FI75433C - Diagnostic method utilizing a specific monoclonal antigen call to human protymocyte antigen and in the method useful composition. - Google Patents

Description

1 75433

Diagnostic method using a monoclonal antibody specific for a human presentation cytogen antigen and a composition of matter useful in the method 5 Divided by application 803757

The invention relates to a diagnostic method for detecting a deficiency or excess of OKT10 + cells in humans and to a composition of matter useful in the method. The method utilizes a monoclonal antibody specific for the human presentation thymocyte antigen. 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 (Nature 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 to try to apply the antibody produced by these hybridomas to scientific use. See for example:

Current Topics in Microbiology and Immunology, vol. 81 - "Lymphosyte Hybridomas", edited by F. Melchers, M.

Potter and N. Warner, Springer-Verlag 1978 and references in Publication 25; C.J. 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 30 News, January 1 (1979) 15-17.

These references show the results obtained and the difficulties encountered in trying 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 produce the desired 2,75433 hybridoma and that, if successful, the hybridoma will produce an antibody or that the antibody thus 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.

Attempts to produce a monoclonal antibody specific for human lymphocyte surface antigens have been made in only a few cases. See, e.g., Current 10 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 upon various surface antigens of all human cells. None of these hybridomas produced an antibody 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, 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).

25 Two main groups of lymphocytes are involved in the immunological functions of the human and animal body. The second of these (a cell derived from the thymus, or T cell) is differentiated in the thymus from hematopoietic stem cells. The differentiating cells in the thymus are termed "ty-30 mosocytes." Mature T cells detach from the thymus and then enter the bloodstream, tissues, and lymphoid tissue. These T cells make up a large part of the small lymphocytes circulating in the body. They are immunologically specific and, as effector cells, are directly involved in cell-mediated immunological responses (e.g., 3,754,333 transplant rejection). Although T cells do not secrete antibodies into body fluids, another group of lymphocytes to be treated later will in some cases require T cells to be able to secrete antibodies themselves. Some T cell species function in 5 other immunological systems from the regulatory. The mechanism of this form of cell interaction is not yet fully understood.

The second group of lymphocytes (cells derived from the bone marrow, or B cells) includes those that secrete 10 antibodies. They also develop from hematopoietic stem cells, but their differentiation is not regulated by the thymus. In birds, they differentiate in an organ analogous to the thymus, called the "Bursa of Fabricius." However, no similar organ-15 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 the reaction or kill (dissolve) foreign cells. These 20 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 145 (1977) 221-232 and L. Chess and S.F. Schlossman - "Functional Analysis of Distinct 25 Human T-cell Subsets Bearing Unique Dirrefentiation Antigens", in "Contemporary Topics in Immunobiology", edited by 0. Stutman, Plenum Press 1977, vol.

7, 363–379.

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 prognosis of certain forms of leukemia and lymphoid tissue differs depending on whether they originate from B or T cells. Thus, in assessing disease prognosis 35, it is important to be able to distinguish between these two groups of lymphocytes. See, e.g., 4,75433 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. Thierfelder et al., Springer, Heidelberg 5 1977, 33-45; and D. Belpomme et al., British Journal of

Hematology 38 (1978) 85.

In certain disease states (e.g., juvenile rheumatoid arthritis, malignant disease states, and certain forms of Leu chemistry), T cell subsets have become unbalanced. It has been suggested that in autoimmune diseases, there is generally an excess of "helper" T cells and a deficiency of certain "attenuator" T cells, whereas agammaglobulinemia is associated with an excess of certain "attenuator" T cells or a lack of "helper" T cells. . In malignant-15 disease states, "attenuator" T cells are usually present in excess. In some forms of leukemia, stagnant T cells are formed in excess. The success of the diagnosis may thus depend on whether it is possible to detect this imbalance or excess or to determine which stage of development occurs in excess. See, e.g., J. Kersey et al., "Surface Markers Define Human Lymphoid Malignancies with Differing Prognoses," in Hematology and Blood Transfusion, vol. 20. Springer-Verlag 1977, 17-23 and references 25 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 hei-30 field 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", significantly reducing immunoglobulin activity. production or suspend production altogether. The type of agamma globulinemia can thus be determined by the presence of an excess of attenuator T cells or a deficiency of helper T cells.

