SI8012392A8 - Method for obtaining monoclonal antibody - Google Patents

Description

PROCEDURE FOR OBTAINING A MONOCLONAL ANTIBODY

FIELD OF THE INVENTION

The invention relates to the field of genetic engineering and relates to a process for the production of novel monoclonal antibodies useful in the diagnosis and therapy of various diseases.

Technical problem

A technical problem solved by the present invention is a method of obtaining a new monoclonal antibody having the characteristics detailed below. The new method uses a hybrid cell line to produce a monoclonal antibody for an antigen found on normal human cytotoxic and suppressor T cells. Said hybrid cell line was obtained by the condensation of splenocytes of CAF ₁ mice with P3x63Ag8Ul myeloma cells.

This solution to a technical problem has not been described in previous literature, so the process of the invention is new and original.

The state of the art

Condensation of mouse myeloma cells for spleen cells from immunized mice by Kohler and Milstein 1975 [Nature 256, 495-497 (1975)] demonstrated for the first time that a continuous cell line producing a homogeneous (so-called monoclonal) antibody could be obtained . After this initial work, much effort was made to produce various hybrid cells (called hybridomas) and to use the antibody made using these hybridomas for various scientific studies. See, for example, Current Topics in Microbiologists and Immunologists, Volume Sl - Lymphocyte Hybridomas, F. Melchers, M. Fotter, and N. Warner, Editors, Springer-Verlag, 1978, and references therein; C. J. Bamstable et al .. Celi, 14, 9-20 (May, 1978); P. Parham and W. F. Bodmer, Nature 276, 397-399 (November 1978); Handbook of Experimental Immunologists, Third Edition, Volume 2, by D. M. Wier, Editor, Blackwell, 1978, Chapter 25; and Chemical and Engineering News, January 1, 1979, 15-17. These references at the same time point to the rewards and complications that arise from attempts to produce a monoclonal antibody from a hybridoma. Although the general technique is well understood conceptually, there are many difficulties encountered and many variations that are needed on a case by case basis. In fact, there is no certainty, before attempting to make a hybridoma, that the desired hybridoma will be obtained, that it will produce an antibody if obtained, or that the antibody produced in this way will have the desired specificity. The degree of success is mainly influenced by the type of antigen used and the choice of technique used to isolate the desired hybridoma.

The attempted production of a monoclonal antibody for human surface lymphocyte cell antigens has been described in only a few cases. See, for example, Current Topics in Microbiologists and Immunologists, ibid, 66-69 and 164-169. The antigens used in these published experiments were cultured cell lines of human lymphoblastoid leukemia and human chronic lymphocytic leukemia. Many of the resulting hybridomas appear to produce antibodies 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 major classes of lymphocytes involved in the human and animal immune systems. The first of these (the cell derived from the thymus or the T cell) differentiates into the thymus from the hemopoietic cell line. While inside the thymus, the differentiating cells are called thymocytes. Mature T cells emerge from the thymus and cycle between tissues, vessels and bloodstream. These T cells form a large portion of the small lymphocyte recirculation space. They have immune specificity and are directly involved in cell-mediated immune responses (such as coil rejection) as effector cells. Although T cells do not secrete humoral antibodies, they are sometimes required to secrete these antibodies using the second class of lymphocytes discussed below. Some guys

T cells have a regulatory function in other aspects of the immune system. The mechanism of this cell cooperation process has not yet been fully understood.

The second class of lymphocytes (cells derived from bone marrow or B cells) are those that secrete the antibody. They also develop from the hemopoietic cell line, but their differentiation is not determined by thymus. In stories, they differentiate into an organ analogous to the thymus, called Bursa iii Fabricius. In mammals, however, no equivalent organ has been detected, 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 (relative) of promoting the reaction, suspending the reaction, or destroying (breaking) foreign cells. These subclasses are well understood for mouse systems, but have only recently been discovered for human systems. See, for example, 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 Contemporarv topics in Immunobiologists, O. Stutman, Editor, Plenum Press, 1977, Volume 7, 363-379.

The ability to identify or suppress class III subclasses of T cells 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 disease prognosis depends on the distinction between the 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 Lvmphomas, S. Thierfelder, et al .. eds, Springer, Heidelberg, 1977, 33-45; and D. Belpomme, et. al .. British Journal of Haemotologv, 1978,38,85.

Certain disease conditions (eg, juvenile rheumatoid arthritis, malignancies and agamaglobulinemia) are accompanied by an imbalance of T cell subclasses. It has been suggested that autoimmune diseases are accompanied by an excess of helper T cells or a lack of certain suppressor T cells, whereas agamaglobulinemia is accompanied by an excess of certain suppressor T cells or a lack of helper T cells. The malignant condition is mostly accompanied by an excess of suppressor T cells.

