FI75233B - DIAGNOSTIC FOERFARANDE, VID VILKET MAN UTNYTTJAR EN NY MONOCLONAL ANTIKROPP SPECIFIK MOT MAENNISKANS MONOCYTANTIGEN OCH I FOERFARANDET ANVAENDBAR KOMPOSITION. - Google Patents

Description

1 75233

A diagnostic method utilizing a novel monoclonal antibody specific for a human monocyte antigen and a composition of matter useful in the method 5

By division separated from application 810027

The invention relates to a diagnostic method for detecting a deficiency or excess of OKM1 + cells in humans and to a composition of matter useful in the method. The method utilizes a monoclonal antibody specific for an antigen present in normal human monocytes and granulocytes. The antibody is made using a new hybrid cell line.

15 The association of mouse myeloma cells with the spleen cells of immunized mice by Kohler and Milstein 1975 Zjiature 256, 495-497 (1975L /) showed for the first time that it was possible to prepare a continuous cell line that produced a homogeneous (so-called "monoclonal") antibody. Since this initial work, much effort has been directed to the production of various hybrid cells (called "hybridomas") and the use of antibodies made with these hybridomas for various scientific studies, see, e.g., Current Topics in Microbiology and Immunology, Volume 81 - "Lymphocyte Hybridomas," F. Melchers, M. Potter, and N. Warner, Towers, Springer-Verlag, 1978, and references therein; CJ Barnstable, et al., Cell, 14, 9-20 (May, 1978); P. Parham and WF Bodmer, Nature 276, 397-399 (November, 1978), Handbook of Experimental Immunology, 3rd Edition, Volume 2, DM Wier, editor, Black-well, 1978, Chapter 25, and Chemical and Engineering News, January 1 , 1979, 15-17. These references simultaneously demonstrate the successes and difficulties in attempting to produce a monoclonal antibody from a hybridoma. While the general method is well understood in theory, many difficulties are encountered and modifications are required for each particular 75233 break. In fact, when attempting to produce a particular hybridoma, there is no certainty that the desired hybridoma will be obtained and that, if successful, it will produce an antibody, or that the antibody so produced will have the desired specificity. Success is mainly influenced by the type of antigen used and the selection method used to isolate the desired hybridoma.

Attempts to produce a monoclonal antibody to human lymphocyte cell surface antigens have been described in only a few cases. See. for example, Current Topics in Microbiology and Immunology, ibid., 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 hyb-15 ridomes obtained appeared to produce antibodies to various antigens present in all human cells. No hybridoma produced an antibody against a particular group of human lymphocytes.

Recently, applicants of this application and others have published articles describing the preparation and testing of hybridomas that produce 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, 4061-25 4065 (1979); and Kung, P.C., et al., Science, 206, 347-349 (1979).

In addition, a description of the preparation of an anti-macrophage-producing clone has recently been presented. See. Springer et al.,

Eur. J. Immunol., 9, 301 (1979).

30 There are two main classes of lymphocytes involved in the human and animal immune systems. One of these (a cell derived from the thymus, i.e., a T cell) differentiates in the thymus from hematopoietic stem cells. When these specialized cells are in the thymus, they are termed "thymocytes." Mature T cells leave the thymus and circulate in the tissues, lymphoid, and 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 transplant-related rejection) as effector cells. Although T cells do not secrete antibodies into body fluids, another class of lymphocytes to be discussed later sometimes needs these T cells to secrete these antibodies. Some T cell types play a regulatory role in other functions of the immune system. The mechanism of this cellular cooperation process is not yet fully understood.

The second class of lymphocytes (cells derived from the bone marrow, or B cells) is one that secretes an antibody. They also develop from hematopoietic stem cells, 15 but their differentiation is not determined by the thymus. In birds, they differentiate in an organ corresponding to the thymus, called the "Bursa of Fabricius." However, no similar organ has been found in mammals and it is thought that these B cells specialize in the bone marrow.

