HRP940830A2 - Hybrid cell line for producing monoclonal antibody to a human t cell antigen, antibody, and methods - Google Patents

Description

This invention relates mainly to new hybrid lines and more specifically to biridic cell lines for the production of a monoclonal antibody to an antigen found on essentially all normal human peripheral T cells and on approximately 95% of normal human thymocytes, to diagnostic procedures and preparations using these antibodies.

Condensation of murine myeloma cells to spleen cells from immunized mice 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 that produces a homogeneous (so-called "monoclonal") antibody . After this initial work, many efforts were made to produce various hybrid cells (called “hybridomas”) and to use the antibody generated by these hybridomas for various scientific research. See, for example, Current Topics in Microbiology and Immunology, Volume 81 - “Lymphocyte Hybridomas”, F. Melchers, M. Pottar, and N. Warner, Editors, 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, 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 the rewards and the complications of attempting to produce a monoclonal antibody from a hybridoma. Although the general technique is well understood conceptually, there are many difficulties encountered and many variations required for each specific case. In fact, there is no certainty, before attempting to make a given hybridoma, that the desired hybridoma will be obtained, that it will produce an antibody if obtained, or that the antibody thus produced will have the desired specificity. The degree of success is influenced mainly by the type of antigen used and the choice of technique used to isolate the desired hybridoma.

Attempted production of monoclonal antibody for human surface lymphocyte antigens has been described 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 human lymphoblastoid leukemia and human chronic lymphocytic leukemia cell lines. Many of the resulting hybridomas appeared to produce antibodies 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 construction and testing of hybridomas that produce 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. Sci., 76, 4061-4065 (1979); and Kung, P:C:, et al., Science, 206, 347-349 (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 the henopoietic cell line. While inside the thymus, the differentiating cells are called "thymocytes". mature T cells leave the thymus and circulate between tissues, lymphatic vessels, and the bloodstream. These T cells form a large part of the space for the recirculation of 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 the 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 cell cooperation is not yet fully understood.

Another class of lymphocytes (cells derived from bone marrow or B cells) are those that secrete antibody. They also develop from the hemopoietic cell line, but their differentiation is not determined by the thymus. In birds, they differentiate into an organ analogous to the thymus, called the Bursa or Fabricius. In mammals, however, no equivalent organ has been discovered, although these B 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", "supersor" and "killer" T cells, which have the function of (respectively) promoting a reaction, suppressing a reaction, suppressing a reaction, or destroying (dissolving) foreign station. These subclasses are well understood for mouse systems, but have only recently been discovered for human systems. See, for example, R. L. Evans et al., Journal of Experimental Medicine, Volume 145, 221-232, 1977, and L. Chess on S. F. Schlosaman-“Functional Analysis 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 suppress 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 Lymphomas”, S. Thierfelder, et al., British Journal of Haemotology, 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 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 "supersor" T cells or a deficiency of "helper" T cells. Malignant conditions are mostly accompanied by an excess of "suppressor" T cells.

In certain leukemias, excess T cells are produced in an arrested phase of development. Diagnosis may then depend on the ability to detect this imbalance or excess. see, for example, J. Kersey, et al., “Surface Markers Define Human Lymphoid Malignancies Transfusion, Volume 20, Springer-Verlag, 1977, 17-24 and references cited therein; and E.L. Reinherz, et al., J. Clin, Invest., 64, 392-397 (1979).

Acquired agammaglobulinemia, a disease state in which immune globulin is not produced, includes at least two distinct types. In type I, the failure to produce immune globulin is due to an excess of suppressor T cells, while type II is due to 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 that are responsible for the actual secretion of antibodies, however, these B cells are either suppressed or "not helped", leading to greatly reduced or scant production of immune globulin. 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.

On the therapeutic side, there are some suggestions, although still unproven, that the administration of antibodies against the T cell subtype in excess may have a therapeutic effect in autoimmune or malignant disease. For example, cancer helper T cells (certain cutaneous lymphoma T cells and certain acute lymphoblastic leukemia T cells) can be treated with an antibody to a helper T cell antigen. Treatment of autoimmune disease caused by an excess of helper cells can also be achieved in the same way. Treatment of diseases (eg, malignancies or acquired agammaglobulinemia type I) due to excess suppressor T cells can be treated by administration of antibodies to suppressor T cell antigens.

