HRP940809A2 - Hybrid cell line for producing complement-fixing monoclonal antibody to human t cells, antibody and methods - Google Patents

Description

This invention generally relates to new arrays of hybrid cells and more specifically to arrays of hybrid cells for the production of a fully established monoclonal antibody, to an antigen found in all normal human T cells and cutaneous T lymphoma cells, to the antibody thus produced and to therapeutic and diagnostic methods and mixtures which they use that body (antibody). The fusion of murine myeloma cells with alezin cells from immunized mice, performed by Kohler and Milstein in 1975/Nature 256, 495-497 (1975)/ showed for the first time that it is possible to obtain a continuous cell line that creates a homogeneous (so-called "monoclonal" ) antibody. Since this initial work, many efforts have been made towards the production of various hybrid cells (called "hybridomas") and the application of antibodies made using these hybridomas for various scientific researches. See, for example, Current Topics in Microbiology and Immunology, vol, 81 - “Lymphocyte Hibridomas”, F. elchers, N. Potter and N. Warner, (editors), Springer Verlag, 1978 and references therein; Cl. J. Ranstable, et al., Cell 14, 9-20 (May, 1978); I.Harham and R.F. Bodmer, Nature 276, 397-399 (November, 1978); Handbook of Experimental Immunology, third edition, vol. 2, D.N. Wier, editor, Blackwell, 1978, chapter 25; and Chemical and Engineering News, 1 January 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 terms of its concept, there are a lot of difficulties encountered and different variations for each specific case. In fact, there is no assurance, prior to attempting to make a given hybridoma, that the desired hybridoma will be obtained, that it will produce an antibody (even if it is 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.

Attempts to obtain a monoclonal antibody for human lymphocyte surface cell antigens have been reported only in a few examples. See, for example, Current Topics in Neurobiology and Immunology, ibid 66-69 and 164-169. Antigens representing cell types of cultured human lymphoblastoid leukemia and human chronic lymphocytic leukemia were used in these reported samples. Many of the resulting hybridomas appear to produce antibodies to different antigens in all human cells. None of these hybridomas produced antibody against a previously defined class of human lymphocytes.

It should be understood that there are two main classes of lymphocytes involved in the immune system of humans and animals. The first of these classes (thymus-derived cells or T cells) is differentiated in the thymus from hematopoietic stem cells. Since they are located in the thymus, these differentiated cells are called "thymocytes". Mature T cells leave the thymus and circulate between tissues, lymphatics and the bloodstream. These T cells form a large part of the reservoir of recirculating 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 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 process of cell cooperation is not fully understood.

Another class of lymphocytes (cells derived from the bone marrow or B cells) are those lymphocytes that secrete an antibody. They also develop from hematopoietic stem cells, but their differentiation is not done by the thymus. In birds, they differentiate into a single organ analogous to the thymus called the sac of fabricius. However, no equivalent organ has been discovered in mammals and these B cells are thought to differentiate in the bone marrow.

It is now understood that T cells are divided into at least several subtypes, designated as “helper”, “suppressor” and “killer” T cells, which function to promote, dampen or destroy (destroy) foreign cells. These subclasses can be well understood and understood for murine (mice and rat) systems, but have only recently been described for human systems. See, for example, R.L. Evans et al., Journal of Experimental Medicine, vol. 145, 221-232, 1977; and L. Chess and S. F. Schlosaman - "Functional Analysis of Distinct Human T-Cell Subsets Bearing Unique Differentiation Antigens", in "Centtemporary Topics in Immunology", published by O. Stutman, Plenum Press, 1977, vol. 7, 363-379.

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

For example, some leukemias and lymphomas have different prognoses depending on whether they are of B or T cell origin. In this way, determining the prognosis of the disease depends on distinguishing 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 Leukemis and Lymphomas", S. Thierfelder et al, publishers, Springer, Heidelberg, 1977, 33-45; and D. Belpomme et al. British Journal of Haemotology, 1978, 38, 85. Some disease states (eg, juvenile rheumatoid arthritis and some leukemias) are associated with an imbalance of T cell subclasses. It has been proposed that autoimmune diseases are generally related to an excess of "helper" T cells or a deficiency of some "suppressor" T cells, while malignant conditions are generally related to an excess of "suppressor" T cells. In some leukemias, excess T cells are produced in a slow stage of development.

