FI75232B - Diagnostic method in order to observe specific T-cell population deficiency or surplus through the use of a specific monoclonal antibody for human thymocyte antigen - Google Patents

Abstract

The subject of this invention is a diagnostic method for observing the deficiency or surplus of cells belonging to a specific T-cell population OKT6<+> in humans. Cells belonging to this T-cell population are seen in amounts below normal in primary cirrhosis of the liver, multiple sclerosis and hyper-IgE and in amounts above normal in acute organ transplant and a complete lack in myasthenia gravis, acute mononucleosis infection, all phases of Hodgkin's disease and psoriasis. The method uses a new IgG-class monoclonal antibody whicha) reacts with about 70% of the normal human thymocytes, but not with normal human peripheral T-cells, B-cells, null cells or bone marrow cells,b) reacts with regular human thymocytes with specific means displayed in the figure,c) reacts with peripheral T-cell thymocytes with specific means displayed,d) reacts with different phased ALL-T-cells with specific means displayed,e) reacts with cell lines CEM and MOLT-4, but not with cell line HSB-2, andf) defines T-cell population OKT6<+>The antibody is produced by a hybridoma, prepared by integrating mouse myeloma cell line cells with spleen cells of a mouse immunized with human thymocytes.

Description

1 75232

Diagnostic method for detecting a lack or excess of a specific T cell population using a monoclonal antibody specific for a human thymocyte antigen 5 Partitioned isolated from application 803755.

The invention relates to a diagnostic method for detecting a deficiency or excess of OKT6 + cells in humans. The method utilizes a monoclonal antibody to 10 surface antigens found in approximately 70% of normal human thymocytes. The antibody is made using a new hybrid cell line.

In 1975, Kohler and Milstein combined mouse myeloma cells into the spleen cells of immunized mice Z ^ ature 15 256 (1975) 495-4977 / when it was first discovered that it was possible to prepare a continuous cell line to produce a homogeneous (so-called "monoclonal") antibody.

Since then, many attempts have been made to prepare various hybrid cells (so-called hybridomas) and attempts have been made to apply the antibody produced by these hybridomas to scientific use. See, e.g., Current Topics in Microbiology and Immunology, vol. 81 - "Lymphosyte Hybridomas", edited by F. Melchers, M. Potter and N. Warner, Springer-Verlag 1978 and references cited therein; C.J.

25 Barnstable et al., Cell 14 (May 1978) 9-20; P. Parham, and W.F. Bodmer, Nature 276 (1978) 397-399; Handbook of Experimental Immunology, 3rd Edition, Vol. 2, edited by D.M. Wier, Blackwell 1978, paragraph 25; and Chemical and Engineering News, January 1 (1979) 15-17.

30 These references describe the results obtained and the difficulties encountered in attempting to prepare a monoclonal antibody from hybridomas. Although the manufacturing method is in principle well known, there are many difficulties and each case is different. It cannot be guaranteed in advance that an attempt will be made to attempt to produce the desired hybridoma and that, if successful, the hybridoma will produce 75232 antibodies or that the antibody formed will have the desired specificity. Success depends mainly on the type of antigen used and the selection technique used to isolate the desired hybridoma.

5 Attempts have been made to produce a monoclonal antibody specific for human lymphocyte surface antigens in only a few cases. See, e.g., Current Topics in Microbiology and Immunology, vol. 81 66-69 and 164-169. The antigens used in these experiments were cultured human lymphoblastoid leukemia cell lines and chronic lymphocyte leukemia cell lines. Many hybridomas thus prepared produced an antibody that acted on different surface antigens of all human cells. None of these hybridomas produced antibodies specific for a particular human lymphocyte species.

Recently, publications have been described describing the preparation and testing of hybridomas that produce an antibody specific for certain T cell antigens. See, e.g., Reinherz, E.L., et al., J. Immunol. 123, 20, 1312-1317 (1979); Reinherz, E.L., et al., Proc. Natl. Acad.

Sci., 76, 4061-4065 (1979); and Rung, P.C., et al., Science, 206, 347-349 (1979).

