HRP940786A2 - Hybrid cell line for production of monoclonal antibody for human cytotoxic and supressor t cells, antibody and process - Google Patents

Description

This invention relates mainly to new hybrid cell lines and more specifically to hybrid cell lines for the production of a monoclonal antibody to an antigen found on normal human cytotoxic and suppressor T cells, then to the antibody so produced, and to therapeutic and diagnostic procedures and preparations using this antibody.

Condensation of murine myeloma cells to spleen cells from immunized mice which Kohler and Milstein 1975/Nature 256, 495-497 (1975)/ demonstrated for the first time that it is possible to obtain a continuous cell line producing a homogeneous (so-called "monoclonal") antibody. After this initial work, many efforts were made to produce various hybrid cells (called "hybridomas") and to use the antibody made using these hybridomas for various scientific research. See, for example, Current Topics in Microbiology and Immunology, Volume 81 - "Lymphocyte Hybridomas", F. Melchers, M. Potter, and N. Warner, Editors, Springer-Verlag, 1978, and references therein; C.J. Barnstable et al., Cell, 14, 9-20 (May, 1978); P. Parham and W. F. Bodmer, Nature 276, 397-399 (November, 1978); Handbook of Experimental Immunology, Third Edition, Volume 2, D. M. Wier, Editor, Blackwell, 1978, Chapter 25; and Chemical and Engineering News, January 1, 1979, 15-17. These references simultaneously point to the rewards and complications of attempting to produce a monoclonal antibody from a hybridoma. Although the general technique is well understood conceptually, there are many difficulties encountered and many variations required for each specific case. In fact, there is no certainty, before attempting to make a given hybridoma, that the desired hybridoma will be obtained, that it will produce an antibody if obtained, or that the antibody thus produced will have the desired specificity. The degree of success is influenced mainly by the type of antigen used and the choice of technique used to isolate the desired hybridoma.

Attempted production of monoclonal antibody for human surface lymphocyte antigens has been described only in a few cases. See, for example, Current Topics in Microbiology and Immunology, ibid, 66-69 and 164-169. The antigens used in these published experiments were cultured human lymphoblastoid leukemia and human chronic lymphocytic leukemia cell lines. Many of the resulting hybridomas appeared to produce antibody to various antigens on all human cells. None of the hybridomas produced produced an antibody against a predefined class of human lymphocytes.

It should be clear that there are two main classes of lymphocytes involved in the immune system of humans and animals. The first of these (thymus-derived cell or T cell) differentiates in the thymus from the hematopoietic cell line. While inside the thymus, the differentiating cells are called "thymocytes". Mature T cells leave the thymus and circulate between tissues, lymphatic vessels and the bloodstream. These T cells form a large part of the space for the recirculation of small lymphocytes. They have immunological specificity and are directly involved in cell-mediated immune reactions (such as graft rejection) as effector cells. Although T cells do not secrete humoral antibodies, they are sometimes required for the secretion of these antibodies by another class of lymphocytes discussed below. Some types of T cells have a regulatory function in other aspects of the immune system. The mechanism of this process of cell cooperation is not yet fully understood.

Another class of lymphocytes (cells derived from bone marrow or B cells) are those that secrete antibody. They also develop from the hemopoietic line. cells, but their differentiation is not determined by the thymus. In birds, they differentiate into an organ analogous to the thymus, called the Bursa or Fabricius. In mammals, however, no equivalent organ has been discovered, although these B cells are thought to differentiate within the bone marrow.

It is now understood that T cells are divided into at least several subtypes, called "helper", "suppressor" and "killer" T' cells, which have the function of (respectively) promoting a reaction, suppressing a reaction, or destroying (destroying) foreign cells. These subclasses are well understood for mouse systems, but have only recently been discovered for human systems. See, e.g., R. L. Evans et al., Journal of Experimental Medicine, Volume 145, 221-232, 1977, and L. Chess and S. F. Schlossman - "Functional Analysis of Distinct Human T-Cell Subsets Bearing Unique Differentiation Antigens," in " Contemporary Topics in Immunobiology", O. Stutman, Editor, Plenum Press, 1977, Volume 7, 363-379.

The ability to identify or suppress T cell classes, or subclasses, is important for the diagnosis or treatment of various immunoregulatory disorders or conditions.

For example, certain leukemias and lymphomas have a different prognosis depending on whether they are of B cell or T cell origin. Thus, the assessment of the prognosis of the disease depends on the distinction between these two classes of lymphocytes. See, for example, A.C. Aisenberg and J.C. Long, The American Journal of Medicine, 58:300 (March, 1975); D.. Belpomme, et al., in "Immunological Diagnosis of Leukemias and Lymphomas", S. Thierfelder, et al., eds, Springer, Heidelberg, 1977, 33-45; and D. Belpomme, et al., British Journal of Haemotology, 1978, 38, 85.

Certain disease states (eg, juvenile rheumatoid arthritis, malignancies, and agammaglobulinemia) are associated with an imbalance of T cell subclasses. It has been suggested that autoimmune diseases are accompanied by an excess of "helper" T cells or a deficiency of certain "suppressor" T cells, while agammaglobulinemia is accompanied by an excess of certain "suppressor" T cells or a deficiency of "helper" T cells. Malignant conditions are mostly accompanied by an excess of "suppressor" T cells.

In certain leukemias, excess T cells are produced in an arrested phase of development. Diagnosis may then depend on the ability to detect this imbalance or excess. See, for example, J. Kersey, et al., "Surface Markers" Define Human Lymphoid Malignancies Transfusion, Volume 20, Springer-Verlag, 1977, 17-24 and references cited therein.

