HRP940824A2 - Hybrid cell line for producing monoclonal antibody to a human prothymocyte antigen, antibody, and methods - Google Patents

Description

Field of invention

This invention generally relates to new hybrid cell lines and more specifically to new cell lines for the production of monoclonal antibodies to an antigen found in approximately 95% of normal human thymocytes, to the antibodies thus produced and to therapeutic and diagnostic procedures as well as preparations containing these antibodies.

Description of the state of the art

The fusion of murine myeloma cells with spleen cells of an immunized mouse by the method of Kohler and Milstein in 1975 (Nature 256, 495-497 (1975)) shows for the first time that it is possible to obtain a continuous cell line for the production of homogeneous (so-called "monoclonal") antibodies. Since then, many efforts have been made to obtain different hybrid cells (called "hybridomas") and to use the antibodies produced by these hybridomas in various scientific tests. See, for example, Current Topics in Microbiology and Immunology, Volume 81 - "Lymphocyte Hybridomas", F.Melchers, M.Potter; and N.Warner, publishers Springer-Verlag, 1978 and cited articles; 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, Vol 2, D.M.Wier, Blackwell Publishers, 1978, Section 25; and Chemical and Engineering News, January 1979, 15-17. The above notes point to successes as well as problems in the intentions of producing monoclonal antibodies from hybridomas. Although the general technique is well known, many problems are encountered and many variations are necessary depending on each individual case. In fact, there is no certainty when planning the preparation of a given hybridoma, that it will be obtained by the desired hybrid, that if it is obtained, it will give antibodies or that the antibodies thus obtained will have the desired properties. The degree of success will depend primarily on the type of antigen used and on the choice of technique used to isolate the desired hybridoma.

Attempts to prepare monoclonal antibodies against lymphocyte cell surface antigens have been reported 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 cultured cell lines of human lymphoblastoid leukemia and human chronic lymphocytic leukemia. It was shown that many of the obtained hybridomas produce antibodies for different antigens on human cells. None of the obtained hybridomas produce antibodies against a previously determined type of human lymphocytes.

More recently, the assigned inventors and others have patented subject matter involving the preparation and testing of hybridomas that produce antibodies to specific T-cell antigens. See, for example, Reinherz, E.L. et al. J. Humanology 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 groups of lymphocytes contained in the immune system of humans and mammals. The first of these (thymus-derived cells or T-cells) is differentiated in the thymus from the hematopoietic cell system. While inside the thymus, the cells are called "thymocytes". Mature T cells leave the thymus and circulate between tissues, lymph and blood. The mentioned T cells form a large proportion of recirculating small lymphocytes. They have immunological specificity and are included directly in the cell-mediated immune response as executive cells. Although T cells do not secrete humoral antibodies, they are sometimes necessary for the secretion of these antibodies by another type of lymphocyte, which is described below. Some types of T cells have a regulatory role in some other aspects of the immune system. The mechanism of these processes of cellular cooperation is still not fully elucidated.

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 cell system, 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, there is no corresponding organ and it is thought that these B cells differentiate within the bone marrow.

It is now known that T cells are divided into at least several subtypes, called "helper", "suppressor" and "killer" T cells that have the role of stimulating the reaction, suppressing the reaction, or killing (lysing) foreign cells. These subspecies are well understood in 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 Chess and S.F. Schlossman "Functional Analysis of Distinct Human T-Cell Subsets Bearing Unique Differentiation Antigens", in "Contemporary Topics in Immunobiology", O . Stuman, publisher Plenum Press, 1977, vol. 7, 363-379. The ability to determine or suppress T cell types or subtypes is important for diagnosing or treating various immunoregulatory diseases or conditions.

For example, different 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. Belpomme et al. "Immunological Diagnosis of Leukemias and Lymphomas", S. Thierfelder et al., ed. Springer, Heidelberg 1977, 33-45; and D. Belpomme et al. British Journal of Haematology, 1978, 38, 85.

Certain disease states (eg, juvenile rheumatoid arthritis, malignancies, and agammaglobulinemia) are associated with an imbalance of T cell subtypes. Autoimmune diseases were generally thought to be associated with an excess of "helper" T cells or a deficiency of certain "suppressor" T cells, while agammaglobulinemia was associated with an excess of certain "suppressor" T cells or a deficiency of "helper" T cells. Malignancies are generally associated with an excess of "suppressor" T cells.

In certain leukemias, excess T cells are produced at an arrested stage of development. Thus, the diagnosis may depend on the ability to detect an imbalance or excess and the ability to determine 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, Vol. 20, Spriger-Verlag, 1977, 17-24, and notes contained therein; and E.L. Reinherz et al., J. CIin. Invest., 64, 392,397 (1979).

