SI8011146A8 - Method for obtaining monoclonal antibody okt3 - Google Patents

Description

The invention provides a method for producing a monoclonal antibody OKT3, class IgG and a subclass of IgG2 that reacts with virtually all normal human peripheral T cells but does not react with normal human peripheral B cells.

The state of the art

Fusion of murine myeloma cells for alesino cells from immunized mice performed by Kohler and Milstein in 1975 [Nature 256, 495-497 (1975)] showed for the first time that a continuous cell array producing a homogeneous (so-called monoclonal) antibody could be obtained. Since this initial work, much effort has been directed toward the production of various hybrid cells (referred to as hybridomas) and the application of antibodies made using these hybridomas for various scientific studies. See, for example, Current Topics in Microbiology and Immunology, vol. 81 - Limphocyte Hybridomes, F. Melchers, M. Potter, and N. Warner, (publishers), Springer-Verlag, 1978 and references given in that section; C. J. Barnstable, et al., Whole 14, 9-20 (May, 1978); P. Pairham and W.F. Bodmer, Nature 276, 397-399 (November 1978); Handbook of Experimental Immunology, Third Edition, vol. 2, D. M. Wier, Publisher, Blackwell,

1978, Chapter 25; and Chemical and Engineering News, January 1, 1979,15-17.

These references simultaneously demonstrate the successes and complications of attempting to produce a monoclonal antibody from a hybridoma. Although the general technique is well understood in its concept, there are many difficulties encountered and different variations for each specific case. In fact, there is no certainty, before attempting to make a given hybridoma, that the desired hybridoma will be obtained, then that it will produce an antibody (even if obtained), or that the antibody thus obtained will have the desired specificity. The degree of success depends, in principle, on the type of antigen used and the selection technique used to isolate the desired hybridoma.

The attempted preparation of a monoclonal antibody for human cell surface lymphocyte antigens has been reported in only a few examples. See, for example, Current Topics in Microbiology and Immunology, ibid, 66-69 and 164-169. In these disclosures, antigens have been used to represent the cell types of cultured human lymphoblastoid leukemia and human chronic lymphocytic leukemia. Many of the resulting hybridomas appear to produce antibodies to different antigens in all human cells. None of these hybridomas produced an antibody against a previously defined class of human lymphocytes.

You should understand that there are two major classes of lymphocytes that are involved in the human and animal immune systems. The first of these classes (the cell originating from the thymus iii T cells) is differentiated in the thymus from the hemopoietic base cells. Because they are located in the thymus, these differentiated cells are referred to as thymocytes. Mature T cells emerge from the thymus and circulate between tissues, lymphatics and blood flow. These T cells form a large portion of the reservoir of recirculating small lymphocytes. They have immune specificity and are directly involved in cell-mediated immune responses (such as graft rejection) as effector cells. Although T cells do not secrete humoral antibodies, they are sometimes necessary for the secretion of these antibodies by another class of lymphocytes, which will be discussed below. Some types of T cells play a regulatory function in other aspects of the immune system. The mechanism of this cell cooperation process is not fully understood.

The second class of lymphocytes (cells that stimulate bone marrow or B cells) are those lymphocytes that secrete an antibody. They also develop from hemopoietic basal cells, but their differentiation is not determined by the thymus. In birds, they differentiate into a single organ that is analogous to the thymus and is called the sac of fabriciius. However, no equivalent organ was detected in mammals and these B cells are thought to differentiate into the bone marrow.

It is now understood that T cells are subdivided into at least several subtypes, designated as aiding, attenuating, and killing T cells, which have the function of promoting, attenuating, or destroying (decomposing) foreign cells. These subclasses can be well understood and understood for murine (mice and rats) systems, but have only recently been described for humane systems. See, for example. R. L. Evans et al., Journal of Experimental Medicine, vol. 145, 221-232,1977; and L. Chees and S. F. Schlossman - Functional Analysis of Distinct Human T-Cell Subsets Bearing Unique Differentiation Antigens, in Contemporary Topics in Immunobiology, edited by O. Stutman, Plenum Press, 1977, vol. 7, 363-379.

The ability to identify or attenuate class iii subclasses of T cells is significant for the diagnosis or treatment of various immunoreagulation diseases or conditions.

