HRP940790A2 - Hybrid cell line for producing monoclonal antibody to a human thymocyte antigen, antibody and method of preparation thereof - Google Patents

Description

This invention relates generally to new lines of hybrid cells and more specifically to lines of hybrid cells for obtaining a monoclonal antibody for an antigen found in approximately 70% of normal human thymocytes, then to the antibody thus obtained and to therapeutic and diagnostic methods and mixtures using this body .

Fusion of murine myeloma cells to spleen cells from an immunized mouse, 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 line of cells by making a homogeneous (so-called "monoclonal") antibody. Since that initial work, many efforts have been made to obtain various hybrid cells (called "hybridomas") and to use the antibodies produced by these hybridomas for various scientific investigations. See, for example, Current Topics in Microbiology and Immunology, volume 81, " Lymphocyte Hybridomas", F. Melchers, M. Potter and N. Warner, publishers, Springer-Verlag, 1978 and references herein; C.J. Barnstable et al, Cell, 14, 9-20 (May, 1978); Handbook of Experimental Immunology, Third Edition, Volume 2, D. M. Wier, Publisher, Blackwell, 1978, Chapter 25, Chemical and Engineering News, January 1, 1979, pp. 15-17 These references indicate both the rewards and complications of attempting to a monoclonal antibody is obtained from a hybridoma. the general technique is easily understood in concept itself, there are many difficulties encountered in this work, and many variants of this technique required for each specific case. In fact, there is no certainty, before attempting that a given hybridoma is made, that the desired hybridoma will be obtained, that it will produce an antibody, if it is obtained, or that the body thus produced will have the desired specificity. The degree of success is essentially influenced by the type of antigen used and the selection technique used to isolate the desired hybridoma.

Attempts to obtain a monoclonal antibody for human lymphocyte cell antigens have been reported only in a few examples. See, for example, Current Topics in Microbiology and Immunology, ibid, 66-69 and 164-169. The antigens used in these reported experiments were cultured on human lymphoblastoid leukemia and human chronic lymphocytic leukemia cell lines. Many of the resulting hybridomas appear to produce antibody to different antigens on all human cells. None of the obtained hybridomas produced an antibody against a previously defined class of human lymphocytes. Recently, these applicants and others have authored articles disclosing the production and testing of hybridomas that make antibody to certain T-cell antigens. See, for example, Reinherz, E. L. et al., J. Immunology, 123, 1312-1317 (1979); Reinherz, E.L. et al., Proc. Natl. Acad. Sci. 76, 4061-4065 (1979) and Kung, P.C. et al, Science, 206, 347-349 (1979).

It should be understood that there are two basic classes of lymphocytes involved in the immune system of humans and animals. The first of these classes (thymus-derived cell or T-cell) is differentiated in the thymus from hematopoietic stem cells. Since they are located inside the thymus, the differentiated cells are called "thymocytes". Mature T-cells leave the thymus and circulate between tissues, lymphatics and blood streams. 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 transplant rejection) as effector cells. Although T-cells do not secrete humoral antibodies, they are sometimes required for the secretion of these antibodies by another class of lymphocytes, discussed below. Some types of T-cells play a regulatory role in other aspects of the immune system. The mechanism of this process of cell cooperation is still not fully understood.

Another class of lymphocytes (bone marrow-derived cells or B-cells) are those that secrete antibodies. They also develop from hemopoietic stem cells, but their differentiation is not determined by the thymus. In birds, they differentiate in a single organ, analogous to the thymus, called the sac of Fabricius. However, no equivalent organ has been found in mammals, and these B-cells are thought to differentiate within the bone marrow.

It is now known that T-cells are divided into at least several subtypes, called "helper", "suppressor" and "killer" T-cells, which have the function of helping a reaction, stopping a reaction or killing (decomposing) foreign cells. These subclasses are well understood for mouse 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. Sdilossman - “Functional Analysis of Distinct Human T-Cell Subsets Bearing Unique Differentiation Antigens,” in "Contemporary Topics in Immunobiology", O. Stutman, publisher, Plenum Press, 1977, vol. 7, 363-379.

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

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

Some disease states (eg, juvenile rheumatoid arthritis, malignancies, and agammaglobulinemia) are associated with an imbalance of T-cell subclasses. It has been suggested that autoimmune diseases are generally related to an excess of "helpers". T-cells or a deficiency of some "suppressor" T-cells, while agammaglobulinemia is related to an excess of some "suppressor" T-cells or a deficiency of "helper" T-cells. Malignancies are generally related to an excess of "suppressor" T-cells.

In some leukemias, excess T-cells are produced in an arrested state. In this way, the diagnosis can depend on the ability to detect this imbalance or excess and determine which developmental stage is in excess. See, for example, J. Kersey et al., "Surface Markers Define Human Lymphoid Malignancies with Differing Prognosis," in Hematology and Blood Transfusion, vol. 20, Springer-Verlag, 1977, 17-24, and references therein, and El. L. Reinherz et al., J. Clin. Invest. 64, 392-397 (1979).