Acquired agammaglobulinemia, a disease state in which immunoglobulin is not produced, includes at least two different types. In type I, immunoglobulin is not produced due to an excess of suppressor T cells, while in type II it is due to a lack of helper T cells. In both cases, there is no disorder or lack of the patient's B cells, the lymphocytes that are responsible for the immediate secretion of antibodies; naturally, these B cells are either suppressed or "unhelped", resulting in greatly reduced or absent immunoglobulin production. The type of acquired agammaglobulinemia can thus be determined by testing for an excess of suppressor T cells or for the absence of helper T cells.

From a therapeutic point of view, there are some suggestions, although still unproven, that the administration of antibodies against a subtype of T cells that are in excess may be therapeutically useful in the case of autoimmune or malignant diseases. 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 an autoimmune disease caused by an excess of helper cells can also be done in the same way. Treatment of diseases (e.g. malignant diseases or acquired agammaglobulinemia type I) caused by an excess of suppressor T cells can be carried out using antibodies for the antigen of suppressor T cells.

The use of an antiserum against an entire group of human T cells (so-called antihuman thymocyte globulin or ATG) has been shown to be useful in patients with transplanted organs. Because the cell-mediated immune response (the mechanism by which transplants are rejected) depends on T cells, the administration of 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 Monitoring," Surgery 40:155-163 (1976) and articles cited.

Identification and suppression of human T cell types and subtypes has previously been accomplished using spontaneous autoantibodies or human T cell-selective antisera obtained by immunizing animals with human T cells, drawing blood from animals to obtain serum, and adsorbing antisera with (for example) autologous but not with allogeneic B cells to remove antibodies of undesirable reactivity. Preparation of said antisera is extremely difficult, especially adsorption and purification. Even so purified and adsorbed antiserum contains many impurities 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 found in all injected human T cells. There is no selective production of antibodies against one antigen. Third, the titer of specific antibodies obtained by such procedures is usually quite low (eg, inactivation occurs at dilutions greater than 1:100) and the ratio of specific to nonspecific antibodies is less than 1/106.

See, for example, the Chess and Schlossman articles cited earlier in the text (on pp. 365ff.), as well as the Chemical and Engineering News articles cited earlier, which describe the disadvantages of antisera obtained by previously known methods and the advantages of monoclonal antibodies .

Summary of the invention

A new hybridoma (designated 0KT10) has been discovered that can produce new monoclonal antibodies against an antigen found in approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E-peripheral mononuclear cells (B cells and Null cells) and in 10-20% of bone marrow cells.

The antibody produced in this way is monospecific for a specific determinant on approximately 95% of normal human thymocytes and contains essentially no other anti-human immunoglobulin, in contrast to antisera obtained by earlier procedures (which are hereditarily contaminated with antibodies reactive to numerous human antigens) and monoclonal antibodies obtained by previously known procedures (which are not specific for human thymocyte antigen). Furthermore, this hybridoma can be cultivated to produce antibodies without the need for immunization and killing of animals, and without the difficult steps of adsorption and purification necessary to obtain even impure antisera by previously known procedures.

It is also an object of the present invention to provide hybridomas that will produce antibodies against an antigen found on about 95% of normal human thymocytes.

Another aspect of the presented invention is to provide procedures for the preparation of said hybridomas.

A further aspect of the present invention is to provide homogeneous antibodies to an antigen found on about 95% of normal human thymocytes.

A further aspect is to provide procedures for treating or diagnosing diseases or identifying T cells or subtypes of thymocytes using said antibodies.

Other aspects as well as advantages of the invention will become clear from the above application.

Happily, this invention has found a new hybridoma that produces new antibodies to an antigen found on approximately 95% of normal human thymocytes, an antibody as such, and diagnostic and therapeutic procedures using the antibody. The hybridoma was prepared generally following the procedure of Milstein and Kohler. After immunizing a mouse with human leukemia cells using acute lymphoblastic leukemia (T-ALL) T cells, spleen cells from the immunized mouse are fused with cells from a mouse myeloma line and the resulting hybridomas are screened for antibodies that selectively bind to normal E rosette-positive human T cells. and/or thymocytes. The desired hybridomas are immediately cloned and labeled. As a result, a hybridoma is obtained that produces antibodies (designated as 0KT10) against antigens on approximately 95% of normal human thymocytes. Not only does this antibody react with about 95% of normal human thymocytes, but it also reacts with about 95% of normal human peripheral T cells, 10% of E-peripheral mononuclear cells (B cells and Null cells) and 10-20% of bone marrow cells.

From the point of view of the indicated difficulties in the previous procedures and the lack of success demonstrated by the use of malignant cell lines as antigens, it is surprising that the presented procedure provides the desired hybridoma. It is important to emphasize that the unpredictable nature of hybrid cell preparation does not allow extrapolations from one antigen or cell system to another. In fact, the inventors have discovered that the use of malignant cell line T cells or purified antigens separated from the cell surface as antigens is completely unsuccessful.

Both the hybrid and produced antibodies are labeled "0KT10" in the text, and the individual materials will be understood from the context. 2l was deposited with the display hybrid. November 1979 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland~20852 and is assigned ATCC accession number CRL 8022.