In this way, 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 Hematology and Blood Transfusion, vol. 20, Springer-verlag, 1977, 17-24, and references therein. .

From a therapeutic point of view, there is a suggestion, which has not yet been definitively proven, that administration of antibodies against a T cell subtype in excess may have therapeutic value in autoimmune diseases or malignant conditions. Antisera against an entire 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 prevents or slows down 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, 400, 155-163 (1976) and references therein. Identification and silencing of classes and subclasses of human T cells has previously been performed using spontaneous autoantibodies or selective antisera for human T cells obtained by immunizing animals with human T cells, bled these animals to obtain serum, and adsorbing the antisera with (for example) autologous but not allogeneic B cells to remove antibodies with unwanted reactivities. 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, due to several reasons. First, serum contains millions of antibody molecules even before T cell immunization. Second, immunization causes the production of antibodies against various antigens found on all human T cells (given by injection).

There is no selective production of antibodies against one 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 ratio of specific to nonspecific antibody is less than 1/106.

See, for example, Chess and Schlossman, the paper referenced above (on pages 365 et seq.) and an article published in Chemical and Engineering News (referenced above), which describe the disadvantages of prior art antisera and the advantages of the monoclonal antibody.

A new hybridoma (designated OKT3 and deposited April 26, 1979 in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, 20852, and ATCC numbered CRL 8001) has been discovered which is capable of producing a new complement determined monoclonal an antibody against an 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 lymphoma cells and does not contain another antihuman immunoglobulin, in contrast to earlier antisera (which are inherently contaminated with antibody reactive for numerous human antigens) and earlier monoclonal antibodies (which are not monospecific for human T cell antigen). Even more, this hybridoma can be cultured to produce antibody without the need to immunize and kill the animals, followed by the laborious adsorption and purification steps necessary to obtain even the impure antisera of earlier practice.

Accordingly, one object of this invention is to provide hybridomas that produce antibodies against an antigen found on virtually all normal human T cells and cutaneous T lymphoma cells.

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

It is a further object of this invention to provide a substantially homogeneous antibody against an antigen found in substantially all normal human T cells and cutaneous T lymphoma cells.

It is a further object of the present invention to provide methods for treating (treating) or diagnosing diseases using these antibodies.

Other objects and advantages of the present invention will be apparent from an examination of this disclosure.

In fulfillment of the foregoing objects and advantages, the present invention provides a novel hybridoma that produces a novel antibody to an antigen found in virtually all normal human T cells and cutaneous T lymphoma cells, the antibody itself, and diagnostic and therapeutic methods utilizing this antibody. The hybridoma was obtained generally according to the procedure of Milstein and Kohler. After immunization of mice with normal E rosette positive human T cells, alezen cells from the immunized mice were fused with cells from a murine myeloma line and the resulting hybridomas were screened for 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 obtained that produced an antibody (designated as OKT3) against an antigen on virtually all normal human T cells. Not only does this antibody fail to react with virtually all normal human peripheral T cells, but it also fails to react with other normal peripheral blood lymphoid cells.

In addition, the surface cell antigen recognized by this antibody is detected only in mature thymocytes and is completely absent in more than 90% of normal human thymocytes.

From the aspect of difficulties, which were pointed out in earlier practice, and lack of success, which was achieved when using malignant cell types as antigen, it was a great surprise that the current 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 a T cell type of malignant cells as an antigen 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 subject hybridoma and the antibody produced (using it) are identified herein by the designation “OKT3”, the specialty material to which it refers will be clear 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.

The procedure for obtaining a hybridoma generally includes the following steps:

A. Immunization of mice with E rosette positive purified normal human peripheral T cells. Although female CAF1 mice (a first generation hybrid between Balb/cJ and A/J mice) have been found to be more suitable, it is understood that other types of mice can be used. The program of immunization and concentration of T cells should be such as to produce applicable quantities of suitably stored splenocytes. Three immunizations at fourteen day intervals with 2 x 107 cells/mouse/injection in 0.2 ml phosphate buffered saline were found to be effective.

B. Removal of spleens from immunized mice and preparation of spleen suspensions in the appropriate medium. About 1 ml of medium on the spleen is enough. These experimental techniques are well known.

