HRP940822A2 - Hybrid cell line for producing complement-fixing monoclonal antibody to a human supressor t cells, antibody and methods - Google Patents

Description

Field of invention

This invention relates generally to a novel set of hybrid cells and more specifically to sets of hybrid cells for the production of complement-binding monoclonal antibodies to an antigen found on normal human suppressor T cells, to the antibodies thus produced. as well as to therapeutic and diagnostic procedures and preparations that use these antibodies.

State of the art

The fusion of murine myeloma cells with spleen cells of 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 set for obtaining homogeneous (so-called "monoclonal") antibodies. Since this initial work, many efforts have been focused on obtaining different hybrid cells (called "hybridomas") and on using the antibodies obtained from these hybridomas for various scientific research. See, for example, the notes in Current Topics in Microbiology and Immunology, Volume 81, "Lymphocyte Hybridomas", F. Melchers, M. Potter, N. Warner, Editors, Springer-Verlag, 1978; 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 articles simultaneously indicate the successes and difficulties of attempts to obtain monoclonal antibodies from hybridomas. easily the general technique of the procedure is well understood, many difficulties arise and variations are required for each individual case. In fact, there is no guarantee during the preparation of obtaining a given hybridoma, that it will be obtained with the desired hybrid, that even if it is obtained, it will produce antibodies or that the antibodies thus obtained will have the desired properties. The degree of success is largely dependent on the type of antigen used and the selection technique used to isolate the desired hybridoma.

Attempts to obtain monoclonal antibodies for antigens on the cell surface of human lymphocytes have been published in only a few cases. See, for example, Current Topics in Microbiology and Immunology, ibid, 66-69 and 164-169. The antigens used in these examples are from cell lines of cultured cells of human lymphoblastoid leukemia and human chronic lymphocytic leukemia. Many resulting hybridomas produce antibodies to different antigens on all human cells. None of the hybridomas produce antibodies to the previously defined group of human lymphocytes. More recently, the inventors of this invention and others have been preparing and testing hybridomas that produce antibodies to specific 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 is understandable that there are two basic classes of lymphocytes found in the immune system of humans and animals. The first of them (thymus-derived cells or T cells) differentiate in the thymus from hematopoietic cells. While located within the thymus, the differentiating cells are called "thymocytes". Mature T cells leave the thymus and circulate between lymph tissue and the bloodstream. These T cells form a large part of the recycling small lymphocytes. They have immunological specificity and are directly involved in cell-mediated immune responses (such as rejection of division) as executive cells. Although T cells do not secrete humoral antibodies, they are sometimes necessary for the secretion of these antibodies by another group of lymphocytes, which is explained below. Some T cells play a regulatory role in other aspects of the immune system. The mechanism of action of such cellular cooperation has not yet been fully explained. Another group of lymphocytes (cells derived from the bone marrow or B cells) are those cells that secrete antibodies. They also develop from the hematopoietic apparatus, but their differentiation is not determined by the thymus. In birds, they differentiate into an organ analogous to the thymus, called the Bursa of Fabricius. In mammals, of course, no equivalent organ has yet been discovered, and for now it is thought that B cells differentiate within the bone marrow.

Now it becomes clear that T cells are divided into at least several sub-types, called "helper", "suppressor" and "killer" T cells, which have the role of supporting the reaction, that is, suppressing the reaction, that is, killing (lysing) foreign cells. These subtypes are well known for closed 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 Chees and S.F. Schlossman "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 disable T cell types and subtypes is important for diagnosing or treating various immunoregulatory disorders and conditions. For example, certain leukemias and lymphomas have different prognoses depending on whether they are of B cell or T cell origin. Thus, the assessment of the prognosis of the disease depends on the ability to differentiate between these two types of lymphocytes. See, for example, A.C. Aisenberg and J.C. Long., The American Journal of Medicine, 58:300 (March, 1975); D. Belpome, et al., in "Immunologist Diagnosis of Leukemias and Lymphomas", 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, malignant diseases, and agammaglobulinemia) are associated with an imbalance of T cell subtypes. It is generally believed that autoimmune diseases are associated with an excess of "helper" T cells or a deficiency of certain "suppressor" T cells, while agammaglobulinemia is associated with an excess of certain "suppressor" T cells or a deficiency of "helper" T cells. Malignant diseases are associated with an excess of "suppressor" T cells. In certain leukemias, excess T cells are produced at an arrested stage of development. The diagnosis may therefore depend on the ability to detect this imbalance or excess and on determining which developmental stage is in the excess. See, for example, J. Kersey, et al. "Surface Markers Define Human Lymphoid Malignancies with Differing Prognoses" in Hematology and Blood Transfusion, Volume 20, Springer-Verlag, 1977, 17-24, and corresponding notes therein; and E.L. Reinherz, et al., J. Clin. Invest., 64, 392-397 (1979).

Acquired agammaglobulinemia, a disease in which immune globulin is not produced, contains at least two different types. In type I there is an error in the production of immunoglobulins due to an excess of suppressor T cells, while in type II the reason is a lack of helper T cells. In both types, there is no disorder or deficiency of the patient's B cells, the lymphocytes responsible for the secretion of antibodies; of course, these B cells are either suppressed or not "helped", resulting in greatly reduced or absent immunoglobulin production. The type of acquired agammagfobulinemia 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, which have not yet been proven, that the use of antibodies against the subtype of T cells that are in excess may have a beneficial effect on autoimmune disease or malignancy. For example, cancer helper T cells (certain cutaneous lymphoma T cells and certain lymphoblastic leukemia T cells) can be treated with antibodies to a helper T cell antigen. Treatment of an autoimmune disease caused by an excess of helper cells can also be carried out in a similar way. Treatment of diseases (eg, malignant diseases or acquired agammaglobulinemia type 1) due to an excess of suppressor T cells can be carried out by the use of antibodies for suppressor T cell antigens.

