HRP940831A2 - Hybrid cell line for producing monoclonal antibody to a human monocyte antigen, natibody, and methods - Google Patents

Description

Field of invention

This invention relates mainly to new hybrid cell lines and more specifically to hybrid cell lines for the production of a monoclonal antibody to an antigen found in monocytes and granulocytes of normal humans, to the antibody thus produced, and to therapeutic and diagnostic procedures and preparations using this antibody.

Description of earlier science

Condensation of murine myeloma cells to spleen cells from immunized mice described by Kohler and Milstein 1975/Nature 256, 495-497 (1975)/ demonstrated for the first time that it is possible to obtain a continuous cell line for making a homogeneous (so-called "monoclonal") antibody. After this initial work, many efforts were made to produce various hybrid cells (so-called "hybridomas") and to use the antibodies made by the hybrid cells (so-called "hybridomas") and to use the antibodies made by these hybridomas for various scientific research. See, for example, Current Topics in Microbiology and Immunology, Volume 81 - "Lymhocyte Hybridomas", F. Melchers, M. Potter, and N. Wamer, Editors, Springer-Verlag, 1978, and references therein; C.J. Bamstable, et al., Cell, 14, 9-20 (May, 1978); P. Parham and W. F. Bodmer, Nature 276, 397-399 (Nov. 1978); Handbook of Experimental Immunology, Third Edition, Volume 2, D. M. Wier, Editor, Blackwell, 1978, Chapter 25; and Chemical and Engineering News, January 1, 1979, 15-17.

These references indicate both successes and complications in attempts to produce a monoclonal antibody from a hybridoma. Although conceptually the general technique is well understood, there are many difficulties and variations are necessary in each particular case. In fact, there can be no assurance, prior to attempting to make a given hybridoma, that the desired hybridomas will be obtained, that they will produce an antibody if obtained, or that the antibody so produced will have the desired specificity. The degree of success is mainly influenced by the type of antigen used and the choice of technique used to isolate the desired hybridoma.

Attempted production of monoclonal antibody for surface antigens of human lymphocyte cells has been reported only in a few cases. See, for example, Current Topics in Microbiology and Immunology, ibid, 66-69 and 164-169-. The antigens used in these published experiments were cultured from human lymphoblastoid leukemia and human chronic lymphocytic leukemia cell lines. It appears that all the resulting hybridomas produced antibody to various antigens on all human cells. None of the hybridomas produced produced an antibody against a predefined class of human lymphocytes.

More recently, the present applicants and others have authored articles describing the making and testing of hybridomas that make antibody 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. ScL, 76, 4061-4065 (1979); and Kung, P.C., et al., Science, 206, 347-349 (1979).

Further, there is recent work on the production of an anti-macrophage producing clone. See, Springer, et al., Eur. J. Immunol., 9, 301 (1979).

It should be clear that there are two main classes of lymphocytes involved in the immune system of humans and animals. The first of these (thymus-derived cell or T cell) differentiates in the thymus from hematopoietic stem cells. While inside the thymus, the differentiating cells are called "thymocytes". Mature T cells leave the thymus and circulate between tissues, lymph and bloodstream. These T cells form a large part of the amount of recirculating small lymphocytes. They have immunological specificity and are directly involved in cell-mediated immune reactions (such as graft rejection) as effector cells. Although T cells do not secrete humoral antibodies, they are sometimes required for secretion of these antibodies by another class of lymphocytes discussed below. Some types of T cells have a regulatory function in other aspects of the immune system. The mechanism of this process of cellular cooperation is not yet fully understood.

Another class of lymphocytes (cells derived from the bone marrow or B cells) are those that secrete an antibody. They also develop from the stalk of hemopoietic cells, but their differentiation is not determined by the thymus. In birds, they differentiate into an organ that is analogous to the thymus and is called the Bursa or Fabricius. In mammals, however, no equivalent organ has been discovered, so these cells are thought to differentiate within the bone marrow.

It is now understood that T cells are divided into at least several subtypes, called "helper", "suppressor" and "killer" T cells, which have a function (whether they promote a reaction, suppress a reaction, or destroy (break down)) foreign cells. . These subclasses are well understood for mouse systems, but have only recently been described for human systems. See, for example, R.L. Evans, et al., Journal of Experimental Medicine, Volume 145, 221-232, 1977; and L. Chess and S. F. Schlossman - "Functional Analysis of Distinct Human T-Cell Subsets Bearing Unique Differentiation Antigens", in Topics in Immunobiology", O. Stitman, Editor, Plenum Press, 1977, Volume 7, 363-379.

The ability to identify suppressed (suppressor) classes or subclasses of T cells is important for the diagnosis or treatment of various immunoregulatory disorders or conditions.

For example, certain leukemias and lymphomas have a different prognosis depending on whether they are of B cell or T cell origin. Thus, the assessment of the prognosis of the disease depends on the distinction between these two classes of lymphocytes. See, for example, A.C. Aisenberg and J.C. Long, The American Journal of Medicine, 58:300 (March, 1975); D. Belpomme, et al., in "Immunological Diagnosis of Leukemias AND Lymphosomas", S. Thierfelder, et al., eds, Springer, Heidelberg, 1977, 33-45; and D. Belpomme, et al., British Journal of Haematology, 1978, 38, 85.

Certain disease states (eg, juvenile rheumatoid arthritis, malignancies, and agammaglobulinemia) are associated with an imbalance of T cell subclasses. It has been suggested that autoimmune diseases are mainly accompanied by an excess of "helper" T cells or a deficiency of certain "suppressor" T cells, while agammaglobulinemia is accompanied by an excess of certain "suppressor" T cells or a deficiency of "helper" T cells. Malignant diseases are mostly accompanied by an excess of "suppressor" T cells.

In certain leukemias, certain T cells are produced in an arrested phase of development. Diagnosis may then depend on the ability to detect this imbalance or excess and to determine which stage of development is in excess. See, for example, J. Kersey at 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. Clm. Invest., 64, 392-397 (1979).

