FI75434B - DIAGNOSTIC FOERFARANDE, VID VILKET MAN ANVAENDER EN MONOCLONAL ANTIKROPP SPECIFIK MOT MAENNINSKANS TIDIGA TYMOCYTANTIGEN OCH I FOERFARANDET ANVAENDBAR KOMPOSITION. - Google Patents

Description

1 75434

Diagnostic method using a monoclonal antibody specific for the human early thymocyte antigen and a composition of matter useful in the method 5 Divided by application 803756

The invention relates to a diagnostic method for detecting a deficiency or excess of OKT9 + cells in humans and to a diagnostic composition useful in the method. The method utilizes a monoclonal antibody to an antigen found in early thymocytes (approximately 10% of normal human thymocytes). The antibody is made using a new hybrid cell line.

The incorporation of mouse myeloma cells into the spleen cells of immunized mice, proposed by Köhler and Milstein in 1975 / Tiature 256, 15 495-497 (1975), showed for the first time that it was possible to produce a continuous cell line that produces a homogeneous (so-called "monoclonal") antibody. Since this initial work, much effort has been directed to the production of various hybrid cells (referred to as "hybridomas") and to the use of antibodies produced by these hybridomas for various scientific studies. See. for example, Current Topics in Microbiology and Immunology, Volume 81 - "Lymphocyte Hybridomas", F. Melchers, M. Pot-25 ter and N. Warner, editor, Springer-Verlag, 1978 and references therein; C.J. Barnstable, et al., Cell, 14, 9-20 (May, 1978); P. Parham and W.F. Bodmer, Nature 276, 397-399 (November, 1978); Handbook of Experimental Immunology, 3rd Edition, Volume 2, D.M. Wier, editor, Blackwell, 30 1978, Chapter 25; and Chemical and Engineering News, January 1, 1979, 15-17. These references describe the results obtained at the same time and the difficulties encountered in trying to prepare a hybridoma monoclonal antibody. Although the general method is well understood in theory, many difficulties are encountered and modifications are required in each special case. In fact, there is no certainty, 2 75434 prior to the preparation of a particular hybridoma, that the desired hybridoma will be obtained and that, if successful, it will produce an antibody, or that the antibody thus prepared will have the desired specificity. Success is mainly influenced by the type of antigen used and the selection technique used to isolate the desired hybridoma.

Attempts to produce a monoclonal antibody to human lymphocyte cells for surface antigens have been described in only a few rare cases. See. for example, 10 Current Topics in Microbiology and Immunology, supra pages 66-69 and 164-169. The antigens used in these described experiments were cultured human lymphoblastoid leukemia and human chronic lymphocyte leukemia cell lines. Many of the hybridomas obtained appeared to produce antibodies to various antigens present in all 15 human cells. None of the hybridomas produced antibodies against a particular class of human lymphocytes.

Recently, Applicants and others in the present application have published articles describing the preparation and testing of hybrids that form antibodies to certain T cell antigens. See. for example, 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 Rung, P.C., et al., Science, 206, 347-349 (1979). 25 There are two main classes of lymphocytes involved in the human and animal immune systems. The first of these (a cell derived from the thymus, or T cell) is differentiated in the thymus from hematopoietic stem cells. While the differentiating cells are still in the thymus, ni-30 is termed "thymocytes." Mature T cells leave the thymus and circulate between the tissues, lymphoid tissues, and the circulatory system. These T cells make up a large proportion of the circulating small lymphocytes. They are immunologically specific and are directly involved in cell-mediated immune responses (such as rejection of transplanted organs) as effector cells. Although T cells do not secrete 75434 antibodies into body fluids, they need a second class of lymphocytes to be understood later in order to secrete these antibodies. Some types of T cells have regulatory functions in other functions of the immune system. The mechanism of this 5 cell interaction process is not yet fully understood.

The second class of lymphocytes (bone marrow-derived cells or B cells) are those that secrete an antibody. They also develop from hematopoietic stem-10 bones, but the thymus does not dictate their differentiation. In birds, they differentiate in an organ analogous to the thymus, known as the "Bursa of Fabricius." However, no similar organ has been found in mammals and it is thought that these B cells differentiate in the bone-15 nucleus.