Acquired agammaglobulinemia, a disease state in which immune globulin is not produced, includes at least two different types. In type I, the inability to produce immune globulin is caused by an excess of suppressive T-cells, while in type II it is caused by a lack of helper T-cells. In both types, there appears to be no defect or deficiency in the patient's B-cells, the lymphocytes responsible for actually secreting the antibodies; however, these B-cells are either suppressed or "unassisted," resulting in greatly reduced or non-existent production of immune globulin. The type of acquired agammaglobulinemia can thus be determined by examining whether there is an excess of suppressor T-cells or the absence of helper T-cells.

On the therapeutic side, there is a suggestion, although not yet definitively proven, that administration of antibodies against a T-cell subtype in excess may have a therapeutic advantage in autoimmune disease or malignancy. For example, helper T-cell cancers (some cutaneous T-cell lymphomas and some T-cell acute lymphoblastoid leukemias) can be treated with an antibody to a helper T-cell antigen. Treatment of an autoimmune disease caused by an excess of helper cells can also be done in the same way. Treatment of diseases (eg, malignancy or acquired agammaglobulinemia type I) caused by an excess of suppressive T-cells can be performed by administering antibodies to the antigen of suppressive T-cells.

It has been reported that antisera against an entire class of human T-cells (so-called antihuman thymocyte globulin or ATG) can be used therapeutically in patients who have received organ transplants. Since the cell-mediated reaction (the mechanism by which transplants are rejected) depends on T-cells, administration of T-cell antibodies prevents or delays this rejection process. See, for example, Cosini et al, "Randomized Clinical Trial of ATG in Cadaver Renal Allograft Recipients: Importance of T Cell Monitoring", Surgery, 40, 155-163 (1976) and references therein.

Identification and suppression of human T-cell classes and subclasses has previously been performed using spontaneous autoantibodies or human T-cell-selective antisera obtained by immunizing animals with human T-cells, bled animals to obtain serum by adsorbing antisera with (for example) autologous but not and allogeneic B-cells to remove antibodies of unwanted reactivity. Obtaining these antisera is extremely difficult, especially in the stages of adsorption and purification. Even adsorbed and purified antisera, in addition to the desired antibody, contain many impurities for various reasons. First, serum contains millions of antibody molecules even before T-cell immunization. Second, immunization causes the production of antibodies against a multitude of different antigens found in all injected human T-cells. There is no selective production of antibodies against a single antigen. Third, the concentration of specific antibody obtained using these methods is usually very low (for example, inactive at dilutions greater than 1:100) and the ratio of specific to nonspecific antibody is less than 1 106.

See, for example, the article by Chess and Schlossman, referred to above (on p. 365 ff.) and the article in Chemical and Engineering News (also referred to above), which describe the disadvantages of earlier antisera and the advantages of the monoclonal antibody. A new hybridoma (designated OKT6) has now been discovered that is able to produce a new monoclonal antibody against an antigen found on approximately 70% of normal human thymocytes but not on normal human peripheral lymphoid cells (T-cells, B-cells or neutrophils). or a bone marrow cell.

The antibody thus produced is monospecific for a single determinant in approximately 70% of normal human thymocytes and practically does not contain any other anti-human immune globulin, in contrast to earlier antisera (which are inherently contaminated with antibodies reactive to numerous human antigens) and earlier monoclonal antibodies (which are not monospecific for human thymocyte antigen). In addition, this hybridoma can be cultured to produce antibody without the need to immunize and kill the animals, followed by laborious adsorption and purification - which was 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 in about 70% of normal human thymocytes.

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

A further object of this invention is to provide a practically homogeneous antibody against an antigen found in about 70% of normal human thymocytes.

It is a further object of this invention to provide methods for the treatment and diagnosis of disease or the identification of a T-cell or thymocyte subclass that utilizes this antibody.

Other objects and advantages of the present invention will be apparent from a review of this disclosure.

In order to provide the above objects and advantages, the present invention provides a new hybridoma that produces a new antibody to an antigen found in approximately 70% of normal human thymocytes (but not in normal human peripheral lymphoid cells or bone marrow cells), then the antibody itself and diagnostic and therapeutic methods using this antibody. This hybridoma was obtained with the general application of Milstein and Kohler. After immunization of mice with normal human thymocytes, spleen cells from the immunized mice were fused with cells from one type of murine myeloma and the resulting hybridomas were sorted to obtain those containing the antibody and which gave selective binding to normal E rosette positive human T-cells or thymocytes. The desired hybridomas were then cloned and characterized. As a result, a hybridoma was obtained that produced an antibody (designated as OKT6) against an antigen in approximately 70% of normal human thymocytes. Not only does this antibody react with about 70% of normal human thymocytes, but it also does not react with normal peripheral blood lymphoid cells or bone marrow cells.

In view of the difficulties indicated for earlier practice and the lack of success related to the use of malignant cell types as antigens, it is surprising that this method yields the desired hybridoma. It should be emphasized that the unpredictable nature of obtaining a hybrid cell does not allow extrapolation from one system of antigens or cells to another system. In fact, these applicants found that the use of a type of T-cell - malignant cells or purified antigens separated from the cell surface as antigens was essentially unsuccessful.