20 It has now been found that T cells are divided into several subtypes, termed "helper", "attenuator" and "killer" T cells, which have the function of promoting, attenuating the reaction or killing (dissolving) foreign cells, respectively. . These subclasses are well understood in the body of mice, but have only recently been described in 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 of Distinct Human T-Cell Subsets Bearing Unique Diffrentation 30 Antigens", in "Contemporary Topics in Immunobiology", O. Stutman, editor, Plenum Press, 1977, Volume 7, 363-379.

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

4,75233

For example, certain leukemias and bone marrow tumors have a different prognosis depending on whether they are derived from B cells or T cells. Thus, the assessment of the prognosis of the disease depends on whether the two groups of lymphocytes can be distinguished from each other. See. for example, A.C. Aisenberg and J.C. Long, The American Journal of Medicine, 58: 300 (March, 1975); D. Belpomme, et al., In "Immunological Diagnosis of Leukemias and Lympomas", S. Thierfelder, et al., Editors, Springer, Heidelberg, 10 1977, 33-45; and D. Belpomme, et al., British Journal of

Hematology, 1978, 38, 85.

Certain disease states (e.g., rheumatoid arthritis, malignancies, and agammaglobulinemia) are associated with an imbalance between T cell subclasses.

15 It has been hypothesized that autoimmune diseases are generally associated with an excess of "helper" T cells or a deficiency of certain "attenuator" T cells, whereas agammaglobulinemia is associated with an excess of certain "attenuator" T cells or a "helper" T cell. lack of. Malignancies are usually associated with an excess of 20 "attenuator" T cells.

In certain forms of leukemia, too many stagnant T cells are formed. Diagnosis thus depends on the ability to detect this imbalance or excess and determine which stage of development is too much. See. for example, J. Ker-25 Sey, 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 different types. In type I, immunoglobulin is not formed due to 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, the lymphocytes responsible for the actual secretion of the antibody, do not appear to have 5 75233 deficiency or deficiency; 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 the presence of abundant attenuator T cells or the absence of helper T cells.

On the therapeutic side, it is assumed, as has not yet been fully proven, that the administration of antibodies against the T cell subtype in excess has a therapeutic effect in autoimmune diseases or malignancies. For example, helper T cell cancer (certain skin T cell lymphoid tumors and certain acute T cell lymphoblastic leukemias) can be treated with an antibody to the helper T cell antigen. Treatment of autoimmune diseases caused by an excess of helper cell-15 can occur in a similar manner. Diseases caused by an excess of attenuating T cells (e.g., malignancies or type I agammaglobulinemia) can be treated by administering an antibody to the attenuating T cell antigen.

Antisera 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 (a mechanism involved in the rejection of transplanted organs) depends on T cells, the administration of an antibody to T cells prevents or slows 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) 30 and references therein.

However, lymphocytes form only one class of white blood cells. Two other classes, granulocytes and monocytes, are also important in the functioning of the immune systems of humans and animals. In particular, macrophages, a species of 35 monocytes, are strongly involved in immune function. For example, although macrophages themselves do not secrete 75233 antibody, they have been found to be essential for T cell cooperation in N cell antibody production.

Macrophages are also needed for the development of cytotoxic T cells and for a proliferative response on T cells in response to mitogens such as PHA and Con A. Although theories about the function of the macrophage in these processes have been proposed, its exact function is not known. See. for example, E.S. Golub, The Cellular Basis of the 10 Immune Response, Sinauer Associates, Sunderland, Massachusetts, 1977, pp. 146-158.

The identification and attenuation of human T cells and monocyte classes and subclasses has heretofore been performed using spontaneous autoantibodies or selective antisera to human T cells obtained by immunizing animals with human T cells, flowing blood from the animals to obtain serum, and adsorbing serum. antiserum (for example) with autologous but non-allogeneic B cells to remove antibodies with undesired reactivity. The preparation of these antisera is extremely difficult, especially in the adsorption and purification steps. Even adsorbed and purified antisera contain many impurities in addition to the desired antibody for several reasons. First, the serum contains 25 million antibody molecules even before immunization with T cells. Second, immunization induces the production of antibodies against many antigens present in all injected human T cells. There is no selective antibody production against a single antigen. Third, the titer of specific antibody obtained by such methods is usually very low (e.g., inactive at dilutions greater than 1: 100) and the ratio of specific antibody to non-specific is less than 1/10®.

