HRP940823A2 - Hybrid cell line for producing monoclonal antibody to a human early thymocite antigen, antibody and method of preparation of this antibody - Google Patents

Description

Field of invention

This invention relates mainly to new hybrid cell lines and more specifically to hybrid cell lines for the production of a monoclonal antibody to an antigen found on early thymocytes (approximately 10% of normal human thymocytes), to the antibody so produced, and to therapeutic and diagnostic procedures and preparations using this antibody.

Description of previous knowledge

Condensation of murine myeloma cells to spleen cells from immunized mice described by Kohler and Milstein in 1975 /Nature 256, 495-497 (1975)/ demonstrated for the first time that it is possible to obtain a continuous cell line for the creation of a homogeneous (so-called "monoclonal" ) antibodies. After this initial work, many efforts were made to produce various hybrid cells (so-called "hybridomas") and to use the antibodies produced by these hybridomas for various scientific research.

See, for example, Current Topics in Microbiology and Immunology. Volume 81 - "Lymhocyte Hybridomas", F. Melchers, M. Potter, and N. Warner", Editors, Springer-Verlag, 1978, and references therein: C. J. Barnstable, et. al., Cell, 14, 9 -20 (May, 1978); P. Parham and W. F. Bodmer, Nature 276, 397-399 (November, 1978); Handbook of Experimental Immunology, Third Edition, Volume 2, D. M. Wier, Editor, Blackwall, 1978, Chapter 25; and Chemical and Engineering News, January 1, 1979, 15-17. These references indicate both successes and complications in attempts to produce a monoclonal antibody from a hybridoma. Although conceptually the general technique is well understood, there are many difficulties and variations are necessary in each particular case. In fact, there is no certainty, prior to attempting to make a given hybridoma, that the desired hybridomas will be obtained, that they will produce the antibody if obtained, or that the antibody so produced will have the desired specificity. The degree of success is usually affected the type of antigen used and the choice of technique used sti to isolate the desired hybridoma.

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

More recently, the present applicants and others have authored articles describing the construction and testing of hybridomas that make antibody to certain T-cell antigens.

See, for example, Reinherz, E. L., et al., J. Immunol, 123, 1312-1317 (1979); Reinherz, E.L., et al., Proc. Natl. Acad. Sci., 76, 4061-4065 (1979); and Kung, P.C., et al., Science, 206, 347-349 (1979).

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

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

It is now understood that T cells are divided into at least several subtypes, called "helper", "suppressor" and "killer" T cells, which have a function (whether they promote a reaction, suppress a reaction, or destroy (destroy)) foreign cells. . These subclasses are well understood for mouse systems, but have only recently been described for human systems.

See, for example, R.L. Evans, et al., Journal of Experimental Medicine, Volume 145, 221-232, 1977; and L. Chess and S. F. Schlossman - "Functional Analysis of Distinct Human T-Cell Subsets Bearing Unique Differentiation Antigens", in Topics in Immunobiology", O. Stitman, Editor, Plenum Press, 1977, Volume 7, 363-379.

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

For example, certain leukemias and lymphomas have a different prognosis depending on whether they are of B cell or T cell origin. Thus, the assessment of the prognosis of the disease depends on the distinction between these two classes of lymphocytes.

See, for example, A. C. Aisenberg and J. C. Long, The American Journal of Medicine 58:300 (March 1975) D. Belpomme, et al., in "Immunological Diagnosis of Leukemias AND 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, malignancies, and agammaglobulinemia) are associated with an imbalance of T cell subclasses. It has been suggested that autoimmune diseases are mainly accompanied by an excess of "helper" T cells or a deficiency of certain "suppressor" T cells, while agammaglobulinemia is accompanied by an excess of certain "suppressor" T cells or a deficiency of "helper" T cells. Malignant diseases are mostly accompanied by an excess of "suppressor" T cells.

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

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

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

Antisera against an entire class of human T cells (so-called antihuman thymocyte globulin or ATG) have been reported to be therapeutically useful in organ transplant patients.

Since the cell-mediated immune response (the mechanism by which transplants are rejected) depends on T cells, administration of antibodies to T cells prevents or delays the rejection process. See, for example, Cosimi, et al., "Randomized Clinical Trial of ATG in Cadaver Renal Aligraft Recipients: Importance of T Cell Monitoring," Surgery 40: 155-163 (1976) and references therein.