Of course, antisera against an entire class of human T cells (so-called antihuman thymocyte globulin or ATG) are 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 this rejection process. see, for example, Cosimi, et al., “Randomized Clinical Trial of ATG in Cadaver Renal Allograft Recipients: Impertance of T Cell Nomitoring,” Surgery 40: 155-163 (1976) and references therein; and Wechterm et al., "Manufacture of Antithymocyte Globulin for Clinical Trials", Transplantation, 28 (4), 303-307 (1979).

Identification and suppression of human T cell classes and subclasses has previously been achieved using spontaneous autoantibodies or human T cell-selective antisera obtained by immunizing animals with human T cells, bleeding the animals to obtain serum, and adsorbing the antisera with (for example) autologous but not allogeneic B cells so that antibodies with unwanted reactivity are removed. The production of 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 elicits the production of antibodies against a variety of antigens found in all human injected 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 low, (eg, inactive at dilutions greater than 1:100), and the ratio of specific to nonspecific antibody is less than 1:106.

See, for example, the article by Chese and Sxhlossman referred to above (on pages 365 et seq.) as well as the article in Chemical and Engineering News referred to above, where the disadvantages and advantages of prior art antisera are described monoclonal antibody.

Extract from the invention

A new hybridoma (designated OKT11) has now been discovered that is capable of producing new monoclonal antibodies against an antigen found on essentially all normal peripheral T cells and on approximately 95% of normal human thymocytes, but on normal human B cells or null cells.

The antibody thus produced is monospecific to a single determinant on essentially all normal human peripheral T cells and contains essentially no other anti-human immune globulin, in contrast to antisera of the prior art (which are inseparably contaminated with antibody reactive to numerous human antigens) and monoclonal antibodies of earlier sciences (which are not monospecific for human T cells and thymocyte antigen). Moreover, this hybridoma can be cultured to produce antibody without the need to immunize and kill the animals, followed by the complicated steps of adsorption and purification required to obtain even the impure sera of earlier science.

Accordingly, one object of the invention is to provide hybridomas that produce antibodies against an antigen found on essentially all normal human peripheral T cells.

A further aspect of the 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 present on substantially all normal human peripheral T cells.

A still further object is to provide methods for treating or diagnosing diseases 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 novel antibodies to an antigen found on substantially all normal human peripheral T cells and on approximately 95% of normal human thymocytes (but not on normal human T cells or null cells). , the antibody itself and diagnostic and therapeutic procedures using antibodies. The hybrid is made mainly according to the procedure given by Milstein and Kohler. After immunization of mice with leukemic cells from humans with T-cell acute lymphoblastic leukemia (T-ALL), spleen cells from the immunized mice were fused with cells of a murine myeloma line and the resulting hybridomas analyzed with saturated liquids for those containing an antibody that selectively binds to E rosette positive human T cells and/or E-human cells. The desired hybridomas were later cloned and characterized. as a result, antibody-producing hybridomas (designated OKT11) against antigens on essentially all normal human peripheral T cells were obtained. Not only does this antibody react with essentially all normal peripheral T cells, but it also reacts with about 95% of normal human thymocytes but does not react with normal human T cells or null cells.

Given the difficulties indicated in the prior art and the lack of success 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 hybrid cells does not allow extrapolation from one antigen or cell system to another.

In fact, the present applicants have found that the use of a cell line of malignant T cells or purified antigens separated from the cell surface as antigens has been largely unsuccessful.

Both the hybridoma in question and the antibody produced here are identified by the designation “OKT11”, and which particular material is in question is obvious from the context. The hybridoma in question was deposited on December 13, 1979, in the American Type Culture Collection, 12301 Parklawa Drive, Reekville, Maryland 20352, and given ATOC accession number CRL 8027.

The construction and characterization of the hybridoma and the resulting antibody will be better understood by reference to the following description and Examples.