35 Cf. for example, the article by Chess and Schlossman, cited above (from page 365 onwards), and the article by Chemi- 7 75233 cal and Engineering News, cited above, which describe the shortcomings of prior art antisera and the advantages of the monoclonal antibody. .

A new hybridoma (called 0KM1) has now been discovered which is capable of producing a novel monoclonal antibody against an antigen present on normal human peripheral blood monocytes and granulocytes but not on normal human peripheral lymphocytes (T cells, B cells or B cells). , thymocytes, 10 lymphoblastoid cell lines, or T or B cell-derived tumor cells.

The antibody thus produced is monospecific for a single determinant in normal human peripheral blood monocytes and granulocytes and essentially does not contain any other anti-human immunoglobulin as opposed to prior art antisera (which inherently react with an antibody ) and prior art monoclonal antibodies (which are not monospecific for the human monocyte gene). In addition, this hybridoma can be cultured to produce an antibody without the need to immunize and kill animals, followed by the time-consuming adsorption and purification steps required to obtain prior art antisera from the contaminant.

Both the present hybridoma and the antibody produced by it are identified herein by the term "OKM1", whichever is meant in each case, as will be apparent from the context. The hybridoma was deposited on December 13, 1979 in the American Type Culture 30 Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 and was assigned ATCC Accession No. CRL 8026.

The preparation of the hybridoma and the antibody produced by it is described in FI patent application 810027.

The diagnostic method of the invention, if the above hybridoma and antibody are used, is characterized by reacting a 75233 monocyte composition taken from this unit with a diagnostically effective amount of a mouse monoclonal antibody produced by a hybrid cell line having an ATCC accession number of CRL 8026 , and which antibody reacts with an antigen present on normal human monocytes and granulocytes, but not on normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblast cell lines, or T or B cell-derived tumor cells, determining the percentage of the monocyte population that reacts with said antibody. Example 1 Characterization of 0KM1 Reactivity A. Isolation of Mononuclear Cell Populations Human mononuclear peripheral blood cells were isolated from healthy volunteer donors (15-40 years old) by Ficoll-Hypaque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, NJ). . J. Clin. Lab. Invest. 21 (Suppl. 97): 77, 1968.

Infectious cells were obtained by incubating mononuclear cells for one hour in polystyrene plates (100 x 20 mm tissue culture dishes) (Falcon, Oxnard, CA) in RPMI 1640 (Grand Island Biological Company, Grand Island, NY) supplemented with 20%: human AB serum. After removal of the non-stick fraction, the sticky cells were detached by incubation at 4 ° C in serum-free MEM medium containing 2.5 mM EDTA, followed by gentle scraping with the rubber tip of a disposable syringe.

Mononuclear cells were separated from E + and E populations by rosetting with sheep erythrocytes and by Ficoll-Hypaque'11a differential centrifugation. Rosacification was performed by centrifuging a mixture of mononuclear cells and sheep erythrocytes for five minutes at 800 rpm and incubating for one hour at 4 ° C. The erythrocyte-35 tilymphocyte ratio was 40: 1. E + cells were harvested by lysing sheep erythrocytes with 0.85% 752339 ammonium chloride solution.

Zero cells were prepared by similarly rosetting with a surface immunoglobulin-negative (sGg) population obtained by Sephadex G-200 anti-human-5 (F (ak>) 2 ~ chromatography, as described by Chess et al., Previously described. , J. Immunol., 113: 1113 (1974) Polymorphonuclear leukocytes were obtained by lysing red cells present in a pellet formed by Ficoll-Hypaque centrifugation of peripheral blood.