It has now been found that T cells are divided into at least several subtypes, termed "helper", "attenuating" and "killer" T cells, which function (respectively) to promote, attenuate or kill a response ( dissolve) foreign cells. These subclasses are well understood in the mouse body, but have only recently been described for the human body. See. for example, R.L. Evans, et al., Journal of Experimental Medicine, Volume 145, 221-232, 1977; and L. Chess and S.F. Schlossman - "Functional Analysis 25 of Distinct Human T-Cell Subsets Bearing Unique Differentiation Antigens", in "Contemporary Topics in Immunobiology", O. Stutman, editor, Plenum Press, 1977, Volume 7, 363-379.

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

For example, certain leukemias and lymphoid tumors have a different prognosis depending on whether they are derived from B cells or T cells. Thus, the assessment of disease prognosis depends on how these two classes of lymphocytes can be distinguished. See. for example 4,75434 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", edited by S. Thierfelder, et al. Springer, Heidelberg, 5 1977, 33-45; and D. Belpomme, et al., Britich Journal of

Hematology, 1978, 38, 85.

Certain disease states (e.g., juvenile rheumatoid arthritis, malignancies, and agammaglobulinemia) are associated with T cell subclass imbalances. It has been hypothesized that 10 immunoimmune diseases are generally associated with an excess of "helper" T cells or a lack of certain "attenuator" T cells, while agammaglobulinemia is associated with an excess of certain "attenuator" T cells or a lack of "helper" T cells. . Malignant diseases are usually associated with an excess of "attenuator" T cells.

In certain leukemias, too many T cells in the stagnant stage of development are formed. Diagnosis may thus depend on the ability to detect this imbalance or excess and determine what stage of development is in excess. See. for example, 20 J. Kersey, et al., "Surface Markers Define Human Lymphoid Malignancies with Differing Prognoses" in Hematology and Blood Transfusion, Volume 20, Springer-Verlag, 1977, 17-24 and references therein; and E.L. Reinherz, et al., J. Clin. Invest., 64, 392-397 (1979).

Agammaglobulinemia, a disease state in which no immunoglobulin is formed, comprises at least two distinct types. In type I, immunoglobulin is not formed due to the excess of attenuator I cells, whereas in type II this is due to the lack of helper T cells. Neither type appears to have any deficiency or deficiency in the patient's B cells, the lymphocytes responsible for the actual secretion of the antibody; however, these B cells are either attenuated or "not assisted," resulting in greatly reduced or absent immunoglobulin production. The type of agammaglobulinemia can thus be determined by testing for an excess of attenuator T cells or a lack of helper T cells.

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On the therapeutic side, it has been suggested, which has not yet been conclusively proven, that the administration of antibodies against an excess of the T cell subtype may be therapeutically advantageous in autoimmune diseases or malignancies. For example, helper T cell cancer (certain skin T cell lymphoid tumors and certain acute forms of T cell lymphoblastic leukemia) can be treated with an antibody to the helper T cell antigen. The treatment of autoimmune diseases caused by an excess of helper cells can also be carried out in the same way. Diseases (e.g., malignancies or type I agammaglobulinaemia) that cause an excess of attenuating T cells can be treated by administering an antibody to the attenuating T cell antigen.

Anti-sera against the whole human T cell class (so-called anti-human thymocyte globulin, or ATG) have been shown to be therapeutically useful in transplant patients. Because the cell-mediated immune response (the mechanism by which transplanted organs are repelled) depends on T cells, administration of the antibody to T cells prevents or slows down this rejection process. See. for example, Cosimi, et al., "Randomized Clinical Trial of ATG in Cadaver Renal Allgraft Recipients: Importance of T Cell Monitoring", Surgery 40: 155-163 (1976) and references therein.