Acquired agammaglobulinemia, a disease state in which immune globulin is not produced, includes at least two types. In type I, the lack of production of immune globulin is the result of an excess of suppressor T cells, while in type II it is the result of a lack of helper T cells. In both types, there appears to be no defect or deficiency in the patient's B cells, the lymphocytes responsible for the actual secretion of antibodies; however, these B cells are either suppressed or "not helped" and this leads to greatly reduced or completely absent immune globulin production. The type of acquired agammaglobulinemia can thus be determined by testing for an excess of suppressor T cells or the absence of helper T cells.

From a therapeutic point of view, there are some suggestions, although this has not been definitively proven, that the administration of antibodies against the T cell subtype in excess can have a therapeutic effect in autoimmune diseases or malignant diseases. For example, helper T cell cancers (certain cutaneous T cell lymphomas and certain lymphoblastic (acute) T cell leukemias) can be treated with an antibody to a helper Th cell antigen. Treatment of autoimmune disease caused by an excess of helper cells can be achieved in the same way. Treatment of diseases (eg, malignancies or acquired agammaglobulinemia type I) due to an excess of suppressor T cells can be achieved by administration of antibodies to the suppressor T cell antigen.

Antisera against an entire class of human T cells (so-called antihuman thymocyte globulin or ATG) have been reported to be therapeutically useful in organ transplant patients. Since the cell-mediated immune response (the mechanism by which transplants are rejected) depends on T cells, administration of antibodies to T cells prevents or delays the rejection process. See, for example, Cosimi, et al., "Randomized Clinical Trial of ATG in Cadaver Renal Allograft Recipients: Importance of T Cell Monitoring," Surgery 40: 155-163 (1976) and references therein.

However, lymphocytes comprise only one class of leukocytes. Two other classes, granulocytes and monocytes, are also important in the function of the immune systems of humans and animals. Certainly, macrophages, which are a type of monocyte, are widely involved in immune function. For example, although macrophages themselves do not secrete antibody, they have been found to be essential for the cooperation of T cells in antibody production by B cells.

Macrophages are also required for the generation of cytotoxic T cells and for the generation of a proliferative response by T cells in response to such mitogens as PHA or Con A. Although there has been some speculation about the macrophage's function in these processes, its precise role is unknown. See, for example, E.S. Golub, The Cellular Basis of the Immune Response, Sinauer Associates, Sunderland, Massachusetts, 1977, pages 146-158.

Identification and suppression of human T cells and classes and subclasses of monocytes has previously been achieved using spontaneous autoantibodies or selective antisera for human T cells obtained by immunizing animals with human T cells, bled animals to obtain serum, and adsorption of antisera with (for example) autologous but not allogeneic B cells to separate antibodies with unwanted reactivities. Making these antisera is extremely difficult, especially in the phases of adsorption and purification. Even adsorbed and purified antisera contain many impurities in addition to the desired antibody, for several reasons. First, serum contains millions of antibody molecules even before T cell immunization. Second, immunization induces the production of antibodies against various antigens found in all injected human T cells.

There is no selective production of antibodies against a single antigen. Third, the titer of the specific antibody obtained by such procedures is usually quite low, (eg, inactive at dilutions greater than 1:100) and the ratio of specific to non-specific antibody is less than 1/106.

See, for example, the article by Chess and Schlossman cited above (on pages 365 et seq.) and the Chemical and Engineering News article cited above, which describe the disadvantages of prior art antisera and the advantages of the monoclonal antibody.

Extract from the invention

Now a new hybridoma /(designated OKM1)/ has been found that can produce a new monoclonal antibody against an antigen found on normal human peripheral blood monocytes and granulocytes but not on normal human peripheral lymphoid cells (T cells, B cells, or null cells). , thymocytes, lymphoblastoid cell lines, or tumor cells of T or B cell lines.

The antibody thus produced is monospecific for a single determinant of normal human peripheral blood monocytes and granulocytes and contains essentially no other anti-human immune globulin, unlike antisera of the prior art (which are inseparably contaminated with antibody reactive to numerous human antigens) and monoclonal antibodies. from earlier science (which are not monospecific for human monocyte antigen). Moreover, this hybridoma can be cultured to produce antibody without the need to immunize and kill the animals, followed by the painstaking steps of absorption and purification necessary to obtain even the impure antisera of earlier science.

Accordingly, one object of the present invention is to provide hybridomas that produce antibodies against antigens found on normal human peripheral blood monocytes and granulocytes.

A further object of the present invention is to provide methods for making these hybridomas.

A further object of the invention is to provide a substantially homogeneous antibody against an antigen found on normal human peripheral blood monocytes and granulocytes.

A further object is to provide methods for the treatment or diagnosis of disease or for the identification of monocyte subclasses using this antibody.

Other objects and advantages of the invention will become apparent upon examination of the present description.

To satisfy the foregoing objects and advantages, provided by the present invention is a novel hybrid that produces a novel antibody to an antigen found on normal human peripheral blood monocytes and granulocytes (but not on normal human peripheral lymphoid cells, thymocytes, lymphoblastoid cell lines, or tumor cells T or B cell lines), then the antibody itself and diagnostic and therapeutic procedures using antibodies. The hybridoma is made mainly according to the procedure of Milstein and Kohler. After immunization of mice with purified normal E rosette human mononuclear cells, the immunized mice were fused with cells from a murine myeloma line and the resulting hybridomas were tested for those with supersaturated fluids containing an antibody that showed selective binding to normal E rosette positive and E rosette negative lymphocytes human peripheral blood populations. The desired hybridomas were later cloned and characterized. Because of this, it was obtained by a hybrid that produces an antibody (designated OKM1) against an antigen on normal human peripheral blood monocytes.

Given the difficulties indicated in the prior art and the reported unsuccessful attempts using malignant cell lines as antigens, it was unexpected that the present procedure provided the desired hybridoma. It should be emphasized that the unpredictable nature of the hybrid cell preparation does not allow extrapolation from one antigen or cell system to another. In fact, the present applicants have found that the use of a malignant cell line of T cells or purified antigens separated from the cell surface as antigens has been largely unsuccessful.