Acquired agammaglobulinemia, a disease state in which immune globulin is not produced, includes at least two distinct types. In type I, the failure to produce immune globulin is due to an excess of suppressor T cells, while type II is due to a lack of helper T cells. In both types, there appears to be no defect or deficiency in the patient's B cells, the lymphocytes that are responsible for the actual secretion of antibodies, however, these B cells are either suppressed or "not helped," leading to greatly reduced or absent production of immune globulin. The type of acquired agammaglobulinemia can thus be determined by testing for an excess of suppressor T cells or the absence of helper T cells.

On the therapeutic side, there are some suggestions, although still unproven, that administration of antibodies against the T cell subtype in excess may have a therapeutic effect in autoimmune or malignant disease. For example, cancer helper T cells (certain cutaneous lymphoma T cells and certain acute lymphoblastic leukemia T cells) can be treated with an antibody to a helper T cell antigen. Treatment of autoimmune disease caused by an excess of helper cells can also be achieved in the same way. Treatment of diseases (eg, malignancies or acquired agammaglobulinemia type I) due to excess suppressor T cells can be treated by administration of antibodies to suppressor T cell antigens.

Antisera against an entire class of human T cells (so-called antihuman thymocyte globulin or ATG) have been reported to be therapeutically useful in organ transplant recipients. Since the cell-mediated immune response (the mechanism by which transplants are rejected) depends on T cells, administration of antibodies to T cells prevents or delays this rejection process.

See, for example, Cosimi, et al., "Randomized Clinical Trial of ATG in Cadaver Renal Allograft Recipients: Importance of T Cell Monitoring," Surgery 40: 155-163 (1976) and references therein.

Identification and suppression of human T cell classes and subclasses has previously been achieved using spontaneous autoantibodies or human T cell-selective antisera obtained by immunizing animals with human T cells, bled animals to obtain serum, and adsorbing antisera with (for example) autologous but not allogeneic B cells so that antibodies with unwanted reactivities are removed. Preparation of these antisera is extremely difficult, especially in the phases of adsorption and purification. Even adsorbed and purified antisera contain many impurities in addition to the desired antibody, for several reasons. First, serum contains millions of antibody molecules even before T cell immunization. Second, immunization elicits the production of antibodies against a variety of antigens found in all human injected T cells. There is no selective production of antibodies against a single antigen. Third, the titer of the specific antibody obtained by such procedures is usually quite low, (eg, inactive at dilutions greater than 1:100) and the ratio of specific to nonspecific antibody is less than 1/106.

See, for example, the article by Chess and Schlossman referred to above (on pages 365 et seq.) as well as the article in Chemical and Engineering News referred to above, where the disadvantages and advantages of prior art antisera are described monoclonal antibody.

One of the T cell subsets identified by such antisera from the prior art has been designated the TH2+ subset and has been shown to contain both cytotoxic effector cells for cell-mediated lympholysis and immunoregulatory suppressor T cells that suppress both T cell and B cell function. This subset contains about 20%-30% of human cytotoxic and suppressor TH2+ T cells.

A further new aim is to provide methods for treating or diagnosing diseases using these antibodies.

Other objects and advantages of the invention will become apparent upon examination of the present description.

To meet the foregoing objectives and advantages, the present invention provides a novel hybridoma that produces a novel antibody to an antigen found on normal human cytotoxic and suppressor TH2+ T cells, then the antibody itself, as well as diagnostic and therapeutic procedures using the body. The hybridorm was made mainly according to the procedure of Milstein Kohler. After immunization of mice with normal human thymocytes, spleen cells from the immunized mice are condensed with cells from a murine myeloma line and the resulting hybridomas are analyzed for those with supernatants containing an antibody that gives selective binding to normal human rosette-positive T cells. The desired hybridomas are later cloned and characterized. Because of this, it was obtained by a hybrid that produces an antibody (designated OKT5) against antigens on normal human cytotoxic and suppressor TH2+ T cells. Not only does this antibody react with normal human peripheral cytotoxic and suppressor TH2+ T cells, but it also does not react with other normal peripheral blood lymphoid cells, including helper T cells. Furthermore, the cell surface antigen recognized by this antibody is detected by approximately 80% of normal human thymocytes.

Given the difficulties indicated in the prior art and the lack of success using malignant cell lines as antigens, it was unexpected that the present procedure provided the desired hybridoma. It should be emphasized that the unpredictable nature of hybrid cell preparations does not allow extrapolation from one antigen or cell system to another. In fact, the present applicants found that using a malignant cell line of T cells as an antigen caused the formation of hybridomas that did not produce the desired antibody. Attempts to use antigens from cell surfaces have also been unsuccessful.

Both the hybridoma in question and the antibody produced herein are identified by the designation "OKT5", and the particular material in question is obvious from the context.

The preparation and characterization of the hybridoma and the resulting antibody will be better understood by reference to the following description and Examples.

Detailed description of the invention

The procedure for creating a hybridoma mainly includes the following stages:

A. Immunization of mice with normal human thymocytes. Although female CAF1 mice are found to be preferred, it is contemplated that other mouse strains may be used. The immunization schedule and thymocyte concentration should be such that useful amounts of suitably primary splenocytes are produced. Three immunizations at 14-day intervals with 2 x 107 cells/mice/injection in 0.2 ml of phosphate-buffered saline were found to be effective.

B. Isolation of spleens from immunized mice and preparation of spleen suspension in appropriate medium. About one ml of medium per spleen is enough. This experimental technique is well known.

