WO1985000750A1

WO1985000750A1 - Hybridoma cell line producing suppressive factor of allergy

Info

Publication number: WO1985000750A1
Application number: PCT/US1984/000594
Authority: WO
Inventors: David H. Katz
Original assignee: Quidel
Priority date: 1983-08-18
Filing date: 1984-04-16
Publication date: 1985-02-28
Also published as: AU2822684A; DK175485A; JPS60502202A; EP0154625A4; NO851539L; AU576499B2; EP0154625A1; DK175485D0; CA1215918A

Abstract

A monoclonal hybridoma cell line was constructed for the production of suppressive factor of allergy (SFA). mRNA isolated from the monoclonal hybridoma cell line was identified by its ability to direct SFA synthesis in Xenopus laevis oocytes. The SFA produced by the monoclonal hybridoma cell line and the translational product of the mRNA in the oocytes was found effective in inhibiting IgE-specific receptor expression in dilutions greater than 1:1,000 of the culture supernatant. A sensitive bioasay for SFA was developed using cultures of thymocytes in which Lyt-2 cells were blocked with monoclonal antibodies, and subsequent IgE-specific receptor expression was determined in Lyt-1 cells.

Description

HYBRIDOMA CELL LINE PRODUCING SUPPRESSIVE FACTOR OF ALLERGY

The immune response to a given antigen is in most cases several distinct types of immune responses supported by multiple cell types of the reticuloendothelial system. For example, humoral immunity is mediated by antibodies produced by B lymphocytes, whereas cellular immunity is mediated by specially sensitized T lymphocytes. The interaction of these cells and others is further modulated by various mediators. Cells that have been stimulated by antigens have been shown to produce antigen-specific mediators, or lymphokines, that regulate humoral and cell-mediated immune responses in both a positive and negative manner, as well as non-antigen-specific mediators that are associated with delayed hypersensitivity and B and T lymphocyte collaboration to produce antibodies. Antigens, although classically defined as substrates capable of stimulating the production of antibodies that will react specifically with a particular antigen, also include molecules that will induce an immune response, that is, a complex biologic process that includes the proliferation of lymphoid cells and the subsequent synthesis of recognition molecules, such as antibodies or cellular receptors. The antibodies produced by antigen stimulation have been classified according to their physiochemical properties and have been grouped in 10 immunoglobulin (Ig) categories, called isotypes, which presently include five (5) classes. The particular class involved in this invention is IgE, and is responsible for the type of reaction generally referred to as immediate hypersensitivity.

The IgE system plays perhaps one of the most important defense roles against certain exogenous agents, particularly those which gain access through mucoepithelial and epithelial linings such as the respiratory and gastrointestinal tracts and skin. Moreover, the IgE system is particularly unique in that the biological effects of IgE antibodies are extraordinarily amplified through widely distributed cells which possess IgE-specific Fc receptors(FcRe). Since IgE molecules bind specifically and avidly to such Fc receptors displayed on the surface membranes of tissue-fixed mast cells and circulating basophils, and since such cells are actually little factories of potent pharmacological mediators, only a small number of IgE molecules are required to exert the biological effects for which this system was designed. It makes sense, therefore, for finely-tuned regulatory mechanisms to evolve with particular emphasis on minimizing the quantities of IgE antibody molecules produced following sensitization. In this way, IgE antibody responses could be sufficient to provide protection to the individual without resulting in undesirable and/or deleterious reactions.

This is precisely how the system has been designed to operate under normal circumstances. However, it also seems that the regulatory control of IgE synthesis is so delicate that it becomes susceptible to certain perturbations that can upset the balance. This can result in production of higher than necessary quantities of IgE, which then translates into symptomatic manifestations. Its regulatory mechanisms are so sophisticated and delicate that it has become more susceptible to external perturbations, perhaps some of which are of a man-made nature, and, therefore, not anticipated by evolution, that allergic diseases have been the consequence.

Studies in experimental animal systems have shown that, (1) control of IgE antibody synthesis is dominated by a suppression, or damping mechanism; (2) certain manipulations, such as low dose irradiation or other immunosuppressive regimens, disturb normal damping of IgE antibody production; (3) several regulatory factors which are very selective in their biological effects on the IgE system have been shown to exist, such as the suppressive factor of allergy (SFA), and enhancing factor of allergy (EFA), both of which have been detected, and shown to function, both in vivo, as well as in vitro (Katz, 1978; Katz, et. al., 1979); and (4) the IgE response phenotype of an individual reflects the relative balance of these opposing regulatory factors at the time of antigen sensitization.