Two main groups of lymphocytes are involved in the immunological functions of the human and animal body. Of these, 25 other (thymus-derived cell, or T-cell) differentiate in the thymus from hematopoietic stem cells. The differentiating cells in the cochlear ear are called "thymocytes." Mature T cells detach from the thymus and then enter the bloodstream, tissues, and lymphoid tissue. 30 These T cells make up a large part of the small lymphocytes circulating in the body. They are immunologically specific and, as effector cells, participate directly in cell-mediated immunological reactions (e.g., transplant rejection). Although 35 T cells do not secrete antibodies into body fluids, another group of lymphocytes to be treated later requires 75232 T cells in some cases to be able to secrete antibodies themselves. Some T cell species act as regulators in other functions of the immune system. The mechanism of this form of cell interaction is not yet fully understood.

5 The second group of lymphocytes (cells derived from the bone marrow, or B cells) includes those that secrete antibodies. They also develop from hematopoietic stem cells, but their differentiation is not regulated by the thymus. In birds, they differentiate in 10 organs analogous to the thymus, called the "Bursa of Fabricius." However, no similar organ has been found in mammals, and thus B cells are thought to differentiate in the bone marrow.

T cells are divided into at least three subspecies, termed "helper", "attenuator", and "killer" T cells, which either accelerate or slow down reactions or kill (dissolve) foreign cells. These subclasses are well known in the body of mice, but only recently have their functions been described in the human body. See, e.g., R.L. Evans et al., Journal of Experimental Medicine 20 145 (1977) 221-232 and L. Chess and S.F. Schlossman - "Functional Analysis of Distinct Human T-cell Subsets Bearing Unique Dirrefentiation Antigens", in "Contemporary Topics in Immunobiology", edited by 0. Stutman, Plenum Press 1977, vol. 7, 363-379.

25 The identification or attenuation of different T cell species or subtypes is important in the diagnosis and treatment of various immunoregulatory disorders.

For example, the disease picture of certain forms of leukemia and lymphoma differs depending on whether they originate from B or T cells. Thus, when assessing the prognosis of the disease, it is important that the two groups of lymphocytes must be able to be distinguished from each other. See, e.g., A.C. Aisenberg and J.C. Long, The American Journal of Medicine 58 (1975) 300; D. Belpomme et al., In Immunological Diagnosis of Leukemias and Lymhomas, edited by S. Thier-Felder et al., Springer, Heidelberg 1977, 33-45; and D. Belpomme et al., British Journal of Hematology 38 (1978) 85.

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In certain disease states (e.g., juvenile rheumatoid arthritis), malignant disease states, and agammaglobulinemia, T cell subsets have become unbalanced. It has been suggested that in autoimmune diseases, "helper" -5 T cells are generally present in excess and certain "attenuator" T cells are deficient, whereas in agammaglobulinemia, certain "attenuator" T cells are present in excess or "helper" T cells. there is a shortage. In some forms of leukemia, an excess of 10 T cells in the stagnant stage of development are formed. The success of the diagnosis may thus depend on whether this imbalance or excess can be detected or determined at which stage of development occurs in excess. See, e.g., J. Kersey et al., "Surface Markers Define Human Lymphoid Malignancies with Differential Prognoses," 15 in "Hematology and Blood Transfusion", vol. 20, Springer-Verlag 1977, 17-23, and references therein, and E.L. Reinberg et al., J. Clin. Invest. 64 (1979) 392-397.

Agammaglobulinemia, a disease state in which no immunoglobulin is formed, belongs to at least two different types.

In type I, immunoglobulin is not formed because of the excess of attenuating T cells, whereas in type II this is due to the lack of helper T cells. In both types, the patient's B cells, or lymphocytes, which are responsible for the actual secretion of the antibody, are completely normal and deficient, whereas these B cells are either impaired or "not helped", which significantly reduces immunoglobulin production or suspend production altogether. The type of agammaglobulinemia can thus be determined by the presence of an excess of attenuating T cells or a deficiency of helper T cells.