C. Condensation of suspended spleen cells with murine myeloma cells using a suitable condensation promoter. The preferred ratio is about 5 spleen cells per myeloma cell. The total volume of about 0.5-1.0 ml of condensation medium corresponds to about 108 splenocytes. Many murine myeloma cell lines are known and available, mostly from members of the academic community at various depository banks, such as the Salk Institute Cell Distribution Center, La Jolla, CA. The cell line used should preferably be of the so-called "drug-resistant" type, so that non-condensed myeloma cells will not survive in the chosen medium, while hybrids will. The most common class of 8-azaguanine resistant cell lines, which lack the enzyme hypoxanthine guanine phosphoribosyl transferase and therefore will not be supported by the HAT (hypoxanthine, aminopterin and thymidine) medium. It is also generally preferred that the myeloma cell line used be of the so-called "non-secretory type", in that it does not produce any antibody itself, although secretory types can also be used. However, in certain cases myeloma secreting lines may be preferred. Although the preferred condensation promoter is polyethylene glycol having an average molecular weight of about 1000 to about 4000 (commercially available as PEG 1000, etc.), other condensation promoters known in the art can be used.

D. Diluting and culturing in separate containers, a mixture of uncondensed spleen cells, uncondensed myeloma cells, and condensed cells in a selective medium that will support the uncondensed myeloma cells for a period sufficient to allow death of the uncondensed cells (about one week). Dilution can be of the limiting type, in which the volume of the diluent is calculated. statistically by isolating a certain number of cells (eg, 1-4) in each separate container (eg, each well of a microtiter plate). The medium is one (eg, HAT medium) that will not support an unfused myeloma cell line that is drug-resistant (eg, resistant to 8-azaguanidine). This is why these myeloma cells disappear. Since non-condensed spleen cells are non-malignant, they have only a limited number of generations. Thus, after a certain period of time (about one week), these uncondensed spleen cells no longer reproduce. Condensed cells, on the other hand, continue to reproduce because they have the malignant quality of primary myeloma and the ability to survive in the selective medium of primary splenic cells.

E. Evaluation of the supernatant in each container (well) containing the hybridoma for the presence of antibodies to E rosette positive purified human T cells.

F. Selection (eg, by limiting dilution) and cloning of a hybridoma that produces the desired antibody.

Once the desired hybrid is selected and cloned, the resulting antibody can be produced in one of two ways. The purest monoclonal antibody is produced in vitro by culturing the desired hybridoma in a suitable medium for a suitable period of time, followed by isolation of the desired antibody from the supernatant. The suitable substrate and suitable length of cultivation time are known and easily determined. This in vitro technique produces essentially a monospecific monoclonal antibody, essentially free of another specific antihuman immune globulin. There is a small amount of other immune globulin present since the medium contains xenogeneic serum (eg, fetal calf serum). However, this in vitro procedure may not produce a sufficient amount or concentration of antibody for some purposes, since the concentration of the monoclonal antibody is only about 50 μg/ml.

To produce a much higher concentration of a slightly less pure monoclonal antibody, the desired hybridoma can be injected into mice, preferably syngeneic or semi-syngeneic mice. The hybridoma will cause the formation of tumors that produce antibodies after a suitable incubation time, which will lead to a high concentration of the desired antibody (about 5-20 mg/ml) in the bloodstream and peritoneal exudate (ascites) of the host mouse. Although these host mice also have normal antibodies in their blood and ascites, the concentration of these normal antibodies is only about 5% of the concentration of the monoclonal antibody. Moreover, since these normal antibodies are not antihuman in their specificity, the monoclonal antibody obtained from pooled ascites or from serum is essentially free of any contaminating antihuman immune globulin. This monoclonal antibody is of high titer (active at dilutions of 1:50,000 or more) and has a high ratio of specific to non-specific immune globulin (about 1/20). The produced immune globulin together with myeloma k light chains are non-specific, "nonsense" peptides that only dilute the monoclonal antibody without reducing its specificity.

Examples

Production of monoclonal antibodies

A. Immunization and somatic cell hybridization

Female CAF1 mice (Jackson Laboratories, 6-8 weeks old) were immunized intraperitoneally with 2 x 107 human thymocytes in 0.2 ml of phosphate-buffered saline at 14-day intervals. Four days after the third immunization, the spleens were removed from the mice and a single-cell suspension was made by pressing the tissue through a stainless steel sieve.

Cell condensation was performed according to the procedure developed by Kohler and Milstein. 1 x 108 splenocytes were condensed in 0.5 ml condensation medium comprising 35% polyethylene glycol (PEG 1000) and 5% dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, NY) with 2 x 107 P3X63Ag8U1 myeloma cells provided by Dr. M. Scharff, Albert Einstein College of Medicine, Bronx, NY. These myeloma cells secrete IgG1 k light chains.

B. Selection and growth of hybridomas

After cell condensation, the cells were cultured in HAT medium (hypoxanthine, aminopterin and thymidine) at 37°C with 5% CO2 in a humid atmosphere. Several weeks later, 40 to 100 μl of supernatant from cultures containing hybridomas is added to a pellet of 106 peripheral lymphocytes that have been separated into E rosette positive (E+) and E rosette negative (E-) populations, prepared from blood from healthy human donors. as described by Mendes (J. Immunol. 111:860, 1973). Detection of mouse hybridoma antibodies binding to these cells was determined by indirect immunofluorescence. Cells incubated with culture supernatants were stained with fluorescent goat-anti-mouse IgG (G/M FITC) (Meloy Laboratories, Springfield, VA; F/p = 2.5) and cells coated with fluorescent antibody were later analyzed on a Cytofluorograf FC200. /4800A (Ortho Institutes, Westwood, MA) as described in Example III. Hybridoma cultures containing antibodies that react specifically with E+ lymphocytes (T cells) were selected and cloned twice using limiting dilution procedures in the presence of batch cells. Later, clones were transferred intraperitoneally by injecting 1 x 10 7 cells of a given clone (0.2 ml volume) into CAF1 mice primed with 2,6,10,14-tetramethylpentadecane, sold by Aldrich Chemical Company under the name Pristine. Malignant ascites from these mice were then used to characterize lymphocytes as described below in Example II. The OKT5 hybrid antibody in question was demonstrated by standard techniques to be of the IgG1 subclass.