Both the subject hybridomas and the antibody produced by them are identified herein by the designation "OKM1", the specific material referred to being apparent from the context of the text. The hybridoma in question was deposited on December 13, 1979, at the American Type Culture Collection, 12301 Parklawn Drive Rockville, Maryland 20852, and given ATCC accession number CRL 8026.

Detailed description of the invention

The procedure for making a hybridoma mainly includes the following stages:

A: Immunization of mice with normal human peripheral blood mononuclear cells. Although female BALB/cJ mice are found to be preferred, it is understood that other types of mice may be used. The immunization schedule and thymocyte concentration should be such that useful amounts of suitably primary splenocytes are produced. Three immunizations at fourteen-day intervals with 2 x 107 cells/mouse/injection in 0.2 ml phosphate buffered solution were found to be effective.

B. Separation of spleen from immunized mice and preparation of suspension in appropriate medium. About one ml per spleen is enough. These experimental techniques are well known.

C. Condensation of suspended spleen cells with murine myeloma cells from an appropriate cell line using an appropriate condensation promoter. The total volume of about 0.5-1.0 ml of condensation medium is suitable for about 108 splenocytes. Many murine myeloma cell lines are known, mostly from members of academic societies of various depository banks, such as the Salk Institute Cell Distribution Center, La Jolla, CA. The cell line used should preferably be of the so-called "drug-resistant" type, so that non-condensed myeloma cells will not survive in the chosen medium, while hybrids will. The most common class are 8-azaguanine-resistant cell lines, which lack the enzyme hypoxanthine guanine phosphoribosyl transferase and therefore will not be supported by HAT (hypoxanthine, aminopterin, and thymidine) media. It is also generally preferred that the myeloma cell line be used as a so-called "non-secreting" type, so that it does not produce any antibody per se, although secreting types may be used. However, in certain cases, secretory-type lines may be preferred. Although the preferred condensation promoter is polyethylene glycol having an average molecular weight of about 1000 to about 4000 (commercially available as PEG 1000, etc.), other condensation promoters known in the art can be used.

D. Diluting and culturing in separate containers, a mixture of uncondensed spleen cells, uncondensed myeloma cells, and condensed cells in a selective medium that will not support uncondensed myeloma cells for a satisfactory period of time to allow death of the uncondensed cells (about one week). The dilution can be of the limiting type, in which the volume of the diluent is statistically calculated to isolate a certain number of cells (eg, 1-4) in each separate container (eg, each well of a microtiter plate). The medium is one (eg, HAT medium) that will not support an unfused drug-resistant (eg, 8-azaguanine-resistant) cell line. Since non-condensed spleen cells are not malignant, they can only have a definite number of generations. Thus, after a certain period of time (about one week), these uncondensed spleen cells will fail to reproduce. On the other hand, condensed cells continue to reproduce because they have the malignant quality of myeloma origin and the ability to survive in the selective medium for the source of spleen cells.

E. Evaluation of the supernatant in each container (well) containing hybridomas for the presence of antibodies to E rosette positive purified or E rosette negative human peripheral blood lymphocytes.

F. Selection, (eg, by limiting dilution) and cloning of hybridomas that produce the desired antibody. Once the desired hybrid has been selected and cloned, the resulting antibody can be produced in one of two ways. The purest monoclonal antibody is produced in vitro by culturing the desired hybridoma in a suitable medium for a suitable length of time, after which the desired antibody is isolated from the supernatant. Suitable substrate and suitable length of cultivation time are known or easily determined. This in vitro technique produces an essentially monospecific monoclonal antibody, essentially free of other specific ethnic human immune globulin.

There is a small amount of other immune globulin present since the medium contains xenogeneic serum (eg, fetal calf serum). However, this in vitro procedure may not produce a sufficient amount or concentration of antibody for the same purposes, since the concentration of the monoclonal antibody is only about 50 μg/ml.

To produce a much higher concentration of a slightly less pure monoclonal antibody, the desired hybridoma can be injected into mice, preferably syngeneic or semi-syngeneic mice. Hybridomas will cause the formation of tumors that produce the antibody after a suitable incubation time, which will lead to a high concentration of the desired antibody (about 5-20 mg/ml) in the bloodstream and peritoneal exudate (ascites) of the host mouse. Although these host mice have normal antibodies in their blood and ascites, the concentration of these normal antibodies is only about 5% of the concentration of the monoclonal antibody. Moreover, since these normal antibodies are not antihuman in their specificity, the monoclonal antibody obtained from harvested ascites or from serum is essentially free of any contaminating antihuman immune globulin. This monoclonal antibody has a high titer (active at dilutions of 1:50,000 or higher) and a high ratio of specific to non-specific immune globulin (about 1/20). Immune globulins produced by the incorporation of myeloma light sequences are non-specific, "nonsense" peptides that only dilute the monoclonal antibody without reducing its specificity.

Example 1

Production of monoclonal antibodies

A. Immunization and hybridization of somatic cells

Female BALB/cj mice (Jackson Laboratories; 6-8 weeks old) were immunized intraperitoneally with 2 x 107 rosette-purified human mononuclear cells in 0.2 ml phosphate-buffered saline at 14-day intervals. Four days after the third immunization, the spleens were separated from the mice and a single cell suspension was made by pressing the tissue through a stainless steel mesh.

Cell condensation was performed according to the procedure developed by Kohler and Milstein. 1 x 108 splenocytes were condensed in 0.5 ml condensation medium containing 35% polyethylene glycol (PEG 1000) and 5% dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, NY) with 2 x 107 P3X63Ag8UI myeloma cells provided by Dr. M. Schraff, Albert Einstein College of Medicine, Bronx, NY. These myeloma cells secrete IgG1 light strings.