Identification and suppression of human T cells and classes and subclasses of monocytes has previously been achieved using spontaneous autoantibodies or selective antisera for human T cells obtained by immunizing animals with human T cells, bled animals to obtain serum, and adsorption of antisera with (for example) autologous but not allogeneic B cells to separate antibodies with unwanted reactivities. The production of these antisera is extremely difficult, especially in the phases of adsorption and purification. Even adsorbed and purified antisera contain many impurities in addition to the desired antibody, for several reasons. First, serum contains millions of antibody molecules even before T cell immunization. Second, immunization induces the production of antibodies against various antigens found in all injected human T cells. There is no selective production of antibodies against a single antigen. Third, the titer of the specific antibody obtained by such procedures is usually quite low (inactive at dilutions greater than 1:100) and the ratio of specific to non-specific antibody is less than 1/106.

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

Extract from the invention

A new hybridoma (designated OKT10) has now been found that can produce a new monoclonal antibody against an antigen found on approximately 10% of normal human thymocytes, but not on normal human peripheral lymphoid cells (T cells, B cells, or null cells) or bone marrow cells. core. These thymocytes with which OKT9 reacts are called "early thymocytes".

The antibody thus produced is monospecific for a single determinant on approximately 10% of normal human monocytes and contains essentially no other antihuman immune globulin, in contrast to prior art antisera (which are necessarily contaminated with antibody that reacts with numerous human antigens) and prior art monoclonal antibodies ( which are not monospecific for human thymocyte antigen). Moreover, this hybridoma can be cultured to produce antibody without the need to immunize and kill the animals, followed by the painstaking steps of adsorption and purification required to obtain even the impure antisera of earlier science.

Accordingly, one object of the present invention is to provide hybridomas that produce antibody against an antigen found on about 95% of normal human thymocytes.

A further invention is to provide methods for making these hybridomas.

A further aim of the invention is to provide a homogeneous antibody against an antigen found on about 10% of normal human thymocytes.

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

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

To satisfy the foregoing objects and advantages, the present invention provides a novel hybrid that produces a novel antibody to an antigen found on approximately 10% of normal human thymocytes (but not on normal human peripheral lymphoid cells or bone marrow cells), the antibody itself, both diagnostically and therapeutically procedures using antibodies. Hybridomas are made mainly according to the procedure given by Milstein and Kohler.

After immunization of mice with leukemic cells from humans with T-cell acute lymphoblastic leukemia, spleen cells from the immunized mice were condensed with cells from a murine myeloma line, and the resulting hybridomas were analyzed for those with supernatants containing an antibody that selectively binds to normal E rosette-positive human cells. T cells and/or thymocytes. The desired hybridomas are later cloned and characterized.

As a consequence of this, a hybridoma is obtained that produces an antibody (designated OKT9) against an antigen on approximately 10% of normal human thymocytes. Not only does this antibody react with about 10% of normal human thymocytes, but it also does not react with normal peripheral blood lymphoid cells or bone marrow cells.

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

Both the subject hybridomas and the antibody produced by them are identified herein by the designation "OKT", the particular material being referred to being apparent from the context of the text. The hybridoma in question was deposited on November 21, 1979, in the American Type Culture Collection, 12301 Porklawn Drive, Rockville, Mary and 20852, and has been given the ATCC accession number CRL 8021. The construction and characterization of the hybridoma and the resulting antibody will be better understood in view of reference following description and Examples.

Detailed description of the invention

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

A. Immunization of mice with leukemic cells from humans with ALL T-cells. Although female CAF1 mice have been found to be preferred, it is understood that other types of mice may be used. The schedule of immunization and the concentration of thymocytes should be such that useful quantities of suitably primed splenocytes are produced. It was found that three immunizations at fourteen-day intervals with 2 x 107 cells / mice / injections in 0.2 ml of phosphate buffered saline solution are effective.

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

C. Condensation of suspended spleen cells with murine myeloma cells from an appropriate cell line using an appropriate condensation promoter. The preferred ratio is about 5 spleen cells per myeloma cell. The total volume of about 0.5 - 1 ml of condensation medium is suitable for about 108 splenocytes. Many murine myeloma cell lines are known, mostly from members of academic societies of various depository banks, such as the Salk Institute Cell Distribution Center, La Jolla, CA. The cell line used should preferably be of the so-called "drug-resistant" type, so that non-condensed myeloma cells will not survive in the chosen medium, while hybrids will. The most common class are cell lines resistant to 8-azaguanine, which lack the enzyme hypoxanthine guanine phosphoribosyl transferase and therefore will not be supported by HAT (hypoxanthine, aminopterin, and thymidine) media. It is also generally preferred that the myeloma cell line be used as a so-called "non-secreting" type, so that it does not produce any antibody by itself, although secreting types may be used. However, in certain cases, secretory-type lines may be preferred. Although the preferred condensation promoter is polyethylene glycol having an average molecular weight of about 1000 to about 4000 (commercially available as PEG 1000, etc.), other condensation promoters known in the art can be used.