Detailed description of the invention

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

A. Immunization of mice with leukemic cells from humans with T-ALL. Although female CAF1 mice are found to be preferred, it is contemplated that other mouse strains may be used. The schedule of immunization and the concentration of thymocytes should be such that useful quantities of appropriately primed splenocytes are produced. Three immunizations at 14-day intervals with 2 x 107 cells/mice/injection in 0.2 ml of phosphate buffered saline were found to be effective.

B. Separation of spleens from immunized mice and preparation of spleen suspension in appropriate medium. About one ml of medium per spleen is enough. This experimental technique is well known.

C. Condensation of suspended spleen cells with murine myeloma cells using a suitable condensation promoter. A desirable ratio of about 5 spleen cells per myeloma cell. The total volume of about 0.5 - 1.0 ml of condensation medium corresponds to about 108 splenocytes. Many murine myeloma cell lines are known and available, mostly from members of the academic community at various depository banks, such as the Salk Institute Coll Distribution Center, La Jolla, Ca. The cell line used should preferably be of the so-called "drug-resistant" type, so that uncondensed myeloma cells will not survive in the chosen environment, 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 preferable that the myeloma cell line used is of the so-called "non-secreting type", in that it does not produce any antibody on its own, although secreting types can also be used. However, in certain cases, myeloma-secreting lines may be desirable. 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 support the uncondensed myeloma cells for a sufficient period to allow death of the uncondensed cells (about one week). The dilution can be of the limiting type, in which the volume of diluent is calculated statistically to isolate a certain number of cells (eg, 1.4) in each separate container (eg, each well of a microtiter plate). A medium is one (eg, HAT medium) that will not support an unfused myeloma line that is drug-resistant (eg, resistant to 8-azaguanidine).

This is why these myeloma cells disappear. Since non-condensed spleen cells are non-malignant, they have only a limited number of generations. Thus, after a certain period of time (about one week) these uncondensed spleen cells no longer reproduce, on the other hand, the condensed cells continue to reproduce because they have the malignant quality of primary myeloma and the ability to survive in the selective medium of primary spleen cells.

E. Evaluation of the supernatants in each container (well) containing the hybridoma for the presence of antibodies to E rosette positive purified human T cells or thymocytes.

A. Selection (eg, by limiting dilution) cloning of a hybridoma that produces the desired antibody

Once the desired hybrid is 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 hybrid medium for a suitable period of time, after which the desired antibody is isolated from the supernatant. The appropriate substrate and the appropriate length of cultivation time are known and easily determined. This in vitro technique produces an essentially monospecific monoclonal antibody, essentially free of other specific antihuman immune globulin. There is a small amount of other immune globulin present since the medium contains serum (eg, fetal calf serum). However, this in vitro procedure may not produce a sufficient amount or concentration of antibody for some 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.

The hybridoma will cause the formation of tumors that produce antibodies 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 extrudate (ascites) of the host mouse. Although these host mice also have normal antibodies in their blood and ascites, the concentration of these normal antibodies. Moreover, since these normal antibodies are not antihuman in their specificity, the monoclonal antibody obtained from collected ascites or from serum is essentially free of any contaminating antihuman immune globulin. This monoclonal antibody is of high titer (active at dilutions of 1:50,000 or more) and has a high ratio of specific to non-specific immune globulin (about 1/20). The produced immune globulin together with myeloma light chains are non-specific, "nonsense" peptides that only dilute the monoclonal antibody without reducing its specificity.

Examples.

Production of monoclonal antibodies

A. Immunization and somatic cell hybridization

Female CAF1 mice (Jackson Laboratories; 6-8 weeks old) were immunized intraperitoneally with 2 x 107 human leukemic T-ALL cells in 0.2 ml of phosphate-buffered saline at 14-day intervals. Four days after the third immunization, the spleens were removed from the mice and a single-cell suspension was made by pressing the tissue through a stainless steel sieve.

Cell condensation was performed according to the procedure developed by Kohler and Milstain. 1 x 108 splenocytes are condensed in 0.5 ml of condensation medium comprising 35% polyethylene glycol (PEG 1000) and 5% dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, NY) with 2 x 107 P3x63Ag8Ul myeloma cells provided by Dr. M. Scharff, Albert Einstein College of Medicine, Bronx, NY. These myeloma cells secrete IgG1 light chains.