The preparation and characteristics of the hybridoma and the resulting antibody will be more understandable from the text and the following descriptions as well as from

For example.

Detailed description of the invention

The hybridoma preparation process generally includes the following steps:

A. Immunization of mice using leukemic T-ALL cells. Although female CAF1 mice have been found to be most suitable, other strains can be used. The immunization schedule and thymocyte concentration should be such as to produce useful amounts of suitable splenocytes. Three immunizations at fourteen-day intervals with 2x107 cells/mouse/injection in 0.2 ml of phosphate-buffered saline proved to be the most effective.

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

C. Fusion of suspended spleen cells with mouse myeloma cells of the appropriate cell line using the appropriate fusion promoter. The preferred ratio is about 5 spleen cells per myeloma cell. The total volume of about 0.5-1.0 ml of fusion medium is adequate for about 108 splenocytes. Many myeloma cell lines are known and available, either from various academic communities or from depository banks such as the Salk Institute Cell Distribution Center, La Jolla, CA.

It is advantageous to use a cell line of the so-called "drug resistant" type, so that unfused myeloma cells do not survive in the selective medium, while hybrids survive. The most common is the 8-azaguanine resistant cell line that lacks the enzyme hypoxanthine guanine phosphoribosyl transferase and therefore cannot survive in HAT (hypoxanthine, aminopterin and thymidine) medium. It is also generally accepted that a myeloma cell line of the so-called non-secretory type should be used, so that it itself does not produce antibodies, although secretory types can also be used. In certain cases, of course, secretory myeloma lines are preferred. Although the preferred fusion promoter is polyethylene glycol having an average molecular weight of about 1000 to about 4000 (commonly available as PEG 1000, etc.), other fusion promoters known in the art can also be used.

D. Diluting and culturing in separate containers, 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 period of time sufficient to kill the unfused cells (about a week). The dilution can be of the limiting type, so that the volume of the dilution agent is statistically calculated to isolate a certain number of cells (eg 1-4) in each separate container (or rather in each well of the microtiter dish). There is only one medium (eg HAT medium) and that is the one that will not support a "drug-resistant" (eg 8-azaguanine resistant) unfused myeloma cell line. Therefore, these myeloma cells die. Since unfused spleen cells are not malignant, they only have a certain number of generations. Thus, after a certain period of time (about a week), these unfused spleen cells stop reproducing. The fused cells, on the other hand, continue to reproduce because they have the malignant properties of the myeloma parent and the ability to survive in the selective medium of the parental spleen cells.

E. Evaluation of the supernatant in each container (dish) containing the hybridoma 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 for production of the desired antibody.

Once the desired hybridoma has been selected and cloned, the resulting antibodies can be produced in one of two ways. The purest monoclonal antibody is produced in vitro by culturing the desired hybridoma in a suitable medium for a suitable period of time, followed by detection of the desired antibody from the supernatant. A suitable medium and a suitable length of time for cultivation are generally known or easily determined. This in vitro technique produces an essentially monospecific antibody, essentially free of other specific antihuman immunoglobulins. If the medium contains xenogeneic serum (e.g. sheep serum) there is also a small amount of other immunoglobulins. Of course, the in vitro procedure does not have to produce a sufficient amount or concentration of antibodies for some specific purposes because the concentration of monoclonal antibodies is only about 50 μg/ml.

In order to produce a significantly higher concentration of slightly less pure monoclonal antibodies, the desired hybrid can be injected into mice, preferably syngenetic or semisyngenic mice. After a certain incubation time, the hybridoma will cause the formation of tumors that produce the antibody, which will result in a higher concentration of the desired antibody (about 5-20 mg/ml) in the bloodstream and peritoneal exudate (ascites) of the host. Although this host also has normal antibodies in his blood and ascites, the concentration of these normal antibodies is only about 5% of the concentration of monoclonal antibodies. Moreover, because these normal antibodies are not antihuman in their specificity, monoclonal antibodies obtained from ascites or serum are essentially free from contamination by antihuman immunoglobulin. These monoclonal antibodies have a higher titer (they are active at a dilution of 1:50,000 or more) and the ratio of specific to non-specific immunoglobulins is higher (about 1/20). Immunoglobulins produced by the incorporation of small myeloma chains are nonspecific, "nonsense" peptides that generally dilute monoclonal antibodies without reducing their value due to specificity.

Examples

Production of monoclonal antibodies

A. Immunization and hybridization of somatic cells

Female CAF1 mice (Jackson Laboratories; 6-8 weeks old) are immunized intraperitoneally with 2x107 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 are removed and a single-cell suspension is prepared by passing the tissue through a steel sieve.

Cell fusion is performed using the procedure developed by Kohler and Milstein. 1x108 splenocytes were fused in 0.5 ml fusion medium containing 35% polyethylene glycol (PEG 1000) and 5% dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, NY) with 2x107 P3X63Ag8Ul myeloma cells obtained from Dr. M. Scharff, Albert Einstein College of Medicine, Bronx, NY. These myeloma cells secrete IgG1 k short chains.

