG coated tissue culture flasks. The culture supernatant was concentrated to a 4-fold concentration and 2 ml of the sample was absorbed in 0.4 ml IgESepharose. The effluent fraction was mixed with 0.5 ml PLA2-Sepharose overnight and the immunosorbent was loaded into a small column. After removing the effluent fraction, the column was washed with DPBS and then 1.0 mL ····

- eluted with 31 glycine-HCl buffers, pH 3.0. Partial purification of GIF antilipomodulin (141B9) on Sepharose was performed by Akasaki et al. (J. Immunol., 186, 3172, 1987).

J) Determination of phospholipase inhibitory activity

The affinity-purified GIF is described by Uede et al. (J. Immunol., 1989, 139, 898) was treated with alkaline phosphatase. Short; 1 ml of the preparation was dialyzed against Tris-HCl buffer (pH 8.2), mixed with one unit of insoluble alkaline phosphatase (bovine intestine, Sigma) and kept at room temperature for two hours. After centrifugation, the supernatant was dialyzed against 0.1 M Tris-HCl pH 8.0. Phospholipase A2 inhibitory activity of alkaline phosphatase-treated samples was determined using ³ H-oleic acid biosynthetically labeled E. coli and porcine pancreas PLA2 (Sigma) (Rothut et al., Biochem. Biophys. Commun., 117, 878, 1983). See the procedure for details. Ohno et al.

Internat. Immunol., JL, 425, (1989). Short; porcine pancreatic PLA2 (1 x 10 ~ ^B units) was mixed with GIF in a final volume of 150 µl, and after 5 minutes at 25 ° C, 50 µl of ³ µl of E. coli suspension (5000 cpm) was added and the mixture was added. ⁰ were incubated at 25 C for five minutes. The reaction was stopped by the addition of 50 µl of 2M HCl and then 50 µl of 100 mg / ml BSA was added. The suspension was centrifuged for one minute at 5500 g and the radioactivity was measured in 250 ul of the supernatant by a scintillation spectrometer.

.52

K) Ion exchange column chromatography The culture supernatant of AC5 cells cultured in serum-free tap medium was concentrated to 25-100 fold by ultrafiltration. After centrifugation for 20 minutes at 10,000 rpm, the supernatant was diluted to eight times its concentration with distilled water.

With Tris pH

Adjusted to 8 and immediately afterwards 10 mM

With Tris-HCl buffer, pH

8.0) balanced with a volume of 3 ml

The effluent

DEAE-Sepharose was applied to a CL-6B (Pharmacia) column.

After removal of the fraction, the column was washed with 4 column volumes of 10 mM Tris-HCl buffer containing 20 mM NaCl solution and the washed wash solution was added to the effluent fraction. The column-bound proteins were successively quenched with 10 mM Tris-HCl buffer (pH = 4) containing 50, 75, 100, 150 and 200 mM NaCl solution.

8.0). All eluate fractions were concentrated and

It was dialyzed against Dulbecco's phosphate buffered saline (DBPS).

L) Gel filtration

One ml of sample (in DPBS) was applied to a Superose 12 column (1.6 x 50 cm, Pharmacia) attached to HPLC (Beckman, System Gold). Proteins were eluted from the column with DPBS at a flow rate of 1 ml / min and the appropriate fractions were collected. Column with human IgE (PS protein, M = 185000), bovine serum albumin (BSA, M = 67000),

- Calibrated with 33 ovalbumin (M = 43000), soybean trypsin inhibitor (M = 20100) and cytochrome C (M = 12500). All standard proteins except IgE were obtained from Sigma. The retention time, in the order of the proteins, was 41.97; 52.08; 55.135; 62,097 and 71.67 minutes respectively.

M) Affinity purification of GIF

The supernatant of the CL3 clone cultured in DME medium was concentrated to 5-fold by ultrafiltration, and the GIF in the supernatant was absorbed on 141B9-Sepharose or anti-GIF Sepharose by resuspending the supernatant in 5 ml overnight. Iwata et al., J. Immunol. 141: 3270 (1988). The immunosorbent was then washed with 20 column volumes of DPBS and the bead-bound proteins were eluted with 0.1 M glycine-HCl buffer (pH 3). Mouse GIF from 231F1 cells was also purified by the above procedure using 141B9-Sepharose.

In order to isolate GIF from the culture supernatant of AC5 cells cultured in protein-free medium, the supernatant was concentrated to 50-100 fold by ultrafiltration. Appropriate fractions of DEAE-Sepharose passage supernatant were concentrated to 5-6 mL and mixed at 4 'C overnight with Affigel 10 immunocorbent coupled with monoclonal anti-GIF antibody. The suspension was filled into a small column and the immunosorbent was 40 column volumes.

- Washed with 34 DPBS and 20 column volumes of PBS containing 0.5 M NaCl. The proteins bound to the immunosorbent were eluted with 0.05 M glycine-HCl buffer (pH 3.0-3.2) containing 0.15 M NaCl.

N) Detection of GIF by SDS-PAGE

The affinity purified GIF was dialyzed against 0.01% SDS in deionized water and lyophilized in a Speed vac (Savant Instruments, Hicksville, NY). The samples were then analyzed by SDS gel electrophoresis on a 15% polyacrylamide gel plate using the Laemmli system (Laemmli, J.K., Naturre, 227,680, 1970). The gel plates were fixed and the protein bands stained with silver (Ochs et al.

Electrophoresis, 2,

304, 1981). Molecular weight standards were obtained from Pharmacia.

0) ELISA assays

For the detection of monoclonal anti-GIF antibodies, with slight modifications, Steele et al. (J. Immunol., 142, 2213, 1989). Short; Immunol I plates (Dynatech) were coated with 100 µl of affinity purified GIF diluted in overnight treatment with 0.1 M carbonate buffer pH 9.6. The plates were washed three times with phosphate buffered saline (PBS) containing 0.05% Tween 20 three times. Plates were exposed to 2% BSA in PBS for 6-9 hours

- 35 recorded. Subsequently, 100 µl of each sample was filled into the pockets of the plates and the plates were kept at 4 ° C overnight. Mouse Ig binding was detected using alkaline phosphatase-linked goat anti-mouse Ig (Zymed Foot, So. San Francisco, CA) and alkaline phosphatase substrate (Sigma). The ELISA signal was determined 30 minutes after substrate addition using a 410 nm filter on an MR 5000 reader (Dynatech Foot). The isotype of the monoclonal antibodies was determined by the ELISA method of the mouse mAb isotyping kit (Zymed Foot).

In order to increase sensitivity, a biotin-avidin signal amplification method (Stanley et al., J. Immunol. Methods, 83, 89, 1985) was used to detect GIF in fractions of affinity purified GIF preparation. Maxi-Sorp microtiter plates (Nunc, Copenhagen, Denmark) were coated with 50 ul of each fraction. After two hours incubation at 37 ° C, the plates were washed with Tween / PBS and fixed overnight (4 ° C) in 2% BSA. After another wash, biotin-linked mAb 141-B9 (200 ng / ml) was added to each pocket and the plates were incubated at 37 ° C for two hours. After washing the plates again, 50 µl of a 1: 1500 dilution of streptavidin-alkaline phosphatase conjugate (Zymed Foot) was added to each pocket.