B. Selection and growth of hybridomas

After cell condensation, the cells were cultured in HAT medium (hypoxanthine, aminopterin and thymidine) at 37ºC with 5% CO2 in a humid atmosphere. Several weeks later, 40 to 100 μl of the supernatant from the hybridoma-containing cultures is added to a pellet of 106 peripheral lymphocytes that have been separated into E rosette positive (E+) and E rosette negative (E-) populations, prepared from the blood of healthy human donors such as was described by Mandes (J. Immunol. 111:860, 1973). Detection of mouse hybridoma antibodies binding to these cells was determined by indirect immunofluorescence. Cells incubated with culture supernatants were stained with fluorescent goat-mouse IgG (G/M FITC) (Meloy Laboratories, Springfield, VA; F/F=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. Cultures of hybridomas containing antibodies that react specifically with E+ lymphocytes (T cells) and/or thymocytes are selected and cloned twice using limiting dilution procedures in the presence of seeded cells. Later, the clones were transferred intraperitoneally by injecting 1 x 10 7 cells of the given clones (0.2 ml volume) into CAF1 mice primed with 2,5,10,14-tetramethylpentadecane, sold by Aldrich Chemical Company under the name Pristine. Malignant ascites from these mice were then used to characterize lymphocytes as described below in Example II. It was demonstrated by standard techniques that the OKT11 hybrid antibody in question was of the IgG1 subclass.

Example II

Characterization of OKT5 reactivity

A. Isolation of the lymphocyte population

Human peripheral blood mononuclear cells were isolated from healthy volunteer donors (aged 15-40) using Picoll-Hypaque density gradient centrifugation (Pharmacia Fins Chemical, Piscataway, NJ) according to the technique described by Boyum, Soand, J. Clin, Lab. . Invest. 21 (Suppl. 97):77, 1968. Unfractionated mononuclear cells were separated into surface Ig+ (B) and Ig-.(T plus none) populations by chromatography on a Sephadex G-200 anti F (ab`)2 column such as previously described by Chesa, et al., J. Immunol. 113:1113 (1974). T cells were isolated using E rosette Ig- Population with 5% sheep erythrocytes (Microbiological Associates, Bethesda, HD). The rosette mixture was layered over Picoll-Hypaque and the isolated E+ was treated with 0.155H NH4Cl (10 ml per 108 cells). The resulting population was 2% EAC rosette positive and 95% E rosette positive as determined by standard procedures. Next, the non-rosetted Ig- (Null cell) population was picked from the Ficoll interface. This later population was 5% E+ and 2% Ig+. The surface Ig+(B) population thus obtained from the Bephadex C-200 column was obtained after elution with normal human gamma globulin as described earlier. This population was 95% surface Ig+ and 5% E+.

B. Isolation of thymocytes

Normal human thymus was obtained from patients aged 2 months to 14 days undergoing corrective heart surgery. Freshly obtained parts of the thymus gland were immediately placed in 5% fetal calf serum in medium 199 (Gibco), where the parts were finely chopped with forceps and scissors, and later a single-cell suspension was made by pressing through a wire sieve. Cells were 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 Cell lines

A transformed Epstein-Barr virus (EBV) B cell line from a normal individual (Laz 156) was provided by Dr. H. Lazarus, Sidney Farber Institute, Boston, HA.

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 conjugated with fluorescein (G/M FITC) (Heloy Laboratories) using a Cytofluorograf FC200/4800A (Ortho Instruments). Briefly, 1 x 106 cells are treated with 0.15 ml of OKT5 at a dilution of 1:500, incubated at 4°C for 30 minutes and washed twice. The 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 three times. The cells are then analyzed on a Cytofluorograph and the fluorescence intensity per cell is recorded on a pull-in height analyzer. A similar pattern of reactivity was seen for the 1:10,000 dilution, but further dilution caused loss of reactivity. Baseline staining was achieved by substituting a 0.15 ml aliquot of 1:500 ascites from CAF1 mice injected intraperitoneally with non-producing hybrid clones.