B. Selection and growth of hybridomas

After cell fusion, cells are cultured in HAT medium (hypoxanthine, aminopterin and thymidine) at 37ºC with 5% CO2 in a humidified atmosphere. After several weeks, 40 to 100 µl of culture supernatant containing hybridomas was added to a pellet of 106 peripheral lymphocytes divided into E rosette positive (E+) and E rosette negative (E-) populations, which were prepared from the blood of healthy donors in the manner described described by Mendes (J. Immunol. 111:860, 1973). Detection of murine hybridoma antibodies that bind to these cells was performed by indirect immunofluorescence. Cells incubated with culture supernatant were stained with fluorescent goat-anti-mouse IgG (G/M FITC) (Meloy Laboratories, Springfield, VA; F/P=2.5) and fluorescent antibody-coated cells and immediately analyzed on a Cytofluorograph FC200/4800A. (Ortho Instruments, Westwood, MA) as described in Example III. Hybridoma cultures containing antibodies that react specifically with E+ lymphocytes (T cells) and/or thymocytes were selected and cloned twice by limiting dilution in the presence of host cells. Immediately thereafter, clones were transferred by intraperitoneal injection of 1x107 cells of the given clone (0.2 ml volume) into CAF1 mice using 2,6,10,14-tetramethylpentadecane from Aldrich Chemical Company trade name Pristine. Malignant ascites from these mice were used for lymphocyte characterization as described hereinafter in Example II Using a standard technique, it was proven that the present hybrid antibodies 0KT10 belong to the IgG1 subtype.

Example II

Characterization of 0KT10 reactivity

A. Isolation of the lymphocyte population

Human peripheral blood mononuclear cells are isolated from the blood of healthy volunteer donors (15-40 years of age) by Ficoll-Hypaque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, NY) following the well-known technique of Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl.97): 77, 1968. Unfractionated mononuclear cells are separated into surface Ig+ (B) and Ig- (T plus Null) populations using a Sephadex G-200 anti-F (ab')2 column for chromatography on method described by Chess, et al., J. Immunol. 113:1113 (1974). T cells were detected by E rosetting of the Ig- population with 5% sheep erythrocytes (Microbiological Associates, Bethesda, Nm). The rosette mixture is treated with the Ficoll-Hypaque method and the exposed E+ pellet is treated with 0.155 M NH4Cl (10 ml per 108 cells). The population of T cells obtained in this way is <2% EAC rosette positive and >95% E rosette positive as determined by usual procedures. In addition, the non-rosetting Ig- (Null cells) population is collected from the Ficoll interstitial. This last population is <5% E+ and <2% sIg+. The surface of the Ig+ (B) population was obtained using a Sephadex G-200 column followed by elution with normal human gamma globulins in the manner previously described. That population is >95% surface Ig+ and <5% E+.

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

B. Isolation of thymocytes

Normal human thymus gland was obtained from patients aged two months to fourteen years who had undergone corrective cardiac surgery. Freshly obtained thymus glands were immediately placed in 5% fetal calf serum in medium 199 (Gibco), finely minced with forceps and scissors, and immediately pressed through a steel sieve to obtain a single-cell suspension. The cells are then plated on Ficoll-Hypaque and processed and washed as previously described in section A. The thymocytes thus obtained are >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 using sheep erythrocytes (>20% E+), and by reactivity with T cell specific heteroantisera anti-HTL (B.K.) and A99 as previously described. Tumor populations were cryopreserved at -196°C using liquid nitrogen vapor phase with 10% DMSO and 20% AB human serum until the time of surface determination. All analyzed tumor populations were more than 90% destroyed as determined by Wright-Giemsa morphology of cytocentrifuged preparations.

Example III

Cytofluorographic analysis and cell division

Cytofluorographic analyzes of monoclonal antibodies with whole cell populations were performed by indirect immunofluorescence with fluoroscein-conjugated goat anti-mouse IgG (G/M FITC) (Meloy Laboratories) using a Cytofluorograf FC200/4800A (Ortho Instruments). Briefly, 1x106 cells were treated with 0.15 ml OKT5 at a 1:500 dilution, incubated at 4ºC for 30 minutes and washed twice. Cells were then reacted with 0.15 ml of a 1:40 G/M FITC dilution at 4ºC for 30 minutes, centrifuged and washed 3 times. The cells were then analyzed on a Cytofluorograph and the fluorescence intensity per cell was recorded on the analyzer. A similar pattern of reactivity was seen at 1:10000 dilution, but further dilution caused loss of reactivity. The medium was obtained by substituting a 0.15 ml aliquot of 1:500 ascites from a CAF1 mouse intraperitoneally injected with a non-producing hybrid clone.

In experiments involving antibodies and complement-mediated lympholysis, thymocytes and peripheral T cells were cultured overnight following a selective lysis procedure and then immediately analyzed on a Cytofluorograph.