Identification and attenuation of human T cell classes and subclasses has previously been performed using spontaneous autoantibodies or selective antisera to human T cells obtained by immunizing 30 animals with human T cells, running blood from the animals to obtain serum, and adsorbing with antiserum, e.g. but with non-allogeneic B cells to remove antibodies with undesired reactivity.

35 The preparation of these antisera is extremely difficult, especially in the adsorption and purification steps. As many as 6,754,34 adsorbed and purified antisera contain many impurities in addition to the desired antibody, for many reasons. First, the serum contains millions of antibody molecules even before immunization with T cells. Second, immunization causes the formation of antibodies against various antigens found in all injected human T cells. No selective antibody production occurs against the individual antigen. Third, the titer of the specific antibody obtained by such methods is usually quite low (e.g., inactive at dilutions greater than 1: 100) and the ratio of the specific antibody to the non-specific antibody is less than 1/106.

See. for example, the article by Chess and Schlossman, cited above (from page 365 onwards), and the article by Chemical and Engineering News, cited above, which describe the shortcomings of prior art antisera and the advantages of the monoclonal antibody.

A new hybridoma (called OKT9) has now been discovered that is capable of producing a novel monoclonal antibody against an antigen present in about 10% of normal human thymocytes but not in normal human peripheral lymphocytes (T cells, B cells, or nullocytes). bone) or bone marrow cells. These thymocytes with which OKT9 is reactive are termed "early thymocytes".

The antibody thus produced is monospecific for a single determinant present on about 10% of normal human thymocytes and contains essentially no other anti-human immunoglobulin as an antibody to prior art antisera (which naturally contain as an impurity an antibody reactive with a number of human antigens) and prior art monoclonal antibodies (which are not monospecific for human thymocyte antigen). In addition, these 35 hybridomas can be cultured to produce an antibody without the need to immunize and kill animals, followed by the adsorption and purification steps required to obtain even prior art crude antisera.

Both the present hybridoma and the antibody produced by it are referred to as "0KT9", which indicates which is meant in each case. The hybridoma was deposited on November 21, 1979 in the American Type Culture Collection, 12301 Parklawn Drive Rockville, Maryland 20852, and was assigned ATCC Accession No. CRL 8021. The preparation of this hybridoma and the antibody produced by it is described in FI Patent Application 803756.

The diagnostic method of the invention using the aforementioned hybridoma and antibody is characterized in that a tai5 cell or thymocyte composition taken from this individual 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. 8021, and which antibody reacts with about 10% of normal human thymocytes but not normal human peripheral T cells, B cells, null cells, or bone marrow cells, and the proportion of peripheral T cells and of the total thymocyte population reacts with said antibody.

Example 1 Characterization of OKT9 Reactivity A. Isolation of Lymphocyte Populations Human peripheral mononuclear blood cells were isolated from healthy volunteer donors (15-40 years old) by Ficoll "Hypaque" density gradient centrifugation method 30 (Pharmacia Fine Chemicals, N, Boy). J. Clin. Lab. Invest. 21 (Suppl. 97): 77, 1968. Unfractionated mononuclear cells were separated into surface Ig + (B) and Ig (T + zero) populations by Sephadex G-200 anti-F (ab ') 2 column chromatography as before. is described by Chess, et al., J. Immunol. 113: 1113 (1974). T cells were harvested by E-rosetting the Ig “population with 5% of 75,434 sheep erythrocytes (Microbiological Associates, Bethesda, MD). The "rosetted" mixture was layered on a Ficoll-Hypaque / Lie and the recovered E + pellet was treated with 0.155 M NH 4 Cl (10 mL / 108 cells). The resulting T cell pop-5 was less than 2% EAC "rosette" positive and more than 95% E- "rosette" positive as determined by standard methods. In addition, a non-"rosetting" Ig (zero cell) population was recovered from the Ficoll interface. This latter population was less than 5% E + and <2% slg +. A surface Ig + 10 (B) population was obtained from a Sephadex G-200 column after elution with normal human gamma globulin as described above. This population was more than 95% surface Ig + and less than 5% E +.

Normal human bone marrow cells were obtained from normal 15 voluntary donors from the posterior intestinal ridge by the puncture method.