For example, some leukemias and lymphomas have different prognoses depending on whether they are of B or T cell origin. Thus, determining 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. Some diseased conditions (eg, juvenile rheumatoid arthritis and some leukemias) are related to imbalance of subclasses of T cells. It has been suggested that autoimmune diseases are generally associated with an excess of helper T cells or the lack of some attenuating T cells, while malignant conditions are generally associated with an excess of attenuating T cells. In some leukemias, excess T cells are produced in a slow stage of development. Thus, the diagnosis may 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 Haematology and Blood Transfusion, vol. 20, Springer-verlag, 1977, 17-24, and references contained therein.

From a therapeutic point of view, there is a suggestion, which has not yet been conclusively proven, that the administration of antibodies against the T cell subtype in excess may have therapeutic value in autoimmune diseases or malignancies. Antisera against the whole class of human T cells (so-called antihuman thymocyte globulin or ATG) have been reported to be therapeutically applicable in patients receiving organ transplants. As the cell-mediated immune response (the mechanism by which transplants are rejected) depends on T cells, administration of antibodies to T cells slows down or slows down this rejection process. See, for example, eosimi, et al., Randomized Clinical Trial of ATG in Cadaver Renal Allgraft Recipients: Importance of T Whole Monitoring, Surgery, 40, 155-163 (1976) and references contained in that paper.

The identification and attenuation of classes and subclasses of human T cells was previously performed using spontaneous autoantibodies or selective antisera for human T cells obtained by immunizing animals with human T cells, draining blood of these animals to obtain serum, and adsorbing antisera with (for example) autologous but not allogeneic B cells to remove antibodies with unwanted reactivity. Obtaining these antisera is extremely difficult, especially in the stages of adsorption and purification. Even adsorbed and purified antisera contain many impurities in addition to the desired antibody, which is due to several reasons. First, serum contains millions of antibody molecules even before T cell immunization. Second, immunization causes the production of antibodies against the various antigens found in all human T cells (given by injection). There is no selective production of antibodies against a single antigen. Third, the titer of the specific antibody obtained by such methods is usually quite low (eg inactive at dilutions greater than 1: 100) and the specificity to the nonspecific antibody is less than 1/10 ⁶ .

See, for example, Chess and Schlossman, a paper cited above (on pages 365 et seq.) And an article published in Chemical and Engineering News (reference above) describing the deficiencies of antisera of earlier practice and the advantages of a monoclonal antibody.

A new hybridoma (designated OKT3 and deposited April 26, 1979, at American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, 20852, and designated ATCC as CRL 8001) has been discovered capable of producing a newly complemented monoclonal antibody against the antigen found in virtually all normal human peripheral T cells and cutaneous T lymphoma cells. The antibody thus produced is monospecific for the determining factor in normal human T cells and cutaneous T lymphomas, and contains no other anti-human immunoglobulin, as opposed to earlier antisera (which are inherently contaminated with an antibody reactive to numerous human antigens) and earlier monoclonal antibodies (which are not monospecific for human T cell antigen). Moreover, this hybridoma can be cultivated to produce an antibody without the need to immunize and kill animals, followed by the painstaking degrees of adsorption and purification necessary to obtain even impure antisera.

It is an object of the present invention to provide a method of obtaining a complementarily established monoclonal antibody for human T cells.

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

In order to meet the foregoing objectives and advantages, the present invention provides a new hybridoma that produces a new antibody for antigen found as practically all normal human T cells and cutaneous T lymphoma cells, then only the antibody and diagnostic and therapeutic methods that employ this antibody. The hybridoma was deposited on April 26, 1979, vol. at America Type Culture Collection, 1301 Parkwan Drive, Rockville, Maryland 20852, and received ATCC accession number CRL 8001. The hybridoma was obtained generally by the Milstein and Kohler procedure. After immunization of mice with normal E rosette positive human T cells, the spleen cells of immunized mice were fused with cells from the mouse myeloma array and the resulting hybridomas were tested to obtain those containing an antibody that selectively binds to normal E rosette positive human T cells. The desired hybridomas were then cloned and characterized. As a result, a hybridoma was produced that produces an antibody (designated OKT3) against the antigen in virtually all normal human T cells. Not only does this antibody not react with virtually all normal human peripheral T cells, but it also does not react with other normal peripheral blood lymphoid cells. In addition, surface cell antigen recognized by this antibody was detected only in mature thymocytes and completely absent in more than 90% of normal human thymocytes.