Example IV

Lysis of lymphoid populations with monoclonal antibody and complement

40x106 peripheral T cells or thymocytes were placed in a 15 ml plastic tube (Falcon, Oxnard, CA). Cell pellets were incubated with 0.8 cm3 OKT3, OKT4, OKT8 or normal control ascites diluted 1:200 in PBS, resuspended and incubated at 20°C for 60 minutes. Immediately afterwards, 0.2 cm3 of rabbit complement was added to the antibody of the treated populations, resuspended and further incubated at 37°C with shaking in a water bath for 60 minutes. At the end of this time cells were pelleted and visible cells were counted using Trypan blue exclusion. After counting, cells were washed 2 times in 5% FCS and placed in medium [RPMI 1640 (Grand Island Biological Company, Grand Island, NY) containing 20% AB+ human serum, 1% penicillin-streptomycin, 200 mM L-glutamine, 25 mM 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 a fluorescence image obtained on a Cytofluorograph after the reaction of normal human thymocytes with 0KT10 and other monoclonal antibodies at a dilution of 1:500 and G/M FITC. Background fluorescence was obtained by incubating each population with a 1:500 dilution of mouse ascites injected with the non-producing clone.

Figure 2 shows the stages of intrathymic differentiation in humans.

The production of hybridomas and the production and characterization of the resulting monoclonal antibodies was carried out in the manner previously described in the examples. Although a large amount of antibody has been prepared by injecting hybridomas intraperitoneally into mice and collecting malignant ascites, it is clear that hybridomas can be cultured in vitro by techniques well known in the art and antibodies can be removed from the supernatant.

Table 1 shows the reactivity of OKT6, OKT8, OKT9 and 0KT10 with different populations of human lymphoid cells. 0KT10 monoclonal antibody is reactive with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E-peripheral mononuclear cells (B cells and Null cells) and with 10-20% of bone marrow cells. This reactivity pattern is a test by which the displayed 0KT10 antibodies can be detected and distinguished from other antibodies.

Figure 1 shows a characteristic fluorescence image obtained using 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 patterns of reactivity were seen with 12 additional tested populations of normal human thymocytes. As can be seen, significant differences exist for both percent reactivity and fluorescence intensity with each of these monoclonal antibodies. For example, OKT9 reacts with approximately 10% of thymocytes at low fluorescence intensity while OKT5, OKT6, OKT8 and 0KT10 react with approximately 70% of thymocytes at higher fluorescence intensity. OKT4, which reacts with 75% of thymocytes, is an intermediate between OKT9 and monoclonal antibodies that give an image of higher fluorescence intensity. Additionally, Figure 1 shows that approximately 15% of thymocytes were detected by OKT3 and indirect immunofluorescence. OKT1, whose reactivity pattern is identical to OKT3 on thymocytes, is not shown. The reactivity image from Figure i is another test by which the OKT6 antibody shown can be detected and distinguished from other antibodies.

Table 2 shows the distribution of antigens determined by different monoclonal antibodies on human peripheral T cells and lymphocytes as determined by a series of lysis experiments in the manner described in Example IV. Because only OKT3, OKT4 and OKT 8 bound complementary monoclonal antibodies, only those three were used.

As shown in Table 2A, the entire T cell population reacts with OKT3 while 60%, 25%, and 34% of cells react with OKT4, OKT5, and OKT8, respectively. Lysis with OKT4 and complement reduces the total number to 62% and specifically abrogates 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+ T cells. These experiments indicate that there is a difference between OKT4+ and OKT5+ and OKT8+ populations. Further support for this conclusion was obtained by lysis of T cells with OKT8 and complement. In this case, the percentage of OKT4+ T cells increases, the absolute number remains the same and the OKT5+ and OKT8+ populations are removed. Furthermore, these results indicate that the OKT8+ population is reciprocal to the OKT4+ population and contains a complete selection of OKT5+ T cells.

Similar experiments with human thymocyte populations yield mixed results. As shown in Table 2B, approximately 75% of thymocytes were OKT4+ or OKT5+. Moreover, after lysis with either OKT4 or OKT8, only 25% of thymocytes remain. Most of the remaining thymocytes were reactive with OKT3, while only a small part was reactive with OKT6. These findings indicate that a major part of the human thyocyte population carries OKT4, OKT5, OKT6 and OKT8 surface antigens on the same cell. Additionally, Table 2 shows that after reactions with OKT8 or OKT4, an increase in the number of mature thymocytes bearing the OKT3 antigen was noted. Thus, the majority of OKT3 reactive thymocytes are already divided into OKT4+ or OKT8+ sub-groups, while most of the remaining cells after OKT4 or OKT8 lysis are OKT3+. If OKT3+ is a sub-population of both OKT4+ and OKT8+ then lysis with one of the monoclonal antibodies removes OKT3 reactive thymocytes.