10 B. Isolation of thymocytes

A normal human thyroid gland was obtained from patients who had undergone corrective heart surgery between the ages of two months and 14 years. The freshly obtained parts of the thyroid gland were immediately placed in 5% fetal calf of calf 15 in medium 199 (Gibco), minced with forceps and scissors, and then made into individual cell suspensions by squeezing through a wire mesh. The cells were then layered on Ficoll-Hypaque 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-Based Cell Lines and T Cell Acute Lymphoblastoid Leukemia Cells T cell lines CEM and HSB-2 and B cell lines Laz 25 156 and Laz 388 were obtained from Dr. H. Lazarus (Sidney Farm Cancer Institute, Boston MA). Leukemia cells were obtained from patients diagnosed with acute myelomonocytic leukemia (OMML; 5 patients) and acute myeloblastic leukemia (AML; 8 patients). Tumor populations were maintained at low -196 ° C in liquid nitrogen vapor phase in 10% DMSO and 20% AB human serum until surface characterization.

Example 2

Cell Fluorographic Analysis and Cell Separation 35 Cell fluorographic analysis of monoclonal antibodies in all cell populations was performed by indirect 75233 10 immunofluorescence with fluorescein-linked goat anti-mouse IgG (G / M FITC) using a fluorescence basic assay (Meloy Laboratories). ) (Beckton, Dickinson, Mountain 5 View, CA) or cell fluorography FC200 / 4800A (Ortho Instruments). Briefly, 1 x 10 cells were treated with 0.15 ml of antibody at a dilution of 1: 500, incubated at 4 ° C for 30 minutes, and washed twice. The cells were then reacted with 0.15 ml of a 1:40 dilution of 10 G / M FITC at 4 ° C for 30 minutes, centrifuged and washed twice. The cells were then analyzed by FACS-I and the intensity of fluorescence per cell was recorded on a pulse height analyzer. Background staining was obtained using 0.15 ml of a 1: 500 diluted aqueous humor from 15 BALB / cH mice injected intraperitoneally with a non-productive hybrid clone.

In experiments involving antibody and complement-mediated lymphysis, thymocytes and peripheral T cells were cultured overnight, followed by selective lysis and subsequent analysis by cell fluorography or FACS.

A. Cytotoxicity studies

Sensitization cultures for cell-mediated lympholysis (CML) were performed by the following method. 5 x 10 5 target cells were treated with 0.2 ml of Cr sodium chromate (292 μCi / ml) (New England Nuclear, Boston, MA) and incubated for 90 minutes at 37 ° C. After two washes, the cells were diluted to 2 x 10 4 / ml in medium containing 10% FCS. Twenty μΐ of labeled cells were dispensed into conical microplate wells with 20 μl of serial dilutions of hybridoma antibody. After one hour of incubation at 4 ° C, 20 μΐ of fresh rabbit serum (1:10 dilution) was added to the wells as a source of complement. The plates were incubated at 37 ° C for 1 hour. 140 μl of medium was then added to the wells and the plates were rotated at 400 g for 10 minutes. One hundred μΐ of supernatant was removed from each of the 11,75233 wells and the activity was counted on a gamma scintillation counter (Packard Instrumentation Company Downer's Grove, II). Specific Cr release was calculated using the following formula: 5 51

% released from specific Cr = Exp - SR

MR - SR X 1UU 'where Exp = mean of triplicate determinations observed, SR = spontaneous release from cells incubated with complement alone, and MR = maximum release obtained by treating cells with Triton X surfactant (l -% solution).

Lysis of larger cell numbers for subsequent proliferative assays was performed by resuspending 15-50 x 10 6 mononuclear cells in 1 ml of OKM1 at a 1: 250 dilution and incubating at 4 ° C for one hour.

0.1 ml of fresh rabbit serum was then added and the cells were incubated at 37 ° C for another hour. After three washes, the cells were counted and their viability was determined with Trypan blue.