D. Diluting and culturing in separate containers, a mixture of uncondensed spleen cells, uncondensed myeloma cells, and condensed cells in a selective medium that will not support uncondensed myeloma cells for a satisfactory period to allow death of the uncondensed cells (about one week). The dilution can be of the limiting type, in which the volume of diluent is statistically calculated to isolate a certain number of cells (eg, 1-4) in each separate container (eg, each well of a microtiter plate). The medium is one (eg, HAT medium) that will not support an unfused drug-resistant (eg, 8-azaguanine-resistant) cell line.

E. Because of this, mouse myeloma cells disappear. Since non-condensed spleen cells are not malignant, they can only have a definite number of generations. Thus, after a certain period of time (about one week), these uncondensed spleen cells will fail to reproduce. On the other hand, the condensed cells continue to reproduce because they have the malignant quality of myeloma origin and the ability to survive in the selective medium for the source of spleen cells.

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

F. Selection (eg, by limiting dilution) and cloning of hybridomas that produce the desired antibody. Once the desired hybrid has been selected and cloned, the resulting antibody can be produced in one of two ways. The purest monoclonal antibody is produced in vitro by culturing the desired hybridoma in a suitable medium for a suitable length of time, followed by isolation of the desired antibody from the supernatant. A suitable substrate and a suitable length of cultivation time are known or easily determined. This in vitro technique produces an essentially monospecific monoclonal antibody, essentially free of other specific antihuman immune globulin. There is a small amount of other immune globulin present since the medium contains xenogeneic serum (eg, fetal calf serum). However, this in vitro procedure may not produce a sufficient amount or concentration of antibody for the same purposes, since the concentration of the monoclonal antibody is only about 50 μg/ml. To produce a much higher concentration of a slightly less pure monoclonal antibody, the desired hybridoma can be injected into mice, preferably syngeneic or semisyngeneic mice. Hybridomas will cause the formation of tumors that produce the antibody after a suitable incubation time, which will lead to a high concentration of the desired antibody (about 5-20 mg/ml) in the bloodstream and peritoneal exudate (ascites) of the host mouse. Although these host mice have normal antibodies in their blood and ascites, the concentration of these normal antibodies is only about 5% of the concentration of the monoclonal antibody. Moreover, since these normal antibodies are not anti-human in their specificity, the monoclonal antibody obtained from harvested ascites or from serum is essentially free of any contaminating anti-human immune globulin. This monoclonal antibody has a high titer (active at dilutions of 1:50,000 or higher) and a high ratio of specific to non-specific immune globulin (about 1/20). Immune globulins produced by the incorporation of myeloma light sequences are non-specific, "nonsense" peptides that only dilute the monoclonal antibody without reducing its specificity.

Examples.

Production of monoclonal antibodies

A. Immunization and hybridization of somatic cells

Female CAF1 mice (Jackson Laboratories; 6-8 weeks old) were immunized intraperitoneally with 2 x 107 human leukemic T-ALL cells (patient J.F.) in 0.2 ml phosphate buffered saline at 14-day intervals. Four days after the third immunization, the spleens were separated from the mice and a single cell suspension was made by pressing the tissue through a stainless steel mesh. Cell condensation was performed according to the procedure developed by Kohler and Milstein.

1 x 108 splenocytes were condensed in 0.5 ml condensation medium containing 35% polyethylene glycol (PEG 1000) and 5% dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, NY) with 2 x 107 P3x63Ag8U1 myeloma cells provided by Dr. M. Scharff, Albert Einstein College of Medicine, Bronx, NY. These myeloma cells secrete IgG1 light strings.

B. Selection and growth of hybridomas

After cell condensation, the cells were cultured in HAT medium (hypoxanthine, aminopterin and thymidine) at 37°C with 5% CO2 in a humid atmosphere. Several weeks later, 40 to 100 1 of the supernatants from the hybridoma-containing cultures are added to a pellet of 10 6 peripheral lymphocytes that have been separated into E rosette positive (E+) and E rosette negative (E-) populations, and which have been prepared from the blood of healthy human subjects. donor as described in Mendes (J. Immunol. 111:860, 1973). Detection of murine hybridoma antibodies that bind to these cells was determined by indirect immunofluorescence.