Both the hybridoma in question and the antibody produced from it are identified herein by the designation “OKT6”, the separate material will be apparent from the context. The subject hybridoma was deposited on November 21, 1979 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and was assigned ATCC accession number CRL8020.

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

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

A. Immunization of mice with normal human thymocytes. female CAF1 mice were easily found to be preferred, it is understood that other mouse strains can be used. The immunization program and thymocyte concentration should be such as to produce usable amounts of suitably primed splenocytes. Three immunizations at two-week intervals with 2x107 cells/mouse/injection in 0.2 ml of phosphate-buffered saline were found to be effective.

B. Removal of spleens from immunized mice and preparation of spleen suspension in 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 adjuvant. A suitable ratio is about 5 spleen cells to one myeloma cell. A total volume of about 0.5 to 1.0 ml of fusion medium corresponds to about 108 splenocytes. Many types of murine myeloma cells are known and can be obtained, generally from members of academic communities (associations) or various depository banks, such as the Salk Institute Cell Distribution Center, La Jolla, CA. The type of cells used should preferably be of the so-called "drug-resistant" type, so that unfused myeloma cells do not survive in the selective medium, while the hybrids will. The most common class is the 8-azaguanine cell type, which lacks the enzyme hypoxanthine guanine phosphoribosyl transferase and will therefore not be supported by HAT (hypoxanthine, aminopterin, and thymidine) media. It is also generally advantageous that the type of myeloma cells used is of the so-called "non-secretory" type, meaning that it does not produce any antibody on its own, although secretory types can also be used. However, in some cases, secretory myeloma types may be of advantage. a readily suitable fusion promoter is polyethylene glycol, which has an average molecular weight of about 1000 to about 4000 (commercially available as PEG1000, etc.), other fusion promoters - which are known in practice - can be used.

D. Diluting and culturing in separate dishes 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 death of the unfused cells (about one week). The dilution may be of the limiting type, in which the volume of diluent is calculated statistically to isolate a certain number of cells (eg 1-4) in each separate container (eg each well of a microtiter dish). The medium is one (eg, HAT medium) that will not support a drug-resistant (eg, 8-azaguanine-resistant) unfused type of myeloma cells. This is where these myeloma cells are lost. As non-fused spleen cells, those that have only a finite number of generations are not malignant. In this way, after a certain period of time (about a week), these unfused spleen cells stop reproducing. On the other hand, the fused cells continue to reproduce because they possess the malignant trait of the myeloma progenitor and the ability to survive in the selective environment of the progenitor trait - the spleen cells.

E. Evaluation of the supernatant in each dish (well) containing the hybridoma for the presence of antibody to E rosette positive purified human T-cells or thymocytes.

F. Selection (eg, by limiting dilution) and cloning of hybridomas that produce the desired antibody.

Once the desired hybridoma has been selected and cloned, the resulting antibody can be obtained using one of two methods. The purest monoclonal antibody is obtained in vitro by culturing the desired hybridoma in a suitable medium for a suitable time interval, followed by extraction of the desired antibody from the upper liquid layer. A suitable culture medium and a suitable period of time for cultivation are known or can be easily determined. This in vitro technique produces a practically monospecific monoclonal antibody, practically without any other specific anti-human immunoglobulin. A small amount of other immune globulin is present because the medium contains xenogeneic serum (eg fetal calf serum). However, this in vitro method may not produce a sufficient amount or concentration of antibody for some purposes, as the concentration of the monoclonal antibody is only about 50 µg/ml.

In order to obtain a much higher concentration of a slightly less pure monoclonal antibody, the desired hybridoma can be injected into mice, preferably syngeneic or semi-syngeneic mice. The hybridoma will cause the formation of tumors that produce the antibody after a suitable incubation period, which will result in a high concentration of the desired antibody (about 5 - 20 mg/ml) in the bloodstream and peritoneal exudate (abdominal dropsy) of the host mouse. easily these host mice also . have normal antibodies in their blood and secretions, the concentration of these normal antibodies is only about 5% of the concentration of the monoclonal antibody. In addition, since these normal antibodies are not antihuman in their specificity, the monoclonal antibody obtained from harvested exudates or from serum is virtually free of any contaminating antihuman immune globulin. This monoclonal antibody has a high concentration (active at dilutions of 1:50000 or even higher dilutions) and a high ratio of specific to non-specific immune globulin (about 1/20). The immune globulin that is produced and incorporates the K light myeloma chains are non-specific, "non-binding" peptides, which only dilute the monoclonal antibody without depriving it of its specificity.

Example 1

Obtaining monoclonal antibodies

A. Immunization and hybridization of body cells

Female CAF1 mice (Jackson Laboratories, 6-8 weeks old) were immunized intraperitoneally with 2x107 human thymocytes in 0.2 ml of phosphate-buffered saline at two-week intervals. Four days after the third immunization, the spleens were removed from the mice and a simple cell suspension was made by pressing the tissue through a stainless steel sieve.