C. Fusion of suspended spleen cells with murine myeloma cells from a suitable cell type using a suitable fusion enhancer. The affected ratio is about 5 allesin cells to one myeloma cell. The total volume of about 0.5-1.0 ml of fusion medium is adequate for about 108 splenocytes. Many types of murine myeloma cells are known and can be found in circulation, generally from members of academic associations or various depository banks, such as the Salk Institute Cell Distribution Center, Le Jolla, CA. The type of cells used should preferably be of the so-called "drug-resistant" type, so that unfused myeloma cells will not survive in the selective medium, while the hybrids will. The most general class are 8-azaguanine-resistant cell types, which lack the enzyme hypoxanthine guanine phosphoribosyl transferase and hence will not be supported by HAT (hypoxanthine, aminopterin, and thymidine) media. It is also generally preferred that the type of myeloma cells used is of the so-called "non-secretory type", so that it does not produce the antibody itself, although secretory types can also be used. However, secretory types of myeloma may be suitable in some cases. Although a suitable fusion promoter is polyethylene glycol having an average molecular weight of about 1000 to about 4000 (commercially available as PEG 1000, etc.), other fusion promoters known in the art can also be used.

D. Diluting and culturing in separate containers, a mixture of unfused spleen cells, unfused myeloma cells, and fused cells in a selective medium that will not support the unfused myeloma cells for a period of time sufficient to allow the unfused cells to die (about one week). The dilution may be of the limiting type, in which the volume of diluent is statistically calculated to isolate a certain number of cells (eg, 1-4) in each separate container (eg, each well of a microtiter well). The medium is such (eg, HAT medium) that it will not support a non-fused type of myeloma cells that is drug resistant (eg, that is resistant to 8-azaguanine). Hence these myeloma cells are lost. As unfused spleen cells are not malignant, they only have a finite number of generations. In this way, after a certain period of time (about a week), these unfused spleen cells cannot reproduce. On the other hand, the fused cells continue to reproduce because they possess the malignant quality of the myeloma progenitor and the ability to survive in the selective medium derived from the progenitor - spleen cells.

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

F. Selection (eg, using limited dilution and cloning of hybridomas that produce 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 isolation of the desired antibody from the supernatant.

A suitable medium and a suitable length of cultivation time are known or can be easily determined. This technique in vitro produces a practically monospecific monoclonal antibody, practically without the presence of another specific antihuman immune globulin. A small amount of second immune globulin is present because the medium contains xenogeneic serum (eg telephi fetal serum). However, this in vitro procedure may not produce an amount or concentration of antibody that is sufficient for some purposes, as 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, which produce the antibody, after some suitable incubation time, which will result in a high concentration of the desired antibody (about 5-20 mg/ml) in the bloodstream and peritoneal exudate (acite) of the host house mouse. Although these host mice also have normal antibodies in their blood and tissues, the concentration of these normal antibodies is only about 5% of the concentration of the monoclonal antibody. Even more, since these normal antibodies are not anti-human in their specificity, the obtained monoclonal antibody from collected peritoneal exudates or from serum is practically free of contaminating anti-human immune globulin. This monoclonal antibody has a high titer (active at dilutions of 1:100,000 or higher) and a high ratio of specific to non-specific immune globulin (about 1/20). Myeloma arrays, which produce immune globulin and incorporate k light, are non-specific, "nonsense" peptides, which only dilute the monoclonal antibody without depriving it of its specificity.

Example 1

Obtaining a monoclonal antibody

A. Immunization and hybridization of somatic cells

Female CAF1 mice (Jackson Laboratories, 6-8 weeks old) were immunized intraperitoneally with 2 x 107 E of rosette-purified T cells in 0.2 ml of phosphate-buffered saline (table salt) 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 mesh.

Cell fusion was performed according to the procedure developed by Kohler and Milstein. 1 x 108 splenocytes were fused in 0.5 ml fusion medium containing 35% polyethylene glycol (PEG 1000) and 5% dimethylsulfoxide in RPNI 1640 medium Gibco, Grand Island, Ny, USA) with 2 x 107 P3x63Ag8W1 myeloma cells obtained from dr. M. Scharff from Albert Einstein College of Medicine, Bronx, Ny. These myeloma cells secrete IgG1 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% CO2 in a humidified atmosphere. Several weeks later, 40 to 100 µl of supernatant from hybridoma-containing cultures was added to a ball of 106 peripheral lymphocytes separated into E rosette positive (E+) and E negative (E ̄) populations, which were obtained from the blood of healthy human donors such as was 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 µl of affinity of purified 125I goat-anti-mouse IgG 106 spm/ug, 500µg/ml). (Details of goat anti-mouse IgG iodination are described by Kung, et al., J. Biol. Chem. 251, (8), 2399 (1976)). Alternatively, 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 fluorescent antibody-coated cells were then analyzed for FC200/4800A Cytofluorograph (Ortho Instruments, Westwood, Ma) as described in Example 3. Hybridoma cultures containing antibodies that react specifically with E+ lymphocytes (T cells) were selected and cloned. The clones were then 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 peritoneal exudates from these mice were then used to characterize lymphocytes as described below in Example 2. The subject OKT3 hybrid antibody was demonstrated by standard techniques to represent the IgG2 subclass and to be complement determined.