Example II

Characterization of OKT5 reactivity

A. Isolation of lymphocyte populations

Human peripheral blood mononuclear cells were isolated from healthy volunteer donors (ages 15-40) using Ficoll-Hypaque density gradient centrifugation (Pharmacia Fine Chemical, Piscataway, NJ) according to the technique described by Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97): 77, 1968. Unfractionated mononuclear cells were separated into surface Ig+ (B) and Ig- (T plus none) populations by chromatography on a Sephadex G-200 anti-F(ab')2 column such as previously described by Chess, et al., J. Immunol. 113:1113 (1974). T cells were isolated by E rosetting of the Ig population with 5% sheep erythrocytes (Microbiological Associates, Bethesda, MD). The rosette mixture was layered over Ficoll-Hypaque and the isolated E+ granule was treated with 0.155M NH4Cl (10 ml per 108 cells). The resulting population was <2% EAC rosette positive and >95% E rosette positive as determined by standard procedures. Next, the unresected Ig- (Null cell) population was skimmed from the Ficoll interface. This later population was <5% E+ and <2% sIg+. The surface Ig+ (B) population thus obtained from the Sephadex G-200 column was obtained after elution with normal human gamma globulin as described earlier. This population was >95% surface Ig+ and <5% E+.

Normal human macrophages were obtained from a mononuclear population by adherence to polystyrene. Thus, mononuclear cells were resuspended in the final culture media (RPMI 1640, 2.5 mM HEPES/4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid/buffer, 0.5% sodium bicarbonate, 200 mM L-glutamine and 1% penicillin -streptomycin, supplemented with 20% heat-inactivated human AB serum) at a concentration of 2 x 106 cells and incubated in plastic petri dishes (100 x 20 mm) Falcon Tissue Culture Dish; Falcon, Oxnard, CA) at 37°C overnight. After extensive washing to separate non-adherent cells, the adherent population is separated by washing (suddenly) with cold serum-free medium containing 2.5 mM. EDTA and occasional scraping with the rubber tip of a disposable syringe cap. More than 85% of the cell population could ingest latex particles and had the morphological characteristics of monocytes based on Wright-Cioms staining.

Isolation of thymocytes

Normal human thymus was obtained from patients aged 2 months to 14 days undergoing collective heart surgery. Freshly obtained parts of the thymus gland were immediately placed in 5% fetal calf serum in medium 199 (Gibco), where the parts were finely minced with forceps and scissors, and later a single-cell suspension was made by pressing through a wire sieve. Cells were further layered over Ficoll-Hypaque and spun down and washed as previously described in section A above. Thus obtained thymocytes were 95% viable and > 90% E rosette positive.

C. Cell line

A transformed Epstein-Barr virus (EBV) B cell line from a normal individual (Laz 156) was provided by Dr. H. Lazarus, Sidney Farber Institute, Boston, MA.

Example III

Cytofluorographic analysis and cell separation

Cytofluorographic analysis of monoclonal antibodies with all cell populations was achieved by indirect immunofluorescence with goat anti-mouse IgG conjugated with fluorescein (G/M FITC) (Meloy Laboratories) using the FC200/4800A Cytofluorograph (Ortho Instruments). Briefly, 1 x 106 cells are treated with 0.15 ml OKT5 at a 1:500 dilution, incubated at 4°C for 30 minutes and washed twice. The cells are then reacted with 0.15 ml of a 1:40 dilution of G/M FITC at 4°C for 30 minutes, centrifuged and washed three times. The cells are then analyzed with a Cytofluorograph and the fluorescence intensity per cell is recorded on a pulse height analyzer. A similar reactivity pattern was seen for a 1:10,000 dilution, but further dilution caused loss of reactivity. Baseline staining was achieved by substituting a 0.15 ml aliquot of 1:500 ascites from CAFI mice injected intraperitoneally with a non-producing hybrid clone. The reactivity of the lymphoid populations with horse anti-TH2 and normal horse IgG was determined as previously described in the above-mentioned articles by Reinherz, eta 1.

In experiments intended to separate T cell subsets, 100 x 10 6 unfractionated T cells were labeled with 4 ml of a 1:500 dilution of OKT4 or OKT5 and developed with G/M FITC. OKT4 has previously been shown to be specifically reactive with 55-60% of peripheral blood T lymphocytes representing human helper subsets. Using a fluorescence-activated cell sorter (FACS-I) (Becton, Dickinson, Mountain View, CA), T cells were separated into OKT4+ and OKT4- subsets as well as OKT5+ and OKT5- subsets from the same individual. Viability after sorting was 95% using Trypan blue exclusion in all cases. The purity of these separate populations was > 95%.

Example IV

Analysis of T cell subsets with horse anti-TH2

In a similar manner to Example III, horse anti-TH2 was used to separate TH2+ and TH2- T cells on FACS by labeling 60 x 10 6 unfractionated T cells with 0.12 ml horse anti-TH2 and 0.1 ml R/H FITC (Cappel Laboratories, Downingtown , PA) as previously described in Reinherz, et al. The purity and viability of the sorted cells were similar to the sorted populations described above. FACS-sorted subsets of T cells isolated with horse anti-TH2, OKT4, or OKT5 were cultured for 48 hours in RPMI 1640 containing 20% human AB serum, 1% penicillin-streptomycin, 200 mM Irglutamine, 25 mM HEPES buffer (Microbiological Associates ) and 0.5% sodium bicarbonate at 37°C in a 5% CO2 moist atmosphere. These cultured cells were then analyzed on a Cytofluorograph as described above. Baseline staining was determined by replacing the specific antibody with normal horse IgG and staining as above.