There are indications that when antibodies specific for the overexpressing 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, 10 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 helper cells can also be treated in the same manner. Diseases (e.g., malignancies or type I agamma globulinemia) caused by an excess of attenuating T cells can be treated by administering to the patient an antibody specific for the attenuating T cell antigen. 20 Antisera that affect all human T cells (so-called anti-human thymocytoglobulin, or ATG) have been shown to be therapeutically useful in the treatment of transplant patients. Because T cells affect cell-mediated immunological responses (transplant rejection mechanism), obtaining an antibody specific for T cells prevents or slows this rejection process. See, e.g., Cosimi et al., "Randomized Clinical Trial of ATG in Cadaver Renal Allgraft Recipients: 30 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 6,75433 serum, and adsorbing antiserum, have been used to identify and attenuate human T cell species and subspecies. with autologous non-allogeneic 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 cells. Second, immunization with im-10 causes the formation of antibodies against several antigens 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 1/106. .

See, for example, the above-mentioned 20 articles 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 10) has now been discovered which is capable of producing a novel monoclonal antibody to an antigen found in approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of peripheral mononuclear E ”cells (B cells and null cells) and 10-20% bone marrow cells. The antibody thus formed is monospecific for a single surface antigenic determinant of normal human T cells and contains virtually no other anti-human immunoglobulins, in contrast to hitherto known antisera (which naturally contain 75433 in purity, 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 an 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 crude antisera.

Both the present hybridoma and the antibody produced by it are referred to herein as "OKTIO", and the context indicates which is meant in each case. The present hybridoma was deposited on October 21, 1979 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and has been assigned ATCC Deposit No. CRL 8022.

The preparation of the hybridoma and the antibody produced by it is described in FI patent application 803757.

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 a murine monoclonal antibody produced by a hybrid cell line having an ATCC accession number of CRL 8022, and which antibody reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of peripheral mononuclear mononuclear cells (B cells and zero cells) and with 10-20% bone marrow cells ,. and determining the percentage of peripheral T cells and thymocytes that react with said antibody in the total number thereof.

Example 1 Characterization of OKTIO Antibody Reactivity A. Isolation of Lymphocyte Populations Peripheral mononuclear blood cells were isolated from the blood of normal volunteer blood donors (age 8,75433, 15-40 years) by the Ficoll-Hypaque density gradient centrifugation method of Ficoll-Boypa, Chemicals, Pharmacia way, Scand. J. Clin. Lab.

5 Invest. 21 (suppl. 97) (1968) 77. Unfractionated monocytes were separated into a surface Ig + (B) and surface Ig (T and zero cell) population using Sephadex G-200 anti-F (ab ') 2 ~ column chromatography by the method previously described by Chess et al., J. Immu-10 nol. 113 (1974). T cells were harvested by E-rosetting the Ig ”population 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 M NH 4 Cl 15 (10 mL per 10® cell). The T cell population thus obtained was <2% EAC-positive and> 95% E-positive as determined by standard methods. In addition, the non-rosette Ig ”(null cell) population was separated from the Ficoll interface. This latter population was <5% E-positive and <2% Slg-positive. The surface Ig + (B) 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-25 positive and <5% E-positive.

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

B. Isolation of Thymocytes 30 A normal human thyroid gland was obtained from a patient who had undergone corrective heart surgery and ranged in age from two months to 14 years. The thyroid 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 was formed by squeezing the mixture through a steel sieve 75433. 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-rosent positive.

5 C. T cell lines and T cell acute lymphoblastic leukemia cells T cell lines CEM, HSB-2 and MOLT-4 were supplied by Dr. H. Lazarus (Sidney Farber Cancer Institute, Boston, MA). Leukemia cells were obtained from 25 of 10 patients with acute T cell lymphoblastic leukemia. These

individual tumors had been found to be T-cell tumors in previous experiments because tumor cells spontaneously formed rosettes with sheep erythrocytes (> 20% E +) and because they responded to T-15 cell-specific heteroant sera, anti-HTL