A very useful experimental model, largely in mice, that has helped reveal some of the crucial steps in the regulatory mechanisms controlling IgE synthesis has been the ability to selectively enhance IgE antibody production by exposure of mice to low dose whole body irradiation. More importantly, irradiation-enhanced IgE responses of genetically low responder inbred mice can be modulated selectively by the circulating proteins SFA and EFA.

Most of the properties of SFA and EFA have been summarized elsewhere (Katz, 1978; 1980a; 1980b; 1983). More recent findings have shown that both SFA and EFA, in addition to their originally described effects on IgE antibody synthesis, inhibit IgE-mediated induction of FcRe on lymphocytes (Katz, et al., 1983b). Moreover, unlike the IgE-selective factors being studied in the laboratories of Drs. Ishizaka and Kishimoto (ϊodoi, et al. 1980 and Hirashima, et al., 1982), neither SFA nor EFA displays any IgE-binding properties (Katz, et. al., 1983b). And finally, SFA has a size in the 30,000 dalton range while EFA falls into the 15,000 dalton range (Katz, et al., 1983b). It is clear that the IgE system can be considered one of the most important and sophisticated antibody defense systems that exists, comprising a finely-tuned network of interacting cells and molecules that regulate quality and quantity of a response. Also, this system is responsible for IgE-mediated allergic diseases, diseases that constitute a major health problem with consequenses that not only effect the physical well-being of one out of every three individuals, but also impose a substantial economic impact on both individuals and society alike. Diseases linked to the IgE system include, for example, the generalized anaphylactic reaction and other allergic states such as hayfever, asthma and urticaria.

The knowledge that SFA is one of the critical mediators in the IgE system, and that, according to the present invention, it can be isolated and economically feasibly produced using either hybridoma technology or recombinant DNA technology, potentially makes available new therapies that can be tailored to recapture the fine tuning of the regulatory network controlling IgE synthesis so that ample, but not excessive, quantities of this important protective lymphokine are produced.

The advantages of SFA therapy, using the inventive concepts set forth herein, over conventional therapy are dramatic. Since the SFA molecule is the natural protein the human body uses to down regulate IgE synthesis and to dampen the allergic response in vivo, SFA therapy should have little or no side effects. SFA inhibits only IgE responses and the amount of SFA can be clinically titrated to reduce the IgE levels so that they still contribute to natural immunity (which appears to be essential for life) without being dramatically overproduced or directed against innocuous antigens, thus leading to allergic reactions. Also, the effects of SFA treatment on the production of IgE directed against defined antigens appears to be very long lasting. Mice which are treated with SFA have dramatically reduced antigen stimulated IgE responses even one year (half a lifetime) after treatment. Of course, antigen stimulated IgG responses remain normal. Further, SFA appears to be able to dampen the allergic response to any allergen so that patients could be clinically desensitized to virtually any existing allergy, or to new allergies as they arise. SUMMARY OF THE INVENTION

In the present invention, techniques suitable for isolating mRNA having the coding sequence capable of producing the protein SFA, and assaying for the activity of SFA are disclosed. A hybridoma T cell line (1C1) was produced by fusing SFA-producing human PBL's with JURKAT cell line JKOT. mRNA was isolated from the 1C1 cell line and identified by its ability to direct SFA synthesis in Xenopus laevis oocytes. A sensitive biological assay for SFA was developed using a combination of Lyt-1 cells and the inhibition of FcRe positive cells.

Thus, in one form, the present invention comprises a hybridoma cell line characterized by the production of SFA, and, more particularly, a cell line comprising a fusion of cells from JURKAT human T cell leukemia and SFA-producing human peripheral blood lymphocytes. The SFA or SFA-containing supernatant of the cell line is at least 50% effective in inhibiting FcRe expression in dilutions exceeding ltl,000, and, more characteristically, in dilutions of 1:10,000 to 1:10⁶.