In certain leukemias, excess T cells are produced at a stopped stage of development. The 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 with Differing Prognoses in Haematologists and Blood Transfusion, Volume 20, Springer-Verlag, 1977, 17-24, and references cited therein.

Agamaglobulinemia, a diseased condition in which no immune globulin is produced, involves at least two distinct types. In type I, the lack of producing 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 B cells of the patient, the lymphocytes responsible for the actual secretion of antibodies; however, these B cells are either repressed or unassisted, leading to severely reduced or absent immune globulin production. The type of agamaglobulinemia obtained can thus be determined by testing the excess suppressor T cells or the absence of helper T cells.

On the therapeutic side, there are some suggestions, though not yet proven, 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 skin lymphoma T cells and certain acute lymphoblastic leukemia T cells) may be treated with the help of an antigen of helper T cells. Treatment of an autoimmune disease caused by excess helper cells can also be achieved in the same way. Treatment of the disease (e.g., malignancies or type I agamaglobulinemia) due to excess suppressor T cells can be treated by administering antigen to suppressor T cells.

Antisera against a whole class of human T cells (so-called antihuman thymocyte globulin or ATG) have been reported to be therapeutically useful in patients receiving organ transplants. Because cell-mediated immune responses (the mechanism of transplant-mediated rejection) depend on T cells, administration of antibodies to T cells prevents or inhibits this rejection process. See, for example, Cosimi, et al. Randomized Clinical Trial of ATG in Cadaver Renal Allgraft Recipients: Importance of T Whole Monitoring, Surgerv 40: 155-163 (1976) and references therein.

The identification and repression of human T cell classes and subclasses was previously accomplished using spontaneous autoantibodies or selective antisera for human T cells obtained by immunization of animals with human T cells, bleeding animals to obtain serum, and adsorption of antisera with (for example) autologous but not allogeneic B cells so that antibodies with unwanted reactivity are removed. Making these antisera is extremely difficult, especially in the adsorption and purification stages. 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 induces the production of antibodies against the various antigens found in all human injected T cells. There is no selective production of antibodies against a single antigen. Third, the titer of a specific antibody obtained by such methods is usually quite low (e.g., inactive at dilutions greater than 1: 100) and the ratio of specific to a non-specific antibody is less than 1/10 ⁶ .

See, for example, the article by Chess and Schlossman discussed above (on pages 365 and beyond) and the article in Chemical and Engineering News discussed above, which describes the disadvantages of antisera from earlier science and advantages monoclonal antibody.

One of the T cell subsets identified by such antisera from earlier science was designated the TH ₂ ⁺ subset and was shown to contain cytotoxic effector cells for lymphoid-mediated cells and immunoregulatory suppressor T cells that suppress the function of both T cells and B cells. This subset contains about 20% - 30% of human peripheral T cells. See, for example, Article EL Reinherz, et al .. in J. Immunol. 123: 83 (1979) and New Engl. J. Med. 300: 1061 (1979).

Description of a solution to a technical problem with examples of execution

A new hybridoma (designated OKT5) has now been discovered that is capable of producing a monoclonal antibody against antigens found on normal human peripheral cytotoxic and suppressor TH ₂ ⁺ cells (about 20% of normal human peripheral T cells). The antibody thus produced is monospecific for one determinant on normal human cytotoxic and suppressor TH ₂ ⁺ cells and essentially contains no other anti-human immune globulin, as opposed to antisera from previous science (which are inseparably contaminated with an antibody that is reactive to numerous human antigens) and monoclonal antibodies from earlier science (which are not monospecific for human cytotoxic / suppressor T cell antigen). Moreover, these hybridomas can be cultured to produce an antibody without the need for immunization and killing of animals, followed by the difficult stages of adsorption and purification that are necessary to obtain even impure antisera from earlier science.

According to the invention, one object of the present invention is to provide hybridomas that produce antibodies against the antigen found on normal human cetotoxic and suppressor TH ₂ ⁺ T cells.

A further aspect of the present invention is to provide methods for making these hybridomas.

It is a further object of the invention to provide a substantially homogeneous antigen antibody located on normal human cytotoxic and suppressor TH ₂ ⁺ T cells.

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

Other objects and advantages of the invention become clear by examining the present description.

To meet the foregoing objectives and advantages, the present invention provides a new hybridoma producing a new antibody for an antigen found on normal human cytotoxic and suppressor TH ₂ ⁺ T cells, then the antibody alone, as well as diagnostic and therapeutic methods using the body. The hybridoma was made mainly according to the procedure of Milstein and Kohler. After immunization of mice with normal human thymocytes, the spleen cells of immunized mice were condensed with cells from the mouse myeloma line, and the resulting hybridomas were analyzed for those with supernatants containing an antibody that gave selective binding to normal human rosette positive T cells. The desired hybridomas are subsequently cloned and characterized. Because of this, it was obtained by an antibody-producing hybrid (designated OKT5) against the antigen 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 that recognizes this antibody is detected in approximately 80% of normal human thymocytes.