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

Based on the above results, it was possible to describe the stages of intrathymic development of human thymocytes. As shown in Figure 2, almost all thymocytes carry the 0KT10 marker. Additionally, early-stage thymocytes carry the OKT9 marker (Thyl and Thy2). This stage determines a smaller part of thymocytes and is about 10% of the unfractionated population. Subsequently, human thymocytes acquire a unique thymocyte antigen determined by OKT6 and simultaneously secrete OKT4, OKT5 and OKT8 (Thy4). This last sub-population represents the greater part of thymocytes and is more than 70-80% of the thymus population. With further maturation, thymocytes lose OKT6 reactivity, acquire OKT3 (and OKT1) reactivity, and divide into OKT4+ and OKT5+'/OKT6+ subtypes (Thy7 and Thy8). Finally, it becomes clear that as thymocytes are secreted into the peripheral T cell compartment, they lose the 0KT10 marker because it is a marker that is missing from almost all peripheral T lymphocytes. Possible transition states between these three major stages of thymic development are labeled Thy3, Thy5, and Thy6 in Figure 2.

Because lymphoblastic leukemia of T origin is thought to arise from immature thymocytes, the relationship between the tumor cells of individuals with T-ALL and these stages of intrathymic differentiation has been determined. Twenty-five tumor cell populations from individuals with T-ALL 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 either with 0KT10 alone or with OKT9 and 0KT10, and did not react with other monoclonal antibodies. Thus, it turned out that 15/25 studied cases have antigens of early thymocytes (stage I).

In contrast, 5/25 cases were reactive with OKT6, thus suggesting the derivation of a more mature thymic population (stage II). This T-ALL group was inherently heterogeneous with respect to OKT4, OKT8, and OKT9 reactivity, as shown in Table 3. Cells from 2/5 patients possessed the most common thymocyte antigens including OKT4, OKT6, and OKT8. It is important to note that OKT5 is not present in any of these five stage II tumors, although OKT8 reactivity was noted. This last result clearly indicates 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 determined by its reactivity with OKT3. This tumor was additionally reactive with OKT5, OKT8 and 0KT10. Out of 25 analyzed leukemic populations, only 4 tumors could not be clearly categorized. Three were positive with OKT4 and OKT8, but lacked OKT3 and OKT6 and most likely represent a transition from Thy4 and Thy7,8. One of the 25 cases was a transition from Thy3 to Thy4 because he had OKT8 and 0KT10 reactivity.

T cell lines derived from the T-ALL tumor population also represent cells of a specific stage of intrathymic differentiation. As shown in Table 4, HSB was reactive exclusively with OKT9 and 0KT10 and should therefore be designated as a tumor population derived from stage I. In contrast, CEM was reactive with OKT4, OKT6, OKT8, OKT9 and 0KT10 and therefore derived from 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 0KT10.

Since all studied patients with T-cell acute lymphoblastic leukemia showed the presence of 0KT10+ cells, the use of 0KT10 antibody enables the diagnosis of T-cell ALL. Patients with Null cell ALL also show 0KT10+ cells.

The relationship shown in Tables 2-4 is a further way in which 0KT10 antibodies can be detected and distinguished from other antibodies.

Other monoclonal antibody-producing hybridomas prepared by the present applicants (designated as OKT1, OKT3, OKT4 and OKT5) are described and protected in the following U.S. Pat. patent applications: SN 22,132 filed on March 20, 1979; SN 33,639 filed on April 26, 1979; SN 33,669 filed on April 26, 1979; SN 76,642 filed on September 18, 1979; and SN 82,515 filed on October 9, 1979. Other monoclonal antibody-producing hybridomas prepared by the applicants shown (designated as OKT6, OKT8 and OKT9) are also protected by U.S. Pat. patent applications filed on an unknown date with the following names:

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

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

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

These applications are included in the above text with notes.

According to the presented invention, a hybrid is provided that can produce an antibody for an antigen found on approximately 95% of normal human thymocytes, a process for obtaining said hybridoma, a monoclonal antibody for an antigen found on approximately 95% of normal human thymocytes, methods for obtaining antibodies and methods and preparations for treatment or diagnosis diseases or for identification of T cells or sub-types of thymocytes using said antibody.

TABLE 1

REACTIVITY OF MONOCLONAL ANTIBODIES ON HUMAN LYMPHOID POPULATION

[image]

*Numbers in parentheses represent the number of treated samples;

values are in %.

TABLE 2

DIFFERENCES IN THE DISTRIBUTION OF ANTIGENS DETERMINED BY MONOCLONAL ANTIBODIES

ON HUMAN PERIPHERAL T CELLS AND THYMOCYTES

[image]

* Untreated populations and populations treated with complement alone were unrecognizable in re-analysis. Non-specific lysis was < 5 % in all cases. The results are the mean values of 6 experiments.

↑ C' = complement

TABLE 3

CHARACTERIZATION OF CELL SURFACES IN ACUTE

LYMPHOBLASTIC LEUKEMIA OF T ORIGIN

[image]

* The remaining 4 tumors could not be easily categorized into stages I-III. See the text for their detailed categorization.