Brief description of the drawing

Figure 1 shows the fluorescence scheme obtained on the Cytofluorograf after the reaction of normal human thymocytes and R + and E- peripheral cells with OKT11 and other monoclonal antibodies at a 1:5 dilution with G/N FITC. Baseline fluorescent staining was achieved by incubating each population with a 1:500 dilution of ascitic fluid from mice injected with the non-producing clone.

Hybridoma production and production and characterization of the resulting monoclonal antibody were performed as described in the Examples above. Although large quantities of the subject antibody have been made by injecting the subject hybridoma intraperitoneally into mice and collecting malignant ascites, it is clear that it is intended that the hybridoma can be cultivated in vitro by well-known techniques in science and the antibody is separated from the saturated liquid.

Table 1 shows the reactivities of OKT1, OKT3-6 and OKT-11 with various human lymphoid cell populations. This reactivity scheme is one test by which the subject OKT11 antibody can be detected and distinguished from other antibodies.

Figure 1 shows a representative scheme of fluorescence obtained on a Cytofluorograf after the reaction of normal human thymocyte suspensions and E- and E + peripheral cells with a 1:500 dilution of OKT11, OKT10, OKT8 and G/M MITC. In contrast to OKT1 and OKT3 T cell antigens (which increase when thymocytes mature into peripheral T cells), OKT11 antigen is competitively decreased. The reactivity scheme in Figure 1 is another test by which the subject OKT11 antibody can be detected and distinguished from other antibodies.

Table 2 shows the antigen phenotypes of human T binding lymphocytes, using OKT11 and other monoclonal antibodies. This phenotype provides a further way to detect the OKT11 antibody and to distinguish it from other antibodies.

Table 3 shows the relationship between peripheral T cell levels and T cell subsets and various disease states. These ratios can be used for various diagnostic purposes (eg, for the detection of acute infectious mononucleosis) by analyzing a blood sample of a suspected individual who may be suffering from these disease states to determine T cell levels AND T cell subsets. These ratios can also be used for therapeutic purposes when the pattern of the disease state is an elevated level of a subset of T cells (eg, acquired Type I agammaglobulinemia). For therapeutic use, administration of the appropriate monoclonal antibody to a patient with elevated levels of a subset of T cells will reduce or eliminate the excess. The relationships shown in Table 3 are a further way in which the OKT11 antibody can be detected and distinguished from other antibodies.

Other antibody-producing monoclonal 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 33,669, filed April 26, 1979; I SN 76,652, submitted on September 18, 1979; I SN 82-515, filed Oct. 9, 1979. Further monoclonal antibody-producing hybridomas made by the present applicants (designated OKT6, OKT8, OKT9, and OKT10) have been described and patented in U.S. Pat. Patent applications filed December 4, 1979, entitled: Hybrid Cell Line for Production of Monoclonal Antibody to Human Thymocyte Antigen, Antibodies and Methods; A hybrid cell line for the production of a complement-fixing monoclonal antibody to human suppressor T-cells, Antibody I procedures; A hybrid cell line for the production of a monoclonal antibody to human early thymocyte antigen, antibody I procedures; I Hybrid cell line for production of monoclonal antibody to human antimyocyte antigen, antibody I procedures. Further hybrid antimyocyte antigen, antibody and procedures. A further hybrid made by the present applicants (designated OKM1) is described and protected in U.S. Pat. The patent application filed on the same day as this I is entitled: Hybrid cell line for production of monoclonal antibody to human monocyte antigen, antibody I methods.

These applications are incorporated herein by reference.

According to the present invention there is provided a hybridoma capable of producing an antibody against an antigen found on essentially all peripheral T cells AND on approximately 95% of normal human thymocytes, a process for producing this hybridoma, a monoclonal antibody against an antigen found on essentially all normal human peripheral T cells I on approximately 95% of all normal human thymocytes, methods for the production of antibodies and methods and preparations for the treatment or diagnosis of disease or for the identification of subclasses of T cells using this antibody.