B. Selection and growth of hybridomas

After cell condensation, the cells were cultured in HAT medium (hypoxanthine, aminopterin and thymidine) at 37°C with 5% CO2 in a humid atmosphere. Several weeks later, 40 to 100 1 of the supernatants from the hybridoma-containing cultures are added to a pellet of 10 6 peripheral lymphocytes that have been separated into E rosette positive (E+) and E rosette negative (E-) populations, and which have been prepared from the blood of healthy human subjects. donor as described in Mendes (J. Immunol. 111: 860, 1973). Detection of murine hybridoma antibodies that bind to these cells was determined by indirect immunofluorescence. Cells incubated with tissue supernatants were stained with fluorescent goat-anti-mouse IgG (G/M FITC) (Meloy Laboratories, Springfield, VA; F/p=2.5) and cells coated with fluorescent antibody were later analyzed on a Cytofluorograph FC200/4800A. (Ortho Instruments, Westwood, MA) as described in Example III. Hybridoma cultures containing antibodies that react specifically with E+ lymphocytes (T cells) and/or E- lymphocytes were selected and cloned twice by limiting dilution procedures in the presence of feeder cells. Later, clones are transferred intraperitoneally by injecting 1 x 10 7 cells of a given clone (0.2 ml volume) into BALB/cj primed mice with 2, 6, 10, 14-tetramethylpentadecane, sold by Aldrich Chemical Company under the name Pristine. Malignant ascites from these mice are then used to characterize mononuclear cells as described below in Example II. A monoclonal antibody produced by one of these clones, which reacted with a fraction of both E+ and E- cells, was named OKM1. The hybrid antibody in question was demonstrated by standard techniques to be of the OKM1 IgG2a subclass.

Example II

Characterization of OKM1 reactivity

A. Isolation of mononuclear cell populations

Human peripheral blood mononuclear cells were isolated from human volunteer donors (aged 15-40 years) using Ficoll-Hypaque density gradient centrifugation (Pharmacia Fune Chemicals, Piscataway, NJ) according to the technique of Boyum, Scand. J. Clin. Lab. Invest. 21 ( suppl. 97); 77, 1968.

Adherent cells were obtained by incubating mononuclear cells in polystyrene dishes (100 x 20 mm tissue culture dishes) (Falcon, Oxnara, CA) for one hour in RPMI 1640 (Grand Island Biological Company, Grand Island, NY) supplemented with 20% pooled human AB serum After separation of the non-adherent fraction, adherent cells were separated by incubation at 4°C in serum-free MEM, 2.5 mM EDTA, followed by gentle scratching with the rubber tip of a disposable syringe stopper.

Mononuclear cells are separated into E+ and E- populations by rosetting with sheep erythrocytes and differential centrifugation over Ficoll-Hypaque. Rosetting is achieved by centrifugation of the mononuclear cell/sheep erythrocyte mixture for 5 minutes at 800 rpm and incubation for 1 hour at 4°C. The erythrocyte: lymphocyte ratio was 40:1. E+ cells were regenerated by breaking up sheep erythrocytes with 0.85% ammonium chloride solution.

Null cells are made by similar rosetting on the surface of the immunoglobulin-negative (sIg-) population using Sephadex G-200 anti-human (F(abl)2 chromatography as previously described in Chess, et al., J. Immunol., 113: 1113 (1974).Polymorphonuclear leukocytes were obtained by breaking up erythrocytes contained in a granule formed during Ficoll Hypaque centrifugation of peripheral blood.

B. Isolation of thymocytes

Normal human thymus was obtained from patients aged 2 months to 14 years who had undergone corrective heart surgery. Freshly obtained thymus sections are immediately placed in 5% fetal calf serum in medium 199 (Gibco), finely crushed with forceps and scissors, and later prepared into single-cell suspensions by pressing through a wire sieve. Cells are further layered over Ficoll Hypaque and spun down and washed as previously described in section A above. Thus obtained thymocytes were >95% viable and ≥90% E rosette positive.

C. Acute lymphoblastic leukemia T cell lines and T cells

T cell lines CEM and HSB-2, and B cell lines Laz 156 and Laz 388 were provided by Dr. H. Lazarus (Sidney Farber Cancer Institute, Boston, MA). Leukemic cells were obtained from a patient diagnosed with acute myelomonocytic leukemia (AMML; 5 patients) and myeloblastic leukemia (AML; 8 patients). Tumor populations were cryopreserved under liquid nitrogen in the vapor phase at -196°C with 10% DMSO and 20% AB human serum from the time of surface characterization.

Example III

Cytofluorographic analysis and cell separation

Cytofluorographic analysis of monoclonal antibodies with all cell populations was performed by indirect immunofluorescence with goat anti-mouse IgG (G/M FITC) conjugated to fluorescein (Meloy Laboratories) using Fluorescence Activated Cell Sorter (FACS-I) instruments (Becton, Dickinson, Mountain View, CA) or Cytofluorograf FC200/4800A (Ortho Instruments). Briefly, 1 x 10 6 cells are treated with 0.15 ml OKMl at a 1:500 dilution, incubated at 4°C for 30 minutes, and washed twice. Cells are then reacted with 0.15 ml of a 1:40 dilution of G/M FITC at 4°C for 30 minutes, centrifuged and washed twice. The cells are then analyzed on a FACS-I and the fluorescence intensity per cell at the pulse height of the analyzer is recorded. Baseline staining is obtained by replacement with a 0.15 ml aliquot of 1:500 ascites from a BALB/cJ mouse injected intraperitoneally with a non-producing hybrid clone.

In experiments involving antibody and complement-mediated lympholysis, thymocytes and peripheral T cells were cultured overnight using selective disruption and later analyzed on a Cytofluorograph or FACS.

A. Cytotoxicity studies

Cell-mediated lympholysis (CML) sensitization cultures were established by the following procedure. Five x 106 desired cells are treated with 0.2 ml of 51Cr sodium chromate (292 μCi/mI) (New England Nuclear, Boston, MA) and incubated for 90 minutes at 37°C. After two washes, the cells are diluted to 2 x 105/ml in media containing 10% FCS. Twenty 1 labeled cells are spread into the wells of conical microplates with 20 μl of serial dilutions of the hybridoma antibody. After one hour of incubation at 4°C, 20 μl of fresh rabbit serum (dilution 1:10) was added to the wells as a source of complement. The plates are incubated at 37°C for 1 hour. Then 140 μl of medium is added to the wells and the plates are spun at 400g for 10 minutes. 100 μl of supernatant was removed from each well and counted with a gamma scintillation counter (Parkard Instrumentation Company, Downer's Grove, II). Specifically 51Cr

release is calculated using the following formula:

% specific 51CR release = (Exp-SR) x100

(MR-SR)

where Exp=average of observed triplicate, SR=spontaneous release from cells incubated with complement alone, and MR=maximum release obtained by treatment of cells with Triton X detergent (1% solution).