Example 2

Characterization of OKT3 reactivity

A. Isolation of lymphocyte populations

Human peripheral blood mononuclear cells isolated from healthy volunteer donors (aged 15-40 years) using Ficoll-Hypaque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, Nj) followed by the technique of Boyum, Scand, J. Clin. Lab. Invest. 21 (Suppl. 97) 77, 1968.

Unfractionated mononuclear cells were separated into surface Ig+ (B) and Ig - (T plus null) populations using Sephadex G-200 anti-F(ah`)2 column chromatography as previously described by Chess et al., J Immunol. 113, 1113 (1974). T cells were isolated by E rosecenting of the Ig population with 5% sheep erythrocytes (Microbiological Associates, Bethesda, MD). The rosette mixture was layered over Ficoll-Bethesed, MD). The rosette mixture was layered over Ficoll-Hyapaque and the separated E+ ball was treated with 0.155 M NH4Cl solution (10 ml per 108 cells). The resulting T cell population was <2% EAC rosette positive and <95% E rosette positive as determined using standard methods. In addition, the non-rotating Ig- (zero cell) population was collected from the Ficoll interface. This latter population is <5% E+ and ≤2% slg+. The surface 1g+ (B) population was recovered from a Sephadex G-200 column followed by elution with normal human gamma globulin as described earlier. This population was >95% surface Ig+ and >5% E+.

Normal human macrophages were obtained from the mononuclear population by non-polystyrene adherence. In this way, the mononuclear cells were resuspended in the final culture medium (RPMI 1640, 2.5 mM HEPES /4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid)/buffer, 0.5% sodium bicarbonate , 200 mK L-glutamine and 1% penicillin-streptomycin, to which 20% heat-inactivated human AB serum was added) at a concentration of 2 x 106 cells and incubated in plastic petri dishes (100 x 20 mm) ( Falcon Tissue Culture Dish; Oxnard, CA) at 37°C overnight. After extensive washing to remove non-adherent cells, the adherent population was dissociated by vigorous washing with cold serum-free medium containing 2.5 mM EDTA and occasional scraping with the rubber swab (cap) of the injection syringe. More than 85% of the cell population was able to take up (ingestrate) latex particles and had morphological characteristics of monocytes when stained by the Wright-Giemsa method.

B. Normal thymus

Normal human thymus was obtained from patients aged 2 months to 14 years who had undergone corrective heart surgery. Freshly obtained thymus sections were immediately placed in 5% fetal calf serum in medium 199 (Gibco), finely minced using surgical forceps and scissors, and then processed into single-cell suspensions by pressing through a wire mesh. These cells were then layered over Ficoll-Hypaque and pelleted and washed as described earlier in Part A above. Thymocytes thus obtained were >.95% viable and ≥90% E rosette positive.

C. Cell types

Epstein-Barr virus (EBV transformed B cell types from four normal individuals (Laz 007, Laz 156, Laz 256 and SB) and described them. T cell types CEM, HJD-1, Laz 191 and HHl, obtained from leukemic patients, were provided by Dr. H. Lazarus, Sidney Farber Cancer Institute, Boston, MA.