B. Isolation of thymocytes

A normal human thyroid gland was obtained from patients aged 2 months to 14 years who had undergone corrective heart surgery. The freshly obtained parts of the thyroid gland were immediately placed in 5% fetal calf serum in medium 199 (Gibco), minced with forceps and scissors, and then made into suspensions containing one cell species by squeezing through a wire mesh. The cells were then layered on a Ficoll "Hypaque'lie" and centrifuged and washed as described in Part A above. The thymocytes thus obtained were more than 95% viable and> 90% E-rosette positive.

C. T Cell Cell Lines and Acute T Cell Lympho-30 Blast Leukemia Cells T cell lines CEM, HSB-2, and MOLT-4 were provided by Dr. H. Lazarus (Sidney Farber Cancer Institute, Boston, MA). Leu chemistry cells were obtained from 25 patients diagnosed with T-cell ALL. These individual tumors had previously been determined to be derived from T cells because they spontaneously formed rosettes with sheep erythrocytes 75434 (> 20% E +) and were reactive with T cell-specific heteroantiserum anti-HTL (BK) and A99, such as described above. Tumor populations were cryopreserved at -196 ° C in the liquid nitrogen vapor phase in 10% DMSO and 20% AB human serum until surface characterization.

All tumor populations analyzed were more than 90% blasts according to the Wright-Giemsa morphology of the cell centrifuge preparations.

Example 2 10 Cell fluorographic analysis and cell separation

Cell fluorographic analysis of monoclonal antibodies in all cell populations was performed by indirect immunofluorescence with fluorescein-conjugated goat anti-mouse IgG (G / M FITC) (Meloy Labora-15 tories) using a cell fluorograph FC200 / 4800A (Ortho Instruments). Briefly, 1 x 10 6 cells were treated with 0.15 ml of OKT5 antibody at a 1: 500 dilution, incubated at 4 ° C for 30 minutes, and washed twice. The cells were then reacted with 0.15 ml of a 1:40-20 dilution of G / M FITC at 4 ° C for 30 minutes, centrifuged and washed three times. The cells were then analyzed with a cell fluorograph and the intensity of fluorescence per cell was recorded on a pulse height analyzer. A similar pattern of reactivity was observed at a dilution of 1: 10000, but further dilution caused a loss of reactivity. Background staining was obtained using 0.15 ml of a 1: 500 diluted aqueous humor obtained from a CAF1 mouse injected intraperitoneally with a non-producing hybrid clone.

In experiments involving antibody- and complement-mediated lymphocytes, thymocytes and peripheral T cells were cultured overnight, followed by selective lysis, and then analyzed on a cell fluorograph.

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Example 3

Degradation of lymphoid populations with monoclonal antibody and complement 40 x 10® peripheral T cells or thymocytes were placed in 5 15 ml plastic tubes (Falcon, Oxnard, CA). The cell pellets were incubated with 0.8 ml of OKT3, OKT4 or OKT8 antibody or a normal waterbath control screen diluted 1: 200 in PBS, resuspended and incubated at 20 ° C for 60 minutes. Thereafter, 0.2 ml of fresh rabbit complement was added to the antibody-treated populations, resuspended, and further incubated at 37 ° C in a shaker water bath for 60 minutes. At the end of this time, the cells were centrifuged and viable cells were determined with Trypan blue. After counting, cells were washed two more times in 5% FCS and placed in final medium RMPI 1640 (Grand Island Biological Company, Grand Island, NY) containing 20% AB + human serum, 1% penicillin-streptomycin, 200 mM L- glutamine, 25 mM HEPES buffer 20 and 0.5% sodium carbonate and incubated overnight in a humidified atmosphere of 5% CO 2 at 37 ° C.

Figure 1 shows the fluorescence spectrum obtained with a cell fluorograph after normal human thymocytes have been reacted with OKT9 and other monoclonal antibodies at a dilution of 1: 500 and G / M FITC. Backgrounds fluorescence staining was obtained by incubating each population in a 1: 500 dilution of an aqueous medium obtained from a mouse injected with a non-producing clone.