Dissociation of a larger number of cells for later polyferative tests was performed by resuspending 20 to 50 x 106 mononuclear cells in 1 ml of OKM1 at a 1:250 dilution and incubating at 4°C for 1 hour.

Later, 0.1 ml of fresh rabbit serum was added and the cells were further incubated at 37°C for 1 hour. After three washes, cells are counted and their viability is assessed by exclusion of Trypan blue.

B. Proliferative reaction testing

The proliferative response to soluble antigens was measured as described previously. Cells were brought to a concentration of 2 x 106 viable cells/ml in RPMI 1640 supplemented with 20% human AB serum and cultured in mitrotiter plates in the presence of tetanus toxoid (TT) (Massachusetts, Department of Public Health Biological Laboratories, Boston, MA), purified protein derivative (PPD) (National Institutes of Health, Bethesda, MD), or CF smallpox antigen (Microbiological Associates, Walkersville, MD). Cultures were pulsed after five days with 0.2 Ci/H-methyl/thymidine (3H TdR)(1.9 4 Ci/mM specific activity) (Schwarz-Mann, Orangeburg, NY) and harvested 18 hours later on a MASH 11 apparatus (Microbiological Associates ). 3H-TdR incorporation was measured on a Packard Liquid Scintillation Counter (Packard Instrumentation Company). Each experimental group was tested in triplicate and results are expressed as average counts per minute (cpm) standard error of the mean.

Brief description of the drawing

Figure 1 shows the fluorescence scheme obtained by FACS after the reaction of unfractionated, non-adherent and adherent human peripheral blood mononuclear cells from donor No. 1 with OKM1 at 1:500 dilution and G/M FITC. Baseline fluorescence staining was obtained by incubating each population with a 1:500 dilution of ascites fluid from a mouse injected with a non-producing clone.

Figure 2 shows fluorescence obtained on FACS after reaction of normal human peripheral blood E+ and E- mononuclear cells with OKM1 and G/M FITC.

Figure 3 shows the fluorescence scheme obtained on FACS after reaction of normal human Ig-, E- non-adherent null cells with OKM1 and G/M FITC.

Figure 4 shows the cleavage capacity of OKM1 and complement for human mononuclear cells.

Hybridoma production and production and characterization of the resulting monoclonal antibody were performed as described in the examples above. Although larger quantities of the subject antibody have been made by injecting the subject hybridoma intraperitoneally into mice and harvesting malignant ascites, it should be understood that the hybridoma can be cultured in vitro by techniques well known in the art and the antibody is separated from the supernatant.

Peripheral blood mononuclear cells from nine normal donors were separated into adherent and nonadherent populations on polystyrene dishes, and each fraction was analyzed by indirect immunofluorescence on FACS. The percentages of cells specifically labeled with OKM1 are given in Table I. The average of positive cells was 27% for unfractionated cells, 18% for nonadherent cells, and 78% for adherent cells. FACS fluorescence profiles of three populations for donor no. 1 are illustrated in Figure 1. As seen in Figure 1A, the fluorescence pattern of OKM1 on unfractionated mononuclear cells was bimodal. Two-dimensional mapping of the same population showed that cells brightly stained with OKM1 were also larger.

A population of smaller cells with lower fluorescence intensity was also detected. The largest number of these smaller cells was found in the non-adherent fraction (Figure 1B). Adherent cells, in contrast, contain a lighter-stained larger population (Figure 1C). This reactivity of OKM1 with adherent populations suggested that the mononuclear cell recognized by the antibody was a monocyte and provides one test by which the subject antibody can be detected and distinguished from the antibody.

The study of lymphoid and myeloid cells of different origins provided further evidence for this specificity. OKM1 was unreactive with B cell lines (Liz 156 and Laz 388), T cell lines (CEM and HSB 2), and with three human thymocyte preparations. Moreover, 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 to 82% of cells from five samples of acute myelomonocytic leukemia (AMML) and with about 35% of cells in two of eight cases of acute myeloblastic leukemia (AML). Two myeloid lines, K562 (Lozzio, et al., Blood 45, 321 (1975) and HL 60 (Collins, et al., Native, 270-347 (1977)) were negative. Isolated polymorphonuclear leukocytes from four normal donors were light-labeled. These results were consistent with the knowledge that granulocytes and monocytes are closely related cells of common origin. Furthermore, the data obtained with AML cells appear to indicate that OKM1 reacts preterentially with monocytic cells of myeloid lineage. This reactivity scheme provides an additional test by which the antibody in question can be detected and distinguished from other antibodies.

The staining pattern observed on unfractionated, adherent, and nonadherent mononuclear cells suggested that the antibody-defined monocyte population was heterogeneous with respect to size and adherent properties. As the surface Ia antigen was described on subpopulations of monocytes, the upper cell fractions (donor No. 1) were tested with a monoclonal anti-Ia antibody, OKI1, in order to further characterize the phenotype of OKM1-identified cells. The percentage of OKI1+ cells in the non-adherent and adherent fractions was 9% and 80%, respectively. Since Ia bearing B lymphocytes explain some of the staining found with OKI1 in nonadherent populations, it appears that the small, nonadherent cell recognized by OKM1 is mostly Ia-. In contrast, large, adherent OKM1+ monocytes carry surface Ia determinants.

The distribution of OKM1 cells within the E+ and E- populations is shown in Table II. Although the highest number of labeled cells was found in the E- population, up to 22% (mean, 13%) of positive cells were present in the E+ fraction. FACS fluorescence profiles for E- and E+ cells are shown in Figure 2. E- cells had a bimodal staining pattern due to the presence of large and small cells described above. The fluorescence intensity on the E+ population was more homogeneous. Positive cells in this fraction were found to be small in size and mostly Ia- (average reactivity with OKI1, 3%). This reactivity scheme is a further test by which the antibody in question can be detected and distinguished from other antibodies.