Antiserum against an entire class of human T cells (also called antihuman thymocyte globulin or ATG) has been usefully administered therapeutically in organ transplant patients. Because the cell-mediated immune response (the mechanism by which transplants are rejected) depends on T cells, changing antibodies to T cells prevents or slows down this rejection process. See, for example, Cosimi et al., "Randomized Clinical Trial of ATG in Cadaver Renal Allograft Recipients: Importance of T Cell Monitoringn", Surgery 40:155-163 (1976) and the notes contained therein.

Identification and suppression of sub-classes and classes of human T cells has previously been accomplished using spontaneous antibodies or selective antisera to human T cells obtained by immunizing animals with human T cells, bled these animals to obtain serum, and adsorptive antisera with (for example ) with autologous but not allogeneic T cells to remove antibodies with undesirable reactivity. The preparation of these antisera is extremely difficult, especially during adsorption and purification. Even adsorbed and purified antisera contain contaminants in addition to the desired antibodies, for several reasons. First, serum contains millions of antibody molecules even before T cell immunization. Second, immunization causes the production of antibodies against various antigens on the injected human T cells. There is no selective production of antibodies against only one antigen. Third, the titer of specific antibodies obtained by these methods is usually quite low, (eg, inactivation at dilutions greater than 1:100) and the ratio of specific to non-specific antibodies is less than 1/106.

See, for example, the notes by Chess and Schlossman (pages 365 et seq.) cited earlier and Chemical and Engineering News cited earlier, which describe the disadvantages of earlier antisera procedures and the advantages of monoclonal antibodies.

Summary of the invention

A new hybridoma (designated OKT8) has been found that can produce novel monoclonal antibodies against an antigen found on normal human suppressor T cells (about 30% of normal human peripheral T cells) and on about 80% of normal human thymocytes, but not on B cells or insignificant cells and on less than 2% of bone marrow cells. In addition, OKT8 monoclonal antibodies bind complement.

The antibody thus produced is monospecific for one determinant on approximately 30% of normal human T cells and almost does not contain any other anti-human immunoglobulin, which is contrary to the antisera obtained by earlier procedures (which are characterized by the fact that they are contaminated with antibodies reactive to numerous human antigens) and with monoclonal bodies obtained by earlier procedures (which are not monospecific for the human suppressor T cell antigen. Furthermore, this hybridoma can be cultured to produce antibodies without the need to immunize and kill the animals, followed by the adsorption and purification necessary to obtain impure antisera according to earlier procedures.

Accordingly, it is one object of the present invention to provide hybridomas that produce antibodies against antigens found on normal suppressor T cells.

A further task of the present invention is to provide methods for the preparation of these hybridomas.

A further object of the present invention is to provide homogeneous antibodies against antigens found on normal human suppressor T cells.

A further object of the present invention is to provide methods for treating or diagnosing a disease or for identifying a sub-class of T cells that contains that antibody.

Other objects and advantages of the invention will become apparent from an examination of the present disclosure.

Happily, this invention provides a novel hybridoma that produces a novel antibody to an antigen found on normal suppressor T cells, then the antibody itself, and diagnostic and therapeutic procedures using the antibody. The hybridoma was prepared using the procedure of Milstein and Kohler. After immunization of mice with normal human thymocytes, spleen cells from the immunized mice were fused with murine myeloma cells and the resulting hybridomas were assayed for those from the supernatant containing antibodies that selectively bind to normal rosette positive T cells and/or thymocytes. The desired hybridomas were immediately cloned and characterized. As a result, it was obtained by a hybrid that produces antibodies (designated as ORT8) against antigens on normal human suppressor T cells (about 30% of normal peripheral T cells). Not only do these antibodies react with about 30% of normal T cells, but they also react with about 80% of normal human thymocytes and less than 2% of bone marrow cells and do not react with B cells or nonessential cells.

In view of the difficulties shown in earlier trials and in view of the lack of success using malignant cell lines as antigens, it is surprising that the procedure presented yields the desired hybridoma. It should be emphasized that the unpredictability of the preparation of cell hybrids does not allow extrapolation of one antigen or cell system to another. In fact, applicants of the present invention have discovered that the use of T cells of a malignant cell line or antigen is completely unsuccessful.

Both the displayed hybrid and the antibodies produced using it are identified here with the designation "OKT8", so that this is clear in the text. Said hybrid was deposited on September 18, 1979 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 and assigned ATCC number CRL 8014.

The preparation and properties of the hybridomas and the resulting antibodies will be more readily understood from the notes of the following descriptions and Examples.

Detailed description of the invention

The hybridoma preparation process generally includes the following steps:

A. Immunization of mice with normal human thymocytes. it was easily found that it was most advantageous to use female CAF1 mice, the use of other strains was also considered. The immunization schedule and concentration of thymocytes should be such that it produces useful amounts of splenocytes. Three fourteen day intervals with 2 x 107 cells/mouse/injection in 0.2 ml of phosphate buffered saline proved to be very effective.

B. Extracting the spleen from the immunized mouse and making a suspension of the spleen in the appropriate medium. About one milliliter of medium per spleen is sufficient. These experimental techniques are well known.