In view of the difficulties noted in earlier science and the lack of success using malignant cell lines as antigens, it was unexpected that the present procedure would provide the desired hybrid. It should be emphasized that the unpredictable nature of hybrid cell preparations does not allow extrapolation from one antigen or steel system to another. In fact, the present applicants have found that the use of a malignant T cell line 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 hybrid and the antibody produced herein have been identified by the code ΌΚΤ5, and which particular material is evident in the context.

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

The process for obtaining a monoclonal antibody comprises the following steps:

A. Immunization of mice with normal human thymocytes.

it is readily found that female CAF ₁ mice are desirable, it is contemplated that other strains of the mice may be used. The immunization schedule and thymocyte concentration should be such as to produce useful amounts of appropriately received splenocytes. Three immunizations were found effective at 14-day intervals with 2 χ 10 ⁷ cells / mice / injection in 0.2 ml of phosphate buffered saline.

B. Separation of spleen from immunized mice and making spleen suspension in appropriate environment. About one ml of middle per spleen is sufficient. This experimental technique is well known.

C. Condensation of suspended spleen cells with murine myeloma cells using a suitable condensation promoter. A preferred ratio is about 5 spleen cells per myeloma cell. A total volume of about 0.5-1.0 ml of condensation medium corresponds to about 10 ⁸ splenocytes. Many mouse myeloma cell lines are known and are available, mainly from members of the academic community of various deposit banks, such as the Salk Institute Celi Distrubution Center, La Jolla, CA. The used cell line should preferably be of the so-called drug resistant type, such that non-condensed myeloma cells will not survive in the selected environment, while hybrids will survive. The most common class is 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 used myeloma cell line be of the so-called non-excretory type, which does not itself produce any antibody, although excretory types may be used. However, in some cases myeloma-secreting lines may be desirable. Although a polyethylene glycol condensation promoter having an average molecular weight of from about 1000 to about 4000 (commercially available as PEG 1000, etc.) is desirable, other condensation promoters known in the art may be used.

D. Dilution and cultivation in special containers, mixtures of non-condensed spleen cells, non-condensed myeloma cells, and condensed cells in a selective medium that will support non-condensed myeloma cells over a satisfactory period to allow the death of non-condensed cells (approximately one week). Dilution may be of a limiting type, in which the volume of the diluent is calculated statistically by isolating a number of cells (e.g., 1-4) in each special container (e.g., each hole in the microtiter plate). The substrate is one (e.g., HAT substrate) that will not support a drug-resistant, non-condensed myeloma cell line (e.g., 8-azaguanidine resistant). As a result, these myeloma cells disappear. Because non-condensed spleen cells are non-malignant, they have only a limited number of generations. Thus, after a period of time (about one week), these non-condensed spleen cells no longer reproduce. Condensed cells, on the other hand, continue to reproduce because they have the malignant quality of the underlying myeloma and the ability to survive in the selective substrate of the underlying spleen cells.

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

F. Selection (e.g., by limiting dilution) and cloning of a hybridoma producing the desired antibody.

Since 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. Suitable substrates and appropriate length of cultivation time are known and easily determined. This in vitro technique produces an essentially monospecific monoclonal antibody substantially free of another specific anti-human immune globulin. There is a small amount of other immune globulin present since the substrate contains xenogenic serum (e.g., calf fetal serum). However, this in vitro procedure may not produce a satisfactory amount or concentration of antibodies for some purposes, since the concentration of the monoclonal antibody is only about 50gg / ml.

For the production of a much higher concentration of a slightly less pure monoclonal antibody, the desired hybrid can be injected with mice, preferably singen or semisenic mice. The hybridoma will induce the production of antibodies-producing tumors after a suitable incubation time, leading 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 lady 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 the collected ascites or from the serum is essentially free from macaque contaminating anti-human immune globulin. This monoclonal antibody is high titre (active at dilutions of 1: 50,000 or more) and has a high specificity ratio to non-specific immune globulin (about 1/20). The immune globulin produced together with the myeloma k light chains are non-specific, nonsense peptides that only dilute the monoclonal antibody without reducing its specificity.

EXAMPLE I

Production of monoclonal antibodies

A. Immunization and somatic hybridization of cells

Female CAFj mice (Jackson Laboratories; 6-8 weeks old) were immunized intraperitoneally with 2 χ 10 ⁷ human thymocytes in 0.2 ml phosphate buffered saline at 14-day intervals. Four days after the third immunization, the spleen was removed from the mice and a single-cell suspension was made by compressing the tissue through a stainless steel sieve.