Cells incubated with tissue supernatants were stained with fluorescent goat anti-mouse IgG (G/M FITC) (Meloy Laboratories, Springfield, VA; F/p = 2.5) and cells coated with fluorescent antibody were later analyzed on a Cytofluorograph FC200/4800A. (Ortho Instruments, Westwood, MA) as described in Example III. Hybridoma cultures containing antibodies that react specifically with E+ lymphocytes (T cells) and/or thymocytes were selected and cloned twice by limiting dilution procedures in the presence of feeder cells. Later, clones were transferred intraperitoneally by injecting 1 x 10 7 cells of a given clone (volume 0.2 ml) into CAF1 mice primed with 2, 6, 10, 14-tetramethylpentadecane, sold by Aldrich Chemical Company under the name Pristine. Malignant ascites from all mice are then used to characterize lymphocytes as described below in Example II. It was demonstrated by standard techniques that the subject hybrid antibody OKT9 belongs to the IgG1 subclass.

Example II.

Characterization of Oct9 reactivity

A. Isolation of lymphocyte populations

Human peripheral blood mononuclear cells were isolated from healthy human volunteers (age 15-40) using Ficoll-Kypaqua density gradient centrifugation (Pharmacia Fino chemicals, Piscataway, NJ) according to the technique of Bayum, Scend. J. Clin. Lab. Invest. 21 (Suppl. 97):77. 1968. Fractionated mononuclear cells are separated into surface Ig+ (8) and Ig- (T plus null) populations by Sephadex G-200 anti-F(ab`)2 column chromatography as previously described in Chess, et al., J. Immunol. 113:1113 (1974). T cells are regenerated by E rosetting the Ig- population with 5% sheep erythrocytes (Microbiological Associatos. Bathasda, MD). The rosette mixture was layered over Ficoll. Hypaque and regenerated E+ sent treated with 0.155M NH4Cl (10 ml per 108 cells). The resulting T cell population was <2% EAG rosette positive and >95% E rosette positive as determined by standard procedures. Further, the non-rosetting Ig- population was separated from the Ficoll interface. This latter population was .<5% E+ and ≤2% sIg+. The surface Ig+ population was obtained from a Sephadex G-200 column after elution with normal human gamma globulin as described earlier. This population was >95% surface Ig+ and <5% E+. Normal human bone marrow cells were obtained from the posterior iliac crest of normal human volunteers by needle aspiration.

B. Isolation of thymocytes

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

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

T cell lines GEM, HSB-2, and MOLT-4 were provided by Dr. H. Lazaruo (Sidnay Farber Cencor Institute, Boston, MA). Leukemic cells were obtained from 25 patients diagnosed with ALL T cells. These individual tumors were previously determined to belong to the T cell lineage by their ability to spontaneously form rosettes with sheep erythrocytes (>20% E+), and reactivity with specific anti-HTL antisera to T cells (B.K) and A99, as described earlier. Tumor populations were cryopreserved at -196°C with liquid nitrogen in the vapor phase with 10% OMSD and 20% AB human serum until the time of surface characterization. All analyzed tumor populations were more than 90% disease using Wright-Giemsa morphology of cytocentri-fused preparations.

Example III.

Cytofluorographic analysis and cell separation

Cytofluorographic analysis of monoclonal antibodies with all cell populations was performed by indirect immunofluorescence with fluoroscein-conjugated goat anti-mouse IgG (G/M FITC) (Maloy Laboratorios) using a Cytofluorograf FC200/4000A (Ortho Instrumenta). Briefly, 1 x 106 cells are treated with 0.15 ml OKT5 at a 1:500 dilution, incubated at 4°C for 30 minutes and washed twice. The cells are then reacted with 0.15 ml of a 1:40 dilution of G/M FITC at 4°C for 38 minutes, centrifuged and washed three times. The cells are then analyzed on the Cytofluorograph, and the fluorescence intensity per cell is recorded on the pulse height analyzer. A similar pattern of reactivity was seen at 1:10,000 dilution, but further dilution caused loss of reactivity. Baseline staining is achieved by substituting a 15 ml aliquot of 1:500 ascites from CAF1 mice injected intraperitoneally with a non-producing hybrid clone.

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

Example IV.

Disruption of lymphoid populations with monoclonal antibody and complement

Forty x 10 6 peripheral T cells or thymocytes are placed in a 15 ml plastic tube (Falcon, Oxnard, CA). Cell materials are incubated with OKT3, OKT4, OKT5 or with normal control ascites diluted 1:200 in PBS, resuspended and incubated at 20°C for 30 minutes. Later, 0.2 of fresh rabbit complement is added to the antibody-treated populations, resuspended, and further incubation is performed at 37°C in shaking water for 60 minutes. At the end of this period, cells are spun down and viable cells are numbered using Trypan blue exclusion. After counting, the cells are washed two more times in 5% FCS and plated in final media (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 the scheme of fluorescence obtained on a Cytofluorograph after the reaction of normal human thymocytes with OKT9 and other monoclonal antibodies at 1:500 dilution and G/M FITC. Basic fluorescent staining was achieved by incubating this population with a 1:500 dilution of ascitic fluid from a mouse injected with na-production clones.