C. Fusion of suspended spleen cells with mouse myeloma cells from an appropriate cell line using an appropriate fusion promoter. A favorable ratio is about 5 spleen cells per myeloma cell. The total volume of about 0.5 - 1.0 ml of fusion medium is suitable for about 108 splenocytes. Many murine myeloma cell lines are known and available, mostly from various repositories 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 unfused myeloma cells do not survive in the selective medium, while hybridomas will. The most common class is the 8-azaguanidine-resistant cell line, which lacks the enzyme hypoxanthine guanidine phosphoribosyl transferase and therefore will not be supported by HAT (hypoxanthine, aminopterin, and thymidine) media. It is also generally favorable that the myeloma cell line used is of the so-called "non-secretory" type, so that it does not produce antibodies on its own, although secretory types can also be used. In certain cases, of course, secretory myeloma formations may be preferred. Although the preferred fusion promoter is polyethylene glycol having an average molecular weight of about 1000 to about 4000 (commercially available as PEG 1000, etc.), other fusion promoters known in the art may be used.

D. Diluting and growing a culture in separate containers of a mixture of unfused spleen cells, unfused myeloma cells, and fused cells in a selective medium that will not support unfused myeloma cells for a time sufficient to allow death of the unfused cells (about a week). The dilution can be of the limiting type so that the volume of the diluent is statistically calculated to isolate a certain number of cells (eg 1-4) in each separate container (eg in each well of the microtiter plate). The medium should be one (eg HAT medium) that does not support resistant non-adherent myeloma cell lines (eg 8-azoguanidine resistant). That's how myeloma cells die. Because unfused spleen cells are not malignant, they only have a certain number of generations. Thus, after a certain time (about one week) these unconnected spleen cells stop reproducing. Adherent cells, on the other hand, continue to reproduce because they have the malignant properties of the primary myeloma and the ability to survive in the selective medium inherited from the spleen cells.

E. Evaluating the supernatant in each container (dish) containing hybridomas for the presence of antibodies to E rosette positive purified human T cells or thymocytes.

F. Selection (eg, by limiting dilution) and cloning of hybridomas that produce the desired antibody.

Once the desired hybridoma is selected and cloned, the resulting antibodies can be produced in one or more ways. The purest monoclonal antibody is produced in vitro by culturing the desired hybridoma in a suitable medium for a suitable period of time, followed by detection of the desired antibody from the supernatant. A suitable medium and suitable duration is known or can be determined. This in vitro technique gives basically monospecific monoclonal antibodies, basically without other specific antihuman immune globulins. There are a small number of other immune globulins that are present because the medium contains xenogenetic serum (eg fetal serum). Of course, this in vitro procedure can produce an insufficient amount or concentration of antibodies for certain needs, because the concentration of monoclonal antibodies is around 50 μg/ml.

To produce a much higher concentration of slightly less pure monoclonal antibodies, the desired hybridoma can be injected into mice, preferably syngenetic or semi-syngenetic mice. Hybridomas will cause the formation of tumors that secrete antibodies after a certain incubation time, which will result in a high concentration of the desired antibody (about 5-20 mg/ml) in the bloodstream and peritoneal exudate (ascites) of the host. Although this host has normal antibodies in his bloodstream and ascites, the concentration of these normal antibodies is only about 5% of the concentration of monoclonal antibodies. Furthermore, because these normal antibodies are not antihuman in their specificity, monoclonal antibodies obtained from pooled ascites or from serum are essentially free of any contaminating antihuman immunoglobulin. This monoclonal antibody has a higher titer (it is active at a dilution of 1:50,000 or more) and a ratio of specific to non-specific immunoglobulin (about 1/20). Immunoglobulins produced by incorporation of myeloma small chains are non-specific, "nonsense" peptides that only dilute monoclonal antibodies without reducing their specificity.

Examples

Production of Monoclonal Antibodies

A. Immunization and somatic cell hybridization

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

Cell fusion is performed according to the procedure developed by Kohler and Milstein. 1 x 108 slenocytes were combined in 0.5 ml of fusion medium containing 35% polyethylene glycol (PEG 1000) and 5% dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, NY) with 2 x 107 P3X63Ag87Ul myeloma cells obtained from Dr. M. Scharff, Albert Einstein College of Medicine, Bronx, NY. These myeloma cells secrete IgG1 K small chains.

B. Selection and growth of Hybridomas

After cell fusion, the cells are cultured in HAT medium (hypoxanthine; aminopterin and thymidine) at 37°C with 5° CO2 in a humidified atmosphere. Several weeks later, 40 to 100 μl of the supernatant from the culture containing the hybridomas is added to a pellet of 106 peripheral lymphocytes separated into E rosette positive (E+) and E rosette negative (E-) populations, which were prepared from the blood of healthy people as described by Mendes (J. Immunol. 111:860, 1973). Detection of murine hybridoma antibodies binding to these cells was determined indirectly by immunofluorescence. Cells incubated with culture supernatant are marked with . fluorescent-coated-anti-mouse IgG (G/M FITC) (Meloy Laboratories, Springfield, VA; F/p = 2.5) and fluorescent antibody-coated cells were immediately analyzed on an FC200/4800A Cytofluorograph (Ortho Instruments, Westwood, MA) as described in Example III. Cultures of hybridomas containing antibodies that specifically react with E+ lymphocytes (T cells) and/or thymocytes were selected and cloned twice using a limiting dilution procedure in the presence of host cells. Immediately thereafter, the clones were transferred intraperitoneally by injecting 1 x 10 7 cells of the given clone (volume 0.2 ml) into CAF1 mice using 2,6,10,14-tetramethylpentadecane from Aldrich Chemical Company under the name Pristine. Malignant ascites from these mice is then used to characterize lymphocytes as described in Example II. The presented OKT8 hybrid antibodies were shown to be of the IgG2a sub-class by the usual procedure.