Cell condensation was performed according to a procedure developed by Kohler and Milstein. 1 χ 10 ⁸ splinocytes were condensed in 0.5 ml condensation medium comprising 35% polyethylene glycol (PEG 1000) and 5% dimethyl sulfoxide in RPMI 1640 substrate (Gibco, Grand Island, NY) with 2 χ 10 ⁷ P3X63Ag8Ul myeloma cells that was by Dr. M. Scharff, Albert Einstein College of Medicine, Bronx, NY. These myeloma cells secrete IgG ₁ k light chains.

B. Hybridoma selection and growth

After condensation of the cells, the cells were cultured in a HAT medium (hypoxanthine, aminopterin and thymidine) at 37 ° C with 5% CO ₂ in a humid atmosphere. A few weeks later, 40 to 100 μΐ of supernatants from hybridoma-containing cultures were added to a pellet of 10 ⁶ peripheral lymphocytes, which were separated into E rosette positive (E ⁺ ) and E rosette negative (E) populations, prepared from the blood of healthy human donors as described by Mendes (J. Immunol. 111: 860, 1973). The detection of hybridoma antibodies of mice that bind to these cells was determined by indirect immunofluorescence. Cells incubated with the 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 the fluorescent antibody were subsequently analyzed on Cytofluorograph FC200 / 4800A (Ortho Instruments, 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 dilution restriction procedures in the presence of batch cells. Subsequently, the clones were transferred intraperitoneally by injecting 1 χ 10 ⁷ cells of a given clone (0.2 ml volume) into CAFj mice primed with 2,6,10,14-tetramethylpentadecane, sold by Aldrich Chemical Company under the name Prishtina. Malignant ascites from these mice were then used to characterize the lymphocytes as described below in Example II. Demonstrirano the standard11 Neem techniques gives subject hybrid antibody 0KT5 subklase IgG _r

EXAMPLE II

0KT5 reactivity characterization

A. Isolation of lymphocyte populations

Human peripheral blood mononuclear cells were isolated from healthy voluntary donors (ages 15-40) using Ficoll-Hypaque density gradient centrifugation (Pharmacia Fine Chemical, Piscataway, NJ) according to the technique described by Boyum, Scand. J. Ciin. Lab. Invest. 21 (Suppl. 97): 77, 1968. Unfractionated mononuclear cells were separated into surface Ig ⁺ (B) and Ig (T plus nothing) 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 using E rosette 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 on 10 ⁸ cells). The population thus obtained was <2% EAC rosette positive and> 95% E rosette positive as determined by standard procedures. Further, the non-rosette Ig '(Zero Cell) population is defended from the Ficoll interface. This later population was <5% E ⁺ and <2% / ilg ⁺ . The surface Ig ⁺ (B) population thus obtained from the Sephadex G-200 column was obtained after elution with normal human gamma globulin as described previously. This population was> 95% surface Ig ⁺ and <5% E ⁺ .

Normal human macrophages were obtained from a mononuclear population adhering to polystyrene. Thus, the 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 χ 10 ⁶ cells and incubated in plastic petri dishes (100 x 20 mm) Falcon Tissue Culture Dish; Falcon, Oxnard, CA) at 37 ° C overnight. After extensive rinsing to separate non-invasive cells, the adherent population was separated by rinsing (abrupt) with cold serum-free medium containing 2.5 mM EDTA and occasional scratching with the rubber tip of the discarded syringe. More than 85% of the cell population was able to ingest latex fragments and had monocyte morphological characteristics based on Wright-Giems staining.

B. Thymocyte isolation

Normal human thymus gland is obtained from patients 2 months to 14 days who undergo corrective heart surgery. The freshly obtained thymus gland sections were currently placed in 5% of the fetal calf serum in base 199 (Gibco), the sections being finely pressed with forceps and scissors, and a single-cell suspension was subsequently made by pressing through a wire sieve. The cells were further layered over Ficoll-Eypaque and rotated and washed as described previously in section A above. The thymocytes thus obtained were 95% viable and> 90% E rosette positive.

C. The cell line

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

EXAMPLES

Cytofluorographic analysis and cell separation

Cytofluorographic analysis of monoclonal antibodies with all cell populations was achieved by indirect immuno-fluorescence with goat anti-mouse IgG fluorescein conjugated (G / M FITC) (Meloy Laboratories) using Cytofluorograph FC200 / 4800A (Ortho Instruments). Briefly, 1 χ 10 ⁶ cells were treated with 0.15 ml OKT5 at a dilution of 1: 500, incubated at 4 ° C for 30 minutes and poured twice. The cells were 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. Cells are then analyzed on a Cytofluorograph and the fluorescence intensity per cell on a pulse height analyzer is recorded. A similar reactivity pattern was seen for a 1: 10,000 dilution, but further dilution caused a loss of reactivity. Basic staining was achieved by replacing an aliquot of 0.15 ml of 1: 500 ascites from CAFj mice that were intraperitoneally injected with a non-productive hybrid clone. The reactivity of lymphoid populations with equine anti-TH ₂ and normal equine IgG was determined as described previously in the above-mentioned Reinhers articles, et al.