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

TABLE 4

REACTIVITY WITH MONOCLONAL ANTIBODIES

[image]

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

Although only one hybridoma that produces one monoclonal antibody against human thymocyte antigen is described, it is understood that the presented invention encompasses all monoclonal antibodies that exhibit the above properties. The shown antibody 0KT10 was determined to belong to the IgG1 subtype, which is one of the four IgG subtypes. These subtypes of immunoglobulin G differ from each other in the so-called "fixed" regions, although an antibody to a specific antigen will have a so-called "variable" region that is functionally identical regardless of the subtype of immunoglobulin G. That is, a monoclonal antibody that shows the above properties can be of the subtype IgG1, IgG2a, IgG2b or IgG3 or IgM, IgA or other known Ig types. Differences between these species and subspecies will have no effect on the selectivity of the reaction pattern of the antibody, but will have an effect on further reactions of the antibody with other materials, such as (for example) complement or anti-mouse antibodies. Although an IgG1-specific antibody is shown, it is understood that antibodies having the shown reactivity pattern are included within the scope of the presented invention regardless of the associated type or subtype of immunoglobulin.

Furthermore, within the presented invention are also the procedures for the preparation of monoclonal antibodies described in the earlier text, which use the described hybridoma technique. Although only one example of a hybridoma is presented in the text, it is understood that experts in the field will be able to follow the immunization, fusion and selection procedures described in the text and thus obtain other hybridomas capable of producing antibodies that have the reactivity properties described in this text. Since an individual hybridoma obtained from a known murine myeloma cell line and spleen cells of known species of mice cannot be further identified except by the hybridoma produced antibody, it is understood that all hybridomas that produce an antibody having the previously described reactivity properties are included in the present invention as well as the methods obtaining these antibodies using hybridoma.

Further aspects of the invention are methods of treating or diagnosing diseases using monoclonal antibody 0KT10 or other monoclonal antibodies that exhibit the reactivity pattern described herein. The disclosed antibody can be used to detect and study intrathymic differentiation as shown in Figure 2. Furthermore, the disclosed antibody can be used to diagnose a disease state involving a deficiency or excess of 0RT10+ cells. These techniques can be used using the 0KT10 antibody alone or in combination with other antibodies (eg OKT3 to 0KT10). Patterns of reactivity with T cell antibody selection and T cell selection will enable more accurate detection of certain disease states than is possible using current diagnostic procedures.

Treatment of disease states (for example, malignancies such as Null cell ALL or T-ALL) manifested by an excess of 0KT10+ cells can be performed. by administering a therapeutically effective amount of 0KT10 antibody to the patient. By selectively reacting with 0KT10+ antigen, an effective amount of 0KT10 antibody will reduce the excess of 0KT10+ cells thereby mitigating the effects of the excess. Diagnostic and therapeutic compositions containing effective amounts of 0KT10 antibodies in admixture with diagnostically or pharmaceutically acceptable carriers are also included in the present invention.

Claims (16)