Example II

Characterization of OKT8 reactivity

A. Isolation of the lymphocyte population

Human peripheral blood mononuclear cells were isolated from healthy volunteer donors (15-40 years) by Ficoll-Hypac graded centrifugation (Farmacia Fine Chemicals, Piscataway, NJ) using the technique of Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97): 77, 1968. Unfractionated mononuclear cells were separated into surface Ig+ (B) and Ig- (T plus Null) populations by Sephadex G-200 anti-F (ab')2 column chromatography as previously described by Chess et al., J. Immunol. 113:1113 (1974). T cells are detected by E rossetting of the Ig- population with 5% sheep erythrocytes (Microbiol. Associates, Bethesda, MD). The mixture is spread over Ficoll-Hypaque and the exposed E+ pellets are treated with 0.155M NH 4 Cl (10 ml per cell). The resulting population of T cells is <2% EAC rosette positive and >95% E rosette negative as determined by standard procedures. Additionally, non-rosetting Ig- (Null cells) were collected from the inside of Ficoll. This latter population was <5% E+ and <2% slg+. The surface of the Ig+ (B) population was obtained from a Sephadex G-200 column eluting with normal human gamma globulin as previously described. This population is >95% surface Ig+ and <5% E+.

Normal human bone marrow cells were obtained from the posterior iliac crests of normal human volunteer donors by needle aspiration.

B. Isolation of thymocytes

A normal human thymus gland was obtained from a patient aged two months to 14 years undergoing corrective cardiac surgery. The freshly obtained parts of the thymus gland are immediately placed in 5% fetal calf serum in medium 199 (Gibco), finely chopped with forceps and scissors, and a cell suspension is immediately prepared by pressing through a steel sieve. Cells are then plated on Ficoll-Hypaque, spun and washed as described in part A. The resulting thymocytes were >95% viable and >90% E rosette positive.

C. Cell lines of T origin and T cells of acute lymphoblastic leukemia

T cell lines CEM, HSB-2 and MOLT-4 were obtained from Dr. H. Lazarus (Sidney Farber Cancer Institute, Boston, MA). Leukemic cells were obtained from 25 patients diagnosed with T cell ALL. These individual tumors were previously determined to be of T cell origin by their spontaneous rosette formation with sheep erythrocytes (>20% E+) and by reactivity with the T cell specific heteroantiserum anti-HTL (B.K.) and A99, as previously described. The tumor population was cryopreserved at -196°C with liquid nitrogen with 10% DMSO and 20% AB human serum until surface characterization. All tumor populations that were analyzed were more than 90% frozen by Wright-Giemsa morphology of cytocentrifugal preparations.

Example III

Cytofluorographic analysis and cell division

Cytofluorographic analysis of monoclonal antibodies with whole cell populations was performed by immunofluorescence with fluoroscein-conjugated anti-mouse IgG (G/M FITC) (Meloy Laboratories) using a Cytofluorograph 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 G/M FITC dilution 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.

A similar reactivity pattern is seen at 1:10,000 dilution, but further dilution causes loss of reactivity. The medium was obtained by replacing a 0.15 ml aliquot of 1:500 ascites from a CAF1 mouse intraperitoneally injected with a non-producing hybrid clone.

In assays involving antibody and complement-mediated lympholysis, thymocytes and peripheral T cells were cultured overnight followed by selective lysis and then directly analyzed on the Cytofluorograph.

Example IV

Lymphoid population lysis with monoclonal antibodies and complement

40 x 10 6 peripheral T cells or thymocytes are placed in a 15 ml plastic tube (Falcon, Oxnard, CA). Cell pellets are incubated with 0.8 cc OKT3, OKT4, OKT8 or S normal ascites diluted 1:200 in PBS, resuspended and incubated at 20°C for 60 minutes. Immediately afterwards, 0.2 cc of fresh rabbit complement was added to the antibody-treated population, resuspended and further incubated at 37°C in a water bath with shaking for 60 minutes. At the end of this time, cells are pelleted and visible cells are counted using Tryptan blue. After counting, cells are washed two more times in 5% FCS and placed in final medium [RPMI 1640 (Grand Island Biological Company, Grand Island, NY containing 20% AB+ human serum, 1% penicillin-streptomycin, 200mM L-glutamine , 25mM HEPES buffer and 0.5% sodium bicarbonate] and incubated overnight in a humid atmosphere with 5% CO2 at 37°C.

Brief description of the drawing

Figure 1 shows the fluorescence pattern obtained with the Cytofluorograph after the reaction of normal human thymocytes with OKT6 and other monoclonal antibodies at a dilution of 1:500 and G/M FITC. A fluorescence background was obtained by incubating each population with a 1:500 dilution of ascites obtained from a mouse injected with a non-producing clone.

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

Production of hybridomas and production and characterization of the resulting monoclonal antibodies was carried out as described in the Examples. easily a large amount of the shown antibodies is prepared by injecting the shown hybridomas intraperitoneally into mice and collecting malignant ascites, it is clear that the hybridomas can be cultured in vitro by well-known procedures in the art and that the antibodies can be removed from the supernatant.

Table 1 shows the reactivity of OKT6, OKT8, OKT9 and 0KT10 with different human lymphoid cell populations. The OKT8 monoclonal antibody is reactive with approximately 30% of normal human T cells, with approximately 80% of normal human thymocytes, with less than 2% of bone marrow cells, and is not reactive with B cells and non-essential cells. This reactivity pattern is one test by which the OKT8 antibody can be detected and distinguished from other antibodies.