In experiments designed to separate T cell subsets, 100 χ 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 was previously shown to be specifically reactive with 55-60% T peripheral blood lymphocytes representing human helper subset. Using fluorescence-activated cell sorter (FACS-I) (Becton, Dickinson, Mountain Wiew, CA), T cells were separated into OKT4 ⁺ and OKT4 'subsets as well as OKT5 ⁺ and OKT5' subsets from the same individual. The ability to live after sorting was 95% by means of turning off Trypan blue in all cases. The purity of all separate populations was> 95%.

EXAMPLE IV

Subset analysis of T cells with equine anti-TH ₂

Similarly to Example III, equine anti-TH ₂ was used to separate TH ₂ ⁺ and TH2 'T cells on FACS by marking 60 χ 10 ⁶ unfractionated T cells with 0.12 ml equine anti-TH2 and 0.1 ml R / N FITC (Cappel Laboratories, Downingtown, PA) as previously described in Reinherz, et al. The purity and ability to live of the sorted cells were similar to the sorted populations described above. FACS sorted T cell subsets isolated with equine anti-TH ₂ , OKT4 or OKT5 were cultured for 48 hours with RPMI 1640 containing 20% human AB serum, 1% penicillin-streptomycin, 200 mM L-glutamine, 25 mM HEPES buffer (Microbiological Associates) and 0.5% sodium bicarbonate at 37 ° C in 5% CO ₂ humidified atmosphere. These cultured cells were then analyzed on a Cytofluorograph as described above. Basic staining was determined by replacing the specific antibody with normal equine IgG and staining as above.

EXAMPLE V

Functional study

A. Proliferative study

The mitogenic response of 10 ⁵ uncoupled and FACS-fractionated T lymphocytes was tested in microculture for optimal doses of Concanavalin A (Con A) (Calbiochem, La Jolla, CA) and phyto hemagglutinin (PHA) (Burroughe-Wellcome Company, Hreenville, NC). . The alloantigen proliferative response was measured competitively for the same populations with mitomycin treated for Laz 156, which is the stimulus for the EBV transformed human B lymphoblastoid cell line. Adherence to tetanus toxoid (Massachusetts

The Department of Public Health Biological Laboratories, Boston, MA) and measles antigen (Microbiological Associates) were tested using 10 μ-g / ml final concentration and 1:20 dilution. Five percent of macrophages obtained in the manner described above were added to all populations when initiating cultures in vitro. Mitogen-stimulated cultures were pulsed after 4 days for 0.2 gCi 3H-thymidine ( ³ H-TdR; 1.9 Ci / mM specific activity) (Schwarz / Mann, Division of Becton, Dickinson, Orangeburg, NY) and were hours later on a MASH II apparatus (Microbiological Associates). ³ H-TdR incorporation was measured on a Packard Scintillatio Counter (Packard Instrument Company, Downer's Grove, IL). Basic ³ H-TdR incorporation was obtained by replacing mitogens with substrates. Solution and cell surface alloantigen cultures were pulsed after 5 days with ³ H-TdE for 18 hours, were harvested and counted as above.

B. Cytotoxicity studies

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

⁵¹ Cr released in experiment- ⁵¹ Cr released spontaneously

- X100 ⁵¹ Cr released by defrosting - ⁵¹ Cr released spontaneously

All samples were done in triplicate and the results were expressed as mean ± standard deviation.

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

Unfractionated T cells were activated with 20 μ-g Concavalin A (Con A) (Calbiochem) on 10 ⁶ cells. These Con A treated cells were cultured upright in 25 cm ² tissue culture balloons (Falcon, Oxnard, CA) in RPMI 1640 (Grand Island Biological Company) containing 20% human AB serum, 1% penicillin15 streptomycin, 200 mM L- glutamine, 25 mM HEPES buffer (Microbiological Associates) and 0.5% sodium bicarbonate for 48 hours at 37 ° C in a humidified atmosphere containing 5% CO ₂ . The untreated control T cells were cultured in an identical manner but without Con. A. Subsequently, the cells were rotated, washed five times, and added to freshly autologous responder cells in one-way mixed lymphocyte (MLC) culture as described above. In the suspension assay, MLC culture was established in round bottom microtiter plates (Linbro Chemical Company, New Haven, CT) with triplicate openings, each containing 0.05 χ 10 ⁶ responder lymphocytes (fully mononuclear); 0.05 χ 10 ⁶ iii non-activated iii Con A activated autologous unfractionated or fractionated T cells; and 0.1 χ 10 ⁶ mitomycin-treated stimulated cells (Laz 150). After five days, the cultures were pulsed with 0.2 / xCi ³ H-TdR and harvested 18 hours later, as above. Then the percentage inhibition of MLC proliferation was calculated using the formula:

cpm Con A ⁺ % inhibition = (1--) X100 cpm where cpm Con A ⁺ represents the results of ³ H-TdR incorporation into MLC when added Con A activated T cells or subset T cells, and where cpm represents results when unactivated autologists of the T cell.