D. T cells of acute lymphoblastic leukemia

(T-ALL) and T cells of chronic lymphocytic leukemia (T-CILL)

Leukemia cells were obtained from 12 patients with T-ALL. These cells from individuals were previously designated as T cell lineages by their spontaneous rosette formation with sheep erythrocytes (>20% E+) and reactivity with T cell-specific heterosera, anti-HTL (anti-B.K.) and A99, as previously described by sides Schlossman-3, et. al., Proc. Nat. Acad. Sci. 73, 1288 (1976). Tumor cells from three individuals were reactive (TH2+) with rabbit and/or horse anti-TH2, while cells from the remaining nine individuals were not reactive (TH2 ̄ ). Leukemia cells from two patients with TH2 ̄ T-CLL were also used. Both types, cells (T cells) of acute and chronic leukemia were preserved at a low temperature of -196°C using the vapor phase of liquid nitrogen and kept in 10% dimethylsulfoxide and 20% AR human serum until the time of surface characterization. The analyzed tumor populations 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-anti-mouse IgG (G/M FITC) Meloy laboratories) conjugated with fluorescein on a cytofluorograph FC200/4800A (Ortho Instruments). Briefly, 1-2 x 106 cells were treated with 0.15 ml OKT3 at a dilution of 1:1000, incubated at 4°C for 30 minutes and washed twice. The cells were then reacted with 0.15 ml G/M FITC (diluted 1:40) at 4°C for 30 minutes, centrifuged and washed three times. These cells were then analyzed with a cytofluorograph and the intensity of fluorescence per cell was registered on the height pulse analyzer. A similar mechanism of reactivity was observed at a dilution of 1:1000,000, but further dilution caused loss of reactivity.

Baseline staining was obtained by replacing a 0.15 ml aliquot of peritoneal exudates (1:1000) from an intraperitoneally immunized Balb/cJ mouse with a non-hybrid producing clone.

S1. 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 PITC. For comparison, Fig. 1-5 shows the results with monoclonal antibodies OKT1 and OKT4, which were obtained under equivalent conditions.

S1.2 shows the fluorescence image obtained on a cytofluorograph after reaction of human thymocytes with OKT3 and G/M FITC.

S1.3 shows the image of fluomescence obtained on a cytofluorograph after the reaction of leukemic cells obtained from patients suffering from chronic lymphoblastic leukemia (B cells) with 0KT3 and G/M FITC.

S1. 4 shows a fluorescence image obtained on a cytofluorograph after the reaction of human T cell type HJD-1 with OKT3 and G/M FITC.

Data and characteristic values given in Fig. 1-5 as well as supplementary data for OKT3 (as well as for OKT1 and OKT4) are collected in Table 1.

Hybridoma production and production and characterization of the resulting monoclonal antibody were performed as described in the examples above. Although large amounts of the subject antibody have been obtained by injecting the subject hybridoma intraperitoneally into mice and collecting malignant peritoneal exudates, it is understood that the hybridoma 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 can react with OKT3, while the entire population of B cells, null cells and macrophages, which are isolated from the same individual, are not reactive with OKT3. Similar results were obtained on lymphocyte populations from 15 other normal individuals. Thus, a monoclonal antibody is characterized by being reactive with an antigen contained on the surface of virtually all normal human peripheral T cells, while being unreactive with any antigens on the surface of the three other cell types discussed above. This differential reactivity is a test by which the subject OKT3 antibody 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 unreactive with OKT3, while about 5 to 10% of thymocytes are reactive. The implication of this finding is that during the differentiation process, which transforms the initial basic cells into mature T cells, thymocytes acquire to some degree the same surface antigen found in T cells, which are reactive in relation to 0KT3. It is believed that these thymocytes are in the later stages of differentiation just before exiting the thymus into the bloodstream. Similar results (5-10% reactivity) were obtained using six additional thymus species (samples) from normal individuals aged 2 months to 19 years. The reactivity image given in Fig. 2 provides another method for detecting the subject OKT3 antibody and distinguishing it from other antibodies.

The subject antibody can also be used to determine the proportion of circulating lymphocytes that are T cells. As shown in Table 1, ≥95% of all T cells reacted with the OKT3 antibody. This invention thus includes a procedure for determining (in an individual) the ratio of circulating lymphocytes that are T cells, which includes mixing OKT3 antibodies with a lymphocyte mixture from this individual and determining how to obtain lymphocytes that are OKT3+ and thus T cells.

Further characterization of the subject OKT3 antibody was shown by its reactivity against different types of human T cells, which is illustrated in Fig. 4 and 5. As can be seen, the reactivity of the subject antigen against the types of human T cells was heterogeneous, it was weak for the type HJD-1 and non-existent for types CEM, Laz 191 and HMI. This differential reactivity of OKT3 to different types of human T cells (which are readily obtainable) provides another method for characterizing and describing the T antibody in question.