Figure 1 shows a typical fluorescence image obtained on a Cytofluorograph after the reaction of a suspension of normal human thymocytes with a 1:500 dilution of OKT3, OKT4, OKT5, OKT6, OKT8, OKT9, 0KT10 and G/M FITC. Similar reactivity patterns are seen with 12 additional. of normal human thymocyte populations that were tested. As shown, there are significant differences in both percent reactivity and fluorescence intensity for each of these monoclonal antibodies. For example, OKT9 reacts with approximately 10% of thymocytes with lower fluorescence intensity while OKT5, OKT6, OKT8 and 0KT10 react with approximately 70% of thymocytes with higher fluorescence intensity. OKT4, which reacts with 75% of thymocytes, is an intermediate between OKT9 and monoclonal antibodies that give an image of stronger fluorescence intensity. Additionally, Figure 1 shows that approximately 15% of thymocytes are detectable with OKT3 indirect immunofluorescence. OKT1, whose pattern of reactivity is almost identical to OKT3 on thymocytes, is not shown. The reactivity image of Figure 1 is another test by which the shown OKT8 antibodies can be detected and distinguished from other antibodies.

Table 2 shows the distribution of antigens determined using different monoclonal antibodies on human peripheral T cells and lymphocytes as determined by a series of experimental lyses described in Example IV. Because only OKT3, OKT4 and OKT8 bound complementary monoclonal antibodies, those three were used.

As shown in Table 2A, the entire T cell population reacts with OKT3 while OKT4, OKT5 and OKT8 react with 60%, 25% and 34% of T cells. Lysis with OKT4 and complement reduces the total number by 62% and specifically deletes the OKT4+ population. Additionally, the percentage of OKT5+ and OKT8+ cells increases and there is no effect on the absolute number of OKT5+ and OKT8+ cells. These experiments suggest that OKT4+ is distinct from OKT5+ and OKT8+ populations. Further support for this claim was obtained by lysis of T cells with OKT8 complement.

In this case, the percentage of OKT4+ T cells increases, the absolute number remains the same, and the OKT8+ and OKT5+ populations are removed. Furthermore, these results indicate that the OKT8+ population is reciprocal to the OKT4+ population and contains a complete OKT5+ cell selection.

Similar tests with a population of human thymocytes give different results. As shown in Table 2B, approximately 75% of thymocytes were OKT4+ or OKT8+. Furthermore, after lysis with either OKT4 or OKT8, only 25% of thymocytes remain. Most of the remaining thymocytes were reactive with OKT3, while only a minority was reactive with OKT6. These findings indicate that the majority of the human thymocyte population carries OKT4, OKT5, OKT6, and OKT8 surface antigens on the same cell. Additionally, Table 2 shows that after treatment with OKT8 or OKT4, an increase in mature thymocytes bearing the OKT3 antigen was recorded. Thus, most OKT3-reactive thymocytes are already divided into OKT4+ or OKT8+ sub-groups, while most remaining cells after OKT4 or OKT8 lysis are OKT3+. In case the OKT3+ subpopulations are both OKT4+ and OKT8+, then lysis with monoclonal antibodies should remove reactive thymocytes.

To further determine the relationship of the OKT3 sub-population of reactive thymocytes to other monoclonal antibodies of a certain thymocyte fraction, thymocytes were treated with OKT3 and complement and the remaining cells were then compared with the untreated thymocyte population. As shown in Table 2B, OKT3 and complement remove 25% of thymocytes. Furthermore, there is no major loss of the OKT4, OKT5, OKT6 or OKT8 reactive population. These findings suggest that the vast majority of thymocytes carrying the OKT6 marker are contained in the OKT3- population. Additionally, thymocytes that simultaneously secrete antigens labeled OKT4, OKT5, and OKT8 are suggested to be restricted to the OKT3 population. It should also be noted that the OKT9 reactive population of thymocytes was not reduced after treatment with OKT3 and complement of unfractionated thymocytes, thus showing that the OKT9+ sub-population is largely restricted to the OKT3- population of thymocytes.

Based on these results, it is possible to describe the stages of development of human thymocytes in the thymus. As shown in Figure 2, almost all thymocytes carry the OKT10 marker. Additionally, thymocytes acquire the OKT9 marker (Thy1 and Thy2) at an early stage of development. This stage determines the minority of thymocytes and amounts to about 10% of the unfractionated population. Immediately thereafter, human thymocytes acquire a unique thymocyte antigen designated as OKT6 and secreted by OKT4, OKT5, and OKT8 (thy 4). This last sub-population represents the majority of thymocytes and is about 70-80% of the thymocyte population. With further maturation, thymocytes lose OKT6 reactivity, acquire OKT3 (and OKT1) reactivity, and divide into OKT4+ and OKT5+/OKT8+ subgroups (Thy7 and Thy 8). Finally, it is shown that as thymocytes are secreted into the peripheral T cell compartment, they lose the OKT10 marker because this antigen is missing from almost all peripheral T lymphocytes. Possible intermediate states between these main states of thymocyte development are labeled Thy3, Thy5 and Thy6 in Figure 2.

Because acute lymphoblastic leukemia of T origin is thought to originate from immature thymocytes, the relationship between tumor cells from individuals with T-ALL and these described differentiation states within the thymus has been determined. Twenty-five tumor cell populations from individuals with TALL and three T cell lines previously studied with common anti-T cell reagents and E rosetting were studied. As shown in Table 3, the majority of T-ALL leukemia cells were reactive with either OKT10 alone or with OKT9 and OKT10 and did not react with other monoclonal antibodies. Thus, 15/25 studied cases show possession of early thymocyte antigens (Stage I).