(B.K) and A99 antisera as described above. Tumor populations were cryopreserved at -196 ° C in the liquid nitrogen vapor phase in 10% dimethyl sulfoxide and 20% human AB serum until they were characterized on their surface. 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 monoclonal antibodies and reaction products of all cell populations was performed by an indirect immunofluorescence method in which cells were stained with fluorescein-coupled goat anti-mouse IgG (G / M 30 FITC). -fluorography FC200 / 4800A (Ortho Instruments). In this case, 1 x 10 6 cells were 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. Cells 35 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 was recorded on a pulse height analyzer. The Reagent-5 oral model was similar at a dilution of 1: 10,000, 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 aqueous humor obtained intraperitoneally from a CAF-^ mouse immunized with a non-productive hybrid clone.

In experiments investigating antibody- and complement-mediated lympholysis, thymocytes and peripheral T cells were cultured overnight, followed by selective lysis, and then the cells were analyzed with a 15-cell fluorograph.

Example 3

Degradation of lymphoid populations by monoclonal antibody and complement.

40 x 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, OKT4 or OKT8 antibody or ordinary aqueous humor (reference substance) 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, the cells were centrifuged and viable cells were determined

Trypan-blue. Cells were counted, washed twice with 5% FCS and placed in final medium RPMI 1640 (Grand Island Biological Company,

Grand Island, NY) containing 20% human AB + -se-35 ug, 1% penicillin-streptomycin, 200 mM L-Glutamine, 25 mM HEPES buffer and 0.5% sodium carbonate 11,75433 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 OKT10 and other monoclonal 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, 0KT9 and OKT10 with different human lymphoid cell populations. The monoclonal antibody OKT10 reacts with approximately 95% of normal human thymocytes, with 5% of normal human peripheral T cells, with 10% of peripheral mononuclear E ~ cells (B cells and null cells), and -20% with bone marrow cells. Among other things, this reactivity formula allows the present antibody OKT10 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, OKT4, OKT5, OKT6, OKT8, OKT9 and OKT10 (all at a dilution ratio of 1: 500) and G / M FITC. 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 low fluorescence intensity, while 0KT5, OKT6, 0KT8, and OKT10 react with about 70% of thymocytes at a higher fluorescence intensity. 0KT4, which reacts with 75% of thymocytes, is located between 0KT9 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 with 0KT3 using an indirect immunofluorescence method. 0KT1, which reacts with thymocytes in virtually the same way as 0KT3, does not appear. Also by the reactivity formula shown in Fig. 10 1, the present antibody OKTIO can be detected and distinguished from other antibodies.

Table 2 shows the distribution of antigens determined by different monoclonal antibodies in 15 human peripheral T cells and thymocytes, the distribution being determined by a series of lysis experiments as described in Example 3. Because only OKT3, OKT4, and 0KT8 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. Lysis with 0KT4 and complement reduced the total number of cells by 62% and specifically destroyed the OKT4 + population. In addition, the percentage of OKT5 + and OKT8 + cells increased, but the total number of 0KT5 + and OKT8 + T cells did not change. From this experiment, it could be concluded that the OKT4 + population differed from the OKT5 + -30 and OKT8 + populations. Further support for this hypothesis 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. Therefore, these experiments showed that the OKT8 + population was in contrast to the OKT4 + population containing the entire OKT5 + T cell fraction.

13 75433

Similar experiments with human thymocytes gave 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 OKT6. These findings indicate that most human thymocytes have 0KT4, 0KT5, 0KT6-10, and OKT8 surface antigens in the same cell. In addition, Table 2 shows that treatment of cells with OKT8 and 0KT4 significantly increases the number of mature thymocytes containing OKT3 antigen. Thus, most of the thymocytes reacting with 0KT3 have already been separated into OKT4 + and OKT8 + fractions, as most of the cells that did not react with OKT4 and OKT8 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.