Figure 2 shows the differentiation of thymocytes in the human thymus.

Table 1 shows the reactivity of antibodies OKT6, 0KT8, OKT9 and OKT10 with different human lymphoid cell populations. The monoclonal antibody OKT9 is reactive with about 10% of normal human thymocytes and not with any of the other lymphoid cells tested. This reactivity model is one assay by which the present antibody 0KT9 can be detected and distinguished from other antibodies.

Figure 1 shows a representative fluorescence model obtained with a cell fluorograph after normal human thymocyte suspensions have been reacted with 0KT3, 0KT4, 0KT5, 0KT6, 0KT8, 0KT9 OKTIOs and G / M FITCtn 1. : With 500 dilutions. Similar reactivity patterns were obtained with 12 other normal human-10 thymocyte populations tested. As observed, there are significant differences in both the percent reactivity and the intensity of fluorescence for each of these monoclonal antibodies. For example, OKT9 reacts with about 10% of thymocytes at a low fluorescence intensity, while OKT5, OKT6, OKT8, and OKTIO react with about 70% of thymocytes at a higher fluorescence intensity. OKT4, which reacts with 75% of thymocytes, has a reactivity between antibody OKT9 and monoclonal antibodies with higher fluorescence intensity.

In addition, Figure 1 shows that approximately 15% of thymocytes are detected by OKT3 by indirect immunofluorescence. The figure does not show the antibody OKT1, whose reactivity pattern is in fact similar to that of OKT3 for thymocytes. The reactivity model in Figure 1 is another assay by which the present antibody 0KT9 can be detected and distinguished from other antibodies.

Table 2 shows the distribution of antigens defined by different monoclonal antibodies into human peripheral T cells and thymocytes, the distribution 30 being determined by a series of lysis experiments described in Example 3. Since only OKT3, OKT4 and OKT8 were complement binding monoclonal antibodies, these three were used. .

As shown in Table 2A, the entire T cell population reacted with OKT3, whereas OKT4, 0KT5, and OKT8 reacted with 60%, 25%, and 34% of T cells, respectively.

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Digestion with 0KT4 and complement reduced the total by 62% and specifically eliminated the 0KT4 + population. In addition, the percentage of 0KT5 + and 0KT8 + cells increased and had no effect on the absolute number of 0KT5 + and 0KT8 + T cells. These experiments showed that 0KT4 + was clearly distinct from the 0KT5 + and 0KT8 + populations. Further support for this conclusion was obtained by lysing T cells with 0KT8 and complement. In this case, the percentage of 0KT4 + T cells increased, the absolute number-10 remained the same, and the OKT8 + and OKT5 + populations disappeared. In addition, these results indicated that the OKT8 + population was inverted to the OKT4 + population and included the entire subset of OKT8 + T cells.

Similar experiments in human thymocyte populations gave different results. As shown in Table 2B, approximately 75% of thymocytes were OKT4 + or OKT8 +. In addition, after lysis with either OKT4 or OKT8, only 25% of thymocytes remained. The majority of the remaining thymocytes were reactive with OKT3, while only a small fraction were reactive with OKT6. These findings indicate that the major population of human thymocytes has OKT4, OKT5, OKT6, and OKT8 surface antigens in the same cell. In addition, Table 2 shows that after treatment with OKT8 or OKT4, there is a significant increase in mature thymocytes with OKT3 antigen. Thus, the majority of OKT3-reactive thymocytes have already separated into 0KT4 + or OKT8 + subgroups, as the majority of the remaining cells after 0KT4 or OKT8 lysis are 0KT3 +. If the OKT3 + subpopulation were both 0KT4 + and 0KT8 +, then digestion with either monoclonal antibody would have removed OKT3-reactive thymocytes.