A representative FACS histogram of OKM1 reactivity with Ig-, E-, non-adherent cell populations is illustrated in Figure 3. 48% of cells stained with OKM1 and 11% with OKI1. Thus, these studies indicate that OKM1+, Ia non-adherent cells were unexpectedly abundant in the null cell population. This reactivity scheme is a further novel test by which the antibody in question can be detected and distinguished from other antibodies.

Complement-mediated disruption assays were performed in several populations described above. Serial dilutions of OKM1 were tested on unfractionated, nonadherent and adherent cells. The values of specific 51Cr release are shown in Figure 4. There was a good correlation between the percentages found by indirect immunofluorescence (donor No. 5, Table I) and complement-mediated cleavage for each population.

E+ and E- cells were also studied and data obtained and presented in Table III. Again, the results of specific 51Cr release corresponded to the labeling found by indirect immunofluorescence (donors No. 8, 9 and 10).

The effect of OKM1 and complement pretreatment on a known monocyte that is dependent on T cell function, ie, proliferation to soluble antigens, was studied. Mononuclear cells from a normal donor were incubated with OKM1 antibody (1:250 dilution) in the presence of complement. A graded amount of autologous adherent cells was added to this pre-treated population prior to activation with soluble antigens. As shown in Table IV, OKM1 and complement-treated mononuclear cells did not proliferate in response to solubilized antigens, while the addition of 1% adherent cells could restore their proliferative response. Since incubation with OKM1 antibodies in the absence of complement did not affect the function of these T cells, the effect seen in the presence of complement could be attributed to the specific destruction of monocytes within the mononuclear cell population.

It should be emphasized that when 10 to 20% adherent cells are added to OKM1 pretreated cells, both baseline and antigen-induced proliferation are increased 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 relationships between peripheral T cell levels and T cell subsets and various disease states.

These ratios can be used for diagnostic purposes (eg, to detect acute infectious mononucleosis) by analyzing a blood sample of individuals suspected of having one of these disease states to determine T cell levels and T cell subsets. These relationships can also be used for therapeutic purposes when the cause of the disease state is an increased level of T cell subsets (eg, acquired agammaglobulinemia Type I). For therapeutic use, administration of the appropriate monoclonal antibody to a patient with increased levels of T cell subsets will reduce or eliminate the excess. The relationships shown in Table V are a further way in which the OKT11 antibody can be detected and distinguished from other antibodies.

Other monoclonal antibody-producing hybridomas have been made by the present applicants (designated OKT1, OKT3, OKT4 and OKT5) and are described and protected in the following U.S. Pat. Patent applications: SN 22, 132, filed March 20, 1979; SN 33, 639; filed April 26, 1979; SN 33, filed April 26, 1979; SN 76, 642, filed September 18, 1979; and SN 82, 515, filed Oct. 9, 1979. Further monoclonal antibody-producing hybridomas made by the present applicants (designated OKT6, OKT8, OKT9, and OKTIO) are described and patented in U.S. Pat. Patent applications filed on December 4, 1979, entitled:

Hybrid Cell Line for Producing Monoclonal Antibody to a Human Thymocyte Antigen, Antibody, and Methods;

Hybrid Cell Line For Producing Complement-Fixing Monoclonal Antibody to Human Suppressor T Cells, Antibody, and Methods;

Hybrid Cell Line For Producing Monoclonal Antibody to Human Early Thymocyte Antigen, Antibody, and Methods; and

Hybrid Cell Line For Producing Monoclonal Antibody to a Human Prothymocyte Antiger, Antibody, and Methods.

A further hybridoma that produces a monoclonal antibody is described and patented in U.S. Pat. To a patent application filed on the same date as this one and entitled: Hybrid Cell Line for Producing Antibody for a Human T Cell Antigen, Antibody, and Methods.

These applications are incorporated herein by reference.

According to the present invention, there is provided a hybrid that can produce an antibody against an antigen found on normal human monocytes, a method for producing this hybridoma, a monoclonal antibody against an antigen found on normal human monocytes, methods for producing antibodies, and methods and preparations for treating or disease diagnosis or monocyte subclass identification using this antibody.

TABLE 1

Percentage of cells reacting with OKM1 by indirect immunofluorescence

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aMononuclear cells were prepared by centrifugation on a Ficoll-Hypaque gradient.

TABLE II

Percentage of cells reacting with OKM1 in E rosette positive and E rosette negative populations

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TABLE III

OKM1 Reactivity by complement-mediated cleavage in E rosette positive and E rosette negative populations

Percentage of specific 51Cr release

Antibody E+

Dilution Donor No. 8 Donor No. 9 Donor No. 10

10-2 11.2±3 9.8±2 12.5±1

10-37.8±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

10-2 71.5±4 68.7±5 74.3±5

10-3 73.0±3 63.3±7 70.6±4

10-4 67.0±5 57.3±4 70.9±3

10-5 56.7±3 52.9±3 60.2±5

TABLE IV

Effect of pretreatment of mononuclear cells with OKM1 and C' on antigenome-induced proliferative reactions

Incorporation of 3H-thymidine

Cell population Substrate TT PPD Boginje

F/H 385±88 1.767±425 5.063±1.010 19.09443.139

F/H OKMl'+C 144±10 142±12 162±23 254±4

F/H OKMl+C'+1% adheren-

stem cells 190±51 1,358±209 5,777±1,013 7,269±1,090

+10% of adherent cells 1,425±259 4,128±921 16,412±2,747 38,5581,680

+20% of adherent cells 1,944±665 5,926±1,429 13,182±3,007 40,065±5,810

Adherent cells 192±69 261±186 224±95 804±42

aAll cultures were performed in triplicate. Results are expressed as mean counts per minute ± standard error of the mean

bTT (Tetanus Toxoid): 10 g/ml

cPPD (Purified protein derivative): 10 g/ml

dGoddess : dilution 1/20

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Although only one hybridoma has been described that produces a monoclonal antibody against human thymocyte antigen, it is understood that the present invention encompasses all monoclonal antibodies that exhibit the characteristics described herein. The OKM1 antibody in question was determined to belong to the IgG2a subclass, which is one of the four mouse IgG subclasses. These immunoglobulin G subclasses are distinguished from each other by so-called "fixed" regions, although an antibody to a specific antigen will have a so-called "variable" region that is functionally identical regardless of the immunoglobulin G subclass to which it belongs. That is, a monoclonal antibody exhibiting the characteristics described herein may be subclass. IgG1, IgG2a, IgG2b or IgG3, or class IgM, IgA or other known Ig classes. Differences between these classes or subclasses will not affect the selectivity of the reaction scheme of the antibody, but may affect the subsequent reaction of the antibody with other materials, such as (for example) complement or anti-mouse antibodies. Although the subject antibody is IgG2a specific, it is intended that antibodies having the reactivity pattern described herein are included in the subject invention regardless of which class or subclass of immunoglobulin they belong to.