Example V

Functional studies

A. Proliferative studies

The mitogenic response of 105 unseparated and FACS-fractionated T lymphocytes was tested in microculture to optimal doses of Concanavalin A (Con A) (Caliviochem, La Jolla, CA) and phytohemagglutinin (PHA) (Burrougs-Wellcome Company, Greenville, NC). The alloantigen proliferative response was measured competitively for the same populations with mitomycin-treated Laz 156, which is a stimulus for an EBV-transformed human B lymphoblastoid cell line. Proliferation to tetanus toxoid (Massachusetts Department of Public Health Biological Laboratories, Boston, MA) and measles antigen (Microbiological Associates) was tested using a 10 μg/ml final concentration and a 1:20 dilution. Five percentages of macrophages obtained as described above were added to all populations when initiating in vitro cultures. Mitogen-stimulated cultures were pulsed after 4 days with 0.2 μCi 3H-thymidine (3HTdR; 1.9 Ci/mM specific activity) (Schwarz/Mann, Division of Becton, Dickinson, Orangeburg, NY) and saved 18 hours later on a MASH II apparatus ( Microbiological Associates). 3H-TdR incorporation was measured on a Packard Scintillatic Counter (Packard Instrument Company, Downer's Grove, IL). Basic 3H-TdR incorporation was obtained by replacing the mitogen with media. Solution and cell surface alloantigen cultures were pulsed after five days with 3H-TdR for 18 hours, defended and counted as above.

B. Cytotoxicity studies

Sensitization of cultures to cell-mediated lympholysis (CML) was established by plating unfractionated T cells with mitomycin-treated stimulator cells, all at 2 x 10 6 cells/ml in multiple wells of a microtiter plate. At the end of the five-day period, unfractionated T cells were fractionated into OKT5+ and OKT5- T cell subsets by FACS. These subsets of T cells were then added to the desired cells labeled with 51Cr sodium chromate and specific cytotoxicity was determined after 6 hours of cell incubation. The percentage of cytotoxicity was determined with the following formula:

51Cr released in the experiment-5lCr released spontaneously

-------------------------------------------------- --------------------------- x 100

51Cr released by thawing-51Cr released spontaneously

All samples were performed in triplicate and results are expressed as mean ± standard deviation.

C. Con A Activation of suppressor cells and suppression of MLC

Unfractionated T cells were activated with 20 μg of Concavalin A (Con A) (Calbiochem) per 106 cells. These Con A-treated cells were cultured upright in 25 cm2 tissue culture flasks (Falcon, Oxnard, CA) in RPMI 1640 (Grand Island Biological Company) containing 20% human AB serum, 1% penicillin-streptomycin, 200 mM Irglutamine, 25 mM HEPES buffer (Microbiological Associates) and 0.5% sodium bicarbonate for 48 hours at 37°C in a humid atmosphere containing 5% CO2. Untreated control T cells were cultured identically but without Con A. Later, the cells were spun down, washed five times, and added to fresh autologous responder cells in a one-way mixing lymphocyte culture (MLC) as described above. In the suppression test, the MLC culture was established in round-bottom microtiter plates (Linbro Chemical Company, New Haven, CT) with triplicate wells, each containing 0.05 x 10 6 responder lymphocytes (mostly mononuclear); 0.05 x 106 of either unactivated or Con A activated autologous unfractionated or fractionated T cells; and 0.1 x 106 mitomycin-treated stimulated cells (Laz 156). After five days, cultures were pulsed with 0.2 μCi 3H-TdR and protected 18 hours later, as above. The percentage of inhibition of MCL proliferation was then calculated using the formula:

% inhibition = . [1- (cpm Con A+: cpm)] x 100

where cpm Con A+ represents the results of 3H-TdR incorporation into MLC when Con A of activated T cells or subsets of T cells is added, and where cpm represents the results when unactivated autologous T cells are added.

Brief description of the drawing

Figure 1 shows the fluorescence scheme obtained on the Cytofluorograph after the reaction of normal human peripheral T cells, B cells, null cells and macrophages with OKT5 at a dilution of 1:1000 and G/N FITC in the upper row. For comparison, results with equine anti-TH2 are shown in the bottom row.

Figure 2 shows the fluorescence scheme obtained on the Citofluorograph after the reaction of human thymocytes with OKT5 and G/M FITC(A) and with horse anti-TH2(B).

Figure 3 shows a scheme of fluorescence obtained on a Cytofluorograph after the reaction of separate cell subsets of horse anti-TH2 T cells with OKT5.

Figure 4 shows the fluorescence scheme obtained on the Citofluorograph after the reaction of OKT4 of separate subsets of T cells with OKT5.

Figure 5 shows the cytotoxic capacity of unfractionated T cells (subsets) separated with OKT5 after allosensitization in MLC.

Hybridoma production and production and characterization of the resulting monoclonal antibody were performed as described in the Examples above. Although large quantities of the subject antibody were made by injecting the subject hybridomas intraperitoneally into mice and harvesting malignant ascites, it is clear that hybridomas that can be cultured in vitro using techniques well known in the art and then the antibody is isolated from the supernatant are included.

As shown in the top row of Figure 1, approximately 20% of the human peripheral blood T cell population of a given normal individual reacts with OKT5, whereas all B cells, Null cells, and macrophage populations isolated from the same individual do not react with OKT5. Similarly, equine anti-TH2 reacts with 24% of peripheral T cells and also does not react with B cells, Null cells and macrophages. A monoclonal antibody is thus characterized by reacting with an antigen found on the surface of approximately 20% of normal human peripheral T cells, while not reacting with any antigens on the surface of the other three cell types discussed above. As will be discussed below, the OKT5+ fraction of the human peripheral T cell population is involved in cytotoxic and suppressor T cell subsets. This differential reactivity is one test by which the subject OKT5 antibody can be detected and distinguished from other antibodies.