TABLE 1

DISTRIBUTION OF OKT ANTIGEN IN CELLS AND TISSUES

[image]

*number of tested samples

** average + IS.D.

+ is not specified

++ five samples of peripheral blood and thymus were tested

TABLE 2

NATIGEN PHENOTYPES OF HUMAN T BINDING LYMPHOCYTES

[image]

W = very weak immunofluorescence

TABLE 3

PERIPHERAL MONONUCLEAR CELL LEVELS IN DISEASE STATES MONONUCLEAR CELL LEVELS

[image]

N = within normal limits;

O = absent;

+ = above normal;

++ = well above normal;

- = below normal;

-- = very below normal;

* = these levels return to normal about one week before clinical symptoms disappear

The numbers in parentheses indicate the number of examined patients.

Although only one hybridoma has been described that produces a single monoclonal antibody against a human thymocyte antigen, it is understood that the present invention encompasses all monoclonal antibodies that exhibit the characteristics described herein.

The subject antibody OKT11 was determined to belong to the IgG1 subclass, which is one of the four subclasses of mouse IgG immune globulin. These immunoglobulin C subclasses differ from each other in so-called "fixed" regions, although an antibody to a specific region will have a so-called variable region that is functionally identical regardless of which immunoglobulin G subclass it belongs to. That is, a monoclonal antibody exhibiting the characteristics described herein may be of the IgG1, IgG2a, IgG2a, IgG2b, or IgG3 subclasses, or of the IgM, IgA, or other Ig classes mentioned. Differences between these classes or subclasses will not affect the selectivity of the reaction scheme of the antibody, but may affect the further reaction of the antibody with other materials, such as (for example) the complement of anti-mouse antibodies. Although the specific antibody is IgG1, it is understood that antibodies having the reactivity described herein are included in the subject invention regardless of which immunological class or subclass they belong to.

Further included within the present invention are methods for making the monoclonal antibodies described above using the hybridoma techniques illustrated herein. Although only one example of a hybridoma is provided herein, it is understood that one skilled in the art can use the immunization, condensation, and selection procedures provided herein to obtain other hybridomas capable of producing antibodies having the reactivity characteristics described herein. Since individual hybridomas produced from a known murine myeloma cell line and spleen cells from known species of mice cannot be further identified except by reference to the antibody produced by the hybrid, it is understood that all hybridomas producing an antibody having the reactivity characteristics described above are included in the subject invention, as well as methods for making this antibody using hybridomas.

Further aspects of the invention are methods for treating or diagnosing disease using the OKT11 monoclonal antibody or any other monoclonal antibody exhibiting the reactivity pattern shown herein. The subject antibody can be used to diagnose disease states using the OKT11 antibody either alone or in combination with other antibodies (eg, OKT3 - OKT10). Reactivity patterns with a panel of antibodies to T cells and subsets of T cells may allow precise detection of certain disease states better than is possible using earlier diagnostic procedures.

Treatment of undesirable conditions that manifest themselves as an excess of OKT11+ cells can be achieved by administering a therapeutically effective amount of OKT11 antibody to an individual in need of such treatment. By selectively reacting with the OKT11+ antigen, an effective amount of OKT11 antibody will reduce the excess of OKT11+ cells, thus mitigating the effects of the excess. Diagnostic and therapeutic preparations comprising effective amounts of OKT11 antibody in admixture with diagnostically or pharmaceutically acceptable carriers are also included in the present invention.

Since peripheral T cells are responsible for rejection of callosum, one example of therapeutic use of OKT11 antibodies is to administer to a callow recipient an amount of OKT11 antibody effective to reduce or eliminate callow rejection. In this way, the OKT11 antibody can be substituted for the anti-thymocyte globulin discussed above with a significant increase in specificity.