In view of the difficulties pointed out in earlier practice and the lack of success achieved with the use of malignant steel species as antigens, it was a great surprise that the present procedure gave the desired hybridoma. It should be emphasized that the unpredictable nature of obtaining a hybrid cell does not allow extrapolation from one antigen or cell system to another system. In fact, the present applicants have found that the use of T cell type of malignant cells as antigens causes the formation of hybridomas that did not produce the desired antibody. Attempts to use purified antigens isolated from the cell surface were also unsuccessful.

Both the hybridoma in question and the antibody produced (assisted) are identified herein by the code OKT3, whereby the specific material to which it relates will be clear in context.

The preparation and characterization of a hybridoma and a resultant antibody will be better understood by reference to the following description and examples.

The process for obtaining a hybridoma generally involves the following steps:

A. Immunization of mice with E rosette positive purified normal human peripheral T cells. it was easily found that female CAFj mice (the first generation of hybrids between Balb (cJ and A / J mice) were found to be more suitable, it being understood that other strains of mice could be used. Immunization and T cell concentrations should be such as to produce applicable amounts of suitable splenocytes were stored.Three immunizations were found to be effective at fourteen day intervals with 2 χ 10 ⁷ cells / mouse / injection in 0.2 ml of phosphate buffered saline.

B. Removal of spleen from immunized mice and making spleen suspensions in appropriate medium. About 1 ml of spleen medium is sufficient. These experimental techniques are well known.

C. Fusion of Suspended Spleen Cells with Mouse Myeloma Cells from a Suitable Cell Type Using a Suitable Fusion Accelerator. A suitable ratio is about 5 spleen cells to one myeloma cell. A total volume of about 0.5-1.0 ml of the fusion medium is adequate for about 10 ⁸ splenocytes. Many types of mouse myeloma cell lines are known and can be found in circulation, in general by members of academic associations or by various deposit banks, such as the Salk Institute Celi Distribution Center, La Jolla, Ca. The type of cell used should conveniently be of the so-called drug-resistant type so that unfused myeloma cells will not survive in selective medium while hybrids will survive. The most general class is a type of 8-azaguanin resistant cell that lacks the enzyme hypoxanthine guanine phosphoribosyl transferase and therefore will not be supported by HAT (hypoxanthine, aminopterin and thymidine) medium. It is also generally advantageous for the types of myeloma cell in use to be of the so-called non-radiation type so that it does not itself produce the antibody, although radiation types can also be used. However, in some cases, types of secreting myeloma may be appropriate. a fusion enhancer polyethylene glycol having an average molecular weight of from about 1000 to about 4000 (commercially available as PEG 1000, etc.) is readily suitable, as well as other fusion enhancers known in the art.

D. Dilution and cultivation in separate containers, mixtures of unfused spleen cells, unfused myeloma cells and fused cells in a selective medium that will not support unfused myeloma cells over a period of time sufficient to allow nonfusion cells to die. Dilution can be of a limiting type, in which the volume of the diluent is statistically calculated to isolate a certain number of cells (eg 1-4) in each separate container (eg each recess in a microtiter tray). The medium is one (eg, HAT medium) that will not support a non-fused drug-resistant type of myeloma cell (eg, 8-azaguanin resistant). Hence, these myeloma cells are lost. As unfused spleen cells are not malignant, they have only a finite number of generations. That way, after a certain period of time (about a week), these unfused spleen cells cannot reproduce. On the other hand, fused cells continue to reproduce because they possess the malignant quality of the myeloma ancestor and the ability to survive in a selective medium that comes from the ancestor - the spleen cell.

E. Examination of the upper layer of liquid in each container (recess) containing the hybridoma, for the presence of antibodies to E rosette positive purified human T cells.

F. Selection (eg by applying a limited dilution) and cloning of the hydrodome produced by the desired antibody.

Once the desired hybridoma has been 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 length of time, followed by separation of the desired antibody from the upper portion of the fluid. Suitable medium and suitable length of cultivation time are known or easily determined. This technique in vitro produces a practically monospecific monoclonal antibody, practically without the presence of another specific anti-human immune globulin. A small amount of other immune globulin is present because the medium contains xenogenic serum (eg, calf fetal serum). However, this in vitro procedure does not need to produce an amount or concentration of antibody, which is sufficient for some purposes, since the concentration of the monoclonal antibody is only about 50 _z ug / ml.