Figure 2 shows the stages of intrathymic differentiation in humans. Hybridoma production and the production and characterization of the resulting monoclonal antibody were carried out as described in the Examples above. Although large quantities of the subject antibody have been made by injecting the subject hybridoma intraperitoneally into mice and harvesting the malignant ascites, it is clearly contemplated that the hybridoma may be cultured in vitro by techniques well known in the art and the antibody then separated from the supernatant.

Table 1 shows the reactivity of OKTE, OKT8, OKT9 and OKT10 with various human lymphoid cell populations. The OKT9 monoclonal antibody reacts with approximately 10% of normal human thymocytes and not with other lymphoid cells tested.

This reactivity scheme is one test by which the subject OKT9 antibody can be detected and distinguished from other antibodies.

Figure 1 shows a representative scheme of reactivity obtained on Cytofluorograf after reaction of normal human thymocyte suspensions with 1:500 dilution of OKT3, OKT4, OKT5, OKT6, OKT8, OKT9, OKT10 and G/M FITC. Similar patterns of reactivity are seen with 12 additional tested normal human thymocyte populations. As shown, there are significant differences in both percent reactivity and fluorescence intensity with each of these monoclonal antibodies.

For example, OKT9 reacts with approximately 10% of thymocytes with low fluorescence intensity while OKT5, OKT8 and OKT10 react with approximately 70% of thymocytes with higher fluorescence intensity. OKT4, which reacts with 75% of thymocytes, is midway between OKT9 and monoclonal antibodies that give a pattern with higher fluorescence intensity. Further, Figure 1 shows that approximately 15% of thymocytes were detected with OKT3 by indirect immunofluorescence. OKT1, whose reactivity scheme is practically identical to OKT3 on thymocytes, is not shown. The reactivity scheme in Figure 1 is another test by which the subject OKT9 antibody can be detected and distinguished from other antibodies.

Table 2 shows the distribution of antigens defined by various monoclonal antibodies on human peripheral T cells and lymphocytes, as determined by a series of pull-down experiments described in Example IV. Since only OKT3, OKT4 and OKT8 were complement-fixed monoclonal antibodies, these three were used.

As shown in Table 2A, the entire T cell population reacts with OKT3 while OKT4, OKT5 and OKT8 react with 50%, 25% and 34% of T cells. Breakdowns with DKT4 and complement reduced the total number by 62% and the OKT4+ population was specifically absent. Furthermore, the percentage of OKT5+ and OKT8+ cells increased and there was no effect on the absolute number of OKT5+ and OKT8+ cells. These experiments suggested that OKT4+ is distinct from OKT5+ and OKT8+ populations. Further evidence for this conclusion was obtained by disrupting T cells with OKT8 and complement. In this case, the percentage of OKT4+ cells increased, while the absolute number remained the same, and the OKT8+ and OKT5+ populations were eliminated. Moreover, the results showed that the OKT8+ population was reciprocal to the OKT4+ population and contained the entire subset of OKT5+ T cells.

Similar experiments with human thymocyte populations yielded mixed results. As shown in Table 2B, approximately 75% of thymocytes were OKT4+ or OKT8+. Moreover, after knocking out either OKT4 or OKT8, only 25% of thymocytes remained. The main part of the residual thymocytes was reactive with OKT3, while only a smaller part reacted with OKT8. These findings demonstrate that a major population of human thymocytes carries OKT4, OKT5, OKT5, and OKT8 surface antigens on the same cell. Further, Table 2 demonstrates that after treatment with OKT8 or OKT4, there is a significant increase in mature thymocytes bearing the OKT3 antigen. Thus, the main part of OKT3 reactive thymocytes was already segregated in OKT4+ or OKT8+ tests, since the main amount of residual cells after OKT4 and OKT8 disruption was OKT3+. If the OKT3+ subpopulation was both OKT4+ and OKT8, then stripping with either monoclonal antibody should separate OKT3 reactive thymocytes.

To further determine the relationship of OKT3-reactive thymocyte subpopulations to another monoclonal antibody that defined thymocyte fractions, thymocytes were treated with OKT3 and complement and residual cells were then compared to untreated thymocyte populations. As shown in Table 2B, OKT3 and complement separated 25% of thymocytes. Moreover, there was no major loss of OKT4, OKT5, OKT6, or OKT8 reactive populations after termination. These findings suggest that a large excess of OKT8-bearing thymocytes is at least in the OKT3- population. Furthermore, they further suggest that thymocytes co-expressing antigens defined by OKT4, OKT5 and OKT6 are similarly restricted to the OKT3 population. It should also be emphasized that the OKT9 reactive thymocyte population was not reduced after treatment with OKT3 and a complement of unfractionated thymocytes, indicating that the OKT9+ subpopulation is mostly limited to the OKT3- thymocyte population.