Further included in the present invention are methods for making the monoclonal antibodies described herein using the hybridoma technique described herein. Although only one example of a hybridoma is provided herein, it is contemplated that one skilled in the art will be able to follow the immunization, condensation and selection procedures provided herein to produce antibodies having the reactivity characteristics described herein. Since an individual hybridoma produced from a known murine myeloma cell line and a spleen cell from a known species of mouse cannot be further identified except by reference to the antibody produced by the hybridoma, it is clear that all hybridomas that produce an antibody having the reactivity characteristics described herein are included in the subject invention, and this also applies to the procedures for making this antibody using hybridomas.

Further aspects of the invention are methods for treating or diagnosing a disease using the OKM1 monoclonal antibody or any other antibody exhibiting the reactivity pattern provided herein. The subject antibody can be used to detect and study monocyte differentiation. Moreover, the subject antibody can be used to diagnose disease states as shown in Table V. These techniques can be used using the OKM1 antibody alone or in combination with other antibodies (eg, OKT3 -OKT11). A reactivity scheme with a panel of antibodies against T cells and T cell subsets and/or monocyte subsets will enable more accurate detection of certain disease states than is possible using earlier diagnostic procedures. AMML and (to a lesser extent) AML can be detected by reacting leukemic cells from an individual with OKM1 antibodies.

Treatment of disease states (eg, malignancies such as AMML) manifested by an excess of OKM1+ cells can be achieved by administering a therapeutically effective amount of OKM1 antibody to a patient in need of such treatment. By selectively reacting with the OKM1+ antigen, an effective amount of OKM1 antibodies will reduce the excess of OKM1+ cells, thus mitigating the effects of the excess. Diagnostic and therapeutic preparations comprising effective amounts of OKM1 antibodies in admixture with diagnostically or pharmaceutically acceptable carriers are also included in the present invention.

Claims (15)