B. Proliferative Response Assay The proliferative response to soluble antigens was determined as described above. Cells were concentrated at 2 x 10 3 viable cells / ml in RPMI 1640 supplemented with 20% human AB serum and cultured in microtiter plates in the presence of tetanus toxoid (TT) (Massachusetts Department of Public Health Biological Laboratories, Boston, MA). or in the presence of a purified protein derivative (PPD) (National Institutes of Health, Bethes-30 da, MD) or mumps CF antigen (Microbiological Associates, Walkersville, MD). After 5 days, cultures were treated with 0.2 Ci: 11a / H-methyl] thymidine 3 (H TdR) (1.9 [mu] Ci / mM specific activity) (Schwarz-Mann, Orangeburg, NY) and harvested 18 hours later with a 35 35 MASH 11 instrument. (Microbiological Associates). H-TdR activity was measured on a Packard Liquid Scintillation Counter-12 75233 scintillation counter (Packard Instrumentation Company). Each experimental group was determined in triplicate and results are expressed as means / min (cpm) + standard deviation from mean.

Figure 1 shows a fluorescence model obtained by FACS after unfractionated, non-adherent, and adherent normal human mononuclear peripheral bloods from donor # 1 were reacted with antibody 0KM1 (at a dilution of 1: 500) and G / M FITC. with.

Background fluorescence was obtained by incubating each population with a 1: 500 dilution of aqueous humor from mice injected with a non-producing clone.

Figure 2 shows the fluorescence obtained by FACS after reacting normal human E + and E mononuclear peripheral blood cells with OKM1 and F / M

With FITC.

Figure 3 shows a fluorescence model obtained by FACS after reacting normal human Ig, E and non-adherent null cells with antibody OKM1 and G / M FITC.

Figure 4 shows the degradability of antibody OKM1 and complement to human mononuclear cells.

Mononuclear peripheral blood cells from nine normal donors were separated into infectious and non-infectious populations in polystyrene plates, and each fraction was analyzed by indirect immunofluorescence by FACS. The percentages of cells specifically labeled with OKM1 are given in Table I. The mean number of positive cells was 27% for unfractionated cells, 18% for non-adherent cells, and 78% for infectious cells. The FACS fluorescence profiles of the three populations of donor # 1 are shown in Figure 1. As seen in Figure 1A, the fluorescence pattern of OKM1 in unfractionated mononuclear cells is bimodal. A two-dimensional mapping of the same population 35 showed that the cells brightly stained with OKM1 are also larger. A population of smaller cells with a lower fluorescence intensity was also observed. Most of these small cells are found in the non-infectious fraction (Fig. IB). Infectious cells internally contain a brightly stained larger population (Fig. 1C). From this reactivity of 50 KM1 with infectious populations, it could be concluded that the mononuclear cell recognized by the antibody was a monocyte, and this reactivity allows the antibody in question to be detected and distinguished from other antibodies.

10 Examination of lymphoid and myeloid cells of different origins provided further evidence of this specificity.

OKM1 did not react with B cell lines (Laz 156 and Laz 388), T cell lines (CEM and HSB 2), and three human thymocyte preparations. In addition, three T-CLL, six B-CLL, three T-ALL, and six non-T-ALL tumor cells were also negative. In contrast, OKM1 reacted strongly with 44–82% of the cells in the five acute myelomonocyte leukemia (AMML) samples and with approximately 35% of the cells in two of the eight cases of acute myeblast leukemia (AML). Two myeloid cell lines, K562 (Lozzio, et al., Blood 45, 321 (1975) and HL 60 (Collins, et al., Native, 270 307 (1977)) were negative. Isolated polymorphonuclear leukocytes from four normal donors were clearly labeled. These results were consistent with the notion that granulocytes and monocytes are closely related cells with a common origin, and data from AML cells appeared to indicate that OKM1 reacts primarily with myeloid-derived monocyte cells. antibody can be detected and distinguished from other antibodies.

From the staining pattern observed with unfractionated, infectious and non-infectious mononuclear cells, it can be concluded that the monocyte-35 population determined by the antibody was heterogeneous in terms of size and adhesion properties. After surface Ia antigens were described in subpopulations of monocyte 75233 14 pathways, the above cell fractions (donor # 1) were tested with the anti-Ia monoclonal antibody OKU to further characterize the phenotype of cells recognized by OKM1. The percentage of OKI1 + cells in non-infectious and infectious fractions was 9% and 80%, respectively. Because law-containing B lymphocytes are responsible for some of the staining observed in OKU-non-infectious populations, it appeared that the small, non-infectious cell recognized by OKM1 was essentially Ia. Instead, large, infectious OKM1 + monocytes contain surface Ia determinants.