In order to produce a much higher concentration of the slightly less pure monoclonal antibody, the desired hybridoma can be injected into mice, preferably into singen or semi-conserved mice. Hybridomas will produce antibody-producing tumors after a suitable incubation time, resulting in a high concentration of the desired antibody (about 5-20 mg / ml) in the blood stream and peritonal exudate (acite) of the host mouse. Although these host mice also have normal antibodies in their blood and sacitis, 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 resulting monoclonal antibody from the collected peritonal exudates or from the serum is virtually free of contaminating antihuman immune globulin. This monoclonal antibody is of high titer (active at dilutions of 1: 100,000 or higher concentrations) and a high ratio specific to non-specific immune globulin (about 1/20). Myeloma arrays, which produce immune gobulin and incorporate k light, are nonspecific, meaningless peptides that only dilute a monoclonal antibody without depriving it of its specificity.

Example 1

Production of monoclonal antibodies

A. Immunization and hybridization of somatic cells

Female CAF ₁ mice (Jackson Laboratories, 6-8 weeks old) were intraperitoneally immunized with 2 χ 10 ⁷ E rosette purified T cells in 0.2 ml of phosphate buffered saline (table salts) at 14 day intervals. Four days after the third immunization, spleens were removed from mice and a single cell suspension was made by compressing tissue through a stainless steel mesh.

Cell fusion was performed according to a procedure developed by Kohler and Milstein. 1 χ 10 ⁸ splenocytes were fused into 0.5 ml of a fusion medium containing 35% polyethylene glycol (PEG 1000) and 5% dimethyl sulfoxide in Gibco RPMI 1640 medium.

Grand island, NY, USA) with 2 χ 10 ⁷ P3X63Ag8Ul myeloma cells obtained from Dr. M. Scharff, from Albert Einstein College of Medicine, Bronx, NY. These myeloma cells secrete IgGj k light chains.

B. Hybridoma selection and development

After cell fusion, the cells were cultured in HAT medium (hypoxanthine, aminopterin and thymidine) at 37 ° C with 5% CO ₂ in a humid atmosphere. Weeks later, 40 to 100 μϊ of the upper fluid layer from hybridoma-containing cultures was added to a ball of 10 ⁶ peripheral lymphocytes separated into E rosette positive (E ⁺ ) and E rosette negative (E) populations, obtained from the blood of healthy human donors as described by Mendes (J. Immunol. 111, 860 (1973)). Detection of murine hybridoma antibodies bound to these cells was determined by radioimmunoassay and / or indirect immunofluorescence. In the first method, cells were initially reacted with 100 μ \ affinely purified ¹²⁵ I goat anti-mouse IgG (10 ⁶ ppm / ug, 500 Mg / ml). (The details of iodination of goat-anti-mouse IgG have been described by Kung, et al, J. Biol. Chem. 251, (8), 2399 (1976). Alternatively, cells incubated with the upper portions of the culture fluid are stained with fluorescent goat anti-mouse IgG (G / M FITC) (Meloy laboratories, Springfield, VA; F / p = 2.5) and fluorescent antibody coated cells were then analyzed on a Cytofluorograph FC200 / 4800A (Ortho Instruments, Westwood, MA) as as described in Example 3. Cultures of hybridomas containing antibodies that react specifically with E ⁺ lymphocytes (T cells) were selected and cloned, then the clones were transferred intraperitoneally by injecting 1 χ 10 ⁷ cells of a given clone (0.2 ml volume) into CAF ₁ mice prepared with 2,6,10,14tetramethylpentadecane, sold by Aldrich Chemical Company under the name Prishtina.Malignant peritonal exudates from these mice were then used to characterize lymphocytes as described in Example 2. Subject Hybrid Antibody OKT3 Demonstrators it is early in the standard technique that it represents the IgG ₂ subclass and is complementarily determined.