The major protein produced by the cell line is the protein SFA further comprising a molecule of molecular weight of approximately 15,000 to 30,000 daltons, having no IgE-binding properties, that is selective in activity for the IgE antibody system, that inhibits IgE antibody synthesis, that inhibits igE-mediated FcRe induction on lymphocytes, that is non-antigen-specific, whose activity is not strain or species-dependent, and that selectively stimulates Lyt-1 cells to produce SEM.

In another aspect the invention comprises the hybridoma cell line 1C1, i.e. the hybridoma cell line comprising a mRNA molecule comprising a RNA sequence coding for SFA. In the invention the mRNA translational product is the protein SFA comprising a molecule of molecular weight of approximately 15 ,000 to 30,000 daltons, having no IgE-binding properties, that is selective in activity for the IgE antibody system, that inhibits IgE antibody synthesis, that inhibits IgE-nediated FcRe induction on lymphocytes, that is non-antigen-specific, whose activity is not strain or species-dependent, and that selectively stimulates Lyt-1 cells to produce SEM.

In still another aspect, the invention includes a process for the detection or determination of SFA comprising treating a culture of mouse splenocytes with a measured amount of soluble monoclonal antibody to the Lyt-2 cells, the addition of SFA or SFA containing supernatant to a pre-determined final concentration, the addition of mouse anti-DNP, and the determination of the percentage of FcRe positive cells using a rosette-forming assay as compared to control cultures without SFA.

In yet another aspect, the invention comprises the product of the cell line and process described. DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the ability of PWM to stimulate IgE synthesis in vitro from 10 different human donors as assayed by two different techniques.

Figure 2 depicts the selective suppression of human IgE synthesis in vitro by human SFA.

Figure 3 depicts the inhibitory activities of SFA and EFA on FcRe induction, and shows that SFA and EFA do not have Ige-binding properties.

Figure 4 compares the ability of SFA produced by conventional mouse ascities, hybridoma cell line ICl, and hybridoma cell line ICl mRNA translation product, to inhibit in vitro FcRe induction and in vivo IgE synthesis. DETAILED DESCRIPTION OF THE INVENTION Definitions

CFA: complete Freund's adjuvant (Difco Laboratories, Detroit, MI). DNP: 2,4-dinitrophenyl.

EEMt enhancing effector molecule.

EFA: enhancing factor of allergy.

EIR: IgE-induced regulants.

Fc : crystallizable fragment of IgE. FCSi fetal calf serum.

FcRe: IgE specific Fc receptors.

GAHE; goat anti-human IgE.

GAHG: goat anti-human IgG.

HAT: hypoxanthine, aminopterin, and thymidine. IgE: imraunoglobulin E.

Lyt-1: T lymphocytes.

Lyt-2: T lymphocytes.

MEM: minimum essential medium.

MLR: mixed lymphocyte reaction. MNC: mononuclear cells.

PBL: peripheral blood lymphocytes.

PEG: polyethylene glycol.

PMC: peripheral mononuclear cells.

PWM: pokeweed mitogen. RiA: radioimmunoaβsay.

SEM: suppressive effector molecule.

SFA: suppressive factor of allergy.

SRBC: sheep red blood cells.

TNBS: 2, 4, 6-trinitrobenzene sulfonic acid (ICN

Pharmaceuticals, Cleveland, OH.) TNP: Trinitrophenyl.

This invention involves the production of SFA, or an SFA-like molecule, having the ability to modify human IgE synthesis, and the isolation of mRNA that translates for an SFA or SFA-like molecule capable of modifying IgE synthesis. More particularly, this invention involves the formation of a hybridoma that produces SFA, and the isolation of the mRNA from the hybridoma that is responsible for the expression of the protein SFA.