To further elucidate the relationship of OKT3-responsive thymocyte subpopulations to thymocyte fractions defined by other monoclonal antibodies, thymocytes were treated with 0KT3 and complement, and the cells remaining in the treatment were then compared to untreated thymocytes. Table 2B shows that 0KT3 and complement removed 25% of thymocytes. In contrast, populations reacting with OKT4, OKT5, 0KT6, 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 35 thymocytes co-expressing antigens defined by OKT4, OKT5, and OKT8 are similarly restricted to the 14,75433 0KT3 “population. It should also be noted that the OKT9-responsive thymocyte population did not decrease when unfractionated thymocytes were treated with 0KT3 and complement, indicating that the 0KT9 + -5 subpopulation is largely confined to the 0KT3 ~ -thymocyte 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 10 thymocytes have the OKTIO marker. In addition, thymocytes receive the OKT9 antigen (Thyl and Thy2) at an early stage. This step comprises a small fraction of thymocytes and comprises about 10% of the unfractionated population.

The human thymocyte then receives the thymocyte antigen defined by OKT6 and simultaneously expresses OKT4, OKT5 and OKT8 (Thy4). The latter subpopulation represents the majority of thymocytes and comprises more than 70-80% of the thymocyte population. As Tymo-20 cytes continue to mature, their OKT6 reactivity disappears, they become reactive with OKT3 (and 0KT1), and separate into OKT4 + and OKT 5 + / 0KT8 + fractions (1hy7 and Thy8). It appears that in the final step, when thymocytes detach from the thymus among the peripheral T cells, their OKTIO marker disappears, as this antigen is absent from virtually all peripheral T lymphocytes. Possible transition steps between these three main development stages are denoted in Figure 2 by Thy3, Thy5 and Thy6.

30 Since 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. Assay 35 used 25 tumor cell populations isolated from individuals with T-ALL leukemia and three T cell lines pre-screened with standard anti-T 7 T cell reagents and E rosetting. Table 3 shows that most T-ALL leukemia cells reacted with either OKTIOr alone or 0KT9 and OKTIOrn, but the cells did not react with other monoclonal antibodies. Thus, 15/25 of the cases studied 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 10 more mature thymocyte populations (step 2). This T-ALL group reacted differently with OKT4, OKT8, and OKT9, as shown in Table 3. Two-fifths of patients had tumor cells that contained most of the common thymocyte antigens, including 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 20 different determinants in the same antigen. Finally, 1/25 of the patients had tumors from a mature thymocyte population (step 3) as determined by OKT3 reactivity. In addition, this person's tumor was reactive with OKT5, OKT8, and OKT10. Of the 25 leukemia populations studied, only four were those that could not be clearly classified. Three of these were OKT4- and OKT8-positive, but did not react with OKT3 or OKT6, making them most likely transition-30 stages between Thy4 and Thy7,8. One of the 25 cases 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 contained cells representing a particular stage of 35 thymocyte development. Table 4 shows that HSB-2 reacts exclusively with 0KT9 and OKTIO, and thus defines the tumor population from step 1. In contrast, the CEM-T cell line reacted with 0KT4, 0KT6, OKT9, and OKT10 and was found to be derived from stage 2 thymocytes. Finally, the cell line MOLT-4 appears to be a leukemia transformation between HSB-2 and CEM, as it expresses OKT6, 0KT8, OKT9, and OKT10.

Because all of the T cell acute lymphoblastic leukemia patients studied have been shown to have OKT10 + cells, the OKTIO antibody can be used to diagnose T cell ALL leukemia. Patients with zero-cell ALL leukemia also had OKT10 + cells.

Also, using the data in Tables 2-4, the OKT10 antibody can be detected and distinguished from other antibodies.

Diagnostic methods using other monoclonal antibody-producing hybridomas (0KT1, 0KT3, OKT4 and OKT5 with 0KT6, OKT8 and OKT9) produced by the same applicants are described in FI patent applications 20 844703, 844705, 844704 and 844706 and 844707, 8544. .

The OKT10 of this antibody was found to belong to the IgG1 subclass, which is one of the four subclasses of mouse IgG. These subclasses of immunoglobulin G differ by 25 so-called from their "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 to 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, e.g., complement or anti-mouse antibodies.

17 75433

The present antibody OKTIO can be used to detect and study the differentiation of thyrocytes, as shown in Figure 2. In addition, the present antibody can be used to diagnose disease states caused by a deficiency or excess of OKT10 + cells. In this case, OKT10 antibody as such or a combination of OKTIO and other antibodies (e.g., OKT3 to OKTIO) can be used. Reactivity formulas obtained by reacting several different antibodies with T cells and T cell fractions allow certain disease states to be determined more accurately than using known diagnostic methods.