The distribution of OKM1 cells into E + and E populations is shown in Table II. Although most of the labeled cells were found in the E population, up to 22% (mean 13%) of positive cells were present in the E + fraction. The FACS fluorescence profiles for E and E + cells are shown in Figure 2. The E ~ cells had a bimodal staining pattern due to the presence of the large and small cells described above. Fluorescence intensity in E + populations was more homo-20 genetic. The positive cells found in this fraction were small in size and largely Ia (average reactivity with OKI1 3%). With this reactivity model, the antibody in question can be further detected and distinguished from other antibodies.

25 Representative FACS histogram of OKM1 reactivity

The Ig, E, and non-adherent cell populations are shown in Figure 3. 84% of the cells were stained with OKM1 and 11% with OKU. Thus, these studies showed that the 0KM1 +, Ia non-infectious cell was surprisingly responsible for the majority of the null cell population. This pattern of reactivity is yet another test by which the antibody in question can be detected and distinguished from other antibodies.

Complement-mediated disintegration orders were made for several of the populations described above. Serial dilutions of OKM1 were tested on unfractionated, non-adherent 51 and adherent cells. The specific Cr release values of 15,75233 are shown in Figure 4. The percentage values obtained by indirect immunofluorescence (donor # 5, Table I) and complement-mediated degradation corresponded well to each population.

5 E + and E cells were also examined and the results obtained are shown in Table III. Again, the specific ur release results corresponded to the labeling results obtained by indirect immunofluorescence (donors # 8, # 9, and # 10).

The effect of 10KM1 and complement pretreatment on known monocyte-dependent T cell function, i.e., proliferation in the presence of soluble antigens, was studied. Mononuclear cells from a normal donor were incubated with OKM1 antibody (1: 250 dilution) in the presence of complement. A sorted number of autologous infectious cells were added to this pretreated population prior to treatment with soluble antigens. As shown in Table IV, mononuclear cells treated with OKMlt and complement did not proliferate in response to soluble anti-20 genes, whereas the addition of infectious cells (1%) was able to restore their proliferative response. Since incubation with OKM1 antibodies in the absence of complement did not affect this T cell function, the effect observed in the presence of complement could be attributed to the specific destruction of monocytes in the mononuclear cell population.

It should be noted that when 10-20% of infectious cells were added to OKM1-pretreated cells, both background and antigen-induced proliferation increased as compared to the untreated population.

The effect of OKM1 on CML is also a test by which the antibody in question can be detected and distinguished from other antibodies.

Table V shows the relationship between the concentrations of peripheral T cells 35 and T cell subgroups and the different disease states. These ratios can be used for diagnostic purposes (e.g., to detect acute infectious mononucleosis) by analyzing a blood sample from an individual suspected of having one of these conditions to determine T cell and T cell subsets. The ratios shown in Table 5a V are another way in which the 0KT11 antibody can be detected and distinguished from other antibodies.

Diagnostic methods using other monoclonal-10 antibody-producing hybridomas (OKT1, OKT3, OKT4 and OKT5 and OKT6, OKT8, OKT9 and OKTIO) prepared by the applicants of this patent application are described in FI patent applications 84 4703, 84 4705, 84 4704 and 84 4706 and 84 4707, 85 0062, 85 0063 and 85 0061.