As shown in Figure 2, approximately 80% of normal human thymocytes from a six-month-old child reacted with OKT5. Similar results (about 80% reactivity) were obtained using supplemental thymus samples from normal individuals aged 2 months to 19 years. This value is the same for horse anti-TH2. The reactivity scheme in Figure 2 provides another method for detecting the subject OKT5 antibody and for distinguishing it from other antibodies.

As shown in Figure 3, the subject antibody reacts with TH2+ but not TH2- T cells. Approximately 5-10% of the TH2+ subset does not react with OKT5. The reactivity scheme in Figure 3 provides a third procedure for detecting the subject OKT5 antibody and for distinguishing it from other antibodies.

As shown in Figure 4, the OKT4+ T cell subset is completely unreactive with OKT5. In contrast, the OKT4- subset of T cells is mainly OKT5+ (6,800 to 10,000 cells tested). These results indicate that the OKT5+ T cell subset, like the previously defined TH2+ subset, is reciprocal and distinct from the OKT4+ subset. While OKT4+ contains helper T cells, the OKT5+ subset (as well as the TH2+ subset) contains cytotoxic and suppressor T cells. This reactivity scheme in Figure 4 provides a complementary procedure for identifying the OKT5 antibody and for distinguishing it from other antibodies.

Figure 5 shows that the OKT5+ subset of T cells induces CML. The degree of shedding mediated by this population is greater than that mediated by the unfractionated T cell population. In contrast, the OKT5- population of T cells has a minimal termination effect. After activation in the MLC protov Laz 156, unfractionated T cells are separated into OKT5+ and OKT5- subsets. Both unfractionated and fractionated T cells were analyzed in CML against 51Cr-labeled Laz 156 targets at various effector:target (E:T) ratios. OKT5+ T cell subsets comprised the effector population in cell-mediated lympholysis. At E:T ratios of 5:1, 10:1, and 20:1, the OKT5+ T cell population performed 40%, 58%, and 77% specific cleavage, respectively. This knockdown effect of the isolated OKT5+ subset was significantly greater than the unfractionated T cell population. Given the earlier premise that a TH2+ subset of T cells in humans causes CML, the present findings support the notion that TH2+ and OKT5+ T cell subsets define similar populations of functionally active T lymphocytes. This differential cytotoxic capacity provides a novel second method of identifying the OKT5 antibody and distinguishing it from other antibodies.

Functional studies were performed on lymphoid populations that were separated on a fluorescence-activated cell separator (FACS). The results of these studies are shown in Tables I through III below and provide further support for the previously described characterization of the subject monoclonal antibody.

In these studies, an unfractionated T cell population is treated with 1:500 diluted OKT5 and G/M FITC and separated by FACS into OKT5+ and OKT5- subsets. At a given purity of the obtained populations (greater than or equal to 95%), 5% macrophages are added to the separate populations before culturing in vitro. The unfractionated T cell population and isolated OKT5+ and OKT5- T cell subsets are then stimulated with PHA, Con A, soluble antigens and alloantigens to assess their proliferative responses in vitro.

The proliferative response of unfractionated T cell populations to PHA and Con A is shown in Table I. The maximal proliferative response using the unfractionated T cell population is achieved with 1 µg PHA per 106 cells with reduced responses occurring at 0.5 μg PHA and 0.1 μg PHA per 106 station. Treatment of unfractionated T cells with OKT5 and goat-mouse FITC without subsequent fractionation did not alter the proliferative response. In contrast, differences in response to PHA were obtained with separate OKT5+ and OKT5- T cell subsets. The OKT5- cell population responded to all doses of PHA in a similar manner to the undifferentiated T cell population. However, the proliferative response of OKT5+ cells was significantly lower in all doses of the tested PHA.

Furthermore, at a PHA dose of 0.1 μg per 106 cells, OKT5+ T cells did not proliferate at all, while OKT5- subset T cells and unfractionated cells still responded. On the other hand, the proliferative response of these subsets to Con A was similar and the two cell subsets could not be distinguished from each other or from the unfractionated T cell population.

Reactions to alloantigen in MLC and to soluble antigens were further examined. As shown in Table II, the unfractionated T cell population, the unfractionated T cell population treated with OKT5 and G/M FITC, and both OKT5+ and OKT5- T cell subsets responded similarly in MLC against Laz 156. In contrast, the proliferative responses to soluble antigens provided the clearest distinction between the two subsets. In all cases tested, the OKT5+ subset of T cells proliferated minimally against soluble tetanus and measles toxoid antigens, while the OKT5- subset of T cells responded well.

Earlier studies showed that the TH2+ lymphocyte population could be induced with Con A to suppress autologous lymphocytes in MLC. To determine whether the OKT5+ subset of T cells can clearly express a suppressor function, T cells were activated for 48 hours with Con A and then sorted with OKT5 and OKT4 monoclonal antibodies into distinct T cell subsets. Later, these separate subpopulations of T cells were added to autologous reactive lymphocytes during MLC initiation. Treatment with monoclonal antibody and G/M FITC did not change this result. This result is shown in Table III, and it can be seen that unfractionated T cells activated with Con A did suppress the proliferation of autologous cells in MLC. Although both OKT4+ and OKT5+ T cells proliferate in Con A, only OKT5+ T cells become suppressive when activated with Con A. The OKT4+ subset was not suppressive. Furthermore, the OKT5 subpopulation, which contains both OKT4+ and OKT4- TH2- subpopulations, was minimally suppressive compared to the highly suppressive OKT5+ population.

Table IV shows the relationship between peripheral T cell levels and T cell subsets and various disease states. These ratios can be used for diagnostic purposes (eg, to detect acute infectious mononucleosis) by analyzing a blood sample from an individual suspected of having the disease to determine levels of T cell subsets and T cell levels. These relationships can also be used for therapeutic purposes when the causative agent of the disease state is an elevated level of a subset of T cells (eg, acquired agammaglobulinemia Type I). For therapeutic use, administration of the appropriate monoclonal antibody to a patient with elevated levels of a subset of T cells will reduce or eliminate the excess. The relationships shown in Table IV are a further way in which the OKT5 antibody can be detected and distinguished from other antibodies.