After one hour of incubation at 37 ° C, the amount of alkaline phosphatase bound to the pockets was determined by a signal amplification system (GIBCO-BRL, Bethesda, MD) (Stanley et al., J. Immunol. Methods, β3, 89, 1985). . ELISA Signals were determined at 490 nm.

Example 2: Characterization of human antigen-specific GIF-producing hybridomas

As described previously, peripheral mononuclear cells (PBMCs) from a patient allergic to bee venom were cultured for 3 days in the presence of 10 lag / ml D-PLA2 and activated T cells in the presence of 3 µl / ml recombinant lipocortin. for four days. Subsequently, T cells were fused with BUC cells to produce hybridomas. Four hybridoma clones were produced during the experiment. Each hybridoma clone was cultured in complete DMEM and the culture supernatants were filtered through a YM100 membrane filter. The filtrates were concentrated to a 10-fold concentration and assayed for the presence of GIF using 12H5 cells. The results shown in Table I indicate that two of the four hybridoma clones secreted significant amounts of

GIF.

- .17 L. tablazat

Selection of GIF-producing hybridomas

jd-oil acid ^ emission hybridoma GIF Activity * Effluent / effluent Emission (Cpm) inhibition (%)

cl 1 0/26 (-) ND - cl 2 2/33 (-) 390 ± 27 4 cl 3 29/0 (+) 257 ± 25 37 cl 7 27/5 (+) 303 ± 17 26 control 0/31 b 408 ± 15 -

“The culture filtrate of each clone was concentrated to a concentration of ten times. Units of filtrate were added to an equal volume of suspension of 12H5 cells and cultured for 24 hours in the presence of 10 µg / ml mouse IgE. The values listed are expressed as a percentage of the rosette inhibition of effluent / eluate fractions from lens lectin Sepharose. In the absence of IgE-BF, the IgE-RFC ratio was 24.4 ± 0.3 (SD)%.

* => 12H5 cells were cultured with mouse IgE alone (10 ng / ml)

38 and IgE-BF in culture tiltrates were fractionated on lens lectin Sepharose.

^c The culture filtrates were fractionated on 141B9-Sepharose and the acid eluates taken from the immunosorbent were concentrated to 1/100 of the original culture supernatant volume. Samples were treated with alkaline phosphatase and the ability of dephosphorylated components to inhibit pancreatic phospholipase A2 was determined. (ND = not determined).

The presence of CD3 determinants on the CL3 hybridoma clone was determined by cytofluorometry and the rosette-forming method. Cells were treated with 8 ng / rnl monoclonal antibody 0KT3 and then stained with anti-mouse Ig, percent of which were stained with antibody, and thus, rosette-forming cells with anti-MGG were stained.

UD3 * cells by fluorescein-treated goat Less than ten cells, and only 6-8% of OKT3 monoclonal, formed with coated erythrocytes. was increased in Example 1. Cells forming rosettes with anti-MGG-coupled erythrocytes were separated from the rest by density gradient centrifugation (Percoll layers) and grown in complete DMEM. The above steps were repeated three times in succession to further increase the CD3 * cell population. Following the above steps, cells were treated with 0KT3 antibody and incubated with antiMGG-coated erythrocytes, showing 80-90% of the cell population showing rosette-forming activity. With 0KT3

- 9 cells treated with cytofluorometry for approx.

% stained. However, if the cell cultures were performed within two weeks of the four selections a

By reducing the proportion of CD3 * cells to about 52 percent (as determined by cytofluorometry), the CD3 * population was further increased by cell sorting and cell culture. The cell classifications were repeated twice to obtain a CL3 population that consistently expressed CD3. Fluorescence staining of the population with 0KT3 and WT31 (ant i-TcRa (3)) showed that virtually all cells expressed CD3 and most cells expressed '1'cRa6. CD3 * and CD3 cells were cultured and the filtrates of the cultures were tested for the presence of GIF using 12H5 cells, and GIF activity was only detected in the filtrate of the culture of CD3 * cells, and the results indicate that CD3 · '· cells serve as a source of GIF.

Because of the unique properties of mouse GIF that monoclonal anti-lipomodulin (141B9) binds lymphokine, we decide to determine whether human CL3-derived

GIF can be absorbed on 141B9-Sepharose. THE

CD3 * CL3 clone was cultured to obtain a liter of culture supernatant.

After filtration through a YM100 membrane filter, the filtrate was concentrated to 1 mL and 1 mL

Fractionated on 141B9-Sepharose. After removal of the effluent fraction, the immunosorbent was washed with 10 column volumes of DPBS and eluted with 5 column volumes of glycine-HCl buffer (pH 3). Following dialysis against DPBS, GIF between fractions was determined using 12H5 cells.

J

- 40 activity breakdowns. II. The results shown in Table 1A show that substantially all of the GIF in the culture filtrate was bound to 141B9-Sepharose, which was then isolated by acid elution.

II. spreadsheet

Anti-lipomodulin Sepharose-affinity purified human GIF from CL3 clone ·

Fractions removed from 141B9-Sepharose ⁰ Dilution GIF activity ⁰ effluents / eluate effluent 1:10 0/31 (-) Washer 1:40 0/35 (-) E'luátum 1:10 42/0 (+) 1:40 45/0 (+) 1:80 39/0 (+) Control medium - 0/34

The culture supernatant of the CL3 clone was filtered through YM100 membranes and the filtrate was concentrated to 200-fold.

5 ml of the concentrated filtrate are 1 ml

141B9-Sepharose-on fractional needles.

I

- the immunosorbent was washed with 5 column volumes of CPBS-sei ^to 41 after removing the effluent fraction, and then eluted with 5 column volumes of glycine-HCl buffer (pH 3.0).

° GIF activity was determined using 12H5 cells as described in Table I. The values listed are the percent inhibition of rosette (formation) of effluent / eluate fractions from lens lectin Sepharose. In this assay, the IgE-RFC ratio in the absence of IgE-BF was 22.9 ± 0.6 (SD)%. (+) Indicates the presence of GIF.

Previous studies have shown that murine GIF is a phosphorylated derivative of a phospholipase inhibitor protein (Uede et al., J. Immunol., 1989, 139, 898). As such, the GIF in the filtrate of the culture of the CL3 clone was purified on 141B9-Sepharose. The culture filtrate of the other three clones (CL1, CL2 and CL7) was similarly fractionated on 141B9-Sepharose. The acidic eluates taken from the immunosorbent were treated with alkaline phosphate stearate and tested for the ability of GIF in the eluate to inhibit the release of ³ H-oleic acid and R Biochem. Biophys. Rés. Commun., 117, 78, 1983). The I.

The results reported in Table II show that the affinity purified GIF from clones CL3 and CL7 showed phospholipase inhibitory activity, whereas similar GIF from clones CL1 and CL2 did not inhibit phospholipase A2.

Example 3: Antigen binding characteristics of GIF

Previous studies have shown that antigen-activated T cells cultured with IL-2 in the presence of lipocortin significantly produced no PLA2 affinity in the uterine venom.