Diagnostic compositions consisting of effective amounts of OKTIO antibody and diagnostically acceptable carriers are also within the scope of the present invention.

table 1

Reactivity of monoclonal antibodies with human lymphoid populations.

20 Monoclonal Peripheral Bone marrow (6) Thymus (22) antibody blood E + E ”OKT6 0% 0% 0% 70% OKT8 30% 0% <2% 80% 25 0KT9 0% 0% 0% <10% OKTIO < 5% 10% <20% 95%

Figures in parentheses indicate the number of samples examined; % values are averages.

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Ai -H «Il ++ I ++ 3 (1)« UM

Ai G X O E Ό G - O

3 φ (tj -H -H to

• H · (—i 3: (0 -H P I C

3 3 C to -n C -H - -H

(0 X O Eh Ή 10 3 H O Ή «Il + + + + I O) (O <l) C>

10 Ai O G tfl Gj Φ tO

10 to C (0

En C tO O -H Ai O tn: 0 -n to> • H £ En> i O -H: t0

P «Il I I I I + E -r-l -H P

p to nj o »o P to 3 3 to -H -H * (0 Φ 3 3 to 3 P - O 'tn a: ra c -ί rH to + φ Ai (0 -H fO Eh 3 Φ --- G> i -> Ai «Il ++ II I 3 P: <0 -HO Ai · G -H -n

C -H G · (N φ X SH

ΦΡΦ GtoGOttd ro Ai Ό oo -H «G -H>

3 nj -H Eh -H O> to G

X φ φ «Il till + tO · 0-H3P

O K G O A; ro-H-H3t0 to Φ 3 I> P to 3 (0 + P (0 X 3 X -H (0

• H - to Or a; to> P

£ H (0 (0 G O -H

φ> 1 (0 -H P -H (0 Oi -H G

Ai X -H P I -H (0 P (0 3 Eh rO p X (0 P -H Ai (0

Φ ^ X> 1 X -H P X (0 E

X Μ> ι <C φ -H O Φ (0

φ -H (0 10 -H I X -H> H P

P P> O PEH-H> tOP

to P £ P (03 (00 (0> i G> i> i G>: (0 3 -ro In to> ι-Η P>, -H> i P tO Φ

• H 10 P to -H (0 P G -H

£ O> i G O> rH P -H> (0>,

3 £> ι ~ Φ £ (0 rH Φ Ai -H i — I P

p> 1 in is e - ^>, ρφρμ-ηγηρ tn rH P O> 1 <N -H TJ- VD 00 P p p -H φ P Φ> 1

H -H £ X X - I | -H -H 10 £ Ai X P

(0 Φ tO> iEh φ X oo oo h · φ: <0 ao Ai Ai Φ I (OOtO

rH PO> 1 φ 3> 1 -h -h -h> xxxx -h ax 3 3 -hx «Cu λ H (0 ·· (0 (0EhEhEhEh (0> i Eh Eh rH P Eh + W> <ff) X + + 75433 20

Table 4

Reactivity of cell lines with monoclonal antibodies

Cell line OKT3 OKT4 OKT5 OKT6 OKT8 OKT9 OKTIO

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

Claims (2)

21 75433 A diagnostic method for detecting a deficiency or excess of OKT10 + cells in humans, characterized in that a T cell or thymocyte sample taken from a patient is reacted with a diagnostically effective amount of a mouse monoclonal antibody produced by a hybrid cell line having an ATCC accession number of CRL. 8022, and which antibody reacts with about 95% of human normal 10 thymocytes, 5% of normal human peripheral T cells, 10% of peripheral mononuclear E cells (B cells and zero cells) and 10-20% bone marrow cells, and the 15% of total peripheral T cells and thymocytes reacting with said antibody are determined. 2. A diagnostic composition for detecting an excess or deficiency of OKT10 + cells, characterized in that it consists of a diagnostically acceptable carrier and an amount of a mouse monoclonal antibody required to detect an excess or excess of OKT10 + cells produced by a hybrid cell line having an ATCC accession number 8022, and which antibody reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of peripheral mononuclear E cells (B cells and null cells), and 10 -20% with bone marrow cells.