Claims (6)

1. Procedure for making a monoclonal antibody that: a) reacts with essentially all normal human peripheral T cells and with approximately 95% of normal human thymocytes, but not with normal human B cells or null cells; shows the scheme of reactivity to normal human thymocytes, E+ and E- cells shown in Figure 1; c) shows the scheme of reactivity to T-binding lymphocyte fomotypes, which is shown in Table 2; d) defines a T cell population (OKT11) that is lower than normal levels in myasthenia gravis and multiple sclerosis; it is higher than normal levels in acute graft vs host reaction, hyper IgE, acute infectious mononucleosis, and primary biliary cytosis; and is completely absent in all stages of Hodgkin's disease and psoriasis, indicated by the fact that it includes the stages: i) immunization of mice with human leukemic T-ALL cells; ii) separating the spleen from the mentioned 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) assessing the saturated liquid in each well containing the hybridoma for the presence of the desired antibody; vi) selection and cloning of hybridomas that produce the desired antibody; vii) isolation of antibodies from the upper saturated liquid above said clones. 2. Procedure for the production of monoclonal antibodies which: a) reacts with essentially normal peripheral T cells and with approximately 95% of normal human thyrocytes but not with normal human B cells or null cells; b) shows the scheme of reactivity to normal human thymocytes, E+ and E- cells shown in Figure 1; c) shows the scheme of reactivity according to T-binding lymocyte phenotypes, which is presented in Table 2; d) defines a T cell population (OKT11) that is lower than normal levels in myasthenia gravis and multiple sclerosis; higher than normal levels in acute cholera vs host reaction, hyper IgE, acute infectious mononucleosis and primary biliary cirrhosis; and is completely absent in the stages of Hodgkin's disease and psoriasis, indicated by the fact that it includes the stages: i) immunization of mice with human leukemic T-ALL cells; separating the spleen from the mentioned 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) dilution and cultivation of condensed cells in special wells in a medium that will not support uncondensed .-nieloma cells; v) assessing the saturated liquid in each vial containing the hybridizer for the presence of the desired antibody; selection and cloning of antibodies produced by the hybridoma; vii) isolation of antibodies from the saturated liquid above said clones; viii) transfer of the mentioned clones intraperitoneally into mice; ix) collection of malignant ascites or serum from the mentioned mice. 3. A process for producing a monoclonal antibody that reacts with substantially all normal human peripheral T cells and with approximately 95% of normal human thymocytes, but not with normal human 3 cells or with null cells, comprising culturing hybridoma ATCC CRL 9027 in a suitable media and isolation of antibodies from the saturated liquid above the mentioned hybridomas. 4. A method for producing a monoclonal antibody that reacts with essentially all normal human peripheral T cells and with approximately 95% of normal human thymocytes but not with normal human B cells or with null cells, characterized in that it comprises injecting into mice the hybridoma ATCC CRL 9027 and isolation of antibodies from malignant ascites or serum from the mentioned mice. 5. A process for producing a monoclonal antibody that reacts with substantially all normal human peripheral T cells and with approximately 95% of normal human thymocytes but not with normal human B cells or null cells, as indicated, comprising the steps of: immunizing mice with human leukemic T-ALL cells; ii) separating the spleen from the mentioned 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) dilution and culturing of condensed cells in special wells in a substrate that will not undermine uncondensed myeloma cells; v) assessment of the saturated liquid in each vial containing the hybridoma for the presence of antibodies to E rosette positive purified human thymocyte T cells; vi) selecting and cloning an antibody produced from the hybridoma that reacts with essentially all normal human peripheral T cells and with approximately 95% of normal human thymocytes, but not with normal human B cells or null cells; and vii) isolation of antibodies from the saturated liquid above said clones. 6. A method for producing a monoclonal antibody that reacts with substantially all normal human peripheral T cells and with approximately 95% of normal human thymocytes, but not with normal human B cells or null cells, comprising: i) immunization of mice with human leukemic and T-ALL cells; ii) separating the spleen from the mentioned mice and making a suspension from spleen cells; iii) condensation of said spleen cells with murine myeloma cells in the presence of a condensation promoter; iv) diluting and culturing the condensed cells in suitable special wells in a medium that will not support uncondensed myeloma cells; v) assessment of the saturated fluid in each vial containing the hybridoma for the presence of antibodies to E rosette positive purified T cells or human thymocytes; vi) selecting and cloning a hybridoma that produces an antibody that reacts with essentially all normal peripheral human T cells and with approximately 95% of normal human thyphocytes, but not with normal human B cells or with null cells; vii) transfer of the mentioned clones intraperitoneally in mice; and viii) collecting malignant ascites from said mice, wherein all ascites or serum contain the desired antibody.