GIF, but crossing the same cells with CD3, resulted in the formation of GIF and IgE-BF with affinity for antigen-linked Sepharose. Based on these experiences, we intended to determine that

Clone CL3 produces antigen-binding GIF and IgE-BF. Cells were treated with 0KT3 antibody at 0 ° C and cultured in treated (1.5 χ 10 ® cells / ml) pockets coated with anti-MGG. As a control, untreated CL3 cells were cultured in anti-MGG coated pockets. The supernatants of the cultures were filtered on YM100 membranes and concentrated to 7X by ultrafiltration. Concentrated filtrates overnight were 1 ml

It was absorbed on IgE-Sepharose and the fraction of unbound proteins was pooled with 2 mL of wash solution. The IgE-Sepharos was thoroughly washed and eluted with glycine-HCl buffer. Subsequently, using RPMI 8866 cells as the source of FceR * cells, it was determined whether the eluate fraction taken from IgE-Sepharose contained IgE-BF.

III. spreadsheet

GIF from clone CL3 is unable to bind bee venom PLA2

GIF activity on PLA2-Sepharose Treatment* IgE-BF * ³ (%) eluate Wash Eluate 0KT3 23 34/0 (+) 21/0 (+) 0/24 (-) no 0 28/0 (+) 22/13 (±) 0/26 (-) * Treatment without (test) or Treated with CD3 antibody

cells were grown in pockets coated with anti-MGG.

^b 30 ml culture supernatant was filtered through YM100 membrane filter, concentrated to 4 ml and the filtrate was concentrated. Samples were absorbed in 1.0 ml IgE-Sepharose. The acid eluate fraction was added to 4.0 mL and the amount of IgE-BF was determined by rosette inhibition. In the absence of IgBF, the IgE-RFC ratio was 37.7 ± 1%.

<= 1.0 ml of the effluent fraction taken from IgE-Speharose was fractionated on PLA2-Sepharose. The effluent, wash and acid eluate fraction was added to 1.3 ml and assayed for GIF activity using 12H5 cells. The values listed are those obtained from lentil lectin Sepharose

• 4

expresses the percentage of inhibition of rosette (formation) of effluent / eluate fractions. In the absence of IgE-BF, IgE-

RFC ratio 21.7 ± 0.6 (SD) was%. results show that In III. table published treated with anti-CD3 cell produced Verb- -BF while it is

cells not treated with antibody did not produce detectable levels of IgE-BF. The effluent fraction from IgE-Sepharose was concentrated to twice the concentration and 1 ml samples were plated on a 0.25 ml PLA2-Sepharose column for fractionation. The GIF activity of the samples was assayed by adding the effluent, wash and eluate fraction to 1.5 ml. As shown in Figure III. GIF from either untreated or anti-CD3-treated cells is not bound to PLA2-Sepharose.

It was thought that GIF from C13 cells treated with anti-CD3 did not bind PLA2 because D-PLA2 was used to activate T cells. To investigate this possibility, several T-cell hybridomas were obtained from mononuclear peripheral blood cells (PBMCs) of bee venom-sensitive patients. The procedure is exactly the same as described above, except that 10 µg / ml CNBr-treated PLA2 is used instead of DPLA2 to stimulate peripheral blood cells. As a result of the experiment, 22 hybridoma clones were obtained. GIF analysis of clone cultures filtrates showed that 10 of the 22 clones produced significant GIF (results not reported). Seven GIF-secreting clones

OKT3 was treated and antibody-treated cells were cultured in anti-MGG coated dishes. The filtrates of the cultures were concentrated to a 4-fold concentration and absorbed on IgE Sepharose.

ARC. spreadsheet

Production of antigen-binding GIF by anti-CD3-treated hybridoma cells®

clone IgE-BFt> (%) GIF activity on PLAz-Sepharose ⁰ effluent eluate AC5 20 0/21 (-) 31/0 (+) AF10 36 19/0 (+) 0/21 (-) BA6 8 29/0 (+) 0/24 (-) BE 12 65 0/31 (-) 25/0 (+) BF5 65 0/27 (-) 20/0 (+) CB7 64 0/28 (-) 17/0 (+) CE5 58 0/28 (-) 35/0 (+)

® · 1.2 x 10 ⁷ db. cells were treated with OKT 3. The cells were resuspended in 8 ml of medium and placed in an anti-MGG-coated flask. The culture supernatant was concentrated to 4X concentration, followed by IgE-Sepharose; .ι absorbed.

Effluent fractions derived from IgE-Sepharose

PLA2-Sepharose was fractionated and the effluent and eluate fractions were determined.

GIF activity.

^b The acid eluate fraction from IgE-Sepharose was assayed for the presence of IgE-BF. In the absence of IgE-BF, the IgE-RFC ratio was 23.6 ± 0.6 (SD)%.

® GIF activity was determined using 12H5 cells. The values listed are the percent inhibition of rosette (formation) of effluent / eluate fractions from lens lectin Sepharose. In the absence of IgE-BF, the IgE-RFC ratio was 26.0 ± 0.7 (SD)%.

As shown in FIG. As shown in Table 6, six of the seven clones contained a detachable amount of IgE-BF in the eluate fraction. The effluent fractions from IgE-Sepharose were subsequently fractionated on PLA2-Sepharose and analyzed for GIF activity in effluent and eluate fractions taken from the immunosorbent. The IV. The results shown in Table 5 show that five of the seven clones produced GIFs, most of which bound to PLA2-Sepharose (which were isolated by elution at acidic pH η). To confirm that CD3 crossing is required for the above clones to produce antigen-binding GIF, the five clones in question were cultured in anti-MGG coated pockets without anti-CD3 treatment. As expected, culture supernatants did not contain IgE-BF and GIF in the supernatants did not bind to PLA2-Sepharose.

The present invention provides a method that permits the development of GIF-producing T cells from peripheral mononuclear cells of patients allergic to PLA2, and promotes the generation of GIF-producing hybridomas from these T cells. Typical hybridomas express CD3 determinants and TCRα6, indicating their T-cell hybridoma. Moreover, the TcR complex on the hybridomas appears to be a functional characteristic. As a result of crossing with CD3, both parental T cells (Carini et al., J. Immunol. Methods, 127, 221, 1990) and most GIF-producing hybridomas (Tables III and IV) produced IgE-BF. -t. Cross-linking of clones CL3 and AC5 using WT31 monoclonal antibody and anti-MGG also resulted in IgE-BF production (results not reported). Further testing of the Typical CD3 * · hybridomas showed that each of clones CL3, BE12, AC5, and CB7 expressed both CD4 and CD8. Because BUC cells used for the production of hybridomas are CD4 * and CD8 (based on a personal statement by Dr. J. Stobo), it is unclear whether the parental T cells of the hybridomas co-expressed CD4 and CD8.

Our experiments showed that some T cell hybridomas produced antigen (PLA2) -binding GIF as a result of crossing with CD3. This experience is consistent

.1 «

with the fact that typical murine GIF-producing hybridomas produced antigen-binding GIF by stimulation with antigen-pulsed syngeneic macrophages or by crossing cells with CD3 (Iwata and Ishizaka, J. Immunol. 141: 3270; 1988; Iwata et al., J.

Immunol., 143. 3917, 1989), and raises the possibility that there may be similarities between antigen-binding GIFs of two species. For mouse GIF, the antigen-binding GIF carrier (antigen) from hybridomas suppressed the in vivo antibody response in a specific manner. It has also been clarified that the antigen-specific GIF produced by the hybridomas consists of an antigen-specific polypeptide chain and a non-specific GIF (Jardieu and Ishizaka, Immune Regulation by Characterized Polypeptides, Goldstein et al., Alan R. Liss, New York, p. 595, 1987), and that the antigen binding chain shares between 14 and 12 antigenic determinants shared by the effector-type suppressor T-cell factor (TseF) (Iwata et al., 1989). Independent experiments have shown that the 141-B9 monoclonal anti-lipomodulin antibody and anti-I-J antibodies bind not only GIF but also TseF and

Also the antigen-binding chain of TsiF (I-J * chain); (Jardieu et al., J. Immunol., 138, 1494, 1986; Steele et al., J. Immunol., 142, 2213, 1989). Taken together, these discoveries raise the possibility that the antigen-binding GIF is identical to TseF.