Example 2

Characterization of OKT3 reactivity

A. Isolation of lymphocyte populations

Human peripheral blood mononuclear cells were isolated from healthy voluntary donors (15-40 years old) using Ficoll-Hypaque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, NJ) followed by the Boyum technique, Scand. J. Ciin. Lab. Invest. 21 (Suppl. 97) 77, 1968. Unfractionated mononuclear cells were separated into surface Ig ⁺ (B) and Ig '(T plus zero) populations using Sephadex G-200 anti-F (ab') 2 column chromatography as before described by Chess et al., J. Immunol. 113, 1113 (1974). T cells were isolated by E rosetting the Ig 'population with 5% sheep arithrocytes (Microbiological Asociates, Bethesda, MD). The rosette mixture was layered over Ficoll-Hypaque and the separated E ⁺ ball was treated with 0.155 M NH4Cl solution (10 ml on 10 ⁸ cells). The T cell population thus obtained was <2% EAC rosette positive and <95% E rosette positive as determined using standard methods. In addition, the non-invasive Ig '(zero cell) population was collected from the Ficoll interface. This latter population is <5% E ⁺ and <2% slg ⁺ . The surface Ig ⁺ (B) population was obtained from a Sephadex G-200 column followed by elution with normal human gamma globulin as described previously. This population was> 95% surface Ig ⁺ and> 5% E ⁺ .

Normal human macrophages were obtained from the mononuclear population by adherence to polystyrene. In this way, the mononuclear cells were resuspended in the final culture medium (RPMI 1640, 2.5 mM HEPES [4- (2-hydroxyethyl) -1piperazinepropanesulfonic acid)] buffer, 0.5% sodium bicarbonate, 200 mM L -glutamine and 1% penicillin-streptomycin, to which was added 20% human AB serum heat inactivated) at a concentration of 2 χ 10 ⁶ cells and incubated in plastic petri dishes (100 x 20 mm) (Falcon Tissue Culture Dish; Oxnard, CA) at 37 ° C overnight. After extensive rinsing to remove non-adherent cells, the adherent population was separated by sharp rinsing with cold serum-free medium containing 2.5 mM EDTA and occasional scratching with a syringe (syringe) for injection. More than 85% of the cell population was able to absorb latex particles and have morphological characteristics of monocytes in the Wright-Giemsa staining method.

B. Normal thymus

Normal human thymus gland was obtained from patients aged 2 months to 14 years who underwent corrective heart surgery. The freshly obtained thymus gland sections were immediately placed in 5% fetal calf serum in medium 199 (gibco), finely chopped using surgical pliers and scissors, and then processed into single-cell suspensions by pressing through a wire mesh. These cells were then layered over Ficoll-Hypaque and spun and washed as previously described in section A. The thymocytes thus obtained were> 95% viable and> 90% E rosette positive.

C. Type of cell

Epstein-Barr's Virus (EBV) transformed B cell species from four normal individuals (Laz 007, Laz 156, Laz 256 and SB) and described them. T cell types CEM, HJD-1, Laz 191 and HF1; obtained from leukemic patients were given by Dr. H. Lazarus, Sidney Farber Cancer Institute, Boston, MA.

D. T cells of acute lymphatic leukemia (T-ALL) and T cells of chronic lymphatic leukemia (T-CLL)

Leukemia cells were obtained from 12 patients with T-ALL. These individual cells were previously designated as T cell cells by their spontaneous rosette formation with sheep erythrocytes (> 20% E ⁺ ) and reactivity with T cell specific anti-HTL (anti-BK) and A99 heteroserums, as described previously by Schlossman, et al., ch. Nat. Acad. Sci. 73, 1288 (1976). Tumors from three individuals were reactive (TH2 ⁺ ) with rabbit and / or equine anti-TH2 while cells from the remaining nine individuals were reactive (TH /). Leukemia cells from two patients with TH ₂ T-CLL were also used. Both strains (T cells) of acute and chronic leukemia were conserved at a low temperature of -196 ° C using vapor phase liquid nitrogen and kept in 10% dimethyl sulfoxide and 20% AB human serum until the time of surface characterization. Tumor populations analyzed were> 90% blasts as determined by Wright-Giemsa morphology in all examples.

Example 3

Cytofluorographic analysis

Cytofluorographic analysis of all cell populations was performed by indirect immunofluorescence with goat-antimouse IgG (G / M FITC) (Meloy Laboratories) conjugated with fluorescein on cytofluorograph FC200 / 4800A (Ortho Instruments).