SHORT DESCRIPTION OF THE DRAWINGS

Figure 1 shows the fluorescence pattern obtained on Cytofluorograph after reaction of normal human peripheral T cells, E cells, Zero cells and macrophages with OKT5 at a dilution of 1: 1000 and G / M FITC in the top row. For comparison, results with equine anti-TH _{2 are} shown below.

Figure 2 shows the fluorescence pattern obtained on Cytofluorograph after reaction of human thymocytes with OKT5 and G / M FITC (A) and with equine anti-TH ₂ (B).

Figure 3 shows the fluorescence scheme obtained on Cytofluorograph after reaction to separate T cell subset of equine anti-TH ₂ with OKT5.

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

Figure 5 shows the cytotoxic capacity of unfractionated T cells (subset) separated by 0KT5 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 amounts of the antibody in question are made by injecting the hybridomas in question intraperitoneally into mice and picking malignant ascites, it is clear that hybridomas that can be cultured in vitro using techniques well known in the art are then separated from the supernatant.

As shown in the top row of Figure 1, approximately 20% of the human peripheral blood T cell population of a given normal individual responds with OKT5, whereas all B cells, zero cells, and macrophage populations isolated from the same individual do not respond with OKT5. Similarly, equine anti-TH ₂ reacts with 24% of peripheral T cells and also does not react with B cells, Zero cells and macrophages. The monoclonal antibody is thus characterized by reacting with the antigen present on the surface of approximately 20% of normal human peripheral T cells, while not reacting with the macaque antigens on the surface of the other three cell types discussed above. As discussed below, the OKT5 ⁺ portion of the human peripheral T cell population is involved in the cytotoxic and suppressor subset of T cells. This differentiated reactivity is one assay by which the OKT5 antibody in question can be detected and distinguished from other antibodies.

As shown in Figure 2, approximately 80% of normal human thymocytes from a six-month-old infant respond with OKT5. Similar results (about 80% reactivity) were obtained using supplemental thymus samples from normal individuals 2 months to 19 years old. This value is the same for equine anti-TH ^ The reactivity scheme in Fig. 2 provides a second method for detecting the OKT5 antibody in question and for distinguishing it from other antibodies.

As shown in Figure 3, the antibody in question reacts with TH ₂ ⁺ but not with TH ₂ 'T cells. Approximately 5-10% of the TH ₂ ⁺ subsets do not react with OKT5. The reactivity scheme of Figure 3 provides a third method for detecting the antibody in question OKT5 and for differentiating it from other antibodies.

As shown in Figure 4, the OKT4 ⁺ subset T cell is completely non-reactive with OKT5. In contrast, the OKT4 'subset of T cells is generally OKT5 ⁺ (6,800 to 10,000 tes17 cell). These results show that the OKT5 ⁺ subset of the T cell, as well as the previously defined TH2 ⁺ subset, is reciprocal and different from the OKT4 ⁺ subset. While the OKT4 ⁺ subset 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 0KT5 antibody and for distinguishing it from other antibodies.

Figure 5 shows that the 0KT5 ⁺ subset of T cells induces CML. The breakdown rate 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 rupture effect. After activation in MLC against Laz 156, unfractionated T cells are separated into OKT5 ⁺ and 0KT5 'subsets. Both unfractionated and fractionated T cells were analyzed in CML against ⁵¹ Cr-labeled Laz 156 targets at various effector.target ratios (E: T). OKT5 ⁺ T cell subsets contained an effector population in lymphoid-mediated cells. 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 effect for breaking the isolated OKT5 ⁺ subsets was significantly greater than the unfractionated T cell population. Given the earlier assumption that TH2 ⁺ subset T cells in humans induce 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 for identifying OKT5 antibodies and differentiating them from other antibodies.

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

In these studies, the unfractionated T cell population was treated with a 1: 500 dilution of OKT5 and G / M FITC and separated on FACS into OKT5 ⁺ and OKT5 ′ subsets. At a given purity of the resulting populations (greater than iii equal to 95%), 5% of macrophages were added to separate populations prior to in vitro cultivation. The unfractionated T cell population and isolated subset of OKT5 ⁺ and OKT5 ′ T cells are then stimulated with PHA, Con A, soluble antigens and alloantigens to evaluate their proliferative responses in vitro.