Cell fusion was performed according to the procedure of Kohler and Milstein. 1x108 splenocytes were fused in 0.5 ml fusion medium containing 35% polyethylene glycol (PEG1000) and 5% dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, NY) with 2x107 P3x63Ag8U1 myeloma cells obtained from Dr. M. Scharff, Albert Einstein College of Medicine, Bronx, NY. These myeloma cells secrete IgG1 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% CO2 in a humid atmosphere. A few weeks later, 40 to 100 μl of supernatant from cultures containing hybridomas was added to a pellet of 106 peripheral lymphocytes separated into E rosette positive (E+) and E rosette negative (E-) populations, which were made from blood of healthy human donors as described by Mendes (J. Immunol. 111, 860 (1973)). The detection of mouse hybridoma antibodies, bound to these cells, was determined by indirect immunofluorescence. Cells incubated with culture supernatants were stained with fluorescent anti-mouse IgG (G/M FITC) (Meloy Laboratories, Springfield, VA; F/p=2.5) and fluorescent antibody-coated cells were then analyzed on an FC200/4800A cytofluorograph ( Ortho Instruments, Westwood, MA) as described in section III. Hybridoma cultures containing antibodies that react specifically with E+ lymphocytes (T-cells) and/or thymocytes were selected and cloned twice using limiting dilution methods in the presence of feeder cells. Afterwards, the extracts were transferred intraperitoneally by injecting 1x107 cells of the given extract (0.2 ml each) into CAF1 mice primed with 2,6,10,14-tetramethylpentadecane (sold by Aldrich Chemical Company under the name Pristine). Malignant exudates from these mice were then used to characterize lymphocytes, as described below in Example 2. The OKT6 hybrid antibody in question was demonstrated to be of the tgG1 subclass by standard techniques.

Example 2

Characterization of OKT6 reactivity

A. Isolation of lymphocyte populations

Human peripheral blood mononuclear cells were 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. Ciin. 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(ab')2 column chromatography, as previously described by Chess et al., in J. Immunol. 113, 1113 (1974). T cells were isolated by E rosetting the Ig population with 5% sheep erythrocytes (Microbiological Associates, 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 per 108 cells). The resulting T-cell population was < 2% EAC rosette positive and > 95% E rosette positive as determined by standard methods. In addition, a non-rosetting Ig- (null cell) population was harvested from the Ficoll interphase. This latter population was < 5% E+ and < 2% with Ig+. The surface Ig+ (B) population was obtained 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 bone marrow cells were obtained from the posterior crest of the small intestine of normal human volunteers using needle aspiration.

B. Isolation of thymocytes

A normal human thymus gland was obtained from a patient aged two months to 14 years who underwent cardiac surgery. Freshly obtained parts of the thymus gland were immediately placed in 5% fetal calf serum in medium 199 (Gipco), finely chopped with forceps and scissors, and then converted into single-cell suspensions by pressing through a wire sieve. Cells were then plated over Ficoll-Hypaque and spun down and washed as described earlier in section A above. Thymocytes thus obtained were > 95% fertile and > 90% E rosette positive.

C. Cell types of T strain and T acute lymphoblastic leukemia cells

T cell types CEM, HSB-2 and MOLT-4 were provided by Dr. H. Lazarus (Sidney Farbar Cancer Institute, Boston, MA). Leukemic cells were obtained from 25 patients with a diagnosis of T cells completely. These individual tumors were previously determined to be of the T-cell strain based on their spontaneous rosette formation with sheep erythrocytes (> 20% E+) and reactivity with the specific anti-HTL (B.K.) and A99 heteroantisera for T-cells, as previously described. . Tumor populations were preserved at a low temperature of -196°C with a vapor phase of liquid nitrogen using 10% DMSO and 20% AB human serum until the moment of surface characterization. All tumor populations (which were analyzed) were more than 90% blasts, which was determined by the morphology of cytocentrifuged preparations according to Wright-Giemsa.

Example 3

Cytofluorographic analysis and cell separation

Cytofluorographic analysis of monoclonal antibodies of all cell populations was performed by indirect immunofluorescence with fluorescein-conjugated goat-anti-mouse IgG (G/M FITC) (Meloy Laboratories) using a Cytofluorograph FC200/4800A (Ortho Instruments). Briefly, 1x106 cells were treated with 0.15 ml OKTS at a 1:500 dilution, incubated at 4ºC for 30 minutes and washed twice. The cells were then reacted with 0.15 ml G/M FITC dilution 1:40 at 4ºC for 30 minutes, centrifuged and washed 3 times. The cells were then analyzed on a cytofluorograph and the fluorescence intensity per cell was recorded on a pulse height analyzer. A similar course of reactivity was found at a dilution of 1:10000, but further dilution caused a loss of reactivity. Background staining was obtained by substituting a 0.15 ml aliquot of 1:500 exudate from a CAF1 mouse injected intraperitoneally with a non-hybrid producing clone. In experiments involving antibody and complete mediated lympholysis, thymocytes and peripheral T cells were cultured overnight followed by selective lysis and then analyzed on a cytofluorograph.