In contrast, 5/25 cases were reactive with OKT6, suggesting derivation from a more mature thymic population (Stage II). This T-ALL group is inherently heterogeneous with respect to OKT4, OKT8 and OKT9 reactivity as shown in Table 3. Cells from 2/5 patients have the most common thymocyte antigens including OKT4, OKT6 and OKT8. It is significant to note that OKT5 is not only present in these 5 Stage II tumors, but was also observed through OKT8 reactivity. This last result clearly shows that OKT5 and OKT8 target different antigens or different determinants on the same antigen. Finally, 1/25 of patients' tumors originate from the mature thymocyte population (Stage III) as demonstrated by reactivity with OKT3. These individual tumor cases were additionally reactive with OKT5, OKT8, and OKT10. Out of 25 leukemic populations analyzed, only four tumors could not be clearly categorized. They were positive with OKT4 and OKT8, but not positive with OKT3 and OKT6, and the majority represented transition from Thy4 and Thy7,8. One of the 25 cases represented a transition from Thy3 to Thy4 because it had OKT8 and OKT10 reactivity.

T cell lines derived from the T-ALL tumor population also represent cells from specific stages of differentiation in the thymus. As shown in Table 4, HSB was reactive exclusively with OKT9 and OKT10 and thus subsumed the tumor population obtained from Stage 1. In contrast, CEM was reactive with OKT4, OKT6, OKT8, OKT9 and OKT10 and was shown to originate from Stage II thymocytes. Finally, MOLT-4 appears to represent leukemic transformation at a stage between HSB-2 and CEM because it secretes OKT6, OKT8, OKT9, and OKT10.

Because patients with late-stage (eg, Stage II) T-cell lymphoblastic leukemia show longer disease survival than those with Stage I ALL, the use of the OKT8 antibody allows inference regarding prognosis for a given patient with T-cell ALL. Table 5 shows the relationship between peripheral T cell levels and T cell selection at different stages of the disease.

These ratios can be used for diagnostic purposes (eg, to determine acute infectious mononucleosis) by analyzing a blood sample of an individual suspected of having some of the above stages to determine T cell levels and T cell selection. These relationships can also be used for therapeutic purposes where the cause of the disease state is an increased level of T cell selection (eg Type 1 agammaglobulinemia). For therapeutic use, administration of the appropriate monoclonal antibody to a patient with increased T cell selection will reduce or eliminate the excess.

The relationships shown in Tables 2-5 are another way in which OKT8 antibodies can be detected and distinguished from other antibodies.

Other hybridoma-producing monoclonal antibodies prepared by the authors of this invention (designated as OKT1, OKT3, OKT4 and OKT5) are described and protected in . following the U.S. patent applications: Sn 22,132, filed on March 20, 1979; SN 33,639 reported on April 26, 1979; SN 33,669 reported on April 26, 1979; SN 76,642 reported on September 18, 1979; and SN 82,515 registered on October 9, 1979. Other hybridoma-producing monoclonal antibodies prepared by the same authors (designated OKT6, OKT9, and OKT10) have also been described and patented in U.S. Pat. patent applications registered under the names:

Hybrid cell setup for the production of monoclonal antibodies to human thymocyte antigen, antibodies and methods;

Hybrid cell setup for production of monoclonal antibodies to human early thymocyte antigen, antibodies and methods;

Hybrid cell setup for the production of monoclonal antibodies to human antimyocyte antigen, antibodies and methods.

These applications are also contained here through notes.

According to the presented invention, there is provided a hybrid capable of producing an antibody against an antigen found in normal human suppressor T cells, a process for the production of these hybridomas, a monoclonal antibody against an antigen found in a normal human suppressor T cell, methods for producing antibodies, and methods and preparations for treatment or diagnosis disease or to identify T cells or thymocyte subclasses that use these antibodies.

TABLE 1

Reactivity of monoclonal antibodies to human lymphoid population

[image]

*Numbers in parentheses represent the number of tested samples; % value.

TABLE 2

differences in antigen distribution determined using monoclonal antibodies on human peripheral T

cells and thymocytes

[image]

* Untreated populations and populations treated only with complement could not be distinguished on re-analysis.

Non-specific lysis was less than <5% in all cases. Results represent the mean of 6 experiments.

C1† = complement

TABLE 3

Cell surface features in acute lymphoblastic leukemia of T-origin

[image]

*An additional four tumors could not be easily categorized into Stages I-III. See text for details of their characterization.

† Notation refers to Figure 2.

++ Positive (+) reactivity is defined as >30% specific fluorescence above the medium, while negative reactivity (-) is unrecognizable from the tumor cell suspension medium.

TABLE 4

Reactivity with monoclonal antibodies

[image]

* The criterion for - and + reactivity is the same as in Table 3.

TABLE 5

Peripheral T cell levels in disease states

[image]

N = within time limits

O = absence

+ = above normal

++ = significantly above normal

- = below normal

-- = significantly below normal

*these levels return to normal about a 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 monoclonal antibody against human thymocyte antigen, it is understood that the present invention encompasses all monoclonal antibodies that exhibit the features described herein. The OKT8 antibody shown was determined to belong to the IgG2a subclass, which is one of the four IgG subclasses. These immunoglobulin subclasses differ from others in so-called "fixed" parts, although an antibody for a specific antigen will have a so-called "variable" part that is functionally the same regardless of which immunoglobulin subclass it belongs to. This means that a monoclonal antibody that exhibits the features described here can be of the IgG1, IgG2a, IgG2b, or IgG3 subclass or of the IgM, IgA, or other known Ig classes. Differences between these classes or subclasses will not affect the selectivity of the reaction pattern of the antibody, but may affect further reactions of the antibody with other materials, such as (for example) complement or anti-mouse antibodies. easily the specified antibody is IgG2a specific, it is important to understand that antibodies having the reactivity scheme specified in this text are included in the subject of this invention regardless of the class or subclass of immunoglobulin to which they belong.