There are indications that when antibodies specific for an excess of 35 T cell species are administered to patients with autoimmune disease or malignancies, the antibodies may have a beneficial effect on the development of the disease. For example, helper T cell cancers (certain skin T cell lymphoma tumors and certain forms of acute T cell lymphoblastic leukemia) can be treated by administering to the patient an antibody specific for the helper T cell antigen. Autoimmune disease caused by an excess of 5 helper cells can also be treated in the same manner. Diseases (e.g., malignancies or type I agammaglobulinemia) caused by an excess of attenuating T cells can be treated by administering to the patient an antibody specific for the attenuated T cell antigen.

Antisera that affect all human T cells (so-called anti-human thymocyte globulin, or ATG) have been shown to be therapeutically useful in the treatment of transplant patients. Because T-cells affect cell-mediated immunological responses (the mechanism of rejection of transplanted organs), obtaining an antibody specific for T cells prevents or slows down this rejection process. See, e.g., Cosimi et al., "Randomized Clinical Trial of ATG in Cadaver 20 Renal Allgraft Recipients: Importance of T Cell Monitoring", Surgery 40 (1976) 155-163, and references therein.

To date, spontaneous autoantibodies or antisera selective for human T cells, prepared by immunizing animals with human T cells, removing blood from animals to obtain serum, and adsorbing antiserum with, for example, autologous non-autologous cells, have been used to identify and attenuate human T cell species and subspecies. -alogeneic B cells to remove antibodies with undesired reactivity. The preparation of these antisera is very difficult, especially in the adsorption and purification steps. Adsorbed and purified antisera also contain many impurities in addition to the desired antibody for a number of reasons. One reason is that the serum contains millions of antibody molecules even before immunization with T-so-35 bones. Second, immunization causes the formation of antibodies that affect a variety of antigen species found in all injected human T cells.

It is not possible to selectively produce an antibody specific for a particular antigen. Third, the titer of the specific antibody prepared by these methods is usually quite low (e.g., at dilutions greater than 1: 100, the antibody is inactive) and the ratio of specific to non-specific antibody is less than 1/10 ^.

See, for example, the above-mentioned article by Chess and Schlossman (from page 365 onwards) and the above-mentioned article in "The Chemical and Engineering News", which describe the shortcomings of the antisera used so far and the advantages of the monoclonal antibody.

A new hybridoma (OKT 6) has now been discovered that is capable of producing a novel monoclonal antibody to an antigen found on about 70% of normal human thymocytes but not on normal human peripheral lymphoid cells (T cells, B- cells or null cells) and not bone marrow cells. The antibody thus formed is monospecific for a single human normal T cell surface antigenic determinant present on about 70% of normal human thymocytes and contains virtually no other anti-human immunoglobulins, in contrast to the past. known antisera (which naturally contain as impurities antibodies that react well with many human antigens) and hitherto known monoclonal antibodies (which are not monospecific for a single human thymocyte antigen). In addition, this hybridoma can be cultured to produce the antibody, so that the use of animals to produce the antibody is not necessary, nor are the complex adsorption and purification steps required in previous methods for preparing impure antisera.

35 Both the present hybridoma and the antibody produced by it are referred to herein as 7 75232 "OKT 6", the context being whichever is meant. The present hybridoma was deposited on November 21, 1979 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and has been assigned ATCC Accession No. CRL 8020.

The preparation of the hybridoma and the antibody produced by it is described in FI patent application 803755.