1. Monoclonal antibody of the IgG type produced using a hybridoma formed by the fusion of cells from a mouse myeloma line and spleen cells of a mouse previously immunized with human leukemic T-ALL cells, indicated that: a) reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E- peripheral mononuclear cells (B cells and Null cells) and with 10 to 20% of bone marrow cells; b) shows the pattern of reactivity with normal human thymocytes shown in Figure 1; c) shows the pattern of reactivity with peripheral T cells and thymocytes shown in Table 2; d) reacts with antigen on leukemic T-ALL cells; e) reacts with HSB-2, CEM and MOLT-4 cell lines. 2. Monoclonal antibody, indicated by the fact that it was produced from a hybridoma with the identification characteristics of ATCC 8022. 3. IgG monoclonal-antibody-producing hybrid formed by fusion of spleen cells of a mouse previously immunized with human leukemic T-ALL cells and cells of a mouse myeloma line, indicated that said antibody: a) reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E- peripheral mononuclear cells (B cells and Null cells) and with 10 to 20% of bone marrow cells; b) shows the pattern of reactivity with normal human thymocytes shown in Figure 1; c) shows the pattern of reactivity with peripheral T cells and thymocytes shown in Table 2; d) shows the pattern of reactivity with T cells of ALL state shown in table 3; e) reacts with HSB-2, CEM and MOLT-4 cell lines. 4. A hybridoma, characterized in that it has the identifying characteristics of ATCC 8022. 5. The procedure for preparing a monoclonal antibody which a) reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E- peripheral mononuclear cells (B cells and Null cells) and with 10 to 20% of bone marrow cells; b) shows the pattern of reactivity with normal human thymocytes shown in Figure 1; c) shows the pattern of reactivity with peripheral T cells and thymocytes shown in Table 2; d) shows the pattern of reactivity with T cells of ALL state shown in table 3; e) reacts with HSB-2, CEM and MOLT-4 cell lines, indicated by the fact that it contains steps: i) immunization of mice with human leukemic T-ALL cells; ii) removal of spleens from said mice and preparation of suspension of spleen cells; or) fusion of said spleen cells with mouse myeloma cells in the presence of a fusion promoter; iv) diluting and culturing the fused cells in separate dishes in a medium that will not support unfused myeloma cells; v) evaluation of the supernatant in each vessel 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. Procedure for preparing a monoclonal antibody which a) reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E- peripheral mononuclear cells (B cells and Null cells) and with 10 to 20% of bone marrow cells; b) shows the pattern of reactivity with normal human thymocytes shown in Figure 1; c) shows the pattern of reactivity with peripheral T cells and thymocytes shown in Table 2; d) shows the pattern of reactivity with T cells of ALL state shown in table 3; e) reacts with HSB-2, CEM and MOLT-4 cell lines, indicated by the fact that it contains steps: i) mouse intimacies with human leukemic T-ALL cells; ii) removal of spleens from said mice and preparation of suspension of spleen cells; iii) fusion of said spleen cells with mouse myeloma cells in the presence of a fusion promoter; iv) diluting and culturing the fused cells in separate dishes in a medium that will not support unfused myeloma cells; v) evaluation of the supernatant in each vessel 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 the mentioned clones; viii) transferring said clones intraperitoneally into mice; you ix) collection of malignant ascites or serum from the mentioned mice. 7. A method for detecting a lack or excess of OKT10+ cells in an individual, indicated by the fact that it contains the reaction of a preparation of T cells or thymocytes from the said individual with a diagnostically effective amount of antibody according to claim 12, and measuring the percentage of the total population of peripheral T cells and thymocytes that react with the said antibody . 8. The method according to claim 7, characterized in that the excess of T cells is ALL or Null cells ALL. 9. A method of treating excess OKT10+ cells in an individual, indicated by the fact that it comprises applying to said individual, an effective amount of antibody according to claim 12 so as to reduce the amount of OKT10+ cells. 10. A diagnostic preparation according to the invention that serves to detect an excess or deficiency of OKT10+ cells, characterized in that it contains, in a mixture with a diagnostically acceptable substrate, an amount of antibody according to claim 12 effective in detecting an excess or deficiency of OKT10+ cells. 11. Therapeutic preparation according to the invention, characterized in that it contains, in a mixture with a pharmaceutically acceptable base, an amount of antibody according to claim 12 effective in reducing the amount of OKT10+ cells in an individual who has an excess of said OKT10+ cells. 12. A murine monoclonal antibody, indicated to react with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E peripheral mononuclear cells (B cells and Null cells) and with 10 to 20% of bone marrow cells. 13. Method of preparing a monoclonal antibody that reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E-peripheral mononuclear cells (B cells and Null cells) and with 10 to 20% of bone marrow cells, indicated by to comprise culturing hybridoma ATCC CRL 8022 in a suitable medium and detecting antibodies from the supernatant above said hybridoma. 14. Method of preparing a monoclonal antibody that reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E-peripheral mononuclear cells (B cells and Null cells) and with 10 to 20% of bone marrow cells, indicated by to comprise injecting hybridoma ATCC CRL 8022 into a mouse and re-detecting the antibody from the malignant ascites or serum of said mouse. 15. Method of preparing a monoclonal antibody that reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E-peripheral mononuclear cells (B cells and Null cells) and with 10 to 20% of bone marrow cells, indicated by to include the steps: i) immunization of mice with human leukemic T-ALL cells; ii) removal of spleens from said mice and preparation of suspension of spleen cells; iii) fusion of said spleen cells with mouse myeloma cells in the presence of a fusion promoter; iv) diluting and culturing the fused cells in separate dishes in a medium that will not support unfused myeloma cells; v) evaluation of the supernatant in each vessel 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 the desired antibody that reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E" peripheral mononuclear cells (B cells and Null cells) and with 10 to 20% of bone marrow cells you vii) detection of antibodies from the supernatant above the mentioned clones. 16. A method of preparing a monoclonal antibody that reacts with approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E" peripheral mononuclear cells (B cells and Nuli cells) and with 10 to 20% of bone marrow cells, indicated by to include the steps: i) immunization of mice with human leukemic T-ALL cells; ii) removal of spleens from said mice and preparation of suspension of spleen cells; iii) fusion of said spleen cells with mouse myeloma cells in the presence of a fusion promoter; iv) diluting and culturing the fused cells in separate dishes in a medium that will not support unfused myeloma cells; v) evaluation of the supernatant in each vessel 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 approximately 95% of normal human thymocytes, 5% of normal human peripheral T cells, 10% of E~ peripheral mononuclear cells (B cells and Null cells) and with 10 to 20% of bone marrow cells; vii) transferring said clones intraperitoneally into a mouse; you viii) collection of malignant ascites or serum from said mice, and said ascites or serum contain the desired antibody.