To further determine OKT3-reactive thymocyte subpopulations to other monoclonal antibody-defined thymocyte fractions, thymocytes were treated with 0KT3 and complement, and the remaining cells were then compared to untreated thymocyte populations. As shown in Table 2B, 0KT3 and complement removed 25% of thymocytes. In addition, there was no greater loss of 0KT4, 0KT5, 0KT6, or 0KT8 reactive populations. From these findings, the conclusion can be drawn that most thymocytes with the 0KT6 marker are included in the 0KT3 ”population. Furthermore, it can be concluded that thymocytes that simultaneously express antigens defined by 0KT4, 0KT5, and 0KT8 are similarly restricted to the 0KT3 “population. It should also be noted that the OKT9-reactive population of thymocytes did not decrease after OKT3 and complement treatment of unfractionated thymocytes, thus indicating that the OKT9 + subpopulation is largely limited to the OKT3® thymocyte drop population.

15 Based on these results, it has been possible to describe the stages of intra-thymus development of human thymocytes. As shown in Figure 2, virtually all thymocytes have an OKT10 marker. In addition, thymocytes receive the OKT9 marker (Thyl and the corresponding -20 ti Thy2) at an early stage. This step defines a minority of thymocytes and makes up about 10% of the unfractionated population.

Human thymocytes then receive a unique thymocyte antigen that defines OKT6 and simultaneously express OKT4, OKT5, and OKT8 (Thy4). This latter subpopulation represents the majority of thymocytes and accounts for 70-80% of the thymus population. Upon further maturation, thymocytes lose OKT6 reactivity, gain OKT3 (and OKT1) reactivity, and differentiate into OKT4 + and OKT5 + / OKT8 + subgroups (Thy7 and Thy8). Finally, it appears that when a thymocyte enters peripheral T cells, it loses the OKTIO marker because this antigen is virtually absent from all peripheral T lymphocytes. Possible intermediates between these three main intra-thymic developmental stages are denoted by Thy3, Thy5, and Thy6 in Figure 2.

35 Because acute T-cell-derived acute lymphoblastic leukemia is thought to originate from immature thymocytes, a relationship was determined between tumor cells from individuals with T-ALL and the proposed stages of thyroid differentiation. Twenty-five tumor cell populations from individuals with T-ALL and three T cell lines previously studied with conventional anti-T cell reagents and E-rosetting were examined. As shown in Table III, the majority of T-ALL leukemia cells were reactive with either antibody OKTIO alone or with 0KT9 and OKTIO, and were unable to react with other monoclonal antibodies. Thus, in 15/25 of the cases examined, there appeared to be early onocyte antigens (Phase I). Instead, 5/25 cases were reactive with OKT6, suggesting that the origin is a more mature thymus population (stage II). This T-ALL group was itself heterogeneous for 0KT4, OKT8, and OKT9, respectively, as shown in Table 3. In cells, 2/5 of patients have the majority of thymocyte antigens such as OKT4, 0KT6, and OKT8. It should be noted that OKT5 is not present in any of these five stage II tumors, although OKT8-20 reactivity was observed. This latter result clearly suggests that OKT5 and 0KT8 define different antigens or different determinants in the same antigen. Finally, tumors from one of the 25 patients studied were from a mature thymocyte population (Phase III) as determined by its reactivity with OKT3. In addition, the tumor of this individual was reactive with OKT5, OKT8, and OKTIO. Of the 25 leukemia populations analyzed, only four tumors could not be clearly classified. Three were positive with OKT4 and OKT8, but not with OKT3 30 and 0KT6, and most likely represented intermediate forms from Thy4 and Thy7.8. One of the 25 cases appeared to be an intermediate between Thy3 and Thy4 because it was reactive with 0KT8 and OKTIO.

T cell lines derived from T-ALL tumor po-35 populations also represented cells that belonged to some specific intrathyroid differentiation step.

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As shown in Table 4, HSB was reactive exclusively with 0KT9 and OKT10 and thus would define the tumor population from step I. In contrast, CEM was reactive with 0KT4, 0KT6, 0KT8, 0KT9. and OKTIOtn 5 and appeared to be derived from a stage II thymocyte. Finally, MOLT-4 appears to represent a leukemic variant between HSB-2 and CEM because it expressed OKT6, OKT8, 0KT9, and OKT10.