The diagnostic method of the invention using said hybridoma and antibody is characterized by reacting a T cell or thymocyte sample from a patient with a diagnostically effective amount of an IgG monoclonal antibody produced by a hybrid cell line. , having an ATCC accession number of CRL 8020, prepared by combining cells of a murine myeloma cell line with spleen cells from a mouse immunized with human thymocytes, and which antibody a) reacts with about 70% of normal human thymocytes but not human normal peripheral T cells, B cells, null cells and bone marrow cells, 20 b) reacts with about 65% of untreated thymocytes, 10% of thymocytes treated with a monoclonal antibody produced by a hybrid cell line The ATCC accession number is CRL 8002, and with complement, 82% of thymocytes treated with mono-25 clonal antibody produced by a hybrid cell line having an ATCC accession number of CRL 8001 and complement and 13% of thymocytes treated with a monoclonal antibody produced by a hybrid cell line having an ATCC accession number of CRL 8014 and complement, 30 c) reacting with with phase II T-ALL cells but does not react with phase I or phase III T-ALL cells, d) reacts with cell lines CEM and MOLT-4 but not with cell line HSB-2, and e) determines the T cell population (OKT6 +), which occurs at lower than normal concentrations in primary cirrhosis of the liver, multiple sclerosis and hyper-IgE and at higher concentrations than 8 75232 in acute transplantation, and which is completely absent in myasthenia gravis, acute stage mononucleosis, determining the percentage of peripheral T cells and thymocytes reacting with said antibody in the total number thereof.

Example 1 Characterization of OKT 6 Antibody Reactivity A. Isolation of Lymphocyte Populations Peripheral mononuclear blood cells were isolated from the blood of normal volunteer blood donors (15-40 years of age) by the Ficoll-Hypaque density gradient centrifugation method (Pharmacia Fine Chemicals, Pia., Pharmacia Fine Chemicals). , Scand. J. Clin. Lab. Invest. 21 (suppl. 15 97) (1968) 77. Unfractionated mononuclear cells were separated into surface Ig + (B) and surface Ig (T and zero cell) populations using Sephadex G-200 anti-F (ab ') 2 Column chromatography by the method previously described by Chess et al., J. Immunol. 113 (1974). T cells were taken from tal-20 tea by E-rosetting Ig ”strain with 5% sheep erythrocytes (Microbiological Associates, Bethesda, MD). The rosetted mixture was centrifuged in a Ficoll-Hypaque centrifuge and the recovered E + pellet was treated with 0.155 mM NH 4 Cl (10 mL per 10® cell). The T cell population thus obtained was <2% EAC-25 rosette positive and> 95% E rosette positive as determined by standard methods. In addition, the non-rosette Ig "(null cell) population was separated from the Ficoll interface. The latter population was <5-% E-positive and ^ 2-% slg-positive. Surface Ig + (B) -30 population was separated from the Sephadex-G-200 column by elution with normal human gamma globulin as previously described.This population was> 95% surface Ig-positive and <5% E-positive.

Normal human bone marrow cells were obtained from the posterior iliac crest of 35 healthy volunteer donors by the puncture method.

9 75232 B. Isolation of thymocytes

Normal human thyroid gland was obtained from patients who had undergone corrective heart surgery and ranged in age from two months to 14 years. The Kate-5 parotid gland was dissected, and the pieces were immediately placed in 199 (Gibco) medium containing 5% fetal calf serum, finely chopped with forceps and scissors, and then a suspension containing one cell species was formed by squeezing the mixture through a steel sieve. Next, the cells were centrifuged in a Ficoll-Hypaque centrifuge and washed as described in A. The thymocytes thus obtained were> 95% viable and 90% E-rosette positive.

C. T Cell Lines and T Cell Acute Lymphoblastileu-15 Chemistry Cells T cell lines CEM, HSB-2, and MOLT-4 were provided by Dr. H. Lazarus (Sidney Farber Cancer Institute, Boston, MA). Leukemia cells were obtained from 25 patients with acute lymphoblastic leukemia. These individual tumors had been shown to be derived from T cells in previous experiments because the tumor cells spontaneously formed rosettes with sheep erythrocytes (> 20% E +) and because they responded to T cell-specific heteroantera, anti-HTL (BK), and A99 antisera. as described above 25. Tumor populations were cryopreserved at -196 ° C in the liquid nitrogen vapor phase in 10% dimethyl sulfoxide and 20% in human serum until surface characterization. All tumor populations analyzed were> 90% blasts in the Wright-Giemsa morphology of the cytocentrifuge preparations.