Based on these results, it is possible to describe the stages of intrathymic development of human thymocytes. As shown in Figure 2, virtually all thymocytes carry the OKT10 marker. Furthermore, thymocytes have an OKT9 marker (that is, Thy1 and Thy 2) at an early stage. This phase defines a smaller number of thymocytes and accounts for approximately 10% of the unfractionated population. Later, human thymocytes acquire a unique thymocyte antigen defined by OKT6 and competitively express OKT4, OKT5 and OKT8 (Thy 4). This last subpopulation represents the main part of thymocytes and explains up to 70-80% of the thymic population. With further maturation, thymocytes lose OKT6 reactivity, acquire OKT3 (and OKT1) reactivity, and divide into OKT4+ and OKT5, OKT8+ subsets (Thy 7 and Thy 8). Finally, it appears that as the thymocyte enters the peripheral T cell compartment, it loses the OKT10 marker since this antigen is missing from virtually all peripheral T lymphocytes. Possible transit phases between these three main phases of development in the thymus are labeled Thy 3, Thy 5, and Thy 6 in Figure 2.

Because T-lineage acute limboblastic leukemia is thought to be derived from immature thymocytes, the relationship between the tumor cells of individuals with T-ALL and these proposed stages of intrathymic differentiation was determined.

25 tumor cell populations from individuals with T-ALL and three T cell lines previously studied with conventional anti-7 cell reagents and E rosetting were examined, as shown in Table 3, the major part of T-ALL leukemic cells reacted or only with OKT10 or with OKT9 and OKT10, and did not react with other monoclonal antibodies. Thus, 15/25 of the studied cases seem to have early thymocyte antigens (Phase I.).

In contrast, 5/25 cases reacted with OKT8, suggesting derivation from a more mature thymic population (Phase 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 a majority of thymocyte antigens including OKT4, OKT6, and OKT8. It is worth mentioning that OKT5 was not present in any of these 5 Stage II tumors even when OKT8 reactivity was observed. This last result suggests that OKT5 and OKT8 define different antigens or different determinants on the same antigen. Finally, the tumors of 1/25 patients came from a mature thymocyte population (Phase III) as defined by reactivity with OKT3. In addition, this individual's tumor reacted with OKT5, OKT8 and OKT10. Of the 25 leukemic populations analyzed, only four tumors could not be clearly categorized. Three were positive with OKT4 and OKT3, but lacked OKT3 and OKT6 and most likely represented transitions from Thy4 and Thy7. One of the 25 cases appeared to be a transition from Thy3 to Thy4 as it possessed OKT3 and OKT10 reactivity.

T cell lines derived from T-ALL tumor populations also represented cells from a specific state of intrathymic differentiation. As shown in Table 4, HSB reacted only with OKT9 and OKT10 and will therefore define a Phase I-derived tumor population. In contrast, CEM reacted with OKT4, OKT6, OKT8, OKT9 and OKT10 and appears to be derived from Phase I thymocytes. II. Finally, MOLT-4 appears to represent leukemic transformation at a stage between HSB-2 and CEM as it expresses OKT5, OKT8, OKT9 and OKT10.

Since patients with later stages (eg, Stage II) of acute lymphoblastic leukemia have been shown to have longer disease-free survival than those with Stage I ALL, the use of the OKT9 antibody allows conclusions regarding the prognosis of a given patient with T-cell ALL.

The relationships shown in Tables 2-4 are a further means by which the OKT9 antibody can be detected and distinguished from other antibodies.

Other monoclonal antibody-producing hybridomas made by the present applicants (designated OKT1, OKT3, OKT4 and OKT5) are described and protected in the following U.S. Pat. patent applications: SN 22,132, filed March 20, 1979, SN 33,639, filed April 28, 1979, SN 33,668, filed April 26, 1979, SN 76,642, filed September 14, 1979, and SN 82,515, filed October 9 , 1979

Further other monoclonal antibody-producing hybridomas made by the present applicants (designated OKT6, OKT8 and OKT10) are described and patented in U.S. Pat. patent applications that were filed on the same date as the present application and have the titles:

Hybrid Cell Line For Producing Complement-Fixing Monoclonal Antibody to Human Suppressor T Cell, Antibody, and Methods, Hybrid Cell Lina For Producing Monoclonal Antibody to Human Thymocyta Antigen, Antibody, and Methods, and Hybrid Cell Lina For Producing Monoclonal Antibody to a Human Protnymocyte Antigen, Antibody, and Mathods.