Parental T-cells of a typical 71B4 mouse Ts hybridoma were homogenized with spleen cells mobilized with ovalbumin.

(Iwata and Ishizaka, J. Immunol., 141, 3270 (1988)). Similarly, the present invention is similar to that of the present invention. a non-specific GIF or PLA2-binding GIF derived from human T-cell hybridomas was bound to 141B9-Sepharose (which has been shown in previous studies to also absorb mouse TsFs; et al., J. Immunol., 141, 2213, 1989) It is possible that PLAz-binding GIF from human T-cell hybridomas may represent human antigen-specific TseF, but it is also possible that the antigen-binding GIF is the equivalent of murine TsiF Experiments in our laboratory have shown that the mouse helper T-cell clone D10.G4.1 is capable of producing antigen-binding GIF when the cells are pre-phospholipase nhibitor and subsequently stimulated with antigen (conalbumin) pulse-treated antigen presenting cells (Ohno et al., Internat. Immunol., 2, 257, 1990).

It has also been discovered that this antigen-binding GIF binds to

14-30 monoclonal antibodies, which are more specific for TsiF (Ferguson and Iverson,

J. Immunol., 136, 2896,

1986) such as

14-12 monoclonal antibodies. Green et al.

(J. Mol. Cell

Immunol., 2, 95,

1987) also described that

Clone D10.G4.1

Upon stimulation with UV-irradiated antigen-pulsed macrophages, it produced antigen-binding TsF, and this factor, together with additional molecules, induced the production of effector-type, antigen-specific Ts. Because mononuclear peripheral blood cells from allergic individuals contain helper T cells, it is possible that antigen binding GIF from human hybridomas represents TsiF rather than TseF.

Takeuchi et al. (J. Immunol., 141, 3010, 1988) have formed Tse clones from mononuclear peripheral blood cells (PBMCs) from multiple inoculated individuals with multiple doses of homologous antigen. Modulin et al. (Natúré, 222, 459, 1986) Ts clones have also been produced from ulcers in patients with leprosy leprosy. However, prior to the present invention, suppressor activity-mediating effector molecules (TsF) from human Ts cells have not been identified. Similarities between human GIF and mouse GIF suggest that PLA2-binding GIF from human T-cell hybridomas represents TsF from human suppressor cells. Antigen-binding GIF-producing T-cell hybridomas promote the biochemical characterization of molecules. In mice, it has been repeatedly shown that Ts and TsF (antigen-binding GIF) were more effective at suppressing the in vivo IgE antibody response than the IgG antibody response (Ishizaka et al., J. Immunol. 114, 110, 1975). If the allergen-binding GIF from human T-cell hybridomas does in fact represent TsF, it is reasonable to assume that T-cell factor suppresses the response of the parental T-cell donor IgE antibody.

- 51 Example 4: Preparation of hybridoma cell lines producing cedar pollen-specific GIF

Japanese cedar pollen is one of the major allergens in Japan and causes seasonal allergic rhinitis and conjunctivitis in a large portion of the population. In order to further investigate the general applicability of the experience of the present invention to other antigens, methods for generating antigen-specific GIF-producing T cells and T cell hybridomas (described above) were applied to mononuclear peripheral blood cells from individuals suffering from Japanese cedar. .

The major allergen of Japanese cedar (Sugi, Cryptomeria japonica) is a 40 kD molecular weight cryj-1 glycoprotein (Yasueda et al., J. Allergy and Clin. Immunol., 71, 77, 1983). For our study, the allergen was isolated from the cedar pollen extracts with minor modifications by the procedure described by the previous authors. Short; pollen was degreased with ether and extracted three times with 0.125 M ammonium bicarbonate. From the extracts, the carbohydrate was removed with hexadecyltrimethylammonium bromide. The proteins in the extracts were precipitated with 80% saturated ammonium sulfate solution and the precipitate was dissolved in 0.05 M Tris-HCl pH 7.8. After thorough dialysis against Tris-HCl buffer, the protein fraction was loaded onto a DEAE cellulose column (DE-52, Whatman) and the fraction fraction collected was collected. This was concentrated with 0.01 M acetate buffer (pH

the ···:

• ·.:. :

5.0) dialysed and applied to a buffer balanced CM cellulose column (CM-52, Whatman). The column was washed with buffer and the bound proteins were eluted with 0.1 M phosphate buffer containing 0.3 M sodium chloride. The proteins in the eluate were further fractionated on a Sephacryl S-200 HR column by gel filtration to recover the major protein fraction containing the cryj-1 glycoprotein. The major protein of the fraction, as determined by SDS-polyacrylamide gel electrophoresis, had a molecular weight of 42 kDa and had the same amino acid sequence as cryj-1. The protein was coupled to Affigel 10 in a 1.5 mg / ml gel.

Instead of recombinant human lipocortin I, a synthetic phospholipase A2 inhibitor, 2- (p-amylcinnamoyl) amino4-chlorobenzoic acid (ONO-RS-082, 0NO Pharmaceutical Co.) was used. Previous experiments have shown that ONORS-82 is a specific inhibitor of phospholipase A2 and promotes the production of GIF-producing cells in mouse spleen cultures (Ohno et al., International Immunology, 1, 425, 1989). Spleen cells of ovalbumin-immunized mice were stimulated with ovalbumin and antigen-activated T cells cultured with IL-2 in the presence of 2 µM ONO-RS-082 or 3 µg / ml recombinant human lipocortin I, producing GIF, antigen. specific T cells were produced. Antigen stimulated

T-cells and T-cell hybridomas were prepared essentially as described above, with the exception that the purified cryj-1 glycoprotein and / or phospholipase as antigen.

- ONO-RS-082 was used as the 53 Α2 inhibitor. Mononuclear blood cells were obtained from peripheral blood of patients with allergies to pollen of Japanese cedar and suspended in RPMI 1640 medium containing 10% fetal calf serum (FCS). A suspension of mononuclear cells (3 x 10 ^e cells / ml) was cultured for 3 days in the presence of 10 µg / ml cryj-1. Non-adherent cells were harvested, resuspended in RPMI medium containing 10% FCS (3 x 10 ⁶ cells / ml) and cultured for 4 days in the presence of 60 units / ml human IL-2 and 2 μΜ ONO-RS-082. Cells cultured in this manner were harvested and fused with BUC cells to produce hybridomas. The hybridomas were treated with the SPB-T3b monoclonal anti-CD3 antibody (Spits et al., Hybridoma, 2, 423, 1983) and tested for the presence of CD3 in cells by immunofluorescence. Only CD3 + hybridomas were subcloned (by limiting dilution).