Briefly, 1-2 x 10 ⁶ cells were treated with 0.15 ml OKT3 at a dilution of 1: 1000, incubated at 4 ° C for 30 minutes and washed twice. Cells were then reacted with 0.15 ml G / M FITC (dilution 1:40) at 4 ° C for 30 minutes, centrifuged and washed three times. These cells were then analyzed on a cytofluorograph and the fluorescence strength per cell was recorded on a height pulse analyzer. A similar reactivity mechanism was observed at a dilution of 1: 100,000, but further dilution caused a loss of reactivity. Basic staining was obtained by replacing 0.15 ml aliquots of peritonal exudates (1: 1000) from Balb (cJ mouse that was intraperitoneally immunized with a non-hybrid sildon).

FIG. 1 shows a fluorescence image obtained on a cytofluorograph after reacting normal human peripheral T cells with OKT3 at a dilution of 1: 1000 and G / M FITC. For comparison, in FIG. 1-5 shows the results with the OKT1 and OKT4 monoclonal antibodies obtained under equivalent conditions.

FIG. 2 shows a fluorescence image obtained on a cytofluorograph after human thymocyte response with OKT3 and G / M FITC.

FIG. 3 shows a fluorescence image obtained on a cytofluorograph after the response of leukemic cells obtained from patients with chronic lymphoblastic leukemia (B cells) with OKT3 and G / M FITC.

FIG. 4 shows a fluorescence image obtained on a cytofluorograph after reacting a HJD-1 human T cell type with OKT3 and G / M FITC.

FIG. 5 shows a fluorescence image obtained on a cytofluorograph after reacting a human TEM cell type CEM with OKT3 and G / M FITC.

The data and characteristic values given in Figs. 1-5 as well as supplementary data for OKT3 (as well as for OKT1 and OKT4) were collected in Table 1.

Preparation of the hybridoma and obtaining and characterizing the resulting monoclonal antibody were performed as described in the examples above. Although large amounts of the antibody in question are obtained by injecting the hybridoma in question intraperitoneally into mice and collecting malignant peritonal exudates, it is clearly understood that hybridomas can be cultured in vitro using techniques well known in the art and practice, and the antibody removed from the upper fluid layer.

As shown in FIG. 1, the entire human peripheral blood T cell population of a given normal individual may react with OKT3, whereas whole B cell populations, zero cells, and macrophages isolated from the same individual are not reactive with OKT3. Similar results were obtained for lymphocyte populations from 15 other normal individuals. Thus, the monoclonal antibody is characterized in that it is reactive with the antigen contained on the surface of virtually all normal human peripheral T cells, while unreactive with any antigens on the surface of the three other cell types discussed above. This different reactivity is a test by which the OKT3 antibody in question can be detected and distinguished from other antibodies.

As shown in FIG. 2, the vast majority of normal human thymocytes from a six-month-old baby are completely non-reactive with OKT3, while about 5 to 10% of thymocytes are reactive. The implication of this finding is that during the differentiation process by which the starting base cells are transformed into mature T cells, thymocytes acquire to some extent the same surface antigen found in T cells that are reactive to OKT3. These thymocytes are believed to be in later stages of differentiation just before they exit the thymus into the blood stream. Similar results (5-10% reactivity) were obtained using six supplemental thymus species (specimens) from normal individuals from 2 months to 19 years old. The reactivity image given in FIG. 2 provides a second method for detecting the OKT3 antibody in question and differentiating it from other antibodies.

The subject antibody can also be used to determine the ratios of circulating lymphocytes that are T cells. As shown in Table 1,> 95% of all T cells react with the OKT3 antibody. The present invention also includes a method for determining (in an individual) the ratio of circulating lymphocytes that are T cells, which involves mixing OKT3 antibodies with a lymphocyte mixture from this individual and determining how lymphocytes to be produced are OKT3 ⁺ and thus T cells.

Further characterization of the OKT3 antibody in question is shown by reactivity to different types of human T cells, as illustrated in FIG. 4 and 5. As can be seen, the reactivity of the antigen in question to human T cell types was heterogeneous, low for HJD-1 species and non-existent for CEM, Laz 191 and HM1 species. This differential reactivity of OKT3 against various types of human T cells (readily obtainable) provides another method for characterizing and characterizing the antibody in question.

The absence of reaction of 0KT3 with human B cell types Laz 007, Laz 156, Laz 256 and SB is shown in Table 1. This further confirms the lack of reactivity of OKT3 with B cells obtained from the peripheral blood of normal human populations and provides another method of characterization and differentiation of the antibody in question OKT3.