Furthermore, included within the scope of this invention are the methods of preparation of monoclonal antibodies described previously, which include the hybridoma technique described herein. Although only one example of a hybridoma is shown here, it is understood that those skilled in the art will be able to obtain other hybridomas capable of producing antibodies that will have the reactivity characteristics described in this text by following the immunization, fusion and selection procedures described in this invention. Although the hybridoma was produced from a known murine myeloma cell line and spleen cells from known species of mice, it cannot be further identified except by the antibody-producing features of the hybridoma, and the antibody-producing hybridomas are believed to have the reactivity features previously described and included. within the scope of this invention, as well as methods for obtaining these antibodies using hybridomas.

Further aspects of the invention are methods of treating or diagnosing diseases involving OKT8 monoclonal antibodies or any other monoclonal antibodies exhibiting the pattern of reactivity set forth herein. The disclosed antibody can be used to detect and study differentiation within the thymus as shown in Figure 2. Furthermore, the disclosed antibodies can be used to diagnose disease states as shown in Table 5. These methods can be used using OKT8 antibodies alone or in combination with other antibodies (eg, OKT3-OKT10). Antibody and T cell reactivity schemes and T cell selection will enable more accurate detection of certain disease states (eg, acute infectious mononucleosis) than was possible using previously known methods.

Treatment of disease states (e.g. acute infectious mononucleosis or malignant diseases such as Stage II ALL) manifested by an excess of OKT8+ cells can be followed by administration of therapeutically effective doses of OKT8 antibodies to the patient. By selectively reacting with OKT8+ antigen, an effective amount of OKT8 antibody will reduce excess OKT8+ cells, thus mitigating the effects of the excess. Within the scope of the presented invention are also diagnostic and therapeutic preparations containing effective amounts of OKT8 antibodies in admixture with diagnostically or pharmaceutically acceptable media.

Claims (17)