The CD3 + hybridoma clones were maintained in complete DME medium containing 10% FCS, and the culture supernatants of each clone were tested for the presence of GIF using 12H5 cells. The results obtained with hybridomas from one patient are shown in Table V. GIF activity was detected in culture supernatants of 31E9, 31B7 and 32B4 hybridomas, while supernatants from 31H6 and 31H3 hybridomas showed weak GIF activity. GIF-producing hybridomas were then treated with anti-CD3 antibody followed by anti-mouse immunoglobulin and cultured for 24 hours. Supernatants from cultures were fractionated on cryj-1-linked immunosorbent. GIF activity in the flow fraction and in the acid eluate fraction was assayed using 12H5 cells. The results shown in Table V show that GIF from 31E9 cells was bound to the cryj-1-Affige immunosorbent and could be detached by elution at acidic pH η, while GIF from 31B7 cells was not bound to the antigen-linked immunosorbent. The results indicate that 31E9 cells, when stimulated with anti-CD3 antibody, produce cryj-1 affinity GIF.

Table V.

Production of Human Cedar Allergen-Specific Hybridomas® ·

GIF activity a cryj- 1 hybridoma % rosette inhibition ¹³ Sepharose -on ^c clone (Effluent / eluate) not knit bound none of them 0/23 0/29 - 31H6 20/13 (±) 5/20 (-) 12/10 (±) 31A11 0/25 (-) ND - 31E9 28/5 (+) 0/22 (-) 20/0 (+) 31H3 23/12 (±) 0/34 (-) 38/16 (±) 31B7 32/5 (+) 20/5 (+) 4/24 (-) 31F7 0/26 (-) ND - 32B4 22/0 (+) 22/14 (1) 38/22 (±)

···· ···: * ··:

The hybridomas are derived from two independent experiments.

^b Culture supernatants of unstimulated hybridomas were assayed for the presence of GIF. Aliquots of 12H5 cells were cultured in culture supernatants of hybridomas in the presence of mouse IgE. The culture supernatant of 12H5 cells was filtered on a CF50A membrane to remove IgE and the filtrates were fractionated on lens lectin Sepharose. The amount of IgE-BF in the effluent and eluate fraction was determined by rosette inhibition. The values listed are the percent rosette inhibition of the effluent / eluate fractions of the lens lectin Sepharose. The (+) and (-) signs indicate the presence or absence of GIF. indicates their absence.

Typical hybridomas were treated with anti-CD3 antibody and culture supernatants were fractionated on cryj-1-linked Affigel. GIF activity in the flow-through (unbound) fraction was determined using 12H5 cells. The filtrate of the culture of 12H5 cells was fractionated on lens lectin Sepharose. The values listed are the percent rosette inhibition of the effluent / eluate fractions of the lens lectin Sepharose. GIF from 31E9 cells was bound to cryj-1-Affigel, which was isolated by elution with acidic pH η. GIF from 31B7 cells was almost non-bound on the cryj-1-Affigei column.

9 · 9 · π6

Example 5: Construction and characterization of hybridoma cell lines producing monoclonal antibodies specific for human GIF.

A) Production and screening of hybridomas

Human GIF in the supernatant of CL3 T cell hybridoma culture was purified using anti-lipomodulin (141-B9) -Sepharose. The affinity-purified GIF was mixed in Freund's complete medium and BALB / c mice were immunized by peritoneal injection of the antigen every two weeks for a total of three times. Two weeks after the last inoculation, spleen cells were harvested from the immunized mice and 1 x ^{7 7} . spleen cells were transferred to 625R γ irradiated syngeneic BALB / c mice. Recipient mice were placed immediately after cell transfer and then two weeks thereafter

We immunized with purified GIF mixed with Freund's incomplete adjuvant.

One week after the booster vaccination, their spleen cells

SP 2 / 0-14AG,

HPRT-deficient

Fused to cell line B. The cells are in HAT medium

Cultured with BALB / c macrophages.

102 different hybridoma clones from culture were selected for murine immunoglobulin production, and the immunoglobulin-forming hybridomas were selected for anti-GIF antibody production by ELISA and 12H5 cells.

Immulon I plates (Dynatech) were coated with affinity purified GIF for ELISA. Control pockets were filled with DPBS. Pocket fastening with 2% BSA

After 57, all pockets were filled with culture supernatants and binding to mouse Ig pockets was determined using alkaline phosphatase-linked anti-mouse Ig antibodies. As shown in FIG. The supernatants of the cultures of 11 hybridoma clones shown in Table 1A gave significant ELISA signals.

VI. spreadsheet

Selection of anti-GIF-producing hybridomas®

Hybridoma lg ELISA GIF activity ^c clone isotype Signal / control ^b Effluent / effluent none of them - 0/0 29/1 (+) 334F IgM 0.195 / 0.003 33/1 (+) 355C IgM 0.388 / 0.012 28/0 (+) 338H IgM 0.316 / 0.050 0/29 (-) 318H IgM 0.149 / 0.046 0/31 (-) 388F IgŰ2a 0.892 / 0.100 0/28 (-) 47 6B IgM 0,100 / 0,020 0/20 (-) 489G IgM 0.174 / 0.00 7/15 (?) 481F IgM 0.460 / 0.092 18/0 (+) 335C IgM 0.203 / 0.073 0/27 (-) 419A IgM 0.542 / 0.15 27/1 (+) 312F IgM 0.533 / 0.029 14/8 (±) medium control - 0/0 0/31

- ο6 “Culture supernatants that tested positive for ELISA were tested for the ability to bind GIF from clone CL3.

^b Ig binding of mouse in culture supernatants of hybridomas to GIF-coated pockets as compared to non-specific binding of Ig in the same supernatants to BSA-coated pockets (optical density at 410 nm).

^c A mixture of culture supernatants of hybridomas with purified GIF was filtered on YM100 membranes and the GIF activity of the filtrates was evaluated. 12H5 cells were cultured with mouse IgE in the presence of each filtrate. The IgE-BF produced by the cells was fractionated on lens lectin Sepharose, and the effluent and / or Sepharose-derived effluent was purified. The IgE-BF content of the eluate fractions was determined by rosette inhibition. Values shown are percent rosette inhibition of effluent / eluate fractions. GIF altered the nature of IgE-BG produced by cells (cf. top and bottom of the column). The (+) sign Indicates the presence of GIF.

The presence of anti-GIF in culture supernatant of 11 hybridoma clone cultures was determined using 12H5 cells (Iwata et al., J. Immunol. 140: 2534, 1988). From the supernatants of the clone culture, the globulin factor was recovered by precipitation with 50% saturated ammonium sulfate solution. After dialysis against phosphate-buffered saline, each fraction was supplemented with 1/5 of the supernatant volume of the original culture. Aliquots of affinity purified GIF from CL3 clone using 141B9-Sepharose were mixed with an equal volume of globulin fraction from each clone and incubated overnight at 4 ° C. The mixtures were then filtered through a YM100 membrane filter and the filtrates assayed for GIF. Aliquots of the 12H5 cell suspension were mixed with equal volumes of filtrates and the cell suspensions were cultured in the presence of 10 µg / ml mouse IgE for 24 hours. To remove IgE, the culture supernatants were filtered on a CF50A membrane and the IgE binding factors in the filtrates were fractionated on lens lectin Sepharose. VI. The results shown in Table 1A show that the culture supernatants of clones 338H, 318H, 388F1, 476B and 335C removed GIF, demonstrating that these hybridomas produced anti-GIF.