These applications are incorporated herein by reference.

According to the present invention, there is provided a hybridoma capable of producing an antibody against an antigen found on approximately 10% of normal human thymocytes, a process for the production of this hybridoma, a monoclonal antibody against an antigen found on approximately 10% of normal human thymocytes, methods for the production of antibodies, and methods and preparations for treating or diagnosing disease or identifying T cells or thymocyte subclasses using this antibody.

Table 1.

Reactivity of monoclonal antibodies to human lymphoid populations

[image]

Numbers in parentheses represent the number of tested samples, % values are averages.

Table 2.

Differences in the distribution of monoclonal antibody-defined antigens on human peripheral T cells and thymocytes

[image]

* Untreated populations and populations treated only with complement could not be distinguished by re-analysis. Non-specific termination was 5% of all cases. Results are shown of 6 experiments ;C` = complement ;Table 3. ;Cell surface characteristics of T-lineage acute lymphoblastic leukemia ;[image] ;* The last four tumors cannot be easily categorized into Stage I-III - See text for details of their characterization .

+ Thy symbol refers to Figure 2

≠ Positive (+) reactivity is defined as 30% specific fluorescence above the baseline control, a

negative (-) reactivity is indistinguishable from baseline staining on tumor cell suspensions.

Table 4.

Reactivity with monoclonal antibodies

[image]

The criteria for - and + reactivity were the same as in Table 3.

Although only one hybridoma has been described that produces a monoclonal antibody against a human monocyte antigen, it is understood that the present invention includes all monoclonal antibodies that exhibit the characteristics described herein. The antibody in question, OKT9, was determined to belong to the IgG1 subclass, which is one of the four subclasses of mouse IgG. These subclasses of immunoglobulin G differ from each other by so-called "fixed" regions, although an antibody to a specific antigen will have a so-called "variable" region that is functionally identical regardless of which class of immunoglobulin G it belongs to.

That is, a monoclonal antibody exhibiting the characteristic described herein may be of the IgG1, IgG2a, IgG2b, or IgG3 subclasses, or of the IgM, IgA, or other known Ig classes. The difference between these classes or subclasses will not affect the selectivity of the reaction scheme of the antibody, but may affect the further reaction of the antibody with other materials, such as, for example, complement or anti-mouse antibodies. Although the subject antibody is IgG1 specific, it is contemplated that antibodies having the reactivity pattern described herein are included in the subject invention regardless of which class or subclass they belong to.

Further included in the present invention are methods for making the monoclonal antibodies described herein using the hybridoma techniques described herein. Although only one example of a hybrid is given here, it is understood that one skilled in the art will be able to follow the immunization, condensation and selection procedures provided herein to obtain other hybridomas capable of producing antibodies having the reactivity characteristics described herein.

Since individual hybridomas produced from a known murine myeloma cell line and spleen cells from a known strain of mouse cannot be further identified except by the antibody produced by the hybridoma, it is understood that all hybridomas producing an antibody having the reactivity characteristics described herein are included in the present invention , as well as procedures for making this antibody using hybridomas.

Further aspects of the invention are methods for treating or diagnosing a disease using the OKT9 monoclonal antibody or any other monoclonal antibody exhibiting the reactivity pattern provided herein. The subject antibody can be used to detect and study intrathymic differentiation as summarized in Figure 2. Abnormalities in Phase I differentiation will be indicated by a deviation of about 10% OKT9+ thymocytes.

Moreover, the subject antibody can be used to diagnose disease states involving a defect or excess of OKT9+ cells. These techniques can be used using OKT9 antibodies alone or in combination with other antibodies (eg, OKT3 - OKR10). A reactivity scheme with a panel of antibodies to T cells and subsets of the T pause will enable precise detection of certain disease states than is possible using earlier diagnostic procedures.

Treatment of disease states (eg, malignancies such as certain cases of Stage I or II ALL) manifested by an excess of OKT9+ cells can be achieved by administering a therapeutically effective amount of OKT9 antibody to an individual in need of such treatment.

By selectively reacting with the OKT9+ antigen, an effective amount of OKT9 antibody will reduce the excess of OKT9+ cells, thus mitigating the effects of the excess. Diagnostic and therapeutic preparations comprising effective amounts of OKT9 antibody in admixture with diagnostically or pharmaceutically acceptable carriers are also included within the present invention.