The proliferative response of unfractionated T cell populations to PHA and Con A is shown in Table I. The maximum proliferative response to the unfractionated T cell population is achieved from 1 pg PHA to 10 ⁶ cells with reduced reactions occurring at 0.5 pg PHA and 0. 1 pg PHA per 10 ⁶ cells. Treatment of unfractionated T cells with OKT5 and goat-mouse FITC without subsequent fractionation did not alter the proliferative response. In contrast, differences in reaction to PHA were obtained with a separate OKT5 ⁺ and OKT5 ′ subset of T cells. The OKT5 ′ cell population responded to all doses of PHA in a similar manner to the uncoupled T cell population. However, the proliferative response of OKT5 ⁺ cells was significantly lower at all doses of PHA tested.

Furthermore, at a PHA dose of 0.1 pg per 10 ⁶ cells, the OKT5 ⁺ T cells did not multiply at all, while the OKT5 subset T cells and unfractionated cells still respond. On the other hand, the proliferative response of these subsets to Con A was similar and the two subset cells could not be distinguished from each other or from the unfractionated population of T cells.

The reactions to alloantigen in MLC and 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 subsets of OKT5 ⁺ and OKT5 'T cells reacted similarly in MLC against Laz 156. In contrast, proliferative reactions to soluble antigens provided the clearest difference between the two subsets. In all cases tested, the OKT5 ⁺ subset of T cells reproduced minimally with respect to soluble antigens of tetanus and measles toxoids, while the OKT5 'subset of T cells responded well.

The earliest proučavanja su showed that TH ₂ ⁺ lymphocyte population able to indukuje with Con A so urged AutoLog lymphocytes in MLC. To determine whether OKT5 ⁺ subset T cells could clearly express suppressor function, T cells were activated for 48 h with Con A and then sorted with OKT5 and OKT4 monoclonal antibodies into distinct T cell subset. Subsequently, these separate T cell subpopulations were added to autologous responsive lymphocytes at the initiation of MLC. Testing with monoclonal antibody and G / M FITC did not alter this result. This result is shown in Table III, and it can be seen that the unfractionated T cells activated by Con A suppressed 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, containing both OKT4 ⁺ and OKT4 'TH ₂ ' subpopulations, was minimally suppressive compared with the highly suppressive OKT5 ⁺ population.

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

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

According to the present invention there is provided a hybrid capable of producing an antibody against an antigen present in human cytotoxic and suppressor T cells, a method for producing this hybridoma, a monoclonal antibody against an antigen found on human cytotoxic and suppressor T cells, methods for producing antibodies. as well as methods and preparations for the treatment or diagnosis of the disease using this antibody.

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TABLE IV

LEVELS OF PERIPHERAL T CELLS IN DISEASES

T cell levels

Ill condition OKT3 ± OKT4 ± OKT5 ± Primary biliary cirrhosis (2) N + - Multiple sclerosis (od- macular disease) (8) - N - Miastemia gravis (wound untreated) (3) 0 0 0 Acute Coil vs Host (3) Odo- - 0 Agamaglobulinemia obtained Tipi + Type II 0 Hyper IgE (4) - N Odo- Acute infectious mono- nucleosis (4) * * + Odo- + + Hodgkins disease Phases I & II N N N Phases III & IV N N

N = within normal limits = absent + = above normal + + = well above normal

- = below normal

- = well below normal * = these levels return to normal about one week before clinical symptoms disappear

Brackets in brackets indicate the number of patients examined.

Although only one hybrid that produces one monoclonal antibody against human cytotoxic and suppressor T cell antigens has been described, it is contemplated that the present invention encompasses all monoclonal antibodies exhibiting the characteristics described herein. The antibody OKT5 was determined to belong to the IgG _r subclass, which is one of four mouse IgG subclasses. These immune globulin G subclasses differ from each other in so-called fixed regions, although an antibody to a specific antigen will have a so-called variable region that is functionally identical regardless of which class of immune globulin G belongs. That is, the monoclonal antibody exhibiting the characteristics described herein may be subclasses of IgG _p IgG ₂ a, IgG ₂ b or IgM ₃ , class III IgM, IgA or other known Ig classes. Differences between these classes or subclasses will not affect the selectivity of the antibody reaction scheme, but may affect the further reaction of the antibody with other agents, such as (for example) anti-mouse antibody complement. Although the subject body is specifically IgG _p, antibodies having the reactivity patterns illustrated herein are contemplated to be included in the present invention regardless of which immune globulin class or subclasses they belong to.