Other monoclonal antibody-producing hybridomas have been made by the present Applicants (designated OKT1, OKT3 and OKT4) and are described and protected in the following U.S. patent applications: SN 22,132, filed March 20, 1979; SN 33,639, filed April 26, 1979; and SN 33,669, filed Apr. 26, 1979. These applications are incorporated herein by reference.

According to the present invention, a hybrid that can produce an antibody against an antigen found in human cytotoxic and suppressor T cells, a method for producing this hybridoma, a monoclonal antibody against an antigen found in human cytotoxic and suppressor T cells, methods for producing antibodies, such as and methods and preparations for treating or diagnosing diseases using this antibody.

TABLE I

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TABLE II

PROLIFERATIVE REACTION OF NON-FRACTIONAL CELLS AND OCT5

OF FRACTIONATED SUBSETS OF CELLS WITH SEPARATE MONOCLONAL ANTIBODY

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TABLE III

SA-MEDIATED MLC SUPPRESSION

OKT55 T CELLS INDUCED WITH CON A

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TABLE IV

LEVELS OF PERIPHERAL T CELLS IN DISEASE STATES

[image] N = within normal limits

O = absent

+ = above normal

++ = well above normal

- = below normal

-- = very below normal

* these levels return to normal about one week before the disappearance of clinical symptoms

"Numbers in parentheses indicate the number of patients examined."

Although only one hybridoma has been described that produces a single monoclonal antibody against human cytotoxic and suppressor T cell antigens, the present invention is intended to encompass all monoclonal antibodies that exhibit the characteristics described herein. The OKT5 antibody in question was determined to belong to the IgG1 subclass, which is one of the four mouse IgG subclasses. These immunoglobulin G subclasses differ from each other in so-called "fixed" responses, although an antibody to a specific antigen will have a so-called "variable" region that is functionally identical regardless of which immunoglobulin G class it belongs to. That is, a monoclonal antibody exhibiting the characteristics described herein may be of the IgGl, IgG2a, IgG2b, or IgG3 subclasses, or of the IgH, IgA, or other known Ig classes. Differences between these classes or subclasses will not affect the selectivity of the reaction scheme of the antibody, but may affect the further reaction of the antibody with other materials, such as (for example) the complement of anti-mouse antibodies. Although the subject antibody is IgG1 specific, antibodies having the reactivity patterns illustrated herein are intended to be included in the subject invention regardless of which class or subclass of immune globulin they belong to.

Further included in the present invention are methods for making the monoclonal antibodies described herein using the hybridoma technique described herein. Although only one example hybridoma is provided herein, it is contemplated that one skilled in the art may use the immunization, condensation, and selection procedures described herein to obtain other hybridomas capable of producing antibodies having the reactivity characteristics described herein. Since individual hybridomas produced from a known murine myeloma cell line and spleen cells from known species of mice cannot be further identified except by the antibody produced by these hybridomas, it is intended that all hybridomas producing an antibody having the reactivity characteristics described herein are included in the subject invention, as well as procedures for making this antibody using hybridomas.

Further aspects of the invention are methods for treating or diagnosing disease using the OKT5 monoclonal antibody or any other monoclonal antibody exhibiting the reactivity pattern provided herein. The antibody in question can be used to detect excess activity of cytotoxic or suppressor T cells by reacting a preparation of T cells from an individual with the OKT5 antibody. Excess cytotoxic or suppressor T cell activity will be indicated by the presence of more than 20-30% of the total peripheral T cell population reacting with OKT5. This diagnostic technique can be used using only the OKT5 antibody or in combination with other antibodies (eg, OKT3 and OKT4) as shown in Table 4. Reactivity patterns with a panel of antibodies against T cells and T cell subsets will allow more accurate detection of certain disease condition than was possible using earlier procedures for diagnosis.

Treatment of disease states (eg, malignant diseases) that manifests as an excess of suppressive activity can be achieved by administering a therapeutically effective amount of OKT5 antibody to an individual in need of such treatment. By selectively reacting with the suppressor antigen of T cells, an effective amount of OKT5 antibody will reduce the excess of suppressor T cells, thus mitigating the effects of excess suppressor T cells. Diagnostic and therapeutic preparations comprising effective amounts of OKT5 antibody in admixture with diagnostically or pharmaceutically acceptable carriers are also included within the present invention.

The subject hybridoma OKT5 was deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 on September 18, 1979 and September 28, 1979, and was assigned ATCC "accession number CRL 8013 and CRL 8016, respectively."

Claims (12)