Example 2

Cell fluorographic analysis and cell separation

Cell fluorographic analysis of the reaction products of monoclonal antibodies and all cell populations was performed by an indirect immunofluorescence method in which cells were stained with fluorescein-coupled goat-silenced anti-mouse IgG (G / M FITC) (48 G / M FITC) Ortho Instruments). In this case, 1 x ΙΟ ** cell was treated with 0.15 ml of antibody at a 1: 500 dilution of OKT5, the cells were incubated at 4 ° C for 30 minutes and washed twice. The cells were then reacted with 0.15 ml of G / M FITC (1:40 dilution) at 4 ° C for 30 minutes, the reaction product was centrifuged and washed three times. These cells were then analyzed with a cell fluorograph in which the fluorescence intensity induced by each cell 10 was recorded on a pulse height analyzer. The reactivity was similar at a dilution of 1: 10000, but at higher dilutions the reaction did not occur. The intensity of background staining was determined with 0.15 ml of a 1: 500 dilution of water-15 peritoneal fluid obtained intraperitoneally from a CAF1 mouse immunized with a non-producing hybrid clone.

In experiments examining antibody- and complement-mediated lympholysis, thymocytes and peripheral T cells were cultured overnight, followed by selective lysis, and then the cells were analyzed by cell fluorography.

Example 3

Degradation of lymphoid populations with monoclonal antibody and complement 25 x 10 10 peripheral T cells or thymocytes were added to a 15 ml plastic tube (Falcon, Oxnard, CA). To the cell pellets, 0.8 ml of OKT3, 0KT4, 0KT8 or normal aqueous humor (reference) diluted 1: 200 in PBS was added, the mixtures were suspended and incubated at 20 ° C for 60 minutes. Thereafter, 0.2 ml of fresh rabbit complement was added to the antibody-treated populations, the mixture was suspended and further incubated at 37 ° C for 60 minutes in a water bath connected to a shaker. At the end of the incubation, 35 cells were centrifuged and viable cells were determined on Trypan blue. Cells were counted, washed twice with 11,75232 5% FCL, and placed in final medium RPMI 1640 (Grand Island Biological Company, Grand Island, NY) containing 20% human AB + serum, 1% penicillin-streptomycin, 200 mM L-glutamine, 25 mM HEPES-pus-5 discipline and 0.5% sodium carbonate and the mixture was incubated overnight at 37 ° C in a humidified atmosphere containing 5% co 2.

Figure 1 shows the fluorescence spectrum obtained from a cell fluorograph for normal human thymocytes reacted with OKT 6 and other monolonial antibodies (1: 500 dilution) and G / M FITC. Background fluorescence staining was determined by adding to each population a 1: 500 dilution of aqueous humor from a mouse injected with a non-producing clone and incubating the mixture.

Figure 2 shows the differentiation of thymocytes in the thymus.

Table 1 shows the reactivity of the monoclonal antibodies OKT6, OKT8, OKT9 and OKT10 with different human lymphoid-20 cell populations. The monoclonal antibody OKT6 reacts with about 70% of normal human thymocytes, but not with any of the other lymphoid cells studied. Among other things, this reactivity formula allows the present antibody OKT6 to be detected and distinguished from other antibodies.

Figure 1 shows the fluorescence spectrum obtained from a cell fluorograph for a suspension of normal human thymocytes reacted with antibodies OKT3, 0KT4, OKT5, OKT6, OKT8, OKT9 and OKT10 (all at a dilution ratio of 1: 500) and G / M FITC with. Similar reactivity formulas were obtained for the 12 other normal human thymocyte populations studied. The figure shows that there are significant differences in reactivity percentage and fluorescence intensity between all monoclonal antibodies studied. For example, OKT9 reacts with about 10% of thymocytes at a lower fluorescence intensity, while 0KT5, 0KT6, 0KT8 and OKTIO react with about 70% of thymocytes at a higher fluorescence intensity. OKT4, which reacts with 75% of thymocytes, is located between OKT9 and monoclonal antibodies with higher fluorescence intensity in terms of fluorescence intensity. In addition, Figure 1 shows that about 15% of thymocytes can be detected by OKT3 using an indirect immunofluorescence method. OKT1, which reacts with thymocytes in virtually the same way as OKT3, does not appear. Also using the reactivity formula shown in Figure 1, the present antibody OKT6 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, the distribution being determined by a series of lysis experiments as described in Example 3. Because only OKT3, OKT4, and OKT8 were complement-binding monoclonal antibodies, these three were used in the assay.