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

Further aspects of the invention are methods of treating or diagnosing a disease using a monoclonal antibody OKT5 or any other monoclonal antibody that exhibits the reactivity scheme provided herein. The subject antibody can be used to detect excess activity of cytotoxic or suppressor T cells by reacting an individual's T cell preparation with an 0KT5 antibody. Excessive activity of cytotoxic or suppressor T cells is indicated by the presence of more than 20-30% of the total population of peripheral T cells reacting with OKT5. This diagnosis technique can be used by using only OKT5 antibodies or in combination with other antibodies (eg, OKT3 and 0K.T4) as shown in Table 4. Reactivity schemes with antibody panel against T cells and T cell subsets allow for more accurate detection of certain disease states than was possible using earlier methods of diagnosis.

Treatment of disease states (e.g., malignancies) that manifest themselves as excess suppressive activity can be achieved by administering a retapeutically effective amount of 0KT5 antibody to an individual in need of such treatment. By selectively reacting with T cell suppressor antigen, an effective amount of OKT5 antibody reduces the excess of suppressor T cells, thus mitigating the effects of excess T suppressor cells. Diagnostic and therapeutic preparations comprising effective amounts of 0KT5 antibodies in admixture with diagnostic or pharmaceutically acceptable carriers are also included within the present invention.

Subject hybrid 0KT5 was deposited at American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 on September 18, 1979, and September 28, 1979, and was given ATCC accession number CRL 8013 and CRL 8016, respectively.

The best way to use the invention in an economy known to the Applicant

The invention can best be prepared when the following procedure is used:

Female CAF ₁ mice (Jackson Laboratories; 6-8 weeks old) were immunized intraperitoneally with 2 χ 10 ⁷ human thymocytes in 0.2 ml phosphate buffered saline at 14-day intervals. Four days after the third immunization, the spleen was removed from the mice and a single-cell suspension was made by compressing the tissue through a stainless steel sieve.

Cell condensation was performed according to a procedure developed by Kohler and Milstein. 1 χ 10 ⁸ splinocytes were condensed in 0.5 ml condensation medium comprising 35% polyethylene glycol (PEG 1000) and 5% dimethyl sulfoxide in RPMI 1640 substrate (Gibco, Grand Island, NY) with 2 χ 10 ⁷ P3X63Ag8Ul myeloma cells that was by Dr. M. Scharff, Albert Einstein College of Medicine, Βγοπχ, NY. These myeloma cells secrete IgG ₁ k light chains.

B. Hybridoma selection and growth

After condensation of the cells, the cells were cultured in a HAT medium (hypoxanthine, aminopterin and thymidine) at 37 ° C with 5% CO ₂ in a humid atmosphere. A few weeks later, 40 to 100 μΐ of supernatants from hybridoma-containing cultures were added to a pellet of 10 ⁶ peripheral lymphocytes, which were separated into E rosette positive (E ⁺ ) and E rosette negative (E ') populations, prepared from the blood of healthy human donors as described by Mendes (J. Immunol. 111: 860, 1973). The detection of hybridoma antibodies of mice that bind 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 subsequently analyzed on Cytofluorograph FC200 / 4800A (Ortho Instruments, 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 dilution restriction procedures in the presence of batch cells. Subsequently, clones were transferred intraperitoneally by injecting 1 χ 10 ⁷ cells of a given clone (0.2 ml volume) into CAF ₁ mice that were η

received with 2,6,10,14-tetramethylpentadecane, sold by Aldrich Chemical Com pany under the name Prishtina. The malignant ascites from these mice were then used to characterize the lymphocytes as described below in Example II. It has been demonstrated by standard techniques that the present OKT5 hybrid antibody subclass IgG _r

Claims (1)

A process for producing a monoclonal antibody that reacts with more than 90% of cytotoxic and suppressor TH ₂ ⁺ T human cells, but not with normal human peripheral B cells, Zero cells or macrophages, comprising the steps of: i) immunization of mice with normal human thymocytes; ii) separating the spleen from said mice and suspending the spleen cell; iii) condensing said spleen cells with murine myeloma cells in the presence of a condensation promoter; iv) diluting and culturing the condensed cells in separate holes in a substrate that will not support the non-condensed myeloma cells; v) evaluation of the supernatant in each hole containing the hybrid, for the presence of antibodies on the E rosette of positively purified T cells; vi) selecting and cloning an antibody-producing hybridoma that responds with more than 90% of cytotoxic and suppressor TH ₂ ⁺ T human cells, but not with normal human peripheral B cells, Zero cells or macrophages; vii) isolating the antibodies obtained by said hybridoma, the isolation method is selected from the group consisting of a) isolating the antibodies from the supernatant above said clones iii b) transferring said clones intraperitoneally to mice and collecting malignant ascites or sera from said mice, whereby ascites iii serum contain the desired antibody.