Claims (20)

1. A mouse monoclonal antibody, characterized in that (i) reacts with approximately 10% of normal human thymocytes; (ii) does not react with any of the normal human peripheral cells from the group consisting of T cells and null cells; and (iii) does not react with bone marrow cells. 2. Monoclonal antibody according to claim 1, characterized in that it belongs to the IgG class. 3. Monoclonal antibody according to claim 1 or 2, characterized in that it shows the form of reactivity towards normal human thymocytes shown in Figure 1. 4. Monoclonal antibody according to any one of claims 1 to 3, characterized in that it shows the form of reactivity against peripheral T cells and thymocytes shown in table 2. 5. Monoclonal antibody according to any one of claims 1 to 4, characterized in that it shows the form of reactivity against T cells of grade ALL shown in table 3. 6. Monoclonal antibody according to any one of claims 1 to 5, characterized in that it defines a T cell antigen that appears on 30 to 50% of peripheral T cells after mitogenic stimulation. 7. Monoclonal antibody according to any one of claims 1 to 6, characterized in that it is produced by means of a hybridoma created by the fusion of cells from a murine myeloma line and spleen cells from a mouse previously immunized with human leukemic T-ALL cells. 8. Monoclonal antibody, characterized in that it is produced from hybridoma ATCC CRL 8021 (OKT9). 9. A hybridoma, characterized in that it produces a monoclonal antibody according to any one of claims 1 to 7, obtained by fusion of spleen cells from a mouse pre-immunized with human leukemic T-ALL cells and cells from a murine myeloma line. 10. Hybridoma, indicated that it is ATCC CRL 8021 (OKT9). 11. The method for producing a monoclonal antibody according to any one of claims 1 to 7, characterized in that it includes the following steps: i) immunization of mice with human leukemic T-ALL cells; ii) extracting the spleen from said mouse and making a suspension of spleen cells; iii) fusing said spleen cells with murine myeloma cells in the presence of a fusion promoter; iv) diluting and culturing the fused cells in separate wells in a medium that does not support unfused myeloma cells; v) evaluating the supernatant in each well containing the hybridoma for the presence of an antibody having the properties set forth in any of claims 1 to 7; vi) selecting and cloning hybridomas that produce the desired antibody; and vii) collection of antibodies from the supernatant above the mentioned clones. 12. The method for producing a monoclonal antibody according to any one of claims 1 to 7, characterized in that it includes the following steps: i) immunization of mice with human leukemic T-ALL cells; ii) extracting the spleen from said mouse and making a suspension of spleen cells; iii) fusing spleen cells with murine myeloma cells in the presence of a fusion promoter; iv) diluting and culturing the fused cells in separate wells in a medium that does not support unfused myeloma cells; v) evaluating the supernatant in each well containing the hybridoma for the presence of an antibody having the properties set forth in any of claims 1 to 7; vi) selecting and cloning hybridomas that produce the desired antibody; vii) transfer of said clones intraperitoneally into mice; and viii) collection of malignant ascites or serum from said mouse. 13. A method for the production of a monoclonal antibody, characterized in that it includes growing the hybridoma ATCC CRL 8021 in a suitable medium and collecting the antibody from the supernatant above said hybridoma. 14. A method for producing a monoclonal antibody, characterized in that it includes injecting hybridoma ATCC CRL 8021 into a mouse and obtaining the antibody from malignant ascites or serum from said mouse. 15. A diagnostic composition of substances for the detection of an excess or lack of OKT9+ cells, characterized in that, in a mixture with a diagnostically acceptable carrier, it contains an amount of antibody according to any of claims 1 to 8 effective for detecting an excess or lack of OKT9+ cells. 16. Therapeutic composition of matter, characterized in that, in a mixture with a pharmaceutically acceptable carrier, it contains an amount of antibody according to any one of claims 1 to 8 effective for reducing the amount of OKT9+ cells in a person who has an excess of said OKT9+ cells. 17. A method of detecting a lack or excess of OKT9+ cells, indicated by the fact that it includes the reaction of a composition of T cells or a thymocyte composition, obtained from a person, with a diagnostically effective amount of antibodies according to any one of claims 1 to 8, or with a composition according to claim 15, and measurement the percentage of T cell composition or thymocyte composition that reacts with the antibody. 18. The method according to claim 17, characterized in that it is an excess of T ALL cells. 19. The monoclonal antibody according to any one of claims 1 to 8, or the composition according to claim 16, characterized in that it is used to treat a person who has an excess of OKT9+ cells. 20. A method for producing a composition according to claim 15 or claim 16, characterized in that it comprises mixing a diagnostically or therapeutically effective amount of antibody according to any of claims 1 to 8 with a diagnostically or therapeutically acceptable carrier.