1. IgG class monoclonal antibody produced by a hybridoma formed by the condensation of cells from a murine myeloma line and spleen cells of a mouse previously immunized with human mononuclear cells, characterized by the fact that: a) the antibody reacts with an antigen found on normal human monocytes and granulocytes, but not with normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines or with tumor cells of T or B cell lines; b) shows the pattern of reactivity to normal human monocytes shown in Figure 1 and Table I; c) shows the pattern of reactivity to normal human E+ and E- peripheral human cells shown in Figure 2 and in Table II; d) shows the scheme of reactivity with normal human Ig-, E- non-adherent null cells shown in Figure 3; e) shows the breaking capacity scheme shown in Figure 4 and Table III; f) reacts with leukemia cells from a patient with AMML; g) defines the necessary population of monocytes for the proliferative reaction to dissolved antigens; h) defines a monocyte population (OKM1+) that is lower than normal levels in primary biliary cirrhosis, acute infectious mononucleosis, and graft-versus-host reaction, and higher than normal levels in multiple sclerosis, myasthenia gravis, and abnormal Hodgkins disease, and has normal levels in hyper IgE, early Hodgkins disease and psoriasis. 2. A monoclonal antibody, characterized in that it is produced from a hybridoma having the identifying characteristics of ATCC CRL 8026. 3. A hybridoma producing a monoclonal antibody formed by the condensation of spleen cells from a mouse previously immunized with human mononuclear cells and cells from a murine myeloma line, characterized in that the antibody: a) reacts with an antigen found on normal human monocytes and granulocytes, but not with normal peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines or tumor cells of T or B cell lines; b) shows the pattern of reactivity with normal human monocytes, which is shown in Figure 1 and in Table I; c) shows the pattern of reactivity with normal human E+ and E- peripheral blood cells which is shown in Figure 2 and in Table II; d) shows the pattern of reactivity with normal human Ig-, E-, non-adherent null cells which is shown in Figure 3; e) shows the breaking capacity scheme shown in Figure 4 and Table III; f) reacts with leukemia cells from a patient with AMML; g) defines the necessary population of monocytes for the proliferative reaction to dissolved antigens; h) defines a monocyte population (OKM1+) that is lower than normal levels in primary biliary cirrhosis, acute infectious mononucleosis and host reaction to transplant, is higher than normal levels in multiple sclerosis, myasthemia gravis and abnormal Hodgkins disease, and is equal to normal levels in hyper IgE, early Hodgkins disease and psoriasis. 4. A hybridoma characterized by having the identifying characteristics of ATCC CRL 8026. 5. Procedure for making a monoclonal antibody which: a) reacts with an antigen found on normal human monocytes and granulocytes, but not with normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines or tumor cells of T or E cell lines; b) exhibits the pattern of reactivity to normal human monocytes which is shown in Figure 1 and Table I; c) exhibits a pattern of reactivity with normal human E+ and E- peripheral blood cells as shown in Figure 2 and Table II; d) shows the pattern of reactivity with normal human Ig-, E-, non-adherent null cells which is shown in Figure 3; e) shows the breaking capacity scheme shown in Figure 4 and Table III; f) reacts with leukemia cells from patients with AMML; g) defines the necessary population of monocytes for the proliferative reaction to dissolved antigens; h) defines the monocyte population (OKM1+) which is lower than normal levels in primary biliary cirrhosis, acute infectious mononucleosis and host reaction to transplant, is higher than normal levels in multiple sclerosis, myasthemia gravis and abnormal Hodgkins disease, and which is normal in hyper IgE, early Hodgkins disease and psoriasis, characterized by the fact that it includes the stages: i) immunization of mice with human mononuclear cells; ii) separating the spleen from the mentioned mice and making a suspension of spleen cells; iii) condensation of spleen cells with murine myeloma cells in the presence of a condensation promoter; iv) diluting and culturing condensed cells in special wells in a medium that will not support uncondensed myeloma cells; v) assessing the supernatant in each well containing the hybridoma for the presence of the desired antibody; vi) selection and cloning of a hybridoma that produces the desired antibody; and vii) isolating the antibody from the supernatant above said clone. 6. Process for making a mononuclear antibody which: a) reacts with an antigen present on normal human monocytes and granulocytes, but not with normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines or tumor cells of T or B cell lines; b) shows the pattern of reactivity on normal human monocytes which is shown in Figure 1 and in Table I; c) shows the pattern of reactivity with normal human E+ and E- peripheral blood cells which is shown in Figure 2 and in Table II; d) shows the pattern of reactivity with normal human Ig-, E-, non-adherent null cells which is shown in Figure 3; e) shows the breaking capacity scheme shown in Figure 4 and Table III; f) reacts with leukemia cells from patients with AMML; g) defines the population of monocytes that is necessary for the proliferative reaction to dissolved antigens; h) defines a monocyte population (OKM1+) that is lower than normal levels in primary biliary cirrhosis, acute infectious mononucleosis and host reaction to transplant, is higher than normal levels in multiple sclerosis, myasthemia gravis and abnormal Hodgkins disease and is equal to normal levels in hyper IgE, early Hodgkins disease and psoriasis, characterized by the fact that it includes the stages: i) immunization of mice with human mononuclear cells; ii) separation of spleen with murine myeloma cells and preparation of spleen cell suspension; iii) condensation of said spleen cells with murine myeloma cells in the presence of a condensation promoter; iv) diluting and culturing condensed cells in special wells in a medium that will not support uncondensed myeloma cells; v) assessing the supernatant in each well containing the hybridoma for the presence of the desired antibody; vi) selection and cloning of a hybridoma that produces the desired antibody; vii) isolation of antibodies from the supernatant above said clones; viii) transfer of said clones intraperitoneally into mice; and ix) collection of malignant ascites or serum from the mentioned mice. 7. A percentage for detecting a lack or excess of OKM1+ cells in a patient, indicated by the fact that it comprises the reaction of a monocyte preparation from said patient with a diagnostically effective amount of antibody from Claim 11 and measuring the percentage of the monocyte population that reacts with said antibody. 8. A method for treating OKM1+ cell excess in a patient in need of such treatment, characterized in that it comprises administering to said patient an amount of the antibody from Claim 11 that is effective for reducing the amount of OKM1+ cells. 9. Diagnostic preparation for the detection of OKM1+ cells, to see if they are in excess or deficit, indicated by the fact that it comprises, in a mixture with a diagnostically acceptable carrier, the amount of antibody from Claim 11 that is effective for detecting the excess or lack of OKM1+ cells. 10. Therapeutic preparation, characterized in that it comprises, in a mixture with a pharmaceutically acceptable carrier, an amount of antibody from Claim 11 that is effective for reducing the amount of OKM1+ cells in a patient who has an excess of OKM1+ cells. 11. A murine monoclonal antibody, characterized in that it reacts with an antigen present on normal human monocytes and granulocytes, but not with normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines, or tumor cells T or B cell lines. 12. A method for making a monoclonal antibody that reacts with an antigen present on normal human monocytes and granulocytes, but not with normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines, or tumor cells of T or B cell lines , characterized in that it comprises culturing hybridoma ATCC CRL 8026 in a suitable medium and isolating antibodies from the supernatant above said hybridoma. 13. A method for making a monoclonal antibody that reacts with an antigen found on all normal human monocytes and granulocytes, but not with normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines, or T or B cell tumor cells line, characterized by the fact that it comprises injecting the ATCC CRL 8026 hybridoma mouse and isolating antibodies from malignant ascites or serum of said mouse. 14. A method for making a monoclonal antibody that reacts with an antigen present on normal human monocytes or granulocytes, but not with normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines, or tumor cells of T or B cell lines , indicated by the fact that it includes the stages: i) immunization of mice with human mononuclear cells; ii) separating the spleen from said mice and making a suspension of spleen cells; iii) condensation of said spleen cells with murine myeloma cells in the presence of a condensation promoter; iv) diluting and culturing condensed cells in special wells in a medium that will not support uncondensed myeloma cells; v) evaluation of the supernatant in each well containing the hybridoma for the presence of antibodies to E+ and E- lymphocytes of human peripheral blood; vi) selection and cloning of a hybridoma that produces an antibody that reacts with an antigen found on normal human monocytes and granulocytes, but not with normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines, or T or B cell tumor cells lines; vii) isolation of antibodies from the supernatant above the mentioned clones. 15. A method for making a monoclonal antibody that reacts with an antigen present on normal human monocytes and granulocytes, but not with normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines, or tumor cells of T or B cell lines , indicated by the fact that it includes the stages: i) immunization of mice with human mononuclear cells; ii) separating the spleen from said mice and making a suspension of spleen cells; iii) condensation of said spleen cells with murine myeloma cells in the presence of a condensation promoter; iv) diluting and culturing condensed cells in special wells in a medium that will not support uncondensed myeloma cells; v) evaluation of the supernatant in each well containing the hybridoma for the presence of antibodies to E+ and E- human lymphocytes; vi) selection and cloning of a hybridoma that produces an antibody that reacts with an antigen found on normal human monocytes and granulocytes, but not with normal human peripheral T cells, B cells, null cells, thymocytes, lymphoblastoid cell lines, or tumor T or B cells cell lines; vii) transfer of said clones intraperitoneally into mice; and viii) collecting malignant ascites or serum from said mice, wherein the ascites or serum contains the desired antibody.