The specific reaction of the OKT3 antibody with the antigen in cutaneous T cell lymphomas is illustrated in Table 2, showing the distinction between OKT1 and OKT4. This antibody thus provides a reagent to confirm the diagnosis of cutaneous T cell lymphoma in a patient suspected of having the disease mentioned. Treatment of cutaneous T cell lymphoma by administering a therapeutically effective amount of OKT3 antibody is also contemplated as part of the present invention.

The present invention provides a hybridoma capable of producing an anti-antigen antibody found in virtually all normal human T cells and cutaneous T lymphoma cells, then a process for producing this hybridoma, a monoclonal anti-antigen antibody found in virtually all human T cells. methods for producing this antibody, and methods and methods for treating or diagnosing diseases using this antibody.

Although only one hybridoma producing one monoclonal antibody against human T cell antigens is easily described, it is believed that the present invention encompasses all monoclonal antibodies that exhibit the characteristics described above. The OKT3 antibody was determined to belong to the IgG ₂ subclass, which is one of four murine (murine) IgG subclasses. These immune globulin G subclasses differ from each other in so-called fixed regions, although the antibody for a specific antigen will have a so-called variable region that is functionally identical regardless of which immune globulin G subclass belongs. Thus, the monoclonal antibody exhibiting the characteristics described herein may be subclasses IgG _p IgG ₂ a, IgG ₂ b or IgG ₃ iii IgM class, IgA or some other known Ig classes. Differences between these classes or subclasses will not affect the selectivity of the reaction image of this antibody, but may affect the further reaction of the antibody with other materials such as (for example) complementary or anti-mouse antibodies. Although the antibody in question is specific for IgG ₂ , it is understood that antibodies having the reactivity mechanisms included herein are encompassed by the present invention regardless of the class or subclass of the immune globulin to which they belong.

Within the scope of the present invention there is provided a method of producing the monoclonal antibodies described above using the hybridoma techniques illustrated above. Although only one example of a hybridoma is given here, it is understood that one skilled in the art would be able to work according to the immunization, fusion and selection methods described above and obtain hybridomas that can produce antibodies with the reactivity characteristics described above. Since an individual hybridoma produced from known mouse myeloma and spleen cell types derived from known mouse species cannot be further identified except by reference to an antibody produced by this hybridoma, it is understood that all hybridomas that produce antibodies with the above described characteristics included in the subject matter of the present invention, as methods for making this antibody using this hybridoma.

Further aspects of the present invention are methods of treating or diagnosing a disease, which use the monoclonal antibody OKT3 or another monoclonal antibody that shows the mechanism of reactivity described above. As discussed above, the subject antibody enables the treatment of patients having certain T cell chronic leukemia lymphoblasters by administering therapeutically effective amounts of this antibody. Administration of a therapeutically effective amount of OKT3 antibody to an individual subject to organ transplantation will reduce or eliminate rejection of this transplant. The present antibody also enables the detection of cutaneous T cell lymphomas in an individual by mixing a lymphoma T cell mixture from the aforementioned individual with a diagnostically effective amount of OKT3 antibodies. The presence of a reaction confirms the identity of this disease. Cutaneous T cell lymphoma can be treated by administering to the individual in need of such treatment a therapeutically effective amount of OKT3 antibody. This antibody will react and reduce the amount of Z lymphoma cells, thereby improving the disease. In view of these diagnostic and therapeutic methods, the present invention additionally includes diagnostic and therapeutic compositions comprising a diagnostic or therapeutically effective amount of an OKT3 antibody in a diagnostic or pharmaceutically acceptable carrier.

Table 1

Monoclonal antibody reactivity and properties.

monoclonal antibodies OKT1 OKT3 OKT4 % reactivity with peripheral T-cells (10 samples) peripheral B-cells > 95% > 95% 55% (10 samples) peripheral zero cells <2% <2% <2% (10 samples) <2% <2% <2% tumocytes * (8 samples) 5-10% 5-10% 80% reactivity with: T-chronic lymphatic leukemia (3 cases) T-acute lymphatic leuke- + + (1), - (2) - mummy (8 cases) with zero acute lymphatic - - leukemia (15 cases) with B-chronic lymphatic - - - leukemia (6 cases) + (4); - (2) - - B-Steel Types ^4- (4) - - - T-Steel Types ^4- HJD-1 + (+) - CEM + - + Laz 191 + - - HM1 + - - IgG subclass IgG _t IgG ₂ IgG ₂ by complementary determination - + + * From patients aged 2 months to 18 years.