Table 2A shows that the entire T cell population reacted with OKT3, whereas OKT4, OKT5, and OKT8 reacted with 60%, 25%, and 34% of T cells, respectively. Degradation with OKT4 and complement reduced the total cell count 62 by 25% and specifically destroyed the OKT4 + population. In addition, the percentage of OKT5 + and OKT8 + cells increased, but the total number of OKT5 + and OKT8 + T cells did not change. From this experiment, it could be concluded that the OKT4 + population differed from the OKT5 + and OKT8 + populations. Further support for these 30 hypotheses was obtained from an experiment in which T cells were lysed with OKT8 and complement. At this time, the percentage of OKT4 + T cells increased, the total number remained the same, and the OKT8 + and OKT5 + populations were destroyed. In addition, these experiments showed that the OKT8 + population was in contrast to the OKT4 + -35 population, including the entire OKT5 + T cell fraction.

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Similar experiments with human thymocyte populations yielded different results. Table 2B shows that approximately 75% of thymocytes were 0KT4-positive or 0KT8-positive. Moreover, when cells were lysed with 0KT4 or 0KT8, only 25% of thymocytes remained. Most of the remaining thymocytes reacted with 0KT3, while a small proportion of them reacted with 0KT6. These findings indicate that most human thymocytes have 0KT4, 0KT5, 0KT6, and 0KT8 surface-10 antigens in the same cell. In addition, Table 2 shows that treatment of cells with 0KT8 and 0KT4 significantly increases the number of mature thymocytes containing the 0KT3 antigen. Thus, most of the thymocytes reacting with 0KT3 have already separated into 0KT4 + and OKT8 + fractions, as most of the cells that did not react with 0KT4 and 0KT8 were OKT3 positive. If the OKT3 + subpopulation were both 0KT4-positive and 0KT8-positive, digestion with either monoclonal antibody would have destroyed OKT3-reactive thymocytes.

20 To further find out with OKT3 rea

ratio of thymocyte subpopulations to thymocyte fractions defined by other monoclonal antibodies, thymocytes were treated with OKT3 and complement, and cells remaining in the treatment were then compared to untreated thymocyte populations. From Table 2B

it appears that OKT3 and complement removed 25% of thymocytes. In contrast, populations reacting with OKT4, OKT5, OKT6, and OKT8 antibodies remained virtually unchanged. From these findings, it can be concluded that the majority of thymocytes containing the OKT6 marker belong to the OKT3 “population. In addition, it can be concluded from the results that thymocytes that simultaneously express antigens defined by OKT4, OKT5, and OKT8 are similarly restricted to the OKT3 ”population. 35 It should also be noted that the thymocyte population reacting with 0KT9 did not decrease when unfractionated thymocytes were treated with 0KT3 and complement, indicating that the 0KT9 + subpopulation is largely limited to the 0KT3 “-tychocyte population.

Based on these results, it has been possible to determine the developmental stages of human thymocytes in the thymus. Figure 2 shows that virtually all thymocytes have the OKTIO marker. In addition, thymocytes receive the 0KT9 antigen (Thyl and Thy2) at an early stage. This step comprises a small fraction of thymocytes and comprises about 10% of the non-fractionated population.

Human thymocytes then receive the thymocyte antigen defined by OKT6 and simultaneously express OKT4, OKt5, and OKT8 (Thy4). The latter subpopulation represents the majority of thymocytes and comprises more than 70-80% of the thymocyte population. As thymocytes continue to mature, their OKT6 reactivity disappears, they become reactive with OKT3 (and OKT1) and separate into OKT4 + and OKT5 + / OKT8 + fractions (Thy7 and Thy8). It appears that in the final step, when the thymocytes detach from the thymus into the peripheral T cells, their OKTIO marker disappears, as this antigen is absent from virtually all peripheral T lymphocytes. Possible transition stages between these three main developmental stages are denoted in Figure 2 by Thy3, Thy5 and Thy6.