The absence of reactivity of OKT3 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 a normal human population and provides yet another method of characterizing and differentiation of the OKT3 antibody in question. The specific reaction of OKT3 antibodies with antigen in cutaneous T cell lymphomas is illustrated in Table 2, which shows the differentiation from OKT1 and OKT4. This antibody thus provides a reagent for confirming the diagnosis of cutaneous T cell lymphoma in a patient suspected of having said disease. Treatment of cutaneous T cell lymphomas by administration of a therapeutically effective amount of OKT3 antibody is also contemplated as part of the present invention.

According to this invention, there is provided a hybridoma that is able to produce an antibody against an antigen found in practically all normal human T cells and cutaneous T lymphoma cells, then a process for obtaining this hybridoma, a monoclonal antibody against an antigen found in practically all human T cells, methods for obtaining this antibody, and methods and methods for the treatment or diagnosis of diseases using this antibody.

Although only one hybridoma has been described that produces a single monoclonal antibody against a human T cell antigen, the present invention is intended to encompass all monoclonal antibodies exhibiting the characteristics described above. The OKT3 antibody in question was determined to belong to the Ig G2 subclass, which is one of the four mouse (murine) IgG subclasses. These immunoglobulin 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 immunoglobulin G subclass it belongs to. In this way, the monoclonal antibody that shows the characteristics described here can be of the IgG1, IgG2s, IG2b or IgG3 subclasses or of the IgM, IgA or some other known Ig classes. Differences between these classes or subclasses will not affect the selectivity of the reaction pattern of this antibody, and may affect the further reaction of the antibody with other materials, such as (for example) complement or anti-mouse antibodies. Although the antibody in question is specifically IgG2, it is understood that antibodies having the mechanisms of reactivity included herein are encompassed by this invention regardless of the class or subclass of immune globulin to which they belong.

Also within the scope of this invention are the methods for obtaining monoclonal antibodies, which are described above, using the hybridoma techniques illustrated above. Although only one example of a hybridoma is provided herein, it is understood that one skilled in the art could work according to the above immunization, fusion and selection methods to obtain hybridomas capable of producing antibodies with the reactivity characteristics described above. Since an individual hybridoma, which is produced from known species of murine myeloma and spleen cells obtained from known species of mice, cannot be further identified except by reference to the antibody produced by this hybridoma, it is understood that all hybridomas, which produce an antibody with the characteristics described above covered by the subject of this invention, as well as methods for making this antibody using this hybridoma.

Further aspects of the present invention are methods of treating or diagnosing diseases, which use the monoclonal antibody 0KT3 or another monoclonal antibody that exhibits the above mechanism of reactivity. As discussed above, the subject antibody enables the treatment of patients having certain T cell chronic lymphoblastic leukemia by administering therapeutically effective amounts of this antibody. Administration of a therapeutically effective amount of OKT3 antibody to an individual subject undergoing organ transplantation will reduce or eliminate rejection of the transplant.

The subject antibody also enables the detection of cutaneous T cell lymphomas in an individual by mixing the lymphoma T cell mixture from the aforementioned individual with a diagnostically effective amount of OKT3 antibody. The presence of a reaction confirms the identity of this disease. Cutaneous T cell lymphoma can be treated by administering to an individual in need of such treatment a therapeutically effective amount of an OKT3 antibody. This antibody will react and reduce the amount of Z lymphoma cells, leading to an improvement in the disease. In terms of these diagnostic and therapeutic methods, the present invention additionally includes diagnostic and therapeutic compositions comprising a diagnostically or therapeutically effective amount of OKT3 antibody in a diagnostically or pharmaceutically acceptable carrier.

Table 1

Monoclonal antibody reactivity and properties.

[image]

* 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, CE1, Laz 191 and H41 obtained from leukemic patients.