Example 4

Decomposition of lymphoid populations with monoclonal antibody and complement

40x106 peripheral T cells or thymocytes were placed in a 15 ml plastic tube (Falcon, Oxnard, CA). Cell pellets were incubated with 0.8 ml OKT3; OKT8 or normal exudate control diluted in PBS 1:200, resuspended and incubated for 60 minutes at 20ºC. 0.2 n of fresh rabbit complement was then added to the antibody-treated populations, resuspended and further incubated at 37ºC in a shaking water bath for a period of 60 minutes. At the end of this time period the cells were spun down and the fertile cells were numbered with Trypan blue exclusion. After counting, the cells were washed twice in 5% FCS and placed in the final medium /RPMI 1640 (Grand Island Biological Company Grand Island, NY) containing 20% AB+ human serum, 1% penicillin-streptomycin, 200 mM L-glutamine , 25 mM HEPES buffer and 0.5% sodium bicarbonate/, incubated overnight in a humid atmosphere with 5% CO2 at 37ºC

Brief description of the drawing

Sl. 1 shows the fluorescence pattern obtained on a cytofluorograph after reacting normal human thymocytes with OKT6 and other monoclonal antibodies at a dilution of 1:500 and G/M FITC. Background fluorescent staining was obtained by incubating each population with exudate fluid (dilution 1:500) from a mouse injected with a non-producing clone.

Sl. 2 shows the stages of intrathymic differentiation in man.

Hybridoma production and the production and characterization of the resulting antibody were performed as described in the examples above. easily large amounts of the subject antibody were obtained by injecting the subject hybridoma intraperitoneally into mice and harvesting the malignant exudates, it is obviously understood that these hybridomas can be cultured in vitro using techniques well known in the art and that the antibody can be removed from the upper fluid layer.

Table 1 shows the reactivity of OKT6, OKT8, OKT9 and 0KT10 against different human lymphoid cell populations. The OKT6 monoclonal antibody is reactive with approximately 70% of normal human thymocytes and not with any other of the lymphoid cells tested. This reactivity model is a test that can detect the subject OKT6 antibody and distinguish it from other antibodies.

Sl. 1 shows a representative fluorescence pattern obtained on a cytofluorograph after reacting suspensions of normal human thymocytes with a 1:500 dilution of OKT3, OKT4, OKT5, OKT6, OKT8, OKT9, 0KT10 and G/M FITC. Similar patterns of reactivity were found with 12 additional populations of normal human thymocytes that were examined. As shown, there is considerable variation in percent reactivity and fluorescence intensity with each of these monoclonal antibodies. For example, OKT9 reacts with approximately 10% of thymocytes at low fluorescence intensity while OKT5, OKT6, OKT8 and OKT10 react with approximately 70% of thymocytes at higher fluorescence intensity OKT4, which reacts with 75% of thymocytes, is an intermediate between the OKT9 monoclonal antibodies that give higher fluorescence intensity model. In addition, Fig. 1 shows that approximately 15% of thymocytes were detected with OKT3 using indirect immunofluorescence. OKT1, whose reactivity model is practically identical to OKT3 in thymocytes, is not shown. The reactivity model in Fig. 1 is another test by which the subject OKT6 antibody can be detected and distinguished from other antibodies

Table 2 shows the distribution of antigens defined by different monoclonal antibodies in human peripheral T cells and lymphocytes, as determined by a series of lysing experiments described in Example 4. Since only OKT3, OKT4, and OKT8 were complement-fixing monoclonal antibodies, these three were used. As shown in Table 2A, the entire T cell population reacts with OKT3 while OKT4, OKT5 and OKT8 react with 60%, 25% and 34% of T cells, respectively. Lysing (digestion with OKT4 and complement reduced the total number by 62% and specifically eliminated the OKT4+ population. In addition, the percentages of OKT5+ and OKT7+ cells increased and there was no effect on the absolute number of OKT5+ and OKT8+ T cells. These experiments suggest that OKT4+ differs of OKT5+ and OKT8+ and populations Further support for this conclusion was obtained by lysing (dissolving) T cells with OKT8+ and complement. In this case, the percentage of OKT4+ T cells increased, while the absolute number remained the same, and OKT8+ OKT5+ were eliminated. In addition, these results demonstrate that the OKT8+ population is reciprocal to the OKT4+ population and contains the entire OKT5+ population of T cells predicted.