B) Purification of human GIF with monoclonal anti-GIF

Of the six hybridoma clones producing the monoclonal antibody to GIF, only 388F1 produced IgG antibody. This hybridoma was subcloned and cultured in Dulbecco's high glucose medium supplemented with 5% FCS. The supernatants of the cultures were concentrated by ultrafiltration and the IgG in the supernatant was recovered using Protein A-Sepharose. To produce an immunosorbent, the monoclonal antibody

- Boo coupled to Tresyl-activated Sepharose. To determine whether the monoclonal antibody is capable of binding molecules that bind to antilipomodulin (141B9) -Sepharose, GIF in culture supernatant was absorbed on 141B9-Sepharose and eluted at acidic pH. The affinity purified GIF was then needled at the Sepharose-on fraction coupled to antiGIF (388F1). After removal of the effluent fraction, the immunosorbent column was washed with 10 column volumes of Dulbecco's phosphate buffered saline (DPBS) and eluted with glycine-HCl buffer (pH 3.0). Serial dilution of effluent and eluate fractions were assayed for GIF activity using 12H5 cells. VII. The results in Table 1A show that the GIF in the acid eluate fraction taken from 141B9-Sepharose bound to the anti-GIF (388F1) -Sepharose immunosorbent, which was isolated by elution with acidic pH η. The results demonstrate that both anti-lipomodulin and anti-GIF bind to human GIF.

VII. spreadsheet

Fractionation of partially purified human GIF

with anti-GIF (388F1) connected Sepharose * Fractions a GIF Activity ^t ' 388F-Sepharose Dilution Effluent / effluent effluent 1:10 0/35 (-) 1:20 0/29 (-) eluate 1:20 39/0 (+) 1:40 26/0 (+) Fraction unionized 1:40 27/0 (+) Medium control 0/27

* GIF in culture supernatants of CL3 clone was purified using anti-lipomodulin Sepharose.

Affinity purified GIF (1.5 mL) was fractionated on 0.75 mL of 388F-Sepharose. After removal of the effluent fraction, the column was washed with 10 column volumes of DPBS and eluted with 3 column volumes of glycine-HCl (pH 3.0).

^to GIF activity using 12H5 cells, IV. was evaluated according to the procedure described in Table II. Values shown are the percent rosette inhibition of effluent / eluate fractions from lens lectin Sepharose. The (+) sign indicates the presence of GIF.

To determine whether anti-human GIF is capable of binding murine GIF, mouse GIF from 231F1 Ts hybridoma cells was purified on 141B9-Sepharose and aliquots of purified mouse GIF on 141B9-Sepharose or 388F- Fractionation on Sepharose. After removal of the effluent fraction, the immunosorbents were washed with 3 column volumes of DPBS and eluted with 3 column volumes of glycine-HCl buffer, pH 3.0. As expected, the GIF was completely absorbed on 141B9-Sepharose, which was removable by acid elution. Neither the effluent nor the wash fraction showed GIF activity. Provided the same GIF preparation

Fractionation on 388F-Sepharose showed weak GIF activity in the effluate fraction. The activity was highest detected in the DPBS wash fraction, whereas the acid eluate fraction showed no GIF activity. Mouse GIF appears to bind to anti-human GIF only with very weak affinity and also dissociate from the immunosorbent upon washing at neutral pH η. These results indicate that the 388F1 monoclonal antibody is specific for human GIF.

- ο3 Ο Purification of human GIF by ion exchange chromatography

C5 cells were subcloned at limit dilution to give CD3 '' clones. These cells were cultured in serum-free ABC medium. Expression of CDS on cultured subclones was confirmed by cytofluorometry. Culture supernatants of CD3 ^- * · subclones

It was concentrated to a concentration of 10-30 fold, and GIF activity was determined in serial dilutions of the formulations.

Based on this, the subclone AC5-23 was selected because a 1: 3 dilution of the supernatant concentrated to a concentration of ten times this subclone altered the glycosylated IgE-BF-forming property of 12H5 cells such that 12H5 cells treated with unglycosylated IgE-BF after treatment. They produced.

Assays were conducted to determine whether human GIF could be purified by ion exchange column chromatography. Supernatant of the AC5 subclone cultured in ABC medium

25 and hair concentrated. Concentrated culture supernatant Aliquot of 10 ml with Tris pH 8.0 We were with distilled water to a volume of eight times

diluted and applied to a DEAE-Sepharose column. Protein bound to the column was eluted with 10 mM Tris buffer containing increasing concentrations of NaCl (see Example 1). Each fraction was concentrated to 10 mL and evaluated for GIF activity.

-

VIII. spreadsheet

Distribution of GIF activity between DEAE-Sepharose fractions

Tris-HCl + Protein- GIF faction NaCl (mM) ^a content (ug) ^e activity ⁰ First 20 65.5 21/0 (+) Second 50 35.0 20/6 (+) Third 75 42.5 7/20 (-) 4th 100 38.5 3/19 (-) 5th 150 41.5 0/21 (-) 6th 200 42.0 0/20 (-) medium control 0/22 (-)

Supernatants from concentrated culture of AC5 cells were diluted to eight times volume with distilled water and applied to a DEAESepharose column. Fraction 1 is a mixture of flow fraction and 10 mM Tris-HCl buffer pH 8.0 containing 20 mM NaCl. The column was eluted stepwise with 10 mM Tris-HCl buffer containing increasing concentrations of NaCl.

^b After concentration of the fractions, the total protein content was determined. After elution with Trispuffer containing 200 mM NaCl, many proteins remained on the column.

^c GIF activity

It was detected using 12H5 cells.

The values listed are the percent rosette inhibition of the effluent / eluate fractions of the lens lectin Sepharose. The

In the absence of IgE-BF, the RFC ratio was 22.6 ± 0.7 (SD)%. The (+) and (-) signs indicate the presence or absence of GIF, respectively.

indicates absence.

As shown in FIG. As shown in Table II, GIF activity was only detectable in the flow fraction and the 50 mM NaCl eluate fraction. Titration of serial dilutions of these two fractions showed that the fraction passed through had greater GIF activity than the 50 mM NaCl fraction.

Independent culture supernatant experiments confirmed the experience of concentrating culture supernatants of AC5 cells to 100-fold concentration, diluting three times with distilled water and passing through a large portion of the culture supernatant GIF on the DEAE-Sepharose column. The flow-through fraction was mixed with 10 mM Tris buffer wash fractions containing 50 mM NaCl and concentrated to the original volume of sample loaded on DEAE-Sepharose. The concentrated

- Titration of the serial dilutions of the culture supernatant from 66 cultures and the passage (50 mM NaCl) fraction showed that a 1:30 dilution of both samples alters the glycosylated IgE-BF production capacity of 12H5 cells, resulting in the non-glycosylated IgE-BF of 12H5 cells. they produce. It was also found that 75-80% of the culture supernatant protein was removed by passage through a DEAE-Sepharose column.

To determine the molecular weight of the GIF, 0.5 ml of the concentrated fraction passing through the DEAE-Sepharose column was loaded onto a Superose-12 column and the proteins were eluted at a flow rate of 1 ml / min. In this experiment, 5 ml fractions were collected and tested for GIF activity using 12H5 cells. GIF activity is shown in Figs. was detected in fraction 9 min. As fractions 6 and 8 showed little activity, further investigation of GIF activity was performed in fractions 6, 8 and 9. GIF was detectable in 1:10 dilution of fraction 9, but not in 1: 2 dilution of the other two fractions. This result indicates that human GIF is likely to have a molecular weight of between 11 kD and 18 kD. In order to more precisely determine the size of the GIF molecules, gel filtration on a Superose-12 column was carried out in a similar manner, except that 1 and 2 g / ml of GIF were used. Fractions of 2.5 ml were collected. The results of three independent experiments showed that most of the GIF appeared in the fraction taken between 68 and 72 minutes. This means that GIF has a molecular weight of 12-18 kD as determined by gel filtration.