25 Because acute T-cell lymphoblastic leukemia is thought to be due to immature thymocytes, the relationship between tumor cells isolated from individuals with T-ALL leukemia and the developmental stages of said putative thymocytes was determined. The assay used 25 tumor cell populations isolated from individuals with T-ALL-30 leukemia and three T cell lines that had been pre-screened with standard anti-T cell reagents and E-rose. Table 3 shows that most T-ALL-Leu chemistry cells reacted with either OKT10 alone or OKT9 and 35 OKT10, but the cells did not react with other monoclonal antibodies. Thus, 15/25 of the 15,75232 cases examined appeared to contain early thymocyte stage antigens (stage 1).

In contrast, 5/25 of the cases reacted with 0KT6, from which it can be concluded that they are derived from 5 mature thymocyte populations (step 2). This T-ALL group reacted differently with 0KT4, 0KT8, and 0KT9, as shown in Table 3. Two-fifths of patients had tumor cells that contained most of the common thymocyte antigens, including 10 OKT4, OKT6, and OKT8. It should be noted that OKT5 is not present in any of these five stage 2 tumors, although OKT8 reactivity was observed. From this latter result, it can be clearly concluded that OKT5 and OKT8 define different antigens or different determinants in the same antigen. Finally, 1/25 of the patients had tumors from a mature thymocyte population (step 3) as determined by 0KT3 reactivity. In addition, this person's tumor was reactive with OKT5, OKT8, and OKT10. Of the 20 25 leukemia populations studied, only four were those that could not be clearly classified. Of these, three were OKT4- and OKT8-positive, but did not react with OKT3 or OKT6, making them most likely transitions between Thy4 and Thy7.8. One of the 25 cases 25 turned out to be a transition phase between Thy3 and Thy4, as it was reactive with 0KT8 and OKT10.

T cell lines derived from T-ALL tumor populations also represented cells belonging to a specific intrathymic differentiation step. Table 4 shows that HSB-2 reacts exclusively with OKT9 and OKT10, and thus defines the tumor population from step 1. In contrast, the CEM-T cell line reacted with OKT4, 0KT6, OKT9, and OKT10 and was found to be derived from stage 2 thymocytes. Finally, the cell line MOLT-4 is apparently a leukemia transformation between HSB-2 and CEM, as it expresses OKT6, OKT8, OKT9 and OKT10.

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Because later stage (e.g., stage II) T cell acute lymphoblastic leukemia subjects have been shown to recover more completely than stage I ALL leukemia patients, the 0KT6 antibody can be used to draw conclusions about the prognosis of a person with T cell ALL leukemia.

Table 5 shows the relationship between peripheral T cell and T cell fraction concentrations and different disease states. These ratios can be used for diagnostic purposes (e.g., for the detection of acute infectious mononucleosis) by taking a blood sample from a person suspected of having one of these diseases and determining the levels of T cells and T cell fractions in the sample. Also, using the ratios shown in Tables 2-5, the OKT6 antibody can be detected and distinguished from other antibodies.

Diagnostic methods using other hydridomes (OKT1, OKT3, OKT4 and OKT5 and OKT8, OKT9 and OKTIO) produced by the same applicants producing monoclonal antibodies are described in FI patent applications 844 703, 20 844 705, 844 704 and 844 706 and in applications 850 062, 850 063 and 850 061.

The OKT6 antibody in question was found to belong to the IgG · ^ subclass, which is one of the four subclasses of mouse IgG. These subclasses of immunoglobulin G differ from the so-called

25 of its "fixed" regions, although the structure of an antibody specific for a particular antigen has a so-called a "variable" region that is functionally similar regardless of which immunoglobulin G subclass it belongs to. That is, a monoclonal antibody having the characteristics described above may belong to the subclasses IgG1-IgG2a, IgG2b or IgG3 or to the classes IgM or IgA or other classes of Ig. Differences in these classes or subclasses do not affect the selectivity of antibody reactivity, but may affect how the antibody subsequently reacts with other agents, for example, complement or anti-mouse antibodies.