Similar experiments with human thymocyte populations yielded mixed results. As shown in Table 2B, approximately 75% of thymocytes were OKT4+ or OKT8+. In addition, subsequent lysis (decomposition) with OKT4 or OKT8 results in only 25% of thymocytes remaining. Most of the remaining thymocytes are reactive to OKT3, while only a small part is reactive to OKT6. These findings indicate that a major part of the human thymocyte population carries OKT4, OKT5, OKT6 and OKT8 surface antigens on the same cell. In addition, Table 2 shows that subsequent treatment with OKT8 or OKT4 leads to a significant increase in mature thymocytes bearing the OKT3 antigen. In this way, the greater part of OKT3 reactive thymocytes has already been separated into OKT4+ or OKT8+ subtypes because the greater part of residual cells, after OKT4 or OKT8 lysis, are OKT3+. If the OKT3+ subpopulation was OKT4+ and OKT8+, then lysing with either antibody would remove OKT3 reactive thymocytes. To further determine the relationship of the OKT3 reactive thymocyte subpopulation to other thymocyte fractions, which were defined as monoclonal antibody (antibody) thymocytes were treated with OKT3 and complement and the remaining cells were then compared with untreated thymocyte populations. As shown in Table 2B, OKT3 and complements removed 25% of thymocytes. In addition, there was no major loss of OKT4, OKT5, OKT6, or OKT8 reactive populations. These findings suggest that the vast majority of thymocytes bearing the OKT6 marker are contained in the OKT3- population. Additionally, they further suggest that thymocytes simultaneously expressing antigens defined by OKT4, OKT5 and OKT8, also restricted to the OKT3- population. It should also be noted that the OKT9 reactive population of thymocytes was not reduced after treatment of unfractionated thymocytes with OKT3 and complement, thus demonstrating that the OKT9+ subpopulation is largely restricted to OKT3- thymocyte population.

Based on these results, it is possible to describe the state of intrathymic development of human thymocytes. As shown in Fig. 2, practically all thymocytes carry the 0KT10 marker. In addition, early-stage thymocytes acquire the OKT9 marker (Thy1 or Thy2). This stage defines a smaller part of thymocytes and accounts for approximately 10% of the unfractionated population. Subsequently, human thymocytes acquire the thymocyte unique antigen defined by OKT6 and competitively express OKT4, OKT5 and OKT8 (Thy4). This last population represents the majority of thymocytes, accounting for about 70 - 80% of the thymic population. With further maturation, thymocytes lose OKT6 reactivity, acquire OKT3 (and OKT1) reactivity, and differentiate into subtypes OKT4+ and OKT5+/OKT8+ (Thy7 and Thy8). Finally, it appears that as the thymocyte is exported to the peripheral T cell compartment, it loses the 0KT10 marker because this antigen is absent from virtually all peripheral T lymphocytes. Possible transitional states between these three main stages of thymic development are marked Thy3, Thy5 and Thy6 in Fig. 2.

Since T-type acute lymphoblastic leukemia is thought to originate from immature thymocytes, the relationship between tumor cells from individuals with T-ALL and those proposed states of intrathymic differentiation was determined. 25 tumor cell populations from individuals with T-ALL and 3 types of T cells previously studied with common Anti-T cell reagents and E rosetting were examined. As shown in Table 3, the majority of T-ALL leukemia cells were reactive with 0KT10 alone or OKT9 and 0KT10, and did not react with other monoclonal antibodies. Thus, 15 of the 25 studied cases showed early thymocyte antigens (grade I). In contrast, 5 of 25 examined cases were reactive to OKT6, suggesting derivation from a more mature thymic population (grade II). This T-ALL group is inherently heterogeneous with respect to OKT4, OKTB, and OKT9 reactivity, as shown in Table 3. Cells from patients 2 to 5 have most of the common thymocyte antigens including OKT4, OKT6, and OKT8. It is worth mentioning that OKT5 was not present in any of these 5 grade II tumors even though OKT8 reactivity was observed. This last result clearly suggests that OKT5 and OKT8 define different antigens or different determinants in the same antigen. Finally, 1 in 25 patient tumors came from a mature thymocyte population (grade III) as defined by its reactivity to OKT3. This individual tumor, in addition, was reactive to OKT5, OKT8 and 0KT10. Out of 25 leukemic populations analyzed, only 4 tumors could not be clearly categorized. Three were positive for OKT4 and OKT8, but did not react with OKT3 and OKT6 and most likely represent transition states from Thy4 and Thy7,8. One of the 25 cases appeared to be a transition from Thy3 to Thy4 because it possessed OKT8 and 0KT10 reactivity.

T cell types, derived from T-ALL tumor populations, also represented cells from a specific state of intrathymic differentiation. As shown in Table 4, HSB was exclusively reactive to OKT9 and OKT10 and hence could define a tumor population originating from stage I. In contrast, CEM was reactive to OKT4, OKT6, OKT8, OKT9 0KT10 and appeared to originate from from stage II thymocytes. Finally, MOLT-4 appeared to represent a leukemic transformation at a stage between HSB-2 and CEM because it expressed OKT6, OKT9, and 0KT10.

Since patients with later stages (eg, stage II) T-cell acute lymphoblastic leukemia have been shown to have a longer disease-free survival than those with stage I ALL, the use of OKT6 antibodies allows conclusions regarding the prognosis for a given patient with T-cell ALL.

Table 5 shows the relationship between the levels of peripheral T cells and T cell subtypes and different disease states. These ratios can be used for diagnostic purposes (eg, to detect acute infectious mononucleosis) by analyzing blood samples from an individual suspected of having one of these disease states to determine T cell levels and T cell subtypes. These relationships can also be used for therapeutic purposes where the cause of the disease state is an elevated level of a T cell subtype (eg type I acquired agammaglobulinemia). For therapeutic purposes, administration of the appropriate monoclonal antibody to a patient with elevated levels of a T cell subtype will reduce or eliminate the excess. The relationships shown in Tables 2 through 5 are a further way in which the OKT6 antibody can be detected and distinguished from

other antibodies.