Experiments were also performed to identify GIF by SDS-PAGE. Two liters of the supernatant of the hybridoma cultured in ABC medium was concentrated to a 100-fold concentration and fractionated on a DEAE-Sepharose column. Similar to the experiments described above, the concentrated supernatant was diluted three times with deionized water and passed through a DEAE-Sepharose column. The flow-through fraction was concentrated, pre-absorbed on human IgG-linked Sepharose, and the GIF in the fraction was purified by affinity chromatography on 388F-Affigel (see Figs.

Example 1). In some experiments, the acid eluate fraction from the immunosorbent was adjusted to pH 8.0 and the affinity purification on 388F-Affigel was repeated. The SDS-PAGE analysis of the affinity purified GIF was performed in reduced and unreduced state. In the reduced state, the major band of the composition was at a site corresponding to a molecular weight of 14 kD, while in the unaltered state at 15 kD. In addition, we often encountered a band of 67 kD. Part of the affinity purified composition was dialyzed against DPBS and the GIF in the composition was titrated. Assuming 100% GIF recovery during dialysis and lyophilization, the sample analyzed by SDS-PAGE had a GIF titre of 1: 250.

To determine the relationship between the 14 kD molecular weight protein and GIF, the following experiment was performed. GIF in two liters of culture supernatant of AC5 clone was purified by DEAE-Sepharose column chromatography and affinity purified on 388F-Affigel. The acid eluate fractions taken from the immunosorbent were adjusted to pH 8.0, then concentrated to 1 ml by ultrafiltration and fractionated on a Superose-12 column. The activity of each 2.5 ml eluate fraction was determined using 12H5 cells. GIF activity is 67.5-70. minutes was the highest in eluate taken. The presence of GIF in the fraction was confirmed by ELISA using biotin-coupled mAb 141B9. Although the ELISA signal was weak, only the GIF-containing fraction showed it. One ml of the GIF-containing fraction was lyophilized and analyzed by SDS-PAGE. The results proved that the 14 kD peptide was present in the GIF-containing fraction.

deposition

The following cell lines were deposited with the American Type Culture Collection (ATCC) (1301 Parklawn Drive, Rockville, MD, United States):

Deposit

Cell line ATCC deposit number date

388F HB 10472 1990th May 31 AC5 HB 10473 1990th May 31 31E9 HB X 1992nd June 2

(not yet available) • ♦ · <1

- 69 Cell lines were deposited under the terms of the Budapest Treaty (International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations). This will ensure the maintenance of viable cultures for 30 years from the date of deposit. Under the terms of the Budapest Treaty, strains should be accessible through the ATCC; the contract shall ensure unrestricted and continuous access to breeding stock for the public.

Our legal representative agreed that if any of the deposited cell lines, under appropriate conditions, would be lost, destroyed, or otherwise damaged, they would be replaced without delay by specimens from the same strain. The availability of deposited strains does not entitle anyone to practice the invention in violation of patent law.

The detailed characterization of the invention described above is considered sufficient to enable those skilled in the art to practice the invention. The present invention is not limited to deposited cell lines as they serve to simply characterize one aspect of the invention and any other cell line functionally equivalent thereto may be used within the scope of the invention. Deposition does not mean that the description of the invention is not sufficient to enable the invention to be used in any manner.

70, and is not to be construed as limiting the claims which represent the detailed description of the invention. In addition to the foregoing aspects, the invention will be apparent to those skilled in the art from a number of variations which are within the scope of the claims.

Claims (68)