Because patients with later stages of acute T-cell lymphocytic blast leukemia (e.g., stage II) have been shown to have a better chance of survival than patients with stage I ALL, the OKT9 antibody can be used to draw conclusions about a particular patient with T-cell ALL, prognosis.

The ratios shown in Tables 2-4 provide another way in which the OKT9 antibody can be detected and distinguished from other antibodies.

It has been found that 30-50% of peripheral T cells became OKT9-positive after mitogen stimulation.

This feature provides an additional means by which the OKT9 antibody can be detected and distinguished from other antibodies.

Diagnostic methods using other monoclonal antibody-producing hybridomas (OKT1, OKT3, 25KKT and OKT5 and OKT6, OKT8 and OKTIO) are described in FI patent applications 844 703, 844 705, 844 704 and 844 706 and 844 707, 850 062 and 850 061.

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table 1

Reactivity of monoclonal antibodies to human lymphoid populations 5 Monoclonal Peripheral Bone marrow Thymus (22) antibody blood (30) *) (6) _E + E ~ _%%%% 0KT6 00 0 70 10 0KT8 30 0 2 80 OKT9 00 0 10 OKT10 5 10 20 95 *) Figures in parentheses represent the number of samples tested 15; % values are averages of 17 g 75434

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Table 4

Reactivity with monoclonal antibodies Cell- OKT3 0KT4 0KT5 0KT6 0KT8 0KT9 OKTIO

5 linla_ HSB-2 - *) ---- + + CEM - + - + + + + MOLT-4 --- ++++ 10 *) The criterion for (-) and (+) reactivity was the same as in the table 3.

The present antibody OKT9 belongs to the subclass IgG-, which is one of the four subclasses of mouse IgG. These subclasses of immunoglobulin G differ from the so-called "fixed" regions, although an antibody to a specific antigen has a so-called "fixed" region. a "variable" region that is functionally similar regardless of which immunoglobulin G subclass it belongs to. Ts. a monoclonal antibody having the properties described herein may belong to subclasses IgG1, IgG2a / IgG2b or I9G3 / or classes IgM,

IgA or other known Ig classes. Differences between these classes or subclasses do not affect the selectivity of the antibody reaction pattern, but may affect the further reaction of the antibody with other agents, such as (e.g., complement) or anti-mouse antibodies.

The OKT9 antibody can be used to detect and study intra-thymic differentiation as outlined in Figure 2. Abnormalities in stage I differentiation would be indicated by a deviation of about 10% of OKT9 + -30 thymocytes. In addition, the antibody can be used to diagnose disease states associated with a decrease or proliferation of OKT9 + cells. These methods can be applied using the OKT9 antibody alone or in combination with other antibodies (e.g., OKT3-OKT10). Reactivity models with 35 groups of antibodies to T cells and subgroups of T cells allow for more accurate detection of certain disease states than is possible using prior diagnostic methods in the art.

20 75434

Diagnostic combinations comprising effective amounts of OKT9 antibody in admixture with diagnostically acceptable carriers are also included within the scope of this invention.

Claims (2)

21 75434 A diagnostic method for detecting a deficiency or excess of 0KT9 + cells in an individual, comprising reacting a T cell or thymocyte composition taken from that individual with a diagnostically effective amount of a murine monoclonal antibody produced by a hybrid cell line having an ATCC deposit. number is CRL 8021, which reacts with about 10% of normal human thymocytes, but not normal human peripheral T cells, B cells, null cells or bone marrow cells, and determines how much of the peripheral The total population of T cells and thymocytes reacts with said antibody. 2. A diagnostic combination for detecting a deficiency or excess of OKT9 + cells, characterized in that it comprises, in admixture with a diagnostically acceptable carrier, the amount of mouse monoclonal antibody produced by a hybrid cell line produced by the ATCC to detect a deficiency or excess of 0KT9 + cells. number is CRL 8021, and which antibody reacts with about 10% of normal human thymocytes, but not normal human peripheral T cells, B cells, null cells, or bone marrow cells.