Obtained from dr. H. Lazarus, Sidney Farber Cancer Center.

B cell types laz 256, 156, 007 and SB obtained by Epstein-Barr viral transformation of human peripheral B cells and HJD-1, CEM, Laz 191 and HM1 determined from leukemic patients.

Table 2

name of patient- jenta cutaneous T-cell- ska lymphoma di- jagnosis trial with monoclonal 0KT1 0KT3 antibody 0KT4 E. McBride Cesar's bluster crisis, PBI + + - 0.0. 0kley we goats fungoids, node - + + Odom we goats fungoids node + + - Mont- albono ? node + + + Cell Source: PBL = peripheral blood lymphocytes

node = lymph node

The best way to economically apply the claimed invention known to the applicant

In the experience of the applicant, the following example can serve as an example.

Immunization and hybridization of somatic cells

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

Cell fusion was performed according to a procedure developed by Kohler and Milstein. 1 χ 10 ⁸ aplenocytes were fused into 0.5 ml of fusion medium containing 35% polyethylene glycol (PEG 1000) and 5% dimethyl sulfoxide in RPMI 1640 middle Gibco, Grand island, NY, USA) with 2 χ 10 ⁷ P3X63Ag8Ul myeloma cells obtained from Dr. M. Scharff, from Albert Einstein College of Medicine, Bronx, NY. These myeloma cells secrete IgG _t k light chains.

Hybridoma selection and development

After cell fusion, the cells were cultured in 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 the culture supernatant containing hybridomas were added to a ball of 10 ⁶ peripheral lymphocytes separated into E rosette positive (E ⁺ ) and E rosette negative (E ') populations, obtained from the blood of healthy human donors such as has been described by Mendes (J. Immunol. 111, 860 (1973)). Detection of murine hybridoma antibodies bound to these cells was determined by radioimmunoassay and / or indirect immunofluorescence. In the first method, the cells initially reacted with 100 μλ affinity purified ¹²⁵ I goat anti-mouse IgG (10 ⁶ cpm / ptg, 500 gg / ml). (The details of iodination of goat-anti-mouse IgG have been described by Kung, et al, J. Biol. Chem. 251, (8), 2399 (1976). Alternatively, cells incubated with the upper portions of the culture fluid are stained with fluorescent goat anti-mouse IgG (G / M FITC) (Meloy laboratories, Springfield, VA; F / p = 2.5) and fluorescent antibody coated cells were then analyzed on a Cytofluorograph FC200 / 4800A (Ortho Instruments, Westwood, MA) as as described in Example 3. The cultures of hybridomas containing an / r titers that react specifically with E ⁺ lymphocytes (T cells) were selected and cloned, then the clones were transferred intraperitoneally by injection of 1 × 10 ⁷ cells of a given clone (0.2 ml). in CAF ₂ mice prepared with 2,6,10,14tetramethylpentadecane sold by Aldrich Chemical Company under the name Pristine The malignant peritonal exudates from these mice were then used to characterize the lymphocytes as described in Example 2. Subject OKT3 hybrid antibody demonstrated is standa The IgG ₂ subclass represents the native techniques and is complementary.

Claims (1)

Claim: A method for producing a monoclonal antibody OKT3, class IgG, a complementary IgG2 subclass that reacts with virtually all normal human peripheral T cells but does not react with normal human peripheral B cells by culturing the hybridoma on a substrate containing hypoxanthine, aminopterin and thymidine wet atmosphere, cultured with ATCC CRL 8001 hybridomas, at a temperature of 37 ° C in an atmosphere with 5% carbon dioxide for several weeks, and then the above-mentioned anti-body body is released from the top layer of the culture fluid. ORTO PHARMACEUTICAL CORPORATION Representative: Abstract of invention The application provides a method for selectively obtaining a monoclonal antibody OKT3, class IgG, a subclass of IgG2 that is complementary established and which reacts with virtually all normal human peripheral T cells but does not react with normal human peripheral B cells, by cultivating hybridomas on a hypoxanthine-containing substrate , aminopterin and thymidine in a humid atmosphere. The process cultivates ATCC CRL 8001 hybridomas at 37 ° C in an atmosphere containing 5% carbon dioxide for several weeks. Said antibody is separated from the top layer of the culture fluid.