Other monoclonal antibodies and their producing hybridomas obtained by these applicants (designated OKT1, OKT3, OKT4 and OKT5) are described and claimed in the following US patent applications: SN 22,132, filed March 20, 1979; SN 33 639, submitted on April 26, 197; SN 33 669, submitted on April 26, 1979, and SN 76 642, submitted on September 18, 1979, and SN 82 515, submitted on October 9, 1979. Other monoclonal antibody-producing hybridomas obtained by these applicants (designated OKTB, OKT9 and 0KT10) are described and claimed in US patent applications filed on the same date as this application and are named:

A type of hybrid cell for obtaining a monoclonal antibody (fixed for complement) for human suppressive T cells, the antibody itself and methods of its administration; Type of hybrid cell for obtaining a monoclonal antibody for human early thymocyte antigen, the antibody itself and methods of its application, and Type of hybrid cell for obtaining a monoclonal antibody for human antithymocyte antigen, only the antibody and methods of its application.

These applications are incorporated by reference in this application.

According to this invention, there is provided a hybridoma capable of producing an antibody against an antigen found in approximately 70% of normal human thymocytes, a method for obtaining this hybridoma, a monoclonal antibody against an antigen found in approximately 70% of normal human thymocytes, methods for obtaining this antibody and methods and compositions for treatment (treatment) or disease diagnosis or identification of T cells or subclasses of thymocytes with the use of this antibody.

Table 1

Reactivity of monoclonal antibodies in human lymphoid populations

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* The numbers in parentheses represent the number of tested samples;

percentage values indicate mean values.

Table 2. Differences in distribution of antigens defined by monoclonal antibody in human peripheral T and thymocytes

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* Untreated populations and populations treated with complement alone cannot be distinguished on reanalysis. Non-specific analysis was < 5% in all cases. The obtained results are representative of 6 experiments.

+C' = complement

Table 3. Cell surface characteristics of T-type acute lymphoblastic leukemia

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* An additional four tumors cannot be easily categorized into stage I-III. See specification text for their characterization.

+ This mark refers to Fig. 2.

++ Positive (+) reactivity is defined as > 30% specific fluorescence above the basic control sample, while negative reactivity is impossible to distinguish from basic (background) staining in tumor suspensions.

Table 4

Reactivity with monoclonal antibodies

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* The criterion for negative (-) or positive (+) reactivity was the same as in table 3.

Table 5.

Peripheral T cell levels in disease states

[image]

N = within normal limits ++ = well above normal

O = absent - = below normal

+ = above normal -- = well below normal

* These levels return to normal a week before clinical symptoms disappear

Numbers in parentheses indicate the number of patients considered.

readily described is only one hybridoma that produces a single antibody against human thymocyte antigen, this invention is intended to encompass all monoclonal antibodies that exhibit the characteristics described above. The antibody in question, OKT6, was determined to belong to the IgG1 subclass, which is one of the four subclasses of mouse IgG. These immunoglobulin G subclasses differ from each other in so-called "fixed" regions, although for a specific antigen an organism will have a so-called "variable" region that is functionally identical regardless of the fact to which immunoglobulin G subclass it belongs. This means that a monoclonal antibody, which exhibits the characteristic described herein, may be of the IgG1, IgG2a, IgG2b, or IgG3 subclasses or of the IgM, IgA, or other known Ig classes. Differences between these classes or subclasses will not affect the selectivity of the reaction flow of the antibody, but may affect the subsequent reaction of the antibody with other substances, such as (for example) complement or anti-mouse antibodies. easily the antibody in question is IgG1 specific, antibodies having the course of reactivity illustrated herein are considered to fall within the scope of this invention, regardless of which class or subclass of immune globulin they belong to.

The present invention further includes methods for obtaining the monoclonal antibodies described above using the hybridoma technique illustrated above. Although only one example hybridoma is provided herein, it is believed that one skilled in the art can follow the immunization, fusion, and selection procedures provided herein and obtain other hybridomas capable of producing antibodies having the reactivity characteristics described above. Since an individual hybridoma derived from a known mouse myeloma cell type cannot be further identified except by reference to the antibody produced by this hybridoma, all hybridomas that produce an antibody with the characteristics described above are considered to be included. within the scope of this invention, as well as procedures for obtaining this antibody, using the mentioned hybridoma.

Further aspects of the present invention are methods of treating or diagnosing disease using the OKT6 monoclonal antibody or other monoclonal antibody, which exhibits the above mechanism of reactivity. The subject antibody can be used to detect and study intrathymic differentiation as summarized in Fig. 2. Abnormalities in stage II differentiation are indicated with a deviation of about 70% of OKT6+ thymocytes. In addition, the subject antibody can be used to diagnose disease states as shown in Table 5. These techniques can be used with the use of the OKT6 antibody alone or in combination with other antibodies (eg, OKT3 - 0KT10). Mechanisms of reactivity with a panel of antibodies for T cells and subtypes of T cells will enable more precise detection of some disease states than was possible using earlier methods of diagnosis.