The description of the invention is divided into the following stages:

I. Biosynthesis of human IgE in vitro.

II. Biosynthesis of human SFA and SFA suppression of human IgE synthesis in vitro.

III. Involvement of FcRe receptors in IgE regulation.

IV. Construction of human T cell hybridomas producing SFA and translation of messenger RNA to SFA in frog oocytes.

I. Biosynthesis of human IgE in vitro The biosynthesis of human IgE in vitro was accomplished by stimulating human peripheral mononuclear cells (PMC) by exposure to pokeweek mitogen (PWM) (Sigma Chemical Company, St. Louis, Missouri). The culture system used has been described in detail (Nonaka, et al., 1981; Zuraw, et al., 1981; Katz, et al., 1982; Katz, 1982), and consists essentially of two stages; the first stage consisting of exposure of cells to stimulating mitogen, and the second stage, in which cells were washed and replated in cultures devoid of any additional mitogen for a further six days. Under such conditions, human mononuclear cells were shown to synthesize significant quantities of IgE. Although various techniques for IgE assay are commercially available, in the preferred embodiment, the inventors use a radioimmunoassay (RIA) based on the preparation of high-titer goat anti-human IgE (GAHE) prepared from the serum of two different patients with IgE myelomas; these are designated IgE (PS) and IgE (DZA) (Zuraw, et al., 1981; Chen, P.-P.,et al., 1983, respectively).

The production of GAHE was prepared by hyperimmunizing a goat with 100 micrograms of IgE (PS or DZA) emulsified in complete Freunds Adjuvant (CFA) (Difco Laboratories, Detroit, MI) every three weeks over a time period of 11 months. Bleedings were accumulated at varying intervals during this immunization schedule and over a several-month period thereafter. The GAHE serum was screened by a quantitive binding analysis. Serial dilutions of the antiserum in normal goat serum were mixed with a known amount of lodine-125-IgE (PS) myeloma protein and were precipitated with rabbit anti-goat serum in order to construct a binding curve; twenty microliters of a 1:2,500,000 dilution were found to bind 0.25ng of IgE protein.

The Ig fraction of the GAHE was obtained by precipitating it twice with amonium sulfate at final concentrations of 40% and 30% saturation. The recovered Ig fraction of GAHE was depleted of anti-light chain antibodies by absorption with normal human serum coupled to Sepharose 4B (Pharmacia, Uppsala, Sweden). The absorbed GAHE was used as the coating protein in the RIA. purified anti-human IgE antibodies were isolated from the normal human serum-absorbed GAHE proteins by affinity chromatography on lgE(PS)-coupled Sepharose 4B and elution with 0.1M glycine-HCl buffer, pH 2,3. Affinity-purified GAHE was labeled with Iodine-125 by solid-state lactoperoxidase. The iodinated GAHE was then separated from free Iodine-125 by passage over a G-25 column (PD-10, Pharmacia). The specific activities of labeled GAHE ranged from 9 to 18 X 10⁶ CPM/microgram. Labeling was generally performed once a week. A conventional solid-phase RIA system was employed to measure IgE levels in culture supernatants (Zollinger, et al . , 1976).

A summary of results obtained with peripheral blood cells from ten different donors stimulated with varying doses of PWM is presented in Figure 1. The distinction between the upper panel A and lower panel B lies in the GAHE reagent (PS or DZA) employed for detecting IgE responses by radioimmunoassay of the culture supernatant fluids. The values shown in the upper panel were obtained with anti-human IgE that had been affinity purified on the same IgE myeloma protein, PS, as that used for hyperimmunization to raise the goat antiserum; the values depicted in the lower panel were those obtained on the same culture supernatant fluids using anti-human IgE that had been affinity-purified on a completely unrelated IgE myeloma protein, DZA, than that used for immunization. The point is that irrespective of which reagent was used for the immunoassay, cultures stimulated with PWM clearly displayed levels of IgE that were significantly above the baseline values of IgE detected in the parallel unstimulated control cultures, and thereby eliminating the possibility of reagent-induced assay artifacts. Such responses represent de novo biosynthesis of IgE as a result of PWM stimulation, since incorporation of the protein synthesis inhibitor, cyclohexemide, totally abolishes the capacity of human lymphoid cells to synthesis IgE in response to PWM (Zuraw, et al., 1981),

II, Biosynthesis of human SFA and SFA suppression of human IgE synthesis in vitro The production of human SFA and its capability of selectively inhibiting IgE synthesis has been described (Zuraw, et al., 1981; Katz, et al., 1982; Katz, 1983). Essentially, SFA is obtained from two-way human mixed lymphocyte reactions (MLR), in which mononuclear cells (MNC), were suspended at 1 X 10⁶ cells/ml in culture medium. One milliliter from each of two unrelated donors was placed in a 2ml culture well. The cultures were placed in 5% CO₂ humidified atmosphere at 37°C for eight days, at which times the supernatants were harvested by centrifugation and stored at -20°C.