1. A substantially pure, human, antigen-specific, glycosylation inhibiting factor. The glycosylation inhibiting factor of claim 1, wherein said antigen is allergenic. The glycosylation inhibitory factor of claim 1, wherein said antigen is an autoimmune antigen. The glycosylation inhibitory factor of claim 2, wherein said allergen is a poison produced by an animal species. The glycosylation inhibiting factor of claim 4, wherein said poison is a honeybee poison. The glycosylation inhibiting factor of claim 5, wherein said honeybee venom is an allergenic phospholipase A. The glycosylation inhibitor according to claim 6, characterized in that it has antigen-binding specificity for the human antigen-specific glycosylation inhibitor produced by AC5 cell line deposited under accession number HB 10473 ATCC. Glycosylation inhibitory factor according to claim 6, characterized in that it is produced by the AC5 cell line deposited under accession number HB 10473 ATCC. The glycosylation inhibitory factor of claim 2, wherein said allergen is pollen. The glycosylation inhibiting factor of claim 9, wherein said pollen is a tree produced by a tree. The glycosylation inhibiting factor of claim 10, wherein said wood pollen is cedar pollen. The glycosylation inhibitory factor of claim 11, characterized in that it has antigen-binding specificity for the human antigen-specific glycosylation inhibitory factor produced by HBE ATCC accession number 31E9. The glycosylation inhibitory factor of claim 11, characterized in that it is produced by the 31E9 cell line deposited under HB X ATCC. 14. A continuous hybridoma cell line capable of producing a human antigen-specific glycosylation inhibitor. The hybridoma of claim 14 wherein said antigen is allergenic. 16. The hybridoma of claim 15, wherein said antigen is an autoimmune antigen. 17. The hybridoma of claim 15, wherein said allergen is a poison produced by an animal species. 18. The hybridoma of claim 17, wherein said venom is a honeybee venom. 19. The hybridoma of claim 18, wherein the honeybee venom is an allergen phospholipase A2. 20. The hybridoma of claim 19, which is the AC5 cell line deposited under ATCC Accession No. HB 10473. 21. The hybridoma of claim 15, wherein said allergen is pollen. 22. The hybridoma of claim 21, wherein said pollen is a tree produced by a tree. 23. The hybridoma of claim 22, wherein said tree pollen is cedar pollen. ί The hybridoma of claim 23, characterized in that it is a 31E9 cell line deposited under HB X ATCC Accession Number. 25. A method of producing a continuous hybridoma cell line capable of producing a human antigen-specific glycosylation inhibitory factor, comprising: (a) generating activated human antigen-mobilized T cells for said antigen; and (b) fusing the activated T cells with the fusion partner cell line to produce human antigen-specific hybridosomes capable of producing glycosylation inhibitors. 26. The method of claim 25, wherein the antigen-activated human antigen-mobilized T cells referred to in (a) are produced as follows: (i) obtaining a sample containing human antigen-mobilized T cells; (ii) activating the T cells by culturing with the antigen to which they have been mobilized; and (iii) culturing the activated T cells in the presence of interleukin-2 and phospholipase A. 27th The method of claim 26, wherein said phospholipase A2 inhibitor is lipocortin. I • · · ··. · - · .. ·.:. : employed. 28th THE Claim 26 procedure, with that characterized in that the said antigen, to which the T cells mobilizing •happened, chemically modified. 29th THE According to claim 28 procedure, with that characterized in that the said antigen guanidine hydrochloride modified by. 30. The method of claim 28, wherein said antigen is modified with cyanogen bromide. 31. The method of claim 25, wherein the human antigen-mobilized T cells are derived from a mononuclear cell fraction of peripheral blood. 32. The method of claim 25, wherein the fusion partner cell line is a human lymphoblastoid T cell line. 33. The method of claim 32, wherein the lymphoblastoid cell line is a BUC cell line. 34. A process for the preparation of a substantially pure, human, antigen-specific, glycosylation-inhibiting factor, characterized in that: 76 (a) culturing a continuous hybridoma cell line capable of producing a human antigen-specific glycosylation inhibitor in order to produce a human antigen-specific glycosylation inhibitor; and b) isolating the substantially pure human antigen-specific glycosylation inhibitory factor from the culture. 35. The method of claim 34, wherein said continuous hybridoma cell line is as defined in claim 25. A process according to claim 1. 36. The method of claim 34, wherein said hybridoma cell line is exposed to antigen-pulsed syngeneic macrophages or antibodies to CD3 or T cell receptors. 37. The method of claim 34, wherein said human antigen-specific glycosylation inhibitor is thoroughly purified by affinity purification using the antigen to which the antigen-specific glycosylation inhibitor is bound. 38. The method of claim 34, wherein the substantially pure human antigen-specific glycosylation inhibitor is isolated as follows: »···» « (i) reacting the hybridoma cell line culture with monoclonal antibodies specifically reactive with human glycosylation inhibiting factor; (ii) eluting the human glycosylation inhibiting factor from the monoclonal antibody; (iii) reacting the eluted glycosylation inhibitor with the antigen to which the human antigen-specific glycosylation inhibitor is bound; (iv) eluting the human antigen-specific glycosylation inhibiting factor from the antigen; and (v) recovering the human antigen-specific glycosylation inhibitor. 39. The method of claim 34, wherein the essentially pure human antigen-specific glycosylation inhibitor is isolated as follows: (i) reacting the hybridoma cell line culture with the antigen to which the human antigen-specific glycosylation inhibitory factor binds; (ii) eluting the human glycosylation inhibiting factor from the antigen; (iii) reacting the eluted glycosylation inhibitor with monoclonal antibodies specifically reactive with human glycosylation inhibitor; (iv) eluting the human antigen-specific glycosylation inhibiting factor from the monoclonal antibody; and (v) recovering the human, antigen-specific, - 78 glycosylation inhibitors. 40. The method of claim 34, wherein the isolation of the substantially pure human antigen-specific glycosylation inhibitor is as follows: (i) contacting the culture of the hybridoma cell line with anion exchange resin; (ii) eluting the human glycosylation inhibitory factor from the ion exchange material; (iii) reacting the eluted glycosylation inhibitor with a monoclonal antibody specifically reactive with the human glycosylation inhibitor or with the antigen to which the human, antigen-specific, glycosylation-inhibiting factor binds, or both; (iv) eluting the human antigen-specific glycosylation inhibitor; and (v) recovering the human antigen-specific inhibitory factor. 41. The method of claim 40, wherein said anion exchange agent is diethylaminoethyl linked to said resin. 42. The method of claim 40, wherein said human glycosylation inhibitory factor has a salt concentration ranging from about 10 mM to about 100 mmM * ········ - Elute with 79. 43. A process according to claim 40 wherein said salt is sodium chloride. 44. The method of claim 40, wherein said hybridoma cell line culture is concentrated to a concentration of about 25 to 1000 times and then diluted to a volume of about 3 to 10 times before being applied to the anion exchange resin. 45. The method of claim 38 or 39, characterized in that a monoclonal antibody having the specificity of a monoclonal antibody produced by the 388F1 cell line deposited under ATCC Accession No. HB 10472 is used. 46. The method of claim 38 or 39, wherein the monoclonal antibody produced by the 388F1 cell line deposited under ATCC Accession No. HB 10472 is used. 47. A method of suppressing a human immune response to an antigen, comprising treating the subject with an immunosuppressive amount of a human glycosylation inhibitor which is capable of specifically binding the human glycosylation inhibitor. 48. The method of claim 47, wherein said antigen treatment is an allergen treatment. 49. The method of claim 47, wherein said antigen treatment is an autoimmune antigen treatment. 50. The method of claim 48, wherein said antigen treatment is an antiseptic treatment of an animal species. 51. The method of claim 50, wherein said anti-poison treatment is an anti-bee venom treatment. 52. The method of claim 51, wherein said anti-honeybee allergen treatment is phospholipase A2. 53. The method of claim 52, wherein said antigen-binding human antigen-binding factor is produced by the human antigen-specific glycosylation inhibitory factor produced by the AC5 cell line deposited under accession number HB 10473 ATCC. 54. The method of claim 52, wherein the human glycosylation inhibitor is produced by the AC5 cell line deposited under ATCC Accession No. HB 10473. - Hl · »· · · ··· is applied. 55. A 4Θ. The process of claim 1, wherein said anti-allergen treatment is an anti-pollen treatment. 56. The method of claim 55, wherein said anti-pollen treatment is treatment of a tree produced pollen. 57. The method of claim 56, wherein the treatment is cedar pollen. 5Θ. The method of claim 57, wherein the antigen-binding factor of a human antigen-specific glycosylation inhibitor is produced by the 31E9 cell line deposited under accession number HB X ATCC. 59. The method of claim 57, wherein the human glycosylation inhibitor is produced by the 31E9 cell line deposited under accession number HB X ATCC. 60. The method of claim 47, wherein said treatment is performed parenterally. 61. The method of claim 60, wherein: ν · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · That parenteral treatment is by subcutaneous, intramuscular, intraperitoneal, intravitreal, transdermal, or intravenous injection. 62. The method of claim 47, wherein said treatment is administered at a dose of about 0.001 to 2 mg / kg / dose. 63. The method of claim 47, wherein said treatment is administered at a dose of about 0.001 to about 0.2 mg / kg / dose. 64. A pharmaceutical composition comprising an immunosuppressively effective amount of a substantially pure, human, antigen-specific, glycosylation inhibitor, and a pharmaceutically acceptable carrier. 65. The pharmaceutical composition of claim 64, wherein said substantially pure, human, antigen-specific glycosylation inhibitory factor is the AC5 cell line deposited under accession number HB 10473 or the 31E9 cell line deposited under accession number HB X ATCC. has the antigen-binding specificity of the human antigen-specific glycosylation inhibitor. 66. The pharmaceutical composition of claim 64, wherein said substantially pure human antigen-specific glycosylation inhibitory factor is HB 10473 ATCC. It is produced by the AC5 cell line deposited under accession number 83 ········· or the 31E9 cell line deposited under accession number HIt ATCC. 67. A continuous hybridoma cell line capable of secreting monoclonal antibodies specifically reactive with a human glycosylation inhibitor. 68. The hybridoma cell line of claim 67, which is the 388F1 cell line deposited under accession number HB 10472 ATCC. 69. A monoclonal antibody that is specifically reactive with human glycosylation inhibiting factor. 70. The monoclonal antibody of claim 69, wherein said monoclonal antibody has the specificity of a monoclonal antibody produced by the 368F1 hybridoma cell line deposited under accession number HB 10472 ATCC. 71. The monoclonal antibody of claim 70, wherein said monoclonal antibody is produced by the 388F1 hybridoma cell line deposited under accession number HB 10472 ATCC.