Treatment of disease states (eg, malignant conditions such as stage II ALL) that manifest as an excess of OKT6+ cells can be performed by administering a therapeutically effective amount of OKT6 antibody to an individual in need of such treatment. By selectively reacting with the OKT6+ antigen, the effective amount of OKT6 antibodies will reduce the excess of OKT6+ cells, thereby correcting (improving) the effects of this excess. Included in this invention are diagnostic and therapeutic mixtures, which contain effective amounts of OKT6 antibodies in a mixture with diagnostically or pharmaceutically acceptable carriers.

Claims (20)

1. A mouse monoclonal antibody, characterized in that (i) reacts with approximately 70% of normal human thymocytes, but (ii) does not react with any of the normal human peripheral cells of the group it forms (iii) T cells and null cells, B cells and null cells; and it does not react with normal human bone marrow cells. 2. Monoclonal antibody according to claim 1, characterized in that it belongs to the IgG class. 3. Monoclonal antibody according to claim 1 or claim 2, characterized in that it shows the form of reactivity against normal human thymocytes shown in Figure 1. 4. Monoclonal antibody according to any one of claims 1 to 3, characterized in that it shows the form of reactivity against peripheral T cells and thymocytes shown in table 2. 5. Monoclonal antibody according to any one of claims 1 to 4, characterized in that it shows the form of reactivity against T cells of grade ALL shown in table 3. 6. Monoclonal antibody according to any one of claims 1 to 5, characterized in that it defines a T cell population (OKT6+) which is lower than normal levels in primary biliary cirrhosis, multiple sclerosis and hyper IgE, higher than normal levels in acute host reaction in in terms of transplant rejection, and completely absent in myasthenia gravis, acute infectious mononucleosis, all degrees of Hodgkins disease and psoriasis. 7. Monoclonal antibody according to any one of claims 1 to 6, characterized in that it is produced using a hybridoma created by the fusion of cells from a mouse myeloma line and spleen cells from a mouse previously immunized with human thymocytes. 8. Monoclonal antibody, characterized in that it was produced by hybridoma ATCC CRL 8020 (OKT6). 9. A hybridoma for the production of a monoclonal antibody according to any of claims 1 to 7, characterized in that it is obtained by fusion of spleen cells from a mouse previously immunized with human thymocytes and cells from a mouse myeloma line. 10. A hybridoma, designated as ATCC CRL 8020. 11. The method for producing a monoclonal antibody according to any of claims 1 to 7, characterized in that it includes the following steps: i) immunization of mice with human thymocytes; ii) extracting the spleen from said mouse and making a suspension of spleen cells; iii) fusing said 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) selection and cloning of hybridomas that produce the desired antibody; and vii) collection of antibodies from the supernatant above the mentioned clones. 12. A method for the production of a mouse monoclonal antibody according to any of claims 1 to 7, characterized in that it includes the following steps: i) immunization of mice with human thymocytes; 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) selection and cloning of hybridomas that produce the desired antibody; vii) transfer of said clones intraperitoneally into mice; and viii) collection of malignant ascites or serum from that mouse. 13. A method for the production of a monoclonal antibody, characterized in that it includes growing the hybridoma ATCC CRL 8020 in a suitable medium and collecting the antibody from the supernatant above the hybridoma. 14. A method for producing a monoclonal antibody, characterized in that it includes injecting hybridoma ATCC CRL 8020 into a mouse and obtaining the antibody from malignant ascites or serum from that mouse. 15. A method of detecting the lack or excess of OKT6+ cells in a person, indicated by the fact that it includes the reaction of T cells or thymocyte composition obtained from that person with a diagnostically effective amount of antibodies according to claim 6, or claim 7 when it is in accordance with claim 6 or claim 8 and measuring the percentage of the total population of peripheral T cells and thymocytes that reacts with that antibody. 16. The method according to claim 7, characterized in that it is an excess of T cells ALL. 17. Monoclonal antibody according to claim 6, or claim 7 when it is in accordance with claim 6 or claim 8, characterized in that it is used for the treatment of excess OKT6+ cells in a person who needs such treatment. 18. Composition, characterized by the fact that, in addition to a diagnostically acceptable carrier, the mixture also contains a monoclonal antibody according to claim 6, or claim 7 when it is in accordance with claim 6 or claim 8 and is used to detect the lack or excess of OKT6+ cells in the composition of T cells or thymocyte deposition. 19. Composition, characterized in that, in addition to a pharmaceutically acceptable carrier, it contains in the mixture an effective amount of the monoclonal antibody according to claim 6, claim 7 when it is in accordance with claim 6, or claim 8, wherein the amount of antibody is effective for reducing or removing excess OKT6+ cells in a person who has such an excess. 20. The method for producing the composition according to claim 18 or claim 19, characterized in that it comprises mixing the monoclonal antibody according to claim 6, claim 7 when it is in accordance with claim 6, or claim 8 with a diagnostically or pharmaceutically acceptable carrier.