Using the system for stimulating human IgE synthesis in vitro as described above, the ability of SFA to suppress the synthesis of IgE was tested. In the experiments, donor MNC were placed in first-stage culture, either alone or in the presence of a given MLR culture supernatant (SFA) at a final concentration of 15%. 24 hours later, PWM (1 microgram) was added to each of the three cultures. Three days later (day 0), the cells in each culture group were washed and plated in micro cultures (10 replicate microwells per group) for six additional days. The effect of SFA on the synthesis of IgG was also studied using goat anti-human IgG (GAHG) prepared as described above for GAHE. The mean levels of IgG (upper panel) and IgE (lower panel) synthesized by each culture group, and the effect of SFA on the synthesis of IgG and IgE, are depicted in Figure 2. As shown in the bottom panel, substantial inhibition of IgE synthesis occurs in culture groups exposed to SFA. Importantly, the inhibitory effects of SFA are selective for the IgE response, since, as shown in the top panel, IgG synthesis in the same cultures is not at all inhibited. This lack of effect on IgG synthesis in the same cultures furthermore rules out the possibility of SFA exerting its inhibitory effects as a result of non-specific toxicity. III. Involvement of FcRe Receptors in

IgE Regulation

Recent studies in experimental animals and man provided new insights on the regulatory mechanisms controlling antibody responses of the IgE class. Although receptors for determinants present on the Fc portion of immunologlobulin molecules have been known for some time to exist on membranes of various cell types, the discovery by Spielberg and colleagues (Gonzalez-Molina and Spiegelberg, 1977; Fritsche and Spiegelberg, 1978), and subsequent confirmation of IgE-specific Fc receptors on lymphocytes of both rodents and humans, by Yodoi and

Ishizaka (1979), Yodoi, et al. (1979) and Chen, S.-S., et al. (1981), has opened new and exciting avenues of exploration concerning the role of such FcRe positive lymphoid cells in the overall regulation of IgE synthesis.

An important advance in this regard was the demonstration by Yodoi, et. al. (1979) in the rat, and by

Chen, S.-S., et al. (1981) in the mouse, that normal lymphoid cells exposed to elevated quantities of IgE in vitro are induced to synthesize and express FcRe molecules on their surface membranes.

Of particular usefulness is the fact that FcRe induction by in vitro exposure to IgE (Liu, et al.,1980) can be modulated, and quite selectively, by both SFA (Chen, S.-S., et al.,1981) and EFA (Katz, et al.,1983b). The percentage of FcRe positive cells is detected by conventional rosette assay techniques. Basically, TNP-derivatized sheep red blood cells (SRBC) were prepared by reaction with TNBS as described by Rittenberg and Pratt (1969). The IgE-coated TNP-SRBC were prepared by incubating the red cell suspension (1% in MEM) with subagglutinating amounts of DNP-specific antibodies in the range of 1 to 10 micrograms/ml. The mixture was incubated for 30 minutes in a 37°C. water bath, followed by washing twice with MEM. The IgE-coated red cells were brought to a 1% suspension in rosetting medium (MEM supplemented with 1% FCS and pennicillin/streptomycin) . Rosettes were prepared by adding 50 microliters of lymphocytes (2.5 X 10⁶ cells) to 12 X 75-mm Falcon tubes that contained 100 microliters of appropriate indicator cells. The cell mixtures were incubated in a 37°C water bath for 15 minutes and centrifuged at 500 rpm for 5 minutes. The pellets were incubated unperturbed for an additional 30 minutes at 4°C and were subsequently gently resuspended either with a 100 microliter Eppendorf or by vortexing. The cells were incubated at 4°C overnight, and Trypan blue (0.3 ml) was added to each tube just before enumeration of the rosettes; the viability of cultured cells was routinely 60% or more. As a rule, the number of rosettes in approximately 400 live lymphocytes in each sample pool was counted three or four times, and the arithmetic means and standard errors were calculated from these values, A rosette-forming cell is defined as a lymphocyte that is tightly surrounded by at least three indicator cells.

The capacity of SFA and EFA to modulate the formation of FcRe positive cells is summarized in Figure 3. Cells cultured for 24 hours in the absence of any inductive stimulus display only baseline levels—usually 2-4%—of FcRe positive cells as detected by the rosette assay. In contrast, as shown by group II, exposure to IgE for that same period of time results in the induction of rather appreciable frequencies of FcRe positive cells. This IgE-induced FcRe expression phenomenon can be inhibited by concomitantly exposing such cultured cells to either SFA, shown by groups III and IV, or EFA, shown by groups V and VI.

Moreover, this inhibitory effect of SFA and EFA is not removed by prior adsorption of the molecular entities on igE-affinity columns, demonstrating the absence of IgE-binding properties on the relevant inhibitory molecules in the SFA and EFA preparations, respectively. This is demonstrated by incubating normal CAF₁ spleen cells in either medium alone, in medium containing 10 micrograms/ml IgE, or in medium containing IgE plus 1% SFA or 10% EFA. SFA and EFA obtained from CFA-induced ascites fluids of A/J mice, were precipitated with ammonium sulfate and then fractionated on Con A-Sepharose. The pass-through fraction recovered from such columns was utilized as SFA and the material eluted from Con A-Sepharose (by α-D-methyl mannoside) was used as EFA. A portion of the respective SFA and EFA-enriched fractions was further subjected to affinity chromatography on IgE-coupled Sepharose. Two ml of each of the factors was adsorbed on 2 ml of Ige-Sepharose constructed with 5,25 mg affinity-purified IgE per gram Sepharose 4B. After 24 hrs., the cells exposed (or not) to the various agents were washed and assayed for FcRe expression by rosette formation with DNP-specific igE-coated TNP-SRBC. Data are expressed as the geometric means (standard errors) of rosette-forming cells, as determined from three counts of approximately 300 viable lymphocytes. Statistically significant differences exist between groups I vs. II-VI (P<0.01), and between groups II vs. III-VI (P<0.01). In addition to SFA and EFA inhibition of FcRe expression, other soluble factors, termed IgE-induced regulants (EIR), have been found to regulate FcRe expression, and are unrelated to carry-over of IgE or fragments therefrom (Marcelletti and Katz, 1983a; 1983b; Katz, 1983). These studies have shown that B cells produce two types of EIR; EIR_a induces FcRe expression in B cells, whereas another form, EIR_T, induces FcRe expression in T cells. Therefore, when unfractionated spleen cells are exposed to IgE resulting in the induction of both T and B cells which express FcRe on their surface membranes, B cells must be present for IgE to induce FcRe expression in

T cells. Further, it was found that the EIR_T was produced by FcRe positive B cells that had been stimulated by ElR_ß.

It has been subsequently found that the EIR_T produced by the FcRe positive B cells interacts with T cells of the Lyt-2 subset to cause expression of FcRe. More importantly, it was found that the B cells in which FcRe expression was induced by ElR_ß then interacted with T cells of the Lyt-1 subset which stimulated the production of SFA, and, that ElR_ß, either alone or in conjunction with a yet undefined separate molecular entity produced by the FcRe positive B cells, could replace the requirement for B cells in providing cooperative signals to the Lyt-1 T cells to produce SFA. The result is that SFA then triggers Lyt-1 T cells to synthesize and secrete a final suppressive effector molecule (SEM) which has the ability to (1) directly inhibit B cell expression of FcRe induced by exposure to IgE, and (2) to suppress IgE antibody synthesis both in the in vivo mouse model and in the in vitro human system.

EFA is involved in this regulatory scheme by its ability to stimulate FcRe expression in Lyt-2 positive T cells causing the secretion of a final enhancing effector molecule (EEM) which, like SEM, is capable of directly inhibiting FcRe expression by B cells following exposure to IgE, and is responsible for marked enhancement of IgE synthesis in vivo or in vitro. In other words, SFA requires Lyt-1 positive T cells, and EFA, in contrast, requires Lyt-2 positive T cells in order to effectively inhibit IgE-mediated FcRe induction in vitro.

Previous assays for SFA (Zuraw, et al., 1981 and Nonaka, et al., 1981) were time consuming, requiring at least three weeks. However, having determined that only Lyt-1 cells respond to SFA, a new biological assay system is disclosed for the determination of SFA activity by measuring the percent of FcRe positive cells, as described above, in cultures in which the Lyt-2 cells have been blocked by first treating the culture to antibodies (American Type Culture Collection, Rockville, MD) to Lyt-2 cells.

IV. Construction of human T cell hybridomas producing SFA and translation of messenger RNA to SFA in frog oocytes Having produced SFA from human cells in vitro, and showing the effect of SFA in the regulation of the IgE system, it becomes necessary to be able to produce large quantities of SFA to more easily assess its clinical applicability, and to subsequently produce SFA in an economically feasible manner. To accomplish this, a human T cell hybridoma line was constructed according to the well-known process as discussed by Milstein and Kohler (1975). The general details of this process are well known and will not be repeated here. Basically, for construction of the present invention, human peripheral blood lymphocytes (PBL) were isolated by standard techniques, and cultured with PWM in RPMI 1640 medium plus 10% FCS (Microbiological Associates, Walkersville, MD) for seven days. PBL's were washed and fused with Jurkat cell line clone JKOT which is resistant to thioguanine and ouabain and is sensitive to HAT. Fusion was accomplished with polyethelyne glycol (PEG) in a solution containing 50% PEG and 50% 150 mM Hepes buffer. The cells were placed in 0.1 ml PEG solution for one minute at 37° C, then diluted with RPMI into 5 ml, washed once, then plated into RPMI plus 10% FCS, plus HAT. These cultures contained murine thymocytes as feeder cells. The population of hybridomas constructed were then screened by standard techniques to isolate the individual clones producing SFA.

Although in the preferred embodiment human SFA is produced, it should be noted that SFA is not specieβ-dependent; animal sources are believed to be effective in the human system, and human SFA is likewise effective in most, if not all,animal systems.

The SFA that is produced by the hybridoma ICl was assayed with respect to its ability to inhibit FcRe induction in vitro, and also its ability to inhibit in vivo IgE synthesis in the mouse, according to the techniques described above. The hybridoma produced SFA was also compared with conventional human SFA, produced as described above. As depicted in Figure 4, as can be seen by comparing group II to group I, conventional human SFA blocked both in vitro IgE-mediated FcRe induction and in vivo IgE antibody synthesis, as expected. Importantly, supernatant fluids from the relevant hybridoma ICl are likewise effective in blocking both bioasβay indicators. Recent studies using these same assays have shown that dilutions of the ICl supernatant of greater than 1:1,000 are effective in reducing FcRe positive cell production by 50%. The invention is further characterized in that dilutions of 1:10,000 to l:10⁶ are likewise effective in reducing by 50% FcRe positive cell production.

The mRNA from hybridoma ICl was subsequently isolated according to technique as described by Berger and Birkenmeier (1979). The ability of the mRNA preparations to direct the synthesis of SFA was determined by microinjection into stage V-VI Xenopus laevis oocytes, according to the procedure described by Gurdon, et al., (1971). Typically, 20-30 nanograms of mRNA in 20-30 microliters were injected/oocyte . Oocytes were incubated in modif ied Barth ' s medium supplemented with 100 units/ml pennicillin-100 raicrograms/ml streptomycin, 10 mM-Hepes, 1 mM phenylmethylsulfonyl fluoride and 0.1% Bovine serum albumin. Viable oocytes were selected 16-18 hours after injection and incubated at 5 oocytes/well in 96-well microtiter dishes (100 microliters of the above medium/well) for a further 40-48 hours at room temperature at which time the culture medium was removed and assayed for SFA activity according to the procedures described above. The translation product of mRNA extracted from this hybridoma line shows identical biological activities as the hybridoma produced SFA, as depicted in Figure 4 by group IV.

INDUSTRIAL APPLICATION

The invention has application as a therapeutic for diseases linked to the IgE system that are generally manifested by allergic reactions, such as hayfever and asthma, or by the anaphylactic reaction. Since SFA is a natural body regulant of IgE synthesis and control of the allergic response, little or no side effects would result from its therapeutic use. Further, SFA can be administered in amounts sufficient to reduce the overproduction of IgE that leads to the allergic reactions, while not eliminating IgE levels necessary to contribute to the natural immunity. Also, the effects of SFA are long-lasting, eliminating the need for continuous therapy,

REFERENCES

Berger, S.L. and Birkenmeier, C.S. (1979), Biochemistry

18, 5143.

Chen, S.-S., Bohn, J.W., Liu, F.-T. and Katz, D.H. (1981). Journal of Immunology 127, 166.

Chen, P.P., O'Hair, C.H., Coen, P-.A., Zuraw, B.L. and

Katz, D.H. (1983) Journal of Immunology, in press.

Fritsche, R. and Spiegelberg, H.L, (1978). Journal of Immunology 121, 472.

Gonzalez-Molina, A. and Spiegelberg, H.L. (1977), Journal of Clinical Investigation 59, 616.

Gurdon, J.B., et al. (1971), Nature 233, 177.

Hirashima, M., Uede, T., Huff, T. and Ishizaka, K. (1982), Journal of Immunology 128, 1909. Katz, D.H. (1978). Immunological Reviews 41, 77. Katz, D.H. (1980a). In "Progress in Clinical Immunology" (R.S. Schwartz, ed.), Vol. 4, p. 127-150, Grune and Stratton, Inc., New York, N.Y.

Katz, D.H. (1980b). Immunology 41,1. Katz, D.H. (1983). Allergy, in press.

Katz, D.H., Bargatze, R.F., Bogowitz, CA. and Katz, L.R. (1979). Journal of Immunology 122,2184. Katz, D.H., Nonaka, M., Zuraw, B.L., Cohen, P.A. and O'Hair, C.H. (1982). In "Human B-Lymphocyte Function: Activation and Immunoregulation", (A.S. Fauci and R.E. Ballieux, eds.), pp. 181-194, Raven Press, New York, N.Y. Katz, D.H., Chen, S.-S., Liu, F.-T, Bogowitz, CA. and Katz, L.A. (1983b). Journal of Molecular and

Cellular Immunology, in press.

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Experimental Medicine, in press. Marcelletti, J.F. and Katz, D.H. (1983b) Journal of Experimental Medicine, in press.

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Journal of Experimental Medicine 153, 1574. Rittenberg, M.D., and Pratt, K.L. (1969), Proc. Soc. Exp, Biol. Med. 132, 57,

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Claims

WHAT IS CLAIMED IS:

1. A hybridoma cell line characterized by the production of SFA.

2. The cell line of Claim 1 comprising a fusion of cells from JURKAT human T cell leukemia and SFA-producing human peripheral blood lymphocytes.

3. The cell line of Claim 1 wherein the SFA or SFA-containing supernatant is at least 50% effective in inhibiting FcRe expression in dilutions exceeding 1:1,000.

4. The hybridoma of Claim 1 wherein the major protein produced is the protein SFA further comprising:

(a) a molecule of molecular weight of approximately 15,000-30,000 daltons; (b) a molecule having no igE-binding properties;

(c) a molecule that is selective in activity for the IgE antibody system;

(d) a molecule that inhibits IgE antibody synthesis;

(e) a molecule that inhibits IgE-mediated FcRe induction on lymphocytes;

(f) a molecule that is non-antigen-specific;

(g) a molecule whose activity is not strain or species-dependent; and

(h) a molecule that selectively stimulates Lyt-1 cells to produce SEM.

5. A hybridoma cell line ICl.

6. A hybridoma cell line comprising a mRNA molecule comprising a RNA sequence coding for SFA.

7. The mRNA of Claim 6 wherein the translational product is the protein SFA further comprising:

(c) a molecule that is selective in activity for the IgE antibody system;

(d) a molecule that inhibits IgE antibody synthesis;

(e) a molecule that inhibits IgE-mediated FcRe induction on lymphocytes;

(f) a molecule that is non-antigen-specific

(g) a molecule whose activity is not strain or species-dependent; and

(h) a molecule that selectively stimulates Lyt-1 cells to produce SEM.

8. A process for the detection or determination of SFA comprising the steps: (a) treating a culture of mouse splenocytes with a measured amount of soluble monoclonal antibody to the Lyt-2 cells;

(b) the addition of SFA or SFA containing supernatant to a pre-determined final concentration;

(c) the addition of mouse anti-DNP; and

(d) the determination of the percentage of FcRe positive cells using a rosette-forming assay as compared to control cultures without SFA