75233 17

Table I

Percentage of cells reactive with 0KM1 as determined by indirect immunofluorescence 5 Donor Percentage of positive cells _F / Ha_F / H non-infectious Infectious 1 32.3 20.4 80.6 2 28.2 23.6 83.4 3 20.4 17.5 67.6 10 4 20.6 16.1 74.4 5 26.1 16.5 82.7 6 28.1 2Q, 2 74.2 7 30.5 ND 84.8 8 27.7 ND 80.7 15 9 28.7 11.2 76.0

Mean 26.9 17.9 78.2 a = Mononuclear cells prepared by Ficoll-Hypaque centrifugation 20

Table II

% Of cells reactive with 0KM1 in E-ro-set-positive and E-rosette-negative populations 25

Donor Percentage of positive cells _E ^ _ET__ 1 18.2 64.2 2 12.1 51.2 30 3 13.8 47.9 4 9.7 67.9 5 14.2 41.5 6 22.2 69 .0 7 11.5 51.2 35 8 10.6 58.6 9 9.4 55.6 10 12.8 68.5

Average 13.5 57.5 18 75233

Table III

Reactivity of OKM1 as determined by complement-mediated degradation in E-rosette-positive and E-rosette-negative populations 5

Specific% Cr release

Antibody E +

Dilution Donor Donor Donor No. 8 No. 9 No. 10 10 10 “2 11.2 + 3 9.8 + 2 12.5 + 1 10" 3 7, 8 + 2 10.7 + 2 11, 7 + 2 10-4 4.4 + 2 9, 0 + 2 11, 2 + 2 10 “5 1.5 + 1 6.7 + 1 5.5 + 2 15 E“ ΙΟ ”2 71, 5 + 4 68, 7 + 5 74 .3 + 5 ΙΟ ”3 73, 0 + 3 63, 3 + 7 70.6 + 4 20 10 ~ 4 67, 0 + 5 57.3 + 4 70.9 + 3 10-5 56, 7 + 3 52 , 9 + 3 60.2 + 5 75233 19 ίΰ

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p3 Ά2 55 s _ 1 = 1 sk 5 .§ g ^ 33 ig Pi ** - ik HS ij: 3 H "5 J (, äin Ί I do -diS ^ Sin -h -h ω P -h -h aj aj tn | · °! | 3 3! Il 111 1 | = Boti ilfaäälll i 2! i; il Hl III li II spi li f * "I ill 21 75233

The antibody 0KM1 in question belongs to the subclass IgG2a, which is one of the four subclasses of mouse IgG.

These subclasses of immunoglobulin G differ from the so-called in "solid" regions, although an antibody to a specific 5 antigen has a so-called a "variable" region that is functionally a hinge regardless of which immunoglobulin G subclass it belongs to. Ts. a monoclonal antibody having the characteristics described herein may belong to the subclass IgG1, IgG2a, IgG2b or IgG3, or to 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 (for example) complement or anti-mouse antibodies.

Antibody 0KM1 can be used to detect and study monocyte differentiation. In addition, this antibody can be used to diagnose disease states as shown in Table V. These methods can be applied using the OKM1 antibody alone or in combination with other antibodies (e.g., OKT3-OKT11). Reactivity models with a group of antibodies to T cells or T cell subsets and / or subtypes of monocytes allow more accurate detection of certain disease states than is possible using previous diagnostic methods. For example, AMML and (to a lesser extent) AML can be detected by reacting patient leukemia cells with an OKM1 antibody.

Diagnostic combinations comprising active or amounts of OKM1 antibody in admixture with diagnostically acceptable carriers are also within the scope of this invention.

Claims (2)

A diagnostic method for detecting a deficiency or excess of OKM1 + cells in an individual, characterized in that a monocyte composition taken from this unit 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. 8026, and which antibody reacts with an antigen present on normal human monocytes and granulocytes, but not on normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines or T or B cell-derived tumor cells, and the percentage of the monocyte population that reacts with said antibody. 2. A diagnostic composition for detecting a deficiency or excess of OKM1 + cells, characterized in that it comprises, in admixture with a diagnostically acceptable carrier, the amount of murine monoclonal antibody produced for the detection of a deficiency or excess of OKM1 + cells produced by a hybrid cell line mero is CRL 8026, and which antibody reacts with an antigen present on normal human monocytes and granulocytes, but not on normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cells or T or B cells. with tumor cells.