Table 2

[image]

Cell source: PBL = peripheral blood lymphocytes

Node = lymph node

Claims (20)

1. A murine monoclonal antibody that fixes complement, characterized in that it (i) reacts with essentially all normal human peripheral T cells, but (ii) does not react with any of the normal human peripheral cells from the group consisting of B cells, null cells and macrophages . 2. Monoclonal antibody according to claim 1, characterized in that it belongs to the IgG class. 3. Monoclonal antibody according to claim 2, characterized in that it belongs to the IgG class. 4. Monoclonal antibody according to any one of claims 1 to 3, characterized in that it reacts with approx. 5 % to approx. 10% of normal human thymocytes. 5. Monoclonal antibody according to any one of claims 1 to 4, characterized in that it reacts with all T cells of cutaneous lymphoma. 6. The monoclonal antibody according to any one of claims 1 to 5, characterized in that it reacts with leukemic cells of a human with T cell chronic lymphoblastic leukemia, but does not react with leukemic cells with T cell acute lymphoblastic leukemia, zero cell acute lymphoblastic leukemia or chronic lymphoblastic leukemia. B cell leukemia. 7. Monoclonal antibody according to any one of claims 1 to 6, characterized in that it is produced using a hybridoma formed by the fusion of spleen cells from a mouse, pre-immunized with human T cells, and cells from a mouse myeloma line. 8. Monoclonal antibody, indicated that it is produced from hybridoma ATCC CRL 8001 (OKT3). 9. Therapeutic composition of matter, characterized in that in admixture with a pharmaceutically acceptable carrier, it contains a therapeutically effective amount of antibody according to any one of claims 1 to 8, which amount is effective for reducing or eliminating transplant rejection in the recipient of the transplanted organ. 10. A hybridoma, characterized in that it produces a monoclonal antibody according to any one of claims 1 to 7, which is obtained by fusion of spleen cells from a mouse, previously immunized with human T cells, and cells from a murine myeloma line. 11. Hybridoma, indicated that it is ATCC CRL 8001 (OKT3). 12. Monoclonal antibody according to any one of claims 1 to 8, characterized in that it is used for the treatment of a transplanted organ recipient to reduce or eliminate allograft rejection of said transplanted organ. 13. A method for determining the proportion of lymphocytes circulating in an individual, which are T cells, characterized in that they include mixing the monoclonal antibody according to any of claims 1 to 8 with a sample of lymphocytes obtained from the person in question and determining the proportion of said lymphocyte sample that reacts with said antibody, and therefore they are T cells. 14. The method for the production of monoclonal antibodies according to any one of claims 1 to 7, characterized in that it includes the following stages: (i) immunization of mice with E rosette positive purified human T cells; (ii) extracting the spleen from said mouse and making a suspension of spleen cells; (iii) fusing spleen cells with murine myeloma cells in the presence of a fusion promoter; (iv) diluting and culturing the fused cells in separate wells in a medium that does not support unfused myeloma cells; (v) evaluating the supernatant in each well containing the hybridoma for the presence of an antibody having the properties set forth in any of claims 1 to 7; (vi) selecting and cloning hybridomas that produce the desired antibody; and (vii) collecting antibodies from the supernatant above said clones. 15. The method for producing a monoclonal antibody according to any one of claims 1 to 7, characterized in that it includes the following steps: (i) immunization of mice with E rosette positive purified human T cells; (ii) extracting the spleen from the mouse and making a spleen cell suspension; (iii) fusion of spleen cells with murine myeloma cells in the presence of a fusion promoter; (iv) diluting and culturing the fused cells in separate wells in a medium that does not support unfused myeloma cells; (v) evaluating the supernatant in each well containing the hybridoma for the presence of an antibody having the properties set forth in any of claims 1 to 7; (vi) selecting and cloning hybridomas that produce the desired antibody; (vii) transferring said clones intraperitoneally into a mouse; and (viii) collecting malignant ascites or serum from said mouse. 16. A method for the production of a monoclonal antibody, characterized in that it includes growing the hybridoma ATCC CRL 8001 in a suitable medium and collecting the antibody from the supernatant above said hybridoma. 17. A method for producing a monoclonal antibody, characterized in that it includes injecting ATCC CRL 8001 into a mouse and obtaining the antibody from malignant ascites or serum from said mouse. 18. Antibody according to claim 5, claim 6 or claim 7, characterized in that, depending on claim 5 or claim 8, it is used to confirm the presence of cutaneous T cell lymphoma in a person. 19. The antibody according to claim 5, claim 6 or claim 7, characterized in that, depending on claim 5 or claim 8, it is used for the treatment of a person with cutaneous T cell lymphoma. 20. A method for producing a therapeutic composition according to claim 9, characterized in that it comprises mixing a pharmaceutically acceptable carrier with an amount of antibody according to any one of claims 1 to 8 effective to reduce or eliminate graft rejection in the recipient of the transplanted organ.