1. Complementary-binding monoclonal antibody of the IgG class, produced by means of a hybridoma obtained by the fusion of mouse myeloma cells and spleen cells from mice previously immunized with human thymocytes, indicated that the antibodies: a) react with suppressor T cells (approximately 30% of normal human T cells), but not less than 2% of normal human bone marrow cells and do not react with B cells and Null cells; b) show the pattern of reactivity towards normal human thymocytes shown in Figure 1, reacting with about 80% of normal human thymocytes; c) show the pattern of reactivity towards peripheral T cells and thymocytes shown in Table 2; d) show the pattern of reactivity with T cells of the ALL condition shown in Table 3; e) they react with CEM and MOLT-4 cell lines, but not with HSB-2 cell lines; you f) determine the T cell population (OKT8+) which is lower than normal levels in primary biliary cirrhosis and multiple sclerosis, but does not appear or is lower than normal in hyper IgE states, is higher than normal levels in acute vaccine reactions, is significantly it is above normal in acute infectious mononucleosis, is completely absent in myasthenia gravis and is at normal levels in all stages of Hodgkinson's disease and psoriasis. 2. A monoclonal antibody, characterized in that it is produced by a hybridoma having the characteristics of ATCC 8014. 3. IgG complement-binding hybridoma that produces monoclonal antibodies and which was formed by fusing mouse spleen cells previously immunized with human thymocytes and mouse myeloma cells, characterized in that the antibodies: a) react with suppressor T cells (approximately 30% of normal human T cells), but not less than 2% of normal human bone marrow cells and do not react with B cells and Null cells; b) show the pattern of reactivity towards normal human thymocytes shown in Figure 1, reacting with about 80% of normal human thymocytes; c) show the pattern of reactivity towards peripheral T cells and thymocytes shown in Table 2; d) show the pattern of reactivity with T cells of the ALL condition shown in Table 3; e) they react with CEM and MOLT-4 cell lines, but not with HSB-2 cell lines; you f) determine the T cell population (OKT8+) which is lower than normal levels in primary biliary cirrhosis and multiple sclerosis, but does not appear or is lower than normal in hyper IgE states, is higher than normal levels in acute vaccine reactions, is significantly it is above normal in acute infectious mononucleosis, is completely absent in myasthenia gravis and is at normal levels in all stages of Hodgkinson's disease and psoriasis. 4. A hybridoma, characterized in that it has the identifying features of ATCC 8014. 5. Preparation procedure of complement-binding IgG monoclonal antibody which: a) reacts with suppressor T cells (approximately 30% of normal human T cells), but not less than 2% of normal human bone marrow cells and does not react with B cells and Null cells; b) shows the pattern of reactivity towards normal human thymocytes shown in Figure 1, reacting with about 80% of normal human thymocytes; c) shows the pattern of reactivity towards peripheral T cells and thymocytes shown in Table 2; d) shows the pattern of reactivity with T cells of the ALL condition shown in Table 3; e) reacts with CEM and MOLT-4 cell lines, but not with HSB-2 cell lines; you f) determines the T cell population (OKT8+) which is lower than the normal level in primary biliary cirrhosis and multiple sclerosis, but does not appear or is lower than normal in hyper IgE states, is higher than normal levels in acute vaccine reactions, is significantly above normal in acute infectious mononucleosis, it is completely absent in myasthenia gravis and is at normal levels in all stages of Hodgkinson's disease and psoriasis, indicated by the fact that it contains steps: i) immunization of mice with human thymocytes; ii) removing spleens from said mice and making a suspension of spleen cells; iii) fusing said spleen cells with myeloma cells in the presence of a fusion promoter; iv) diluting and culturing the confluent cells in separate dishes in a medium that will not support non-confluent myeloma cells; v) evaluation of the supernatant in each vial containing the hybridoma for the presence of the desired antibody; vi) selection and cloning of hybridomas that produce the desired antibody; you vii) detection of antibodies from the supernatant above the mentioned clones. 6. Preparation procedure of complement-binding IgG monoclonal antibody which: a) reacts with suppressor T cells (approximately 30% of normal human T cells), but not less than 2% of normal human bone marrow cells and does not react with B cells and Null cells; b) shows the pattern of reactivity towards normal human thymocytes shown in Figure 1, reacting with about 80% of normal human thymocytes; c) shows the pattern of reactivity towards peripheral T cells and thymocytes shown in Table 2; d) shows the pattern of reactivity with T cells of the ALL condition shown in Table 3; e) reacts with CEM and MOLT-4 cell lines, but not with HSB-2 cell lines; you f) determines the T cell population (OKT8+) which is lower than the normal level in primary biliary cirrhosis and multiple sclerosis, but does not appear or is lower than normal in hyper IgE states, is higher than normal levels in acute vaccine reactions, is significantly above normal in acute infectious mononucleosis, it is completely absent in myasthenia gravis and is at normal levels in all stages of Hodgkinson's disease and psoriasis, indicated by the fact that it contains steps: i) immunization of mice with human thymocytes; ii) removing spleens from said mice and making a suspension of spleen cells; iii) fusing said spleen cells with myeloma cells in the presence of a fusion promoter; iv) diluting and culturing the confluent cells in separate dishes in a medium that will not support non-confluent myeloma cells; v) evaluation of the supernatant in each vial containing the hybridoma for the presence of the desired antibody; vi) selection and cloning of hybridomas that produce the desired antibody; vii) detection of antibodies from the supernatant above said clones; viii) transferring said clones intraperitoneally into mice; and ix) collection of malignant ascites or serum of the mentioned mice. 7. A method for detecting a lack or excess of OKT8+ cells in individuals, indicated by the fact that it contains the reaction of a preparation of T cells from said individuals with a diagnostically effective amount of antibody according to Claim 13 and measuring the percentage of the total peripheral T population that reacts with said antibody. 8. The method according to Claim 13, characterized in that there is an excess of T cells ALL. 9. The method of treating an excess of OKT8+ cells in an individual, characterized in that it includes the application to the individual of an amount of antibody according to Claim 13, which will be effective in reducing the number of OKT8+ cells. 10. Diagnostic preparation according to the invention for detecting an excess or deficiency of OKT8+ cells, characterized in that it contains, in a mixture with a diagnostically acceptable substrate, an amount of antibody according to Claim 13 that will be effective in detecting an excess or deficiency of OKT8+ cells. 11. Therapeutic preparation according to the invention, characterized in that it contains, in admixture with a pharmaceutically acceptable carrier, an amount of antibody according to Claim 13 effective to reduce the amount of OKT8+ cells in an individual who has an excess of said OKT8+ cells. 12. A method for detecting acute infectious mononucleosis in individuals, indicated by the fact that it comprises the reaction of a preparation of T cells from said individuals with a diagnostically effective amount of at least one monoclonal antibody selected from the group consisting of OKT3, OKT4, OKT6 and OKT8 and measuring the percentage of the total peripheral population T lymphocytes that react with each of the mentioned antibodies. 13. A mouse monoclonal antibody, characterized in that it reacts with all normal human suppressor T cells, but less than 2% of normal human bone marrow cells and does not react with peripheral B cells or Null cells. 14. A process for the preparation of monoclonal antibodies that react with almost all normal human suppressor T cells, but with less than 2% of normal bone marrow cells and do not react with normal human peripheral B cells or Null cells, indicated by the fact that it contains hybridoma cultivation ATCC CRL 8014 in a suitable medium and detection of antibodies from the supernatant above the hybridoma. 15. A process for the preparation of monoclonal antibodies that react with almost all normal human suppressor T cells, but with less than 2% of normal bone marrow cells and do not react with normal human peripheral B cells or Null cells, characterized in that it contains the injection into the mouse of hybridoma ATCC CRL 8014 and detection of antibodies from malignant ascites or serum of the mentioned mouse. 16. A process for the preparation of monoclonal antibodies that react with almost all normal human suppressor T cells, but with less than 2% of normal bone marrow cells and do not react with normal human peripheral B cells or Null cells, characterized in that it contains the steps: i) immunization of mice with human thymocytes; ii) removing spleens from said mice and making a suspension of spleen cells; iii) fusing said spleen cells with myeloma cells in the presence of a fusion promoter; iv) diluting and culturing the confluent cells in separate dishes in a medium that will not support non-confluent myeloma cells; v) evaluation of the supernatant in each dish containing the hybridoma for the presence of antibodies against E rosette positive purified T cells or human thymocytes; vi) selection and cloning of hybridomas that produce an antibody that reacts with almost all normal human suppressor T cells, but with less than 2% of normal bone marrow cells and does not react with normal human peripheral B cells or Null cells; and vii) detection of antibodies from the supernatant above the mentioned clones. 17. A process for the preparation of monoclonal antibodies that react with almost all normal human suppressor T cells, but with less than 2% of normal bone marrow cells and do not react with normal human peripheral B cells or Null cells, characterized in that it contains the steps: i) immunization of mice with human thymocytes; ii) removing spleens from said mice and making a suspension of spleen cells; iii) fusing said spleen cells with myeloma cells in the presence of a fusion promoter; iv) diluting and culturing the confluent cells in separate dishes in a medium that will not support non-confluent myeloma cells; v) evaluation of the supernatant in each dish containing the hybridoma for the presence of antibodies against E rosette positive purified T cells or human thymocytes; vi) selection and cloning of hybridomas that produce an antibody that reacts with almost all normal human suppressor T cells, but with less than 2% of normal bone marrow cells and does not react with normal human peripheral B cells or Null cells; vii) transferring said clones intraperitoneally into mice; you viii) collection of malignant ascites or serum containing the desired antibody from said mice.