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The present antibody OKT6 can be used to detect and study thymocyte differentiation, as shown in Figure 2. If the number of thymocytes reacting with OKT6 differs from 70%, abnormalities in thymocyte developmental stage II occur. In addition, the present antibody can be used to diagnose various disease states as shown in Table 5. In this case, the OKT6 antibody alone or a combination of OKT6 and other antibodies (e.g., OKT3-OKT10) can be used. Reactivity patterns obtained by reacting a variety of antibodies with T cells and T cell fractions allow certain disease states to be detected more accurately than using known diagnostic methods.

The OKT6 antibody can be included in diagnostic compositions consisting of effective amounts of the OKT6 antibody and diagnostically acceptable carriers.

table 1

Reactivity of monoclonal antibodies with human lymphoid populations

Monoclonal Peripheral Bone marrow (6) Thymus antibody blood (22) E + E "OKT6 0% 0% 0% 70% 25 0KT8 30% 0% <2% 80% 0KT9 0% 0% 0%« 10% OKT10 <5 % 10% «20% 95%

Figures in parentheses indicate the number of samples examined; % values are averages of 18,75232

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Table 4

Reactivity of cell lines with monoclonal antibodies

Cell line 0KT3 0KT4 0KT5 0KT6 0KT8 0KT9 OKTIO

5 HSB-2 -X - - - - + + CEM - + - + + + + MOLT-4 --- + + + + x The criteria for (-) and (+) reactivity were the same as in Table 3.

21 75232

Table 5

Peripheral T cell concentrations in different disease states - T cell concentrations

Disease status 0KT3 + OKtT * ÖKT5 PKT8 + PKT 6 *

Primary liver N + cirrhosis (2)

Multiple sclerosis (advanced) (8) - N - - -

Myasthenia Gravis 10 (not treated at an early stage) (3)

Acute transplantation G. ..- - p ++

Agammaglobulinemia 15 Type I +

Type II G

Hyper IgE (4) - N O * * · - O · * · -

Acute mononucleosis- + O ...— ++ ++ G

2Q infection (4) x

Hodgkins disease

Steps I & II N N N N G

Steps III & IV - N N N O

2 ^ Psoriasis (3/5) N + ··· ++ N N O

N = normal concentration O = absent 30 + = significantly higher than normal ++ = significantly higher than normal _ = lower than normal _ significantly lower than normal 2 ^ x These concentrations return to normal approximately one week before the clinical symptoms disappear.

Values in parentheses indicate the number of patients studied.

Claims (1)

A method for detecting a deficiency or excess of 0KT6 + cells in humans, characterized in that a T cell or thymocyte sample taken from a patient is reacted with a diagnostically effective amount of an IgG class monoclonal antibody produced by a hybrid cell line produced by the ATCC. accession number CRL 8020, prepared by combining cells of a mouse mye-10 animal cell line with spleen cells of a mouse immunized with human thymocytes, and which antibody a) reacts with about 70% of normal human thymocytes but not normal human peripheral T cells, B- b) reacts with about 65% of untreated thymocytes, with 10% of thymocytes treated with a monoclonal antibody produced by a hybrid cell line with ATCC accession number CRL 8002, and with 20 complements. , 82% of thymocytes treated with a monoclonal antibody extracting a hybrid cell line having an ATCC accession number of CRL 8001 and complement and 13% of thymocytes treated with a monoclonal antibody produced by a hybrid cell line having an ATCC accession number of CRL 8014 and complement, c) reacting with step II with T-ALL cells but does not react with phase I or phase III T-ALL cells, d) reacts with cell lines CEM and MOLT-4, but not with cell line HSB-2, and e) determines the T cell population (OKT6 +), which occurs at lower than normal concentrations in primary cirrhosis of the liver, multiple sclerosis and hyper-IgE and at higher than normal concentrations in acute transplantation and is completely absent in myasthenia gravisin-23 752 32 sa, acute mononucleosis in all mononucleosis. phases and psoriasis, and determining the percentage of total peripheral T cells and thymocytes that react with said antibody.