1. Monoclonal antibody from the IgG class produced using a hybridoma formed by the condensation of cells from a mouse myeloma line and spleen cells from mice that were previously immunized with human thymocytes /and cells from a mouse myeloma line/, indicated by the fact that a) reacts with more than 90% of cytotoxic and suppressor TH2+ T cells (which are about 20-30% of all normal human peripheral T cells), but not with normal human peripheral B cells, Null cells or macrophages; b) reacts with about 80% of normal human thymocytes; c) does not react with TH2- T cells or OKT4+ cells, but reacts with about 68% of OKT4- T cells; d) defines a population of T cells (OKT5+) that is highly cytotoxic and that is lower than normal levels in primary biliary cirrhosis, multiple sclerosis, and hyper IgE, than normal levels in all stages of Hodgkins disease and higher than normal levels in acquired agammaglobulinemia Type I and acute infectious mononucleosis. 2. Monoclonal antibody according to Claim 1, characterized in that it is of subclass IgG1. 3. Monoclonal antibody according to Claim 1, characterized in that it is produced from hybridomas formed by the condensation of P3X63Ag8U1 myeloma cells and spleen cells from CAF1 mice previously immunized with human thymocytes 4. Procedure for making a monoclonal antibody which a) reacts with more than 90% of cytotoxic and suppressor TH2+ T cells (which are about 20-30% of all normal human peripheral T cells), but not with human peripheral B cells, Null cells or macrophages; b) reacts with about 80% of normal human thymocytes; c) does not react with TH2- T cells or OKT4+ T cells, but they react with about 68% of OKT4- T cells; d) defines a T cell population (OKT5+) that is highly cytotoxic and that is lower than normal levels in primary biliary cirrhosis, multiple sclerosis, and hyper IgE, than normal levels in all stages of Hodgkins disease and higher than normal levels in acquired agammaglobulinemia Type I and acute infectious mononucleosis, indicated by the fact that it includes: i) immunization of mice with human thymocytes; ii) separating the spleen from the mentioned mice and making a suspension of spleen cells; iii) condensation of said spleen cells with murine myeloma cells in the presence of a condensation promoter; iv) diluting and culturing condensed cells in special wells in a medium that will not support uncondensed myeloma cells; v) evaluating the supernatant in each well containing the hybridoma for the presence of the desired antibody; vi) selection and cloning of a hybridoma that produces the desired antibody; vii) isolation of antibodies from the supernatant above the mentioned clones. 5. The method according to Claim 4, characterized in that said mice are CAF1 type and said myeloma cells are P3X63Ag8U1. 6. Procedure for making a monoclonal antibody which a) reacts with more than 90% of cytotoxic and suppressor TH2+ T cells (which are about 20-30% of all normal human peripheral T cells), but not with normal human peripheral B cells, Null cells or macrophages; b) reacts with about 80% of normal human thymocytes; c) does not react with TH2- T cells or OKT4+ T cells, but reacts with about 68% of OKT4- T cells; d) defines a population of T cells (OKT5+) that is highly cytotoxic and that is lower than normal levels in primary biliary cirrhosis, multiple sclerosis, and hyper IgE, than normal levels in all stages of Hodgkins disease and higher than normal levels in acquired agammaglobulinemia Type I and acute infectious mononucleosis, indicated by the fact that it includes: i) immunization of mice with human thymocytes; ii) separating the spleen from the mentioned mice and making a suspension of spleen cells; iii) condensation of said spleen cells with murine myeloma cells in the presence of a condensation promoter; iv) diluting and culturing condensed cells in special wells in a medium that will not support uncondensed myeloma cells; v) assessing the supernatant in each well containing the hybridoma for the presence of the desired antibody; vi) selection and cloning of a hybridoma that produces the desired antibody; and vii) isolation of antibodies from the supernatant above said clones; viii) transfer of said clones intraperitoneally into mice; and ix) collecting malignant ascites or serum from said mice, wherein the ascites or serum contains the desired antibody. 7. Method according to Claim 6, characterized in that said mice are CAF1 type and said myeloma cells are P3X63Ag8U1. 8. A murine monoclonal antibody, characterized in that it reacts with more than 90% of cytotoxic and suppressor TH2+ human T cells but not with normal human peripheral B cells, Null cells or macrophages. 9. A method for the production of a monoclonal antibody that reacts with more than 90% of cytotoxic and suppressor TH2"1" human T cells but not with normal human peripheral B cells, Null cells or macrophages, indicated by this, which is cultured with hybrid ATCC CRL 8016 in a suitable medium and the antibody is isolated from the supernatant of the above-mentioned hybridoma. 10. A method for producing a monoclonal antibody that reacts with more than 90% of cytotoxic and suppressor TH2+ human T cells but not with normal human peripheral B cells, Null cells or macrophages, characterized by the fact that it comprises injecting hybridoma ATCC CRL 8016 mice and isolating the antibody from malignant ascites serum of the mentioned mice. 11. A method for producing a monoclonal antibody that reacts with more than 90% of cytotoxic and suppressor TH2+ human T cells but not with normal human peripheral B cells, Null cells or macrophages, characterized in that it comprises: i) immunization of mice with normal human thymocytes; ii) separating the spleen from the mentioned mice and making a suspension of spleen cells; iii) condensation of said spleen cells with murine myeloma cells in the presence of a capacitor promoter; iv) diluting and culturing condensed cells in special wells in media that will not support uncondensed myeloma cells; v) assessing the supernatant in each well containing the hybridoma for the presence of antibodies to E rosette positive purified T cells; vi) selection and cloning of a hybridoma that produces an antibody that reacts with more than 90% of cytotoxic and suppressor TH2+ human T cells but not with normal human peripheral B cells, Null cells or macrophages; and vii) isolation of antibodies from the supernatant of the above-mentioned clones. 12. A method for making a monoclonal antibody that reacts with more than 90% of cytotoxic and suppressor TH2+ human T cells but not with normal human peripheral B cells, Null cells or macrophages, characterized in that it includes the steps: i) immunization of mice with normal human thymocytes; ii) separating the spleen from the mentioned mice and making a suspension of spleen cells; iii) condensation of said spleen cells with murine myeloma cells in the presence of a condensation promoter; iv) diluting and culturing condensed cells in special wells in media that will not support uncondensed myeloma cells; v) assessing the supernatant in each well containing the hybridoma for the presence of antibodies to E rosette positive purified T cells; vi) selection and cloning of a hybridoma that produces an antibody that reacts with more than 90% of toxic and suppressor TH2+ human T cells but not with normal human peripheral B cells, Null cells or macrophages; and vii) transfer of said clones intraperitoneally into mice; and viii) collecting malignant ascites or serum from said mice, wherein the